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Effects of doxycycline on heartworm embryogenesis, transmission, circulating microfilaria, and adult worms in microfilaremic dogs
... As a follow-up to the above studies, another study was conducted to determine the effects of doxycycline administered orally, twice daily (bid) at 10 mg/kg for 30 days on heartworm embryogenesis, transmission, circulating mf and adult worms in microfilaremic dogs [12]. The study was designed to determine whether heartworm mf collected at later times after treatment would regain the ability to develop to L 3 which could complete normal development in a dog. ...
... Considering that circulating mf are present for weeks to months after doxycycline plus ML treatment is started, the question arises as to whether these lingering mf are capable of infecting susceptible mosquito vectors and, if so, whether the L 3 from these mosquitoes will develop normally and migrate to the heart and pulmonary arteries of dogs after transmission. Earlier work in this laboratory demonstrated that L 3 collected from mosquitoes fed on blood from dogs 4 days or later after 6 weeks of doxycycline administered orally at 10 m/kg sid with no ML [11] or 43-134 days after 30 days of doxycycline administered orally at 10 mg/kg bid for 30 days with no ML [12] were not capable of developing and migrating to the heart and pulmonary arteries of heartworm-naïve dogs. ...
... The data from this study strongly support the inclusion of doxycycline and an ML in the American Heartworm Society recommended heartworm adulticide protocol, as it adds the significant epidemiological benefit of blocking the normal development of heartworm L 3 , even MLresistant biotypes [12], after they are transmitted under natural conditions to the animal host. ...
Background
This study was conducted to determine whether heartworm infective larvae (L3) collected from mosquitoes fed on dogs during low-dose, short-treatment-regimen doxycycline and ivermectin could develop normally in dogs.
Methods
Twelve Beagles in a separate study were infected with 10 pairs of adult male and female Dirofilaria immitis by IV transplantation and randomly allocated to three groups of four dogs. Starting on Day 0, Group 1 received doxycycline orally at 10 mg/kg sid for 30 days plus ivermectin (min., 6 mcg/kg) on Days 0 and 30; Group 2 received doxycycline orally at 10 mg/kg sid until individual dogs became microfilaria negative (72–98 doses) and ivermectin every other week for six to seven doses. These dogs served as microfilaremic blood donors for the current mosquito studies.
Aedes aegypti were allowed to feed on group-pooled blood samples from treated Groups 1-M and 2-M and untreated control Group 3-M on Days 22 (Study M-A) and 42 (Study M-C) and from Groups 1-M and 2-M on Day 29 (Study M-B) after treatment was started. From the Day 22 mosquito feeding, two dogs in Groups 1-M and 2-M and one dog in Group 3-M were given 50 L3 by SC inoculation. From the Day 29 feeding, two dogs in Groups 1-M and 2-M were given 50 L3. From the Day 42 feeding, two dogs in Group 1-M received 30 L3, while two dogs in Group 2-M and one dog in Group 3-M received 40 L3. All 14 dogs were necropsied for recovery and enumeration of adult heartworms 163–183 days PI.
Results
None of the 12 dogs that received L3 from mosquitoes fed on blood from treated dogs 22, 29 or 42 days after treatment started had any adult heartworms at necropsy, while the two control dogs had a total of 26 and 43 heartworms, respectively.
Conclusions
Treatment of microfilaremic dogs with doxycycline plus an ML, which later renders the L3 incapable of normal development in the animal host, widens the scope of the multimodal approach to heartworm prevention in reducing the spread of heartworm disease.
Graphical Abstract
... In regard to D. immitis, the tetracycline drug doxycycline, administered with or without a macrocyclic lactone (ML) preventive, has been shown to adversely affect every life-cycle stage: circulating microfilariae (mf ) [3,[19][20][21][22][23][24][25][26], infective third-stage larvae (L3) and fourth-stage larvae (L4) [23,27], juveniles [20,27], young adults [20], and mature adults [19, 21-23, 25, 26, 28]. ...
... In regard to D. immitis, the tetracycline drug doxycycline, administered with or without a macrocyclic lactone (ML) preventive, has been shown to adversely affect every life-cycle stage: circulating microfilariae (mf ) [3,[19][20][21][22][23][24][25][26], infective third-stage larvae (L3) and fourth-stage larvae (L4) [23,27], juveniles [20,27], young adults [20], and mature adults [19, 21-23, 25, 26, 28]. ...
... The ML is expected to prevent further infection and gradually reduce microfilaremia levels. However, circulating mf can be detected in some dogs for several months after starting the administration of doxycycline or ivermectin alone or doxycycline plus an ML [22,23,25]. Monthly prophylactic doses of ivermectin along with a month of daily doses of doxycycline administered orally at 10 mg/kg twice daily (bid) can significantly reduce Wolbachia levels, reduce pulmonary infiltrates, reduce worm biomass, decrease post-treatment complications and morbidity [30,31], and impede the spread of both ML-resistant and ML-susceptible heartworms [22,23]. ...
Background
Microfilarial (mf) counts were monitored over 21.3 months for any rebound that might occur in counts, and adulticidal efficacy was assessed following administration of low dosage with short- and long-treatment regimens of doxycycline and ivermectin to heartworm-microfilaremic dogs.
Methods
Twelve heartworm-naïve beagles infected with 10 pairs of adult Dirofilaria immitis by intravenous transplantation were randomly allocated to three groups of four dogs. All treatments started on day 0. On day 0, Group 1 (short-treatment regimen) received doxycycline orally at 10 mg/kg once daily for 30 days plus ivermectin orally (minimum, 6 mcg/kg) on days 0 and 30. Group 2 (long-treatment regimen) received doxycycline orally at 10 mg/kg once daily until individual dogs became mf-negative (72–98 days) and ivermectin every other week until individual dogs became mf-negative (6–7 doses). Group 3 was the untreated control. Mf counts and antigen (Ag) tests were conducted. Dogs were necropsied for recovery and enumeration of heartworms on day 647.
Results
Day −1 mean mf counts were 15,613, 23,950, and 15,513 mf/ml for groups 1, 2, and 3, respectively. Mean counts for Groups 1 and 2 declined until days 239 and 97, respectively, when all were negative. Group 3 had high mf counts throughout the study. There was not a rebound in mf counts in any of the treated dogs after they became amicrofilaremic. All dogs in group 1 and group 3 were Ag-positive throughout the study and had at least one live female worm at necropsy. All dogs in treated Group 2 were positive for Ag through day 154, but were antigen-negative on days 644 and 647, as all had only male worms. Mean live adult worm recoveries for Groups 1, 2, and 3 were 6.8 (range, 5–8), 3.3 (range, 1–6), and 16.0 (range, 14–17), respectively, with a percent reduction in adult worm counts of 57.5% for Group 1 and 79.3% for Group 2.
Conclusions
These data lend support to the use of the American Heartworm Society Canine Guidelines for adulticide therapy recommending the initiation of doxycycline plus a macrocyclic lactone (ML) at the time of the heartworm-positive diagnosis.
Graphical Abstract
... In regard to D. immitis, the tetracycline drug doxycycline, administered with or without a macrocyclic lactone preventive, has been shown to adversely affect every life cycle stage: circulating micro lariae (mf) [3,[21][22][23][24][25][26][27][28], L3 and L4 stages [25,29], juveniles [22,29], young adults [22], and mature adults [21, 23-25, 27, 28, 30]. ...
... In regard to D. immitis, the tetracycline drug doxycycline, administered with or without a macrocyclic lactone preventive, has been shown to adversely affect every life cycle stage: circulating micro lariae (mf) [3,[21][22][23][24][25][26][27][28], L3 and L4 stages [25,29], juveniles [22,29], young adults [22], and mature adults [21, 23-25, 27, 28, 30]. ...
... The ML is expected to prevent further infection and gradually reduce micro laremia levels. However, circulating micro lariae can be detected in some dogs for several months after starting the administration of doxycycline or ivermectin alone or doxycycline plus an ML [24,25,27]. Monthly prophylactic doses of ivermectin along with a month of daily doses of doxycycline administered orally at 10 mg/kg twice daily (bid) can signi cantly reduce Wolbachia levels, reduce pulmonary in ltrates, reduce worm biomass, decrease post treatment complications and morbidity [32,33], and impede the spread of ML-resistant as well as ML-susceptible heartworms [24,25]. ...
Background: Microfilarial (mf) counts were monitored over 21.3 months for any rebound that might occur in counts and adulticidal efficacy was assessed following administration of low dosage with short- and long-treatment regimens of doxycycline and ivermectin to heartworm microfilaremic dogs.
Methods:Twelve heartworm-naïve Beagles infected with 10 pairs of adult Dirofilaria immitis by IV transplantation were randomly allocated to 3 groups of 4 dogs. All treatments started on Day 0. On Day 0, Group 1 (short-treatment regimen) received doxycycline orally at 10 mg/kg once daily for 30 days plus ivermectin orally (min., 6 mcg/kg) on Days 0 and 30. Group 2 (long-treatment regimen) received doxycycline orally at 10 mg/kg once daily until individual dogs became mf negative (72-98 days) and ivermectin every other week until individual dogs became mf negative (6-7 doses). Group 3 was the untreated control. Mf counts and antigen (Ag) tests were conducted. Dogs were necropsied for recovery and enumeration of heartworms on Day 647.
Results: Day -1 mean mf counts were 15,613, 23,950 and 15,513 mf/mL for Groups 1, 2 and 3, respectively. Mean counts for Group 1 and 2 declined until days 239 and 97, respectively, when all were negative. Group 3 had high mf counts throughout the study. There was not a rebound in mf counts in any of the treated dogs after they became amicrofilaremic. All dogs in Group 1 and Group 3 were Ag positive throughout the study and had at least 1 live female worm at necropsy. All dogs in treated Group 2 were positive for Ag through Day 154, but were antigen-negative on days 644 and 647, as all had only male worms.
Mean live adult worm recoveries for Groups 1, 2 and 3 were 6.8 (range, 5-8), 3.3 (range, 1-6) and 16.0 (range, 14-17), respectively, with a percent reduction in adult worm counts of 57.5% for Group 1 and 79.3% for Group 2.
Conclusions: This data lends support to the use of the American Heartworm Society Canine Guidelines for adulticide therapy recommendation of initiating doxycycline plus an ML treatment at the time of the heartworm-positive diagnosis.
... The second and third dose are administered 30 and 31 days, respectively, following the first [32,33]. The pathology associated with killing large worms in situ appears to be ameliorated by removal of the Wolbachia symbiont, especially when combined with pretreatment with IVM [36][37][38]. Doxycycline therapy has the added benefit of rendering circulating microfilariae incapable of developing to infectious L3 larvae in mosquitoes [36,38]. This feature could help to reduce the transmission and spread of macrocyclic lactone-resistant strains from infected dogs. ...
... The pathology associated with killing large worms in situ appears to be ameliorated by removal of the Wolbachia symbiont, especially when combined with pretreatment with IVM [36][37][38]. Doxycycline therapy has the added benefit of rendering circulating microfilariae incapable of developing to infectious L3 larvae in mosquitoes [36,38]. This feature could help to reduce the transmission and spread of macrocyclic lactone-resistant strains from infected dogs. ...
... Doxycycline is administered to heartworm-infected dogs prior to treatment with the macrofilaricide melarsomine; reducing Wolbachia populations reduces the incidence and severity of side effects associated with the killing of adult parasites, presumably by minimizing the release of Wolbachia antigens upon parasite death [36,38]. Doxycycline also prevents the development of L3 and L4 stages of D. immitis to adults, an effect also observed in some animal models of filariasis [102]. ...
Filarial parasites of humans and animals represent a heterogeneous group of pathogenic nematodes, interacting throughout their life cycles with two hosts and, for most species, a bacterial endosymbiont. Various control strategies have been implemented since the mid‐twentieth century, including vector control in endemic zones, mass drug administration campaigns, and preventive chemotherapy in companion animals. The challenges posed by the incompletely understood biology of these long‐lived pathogens, their restricted accessibility, discrepant life stage‐dependent sensitivities to drugs, as well as the potential for severe adverse events, have greatly complicated progress toward elimination of human filariases. This chapter chronicles the history of antifilarial medicines and reviews current understanding of their mechanisms of action, highlighting persisting knowledge gaps some 70 years after the first efforts were undertaken to control filarial infections.
... While our observations could apply to previous studies on sheathed filarial species (B. malayi, B. pahangi, and L. sigmodontis that showed reduced rates of L3 recovery [17][18][19]), they do not account for the different developmental impacts observed in species without a sheath, such as O. volvulus (20) and D. immitis (44,45). Albers et al. (20) observed a different dynamic in the onset of larval development inhibition, which began 3 mo after the end of a 6-wk course of doxycycline and continued at 4 to 5 mo after treatment, with a reduced proportion of L3 larvae and an increasing proportion of L1 and L2 larvae. ...
... Albers et al. (20) observed a different dynamic in the onset of larval development inhibition, which began 3 mo after the end of a 6-wk course of doxycycline and continued at 4 to 5 mo after treatment, with a reduced proportion of L3 larvae and an increasing proportion of L1 and L2 larvae. In the veterinary heartworm D. immitis, McCall et al. (44,45) showed that mf from doxycycline-treated dogs are able to develop to the infective L3 stage within their mosquito vector, but, importantly, these L3 are unable to develop to adult infections in the dog host. These nematodes are known to develop within the Malpighian tubules of the mosquito vector, and do not penetrate through the midgut. ...
... Our reasoning is that Wolbachia-depleted mf empirically demonstrate a profound 79 to 100% block in transmission compared to undepleted controls. This should also apply to unsheathed species, as L3 derived from Wolbachia-depleted mf are unable to develop to adult infections in the definitive host (20,45). Thus, Wolbachia depletion from mf shows an ability to exert both immediate and long-term suppression of transmission, with the added benefit of avoiding the side effects of more traditional microfilaricide treatment (21,24). ...
Significance
Lymphatic filariasis caused by Wuchereria bancrofti , Brugia malayi , and Brugia timori affects 51 million people, leading to severe physical and mental disabilities. A mutualistic symbiosis between these filarial nematodes and Wolbachia bacteria has been exploited as a new curative treatment. Epidemiological modeling of anti- Wolbachia treatment assumes that transmission persists due to the lag phase before microfilariae (mf) become removed from circulation. Here, we show that Wolbachia -depleted mf cannot develop within the mosquito vector—a phenotype associated with down-regulation of B. malayi mf-specific chitinase, an enzyme essential for exsheathment. Our findings add to the broad range of host biological processes dependent on Wolbachia and suggest that anti- Wolbachia treatment mediates a more accelerated impact on elimination of lymphatic filariasis than currently predicted.
... Doxycycline has become a necessary component of heartworm treatment (2), because of its effects on the filarial endosymbiont bacteria, Wolbachia (78,79). The addition of doxycycline markedly reduces the pulmonary pathology associated with dead and dying worms, reduces the risk of thromboembolism and disrupts heartworm development and transmission (33,(80)(81)(82). When given alone to dogs experimentally infected with third stage D. immitis larvae, doxycycline prevented the development of adult worms, with decreased effectiveness against older immature stages (83). ...
... A 28-or 30-day course of doxycycline has demonstrable effects on treatment outcome and either 10 or 20 mg/kg/day is acceptable. For dogs that remain antigen positive 12 months after completing a course of doxycycline, the course should be repeated (2,80); there are no known clinical benefits to repeating the treatment prior to that time point. ...
... The addition of doxycycline to treatment protocols has further reduced the risk of creating ML resistance, by interrupting embryogenesis and larval transmission (56,58,80,115). Moxidectin is also directly microfilaricidal at the topical dose (69). Microfilariae are rapidly eliminated by moxi-doxy, frequently within 3-4 weeks (48,49,52,53,55). ...
Canine heartworm infection, caused by the filarial parasite Dirofilaria immitis, represents a serious and expanding animal welfare concern that is expected to increase due to the effects of climate change and the COVID-19 pandemic. A body of evidence has emerged to support the use of a non-arsenical adulticide treatment protocol, using moxidectin and doxycycline to kill adult heartworms over a prolonged period. While a three-dose protocol using the arsenical drug melarsomine is currently the safest and most effective treatment for heartworm infection, this drug is not available in some countries and is inaccessible for many owners and animal shelters. Moxidectin-doxycycline (moxi-doxy) provides a viable alternative to no treatment at all, in cases where arsenical treatment is not possible. Based on current evidence, the most effective non-arsenical treatment regimen is doxycycline 10 mg/kg PO q 12 or 24 h for 28 days, combined with topical moxidectin at label dose. Moxidectin is repeated monthly until no antigen detected (NAD) status is confirmed. Sustained release injectable moxidectin, in combination with doxycycline, may provide an alternative in remote regions or in settings where significant compliance or accessibility concerns exist, but more studies are needed. In moxi-doxy protocols, doxycycline should be repeated annually until NAD. This review summarizes the safety and efficacy of moxi-doxy, addresses controversies surrounding this treatment approach, and provides detailed recommendations for treatment regimens and post-treatment testing.
... It is seen from these studies that, when used in conjunction with doxycycline, the required duration of ML therapy in a non-arsenical treatment strategy is truncated. The likely mechanism of this beneficial effect is that doxycycline greatly reduces numbers of the endosymbiotic Wolbachia in heartworms, rendering tissue larval stages unable to molt/ survive and adults unable to reproduce and thrive McCall et al., 2014). The exact timing of this process is unclear, but debilitation of the worm not only shortens its life expectancy but also causes degeneration of the reproductive tract, thereby reducing both the HW and Wolbachia antigens released upon worm destruction and lessening the host's inflammatory reaction to worm death Yoon et al., 2016). ...
... Elimination of microfilariae (Mf) reduces the risk of resistance selection during ML use in HW-infected dogs. Doxycycline further reduces this concern by rendering the L3 non-infective, preventing the transmission of resistant strains (McCall et al., 2014). The microfilaricidal effect of M/I effect is enhanced by the concurrent use of doxycycline (Rossi et al., 2010). ...
... Microfilaria testing should be performed in all HW-infected dogs and it is important that they be eradicated if a ML is to be initiated whether as a preventive used along with an arsenical or as part of a non-arsenical protocol such as MOXY. This decreases the chance for selection of resistance amongst Mf exposed to ML. Doxycycline further reduces the concern of resistance selection and propagation by rendering the L3 non-infective, preventing the transmission of resistant strains (McCall et al., 2014). Furthermore, giving M/I at an twice monthly dosing interval may not be necessary. ...
This prospective case series evaluated the adulticidal efficacy of topical 10% moxidectin/2.5% imidacloprid (M/I; Advantage Multi®, Bayer, Shawnee Mission, KS, USA) and doxycycline in dogs with naturally occurring heartworm infection (HWI). Twenty-two dogs with HWI whose owners declined melarsomine were treated with M/I at the preventive dosage twice monthly for 90 days then monthly thereafter and doxycycline (median [interquartile range; IQR] dosage 12.6 [12.0-16.1] mg/kg/day) for the first 15 days. Although strict activity restriction was not imposed, owners were asked to prevent their dogs from exercising strenuously. This protocol was referred to as the MOXY protocol. Antigen testing was performed every 30 to 60 days, until dogs had ‘no antigen detected’ (NAD). Twenty-one of the 22 dogs ultimately converted to NAD by 434 days (median [IQR]), 234 (179-303). One dog remained positive 701 days after MOXY initiation and was considered a treatment failure. All sera which converted to NAD on HW antigen testing were retested after heat-treatment. Twelve dogs had NAD on the heat-treated test on the same day as having their first NAD on the conventional test. Six of 9 dogs testing positive after heat-treatment were retested and all 6 had NAD on a heat-treated test within 2 to 3 months. Microfilaremia was cleared in all 8 dogs re-tested. Four dogs required treatment for cough, thought due to heartworm (HW) death, an average of 89 days after initiation of MOXY. This cough was most likely due to pneumonitis with heartworm-pulmonary thromboembolism. One dog required hospitalization for 24 -hs and recovered fully with corticosteroid therapy and supportive care and 2 dogs were treated in an outpatient fashion with steroids. The MOXY protocol was tolerated and 96% (21/22) of dogs converted to NAD, though 2 dogs required greater than 1 year to achieve this result. Nonaresenical-adulticide therapy may result in pneumonitis and heartworm-pulmonary thromboembolism at unpredictable times, potentially months after initiation of macrocyclic lactone therapy and exercise restriction should be considered when using a nonarsenical protocol. Although not currently recommended by the American Heartworm Society (AHS), non-arsenical strategies are in use and the goal of this study was to evaluate the efficacy, duration of therapy, and safety of an accelerated dosing protocol of M/I with doxycycline.
... Interrogation of the genomes of suspected ML-resistant strains of D. immitis has revealed that these strains are genetically distinct from wildtype strains (Bourguinat et al., 2011b(Bourguinat et al., , 2015(Bourguinat et al., , 2017bGeary et al., 2011;McCall et al., 2014b;Pulaski et al., 2014). We have previously identified a number of single nucleotide polymorphisms (SNPs) that are highly correlated with ML resistance as defined by prophylaxis failure (Bourguinat et al., 2015(Bourguinat et al., , 2017a; although no individual SNP identifies a single gene that accounts for the mechanism of ML resistance, a set of 4-5 SNPs is highly correlated with the phenotype (Bourguinat et al., 2017a). ...
... We have previously identified a number of single nucleotide polymorphisms (SNPs) that are highly correlated with ML resistance as defined by prophylaxis failure (Bourguinat et al., 2015(Bourguinat et al., , 2017a; although no individual SNP identifies a single gene that accounts for the mechanism of ML resistance, a set of 4-5 SNPs is highly correlated with the phenotype (Bourguinat et al., 2017a). A diagnostic test based on SNP markers of ML resistance would be of significant value in distinguishing clinical cases of heartworm disease due to ML resistance from those due to drug compliance failure, as well as for monitoring the extent and spread of resistant genotypes and determining the influence of alternative chemotherapeutic strategies, such as the administration of doxycycline (McCall et al., 2014b), on the spread of ML resistance. However, molecular markers require clinical validation to establish a correlation between genotype and phenotype. ...
... A diagnostic test based on SNP markers of ML resistance could help distinguish clinical cases of heartworm infection due to ML resistance from those due to failure of adequate compliance and for monitoring the extent and spread of resistant isolates in epidemiological surveys. Such a test could also determine the influence of alternative chemotherapeutic strategies on the spread of ML resistance, such as the deployment of doxycycline or melarsomine dihydrochloride (Bowman and Drake, 2017;McCall et al., 2014b). Previously, we identified a set of 10 SNPs that were highly predictive of ML resistance (Bourguinat et al., 2017a). ...
Prophylaxis with macrocyclic lactone (ML) endectocides is the primary strategy for heartworm control. Recent evidence has confirmed that ML-resistant Dirofilaria immitis isolates have evolved. Comparison of genomes of ML-resistant isolates show they are genetically distinct from wild-type populations. Previously, we identified single nucleotide polymorphisms (SNPs) that are correlated with phenotypic ML resistance. Since reliable in vitro assays are not available to detect ML resistance in L3 or microfilarial stages, the failure to reduce microfilaraemia in infected dogs treated with an ML has been proposed as a surrogate clinical assay for this purpose. The goal of our study was to validate the genotype-phenotype correlation between SNPs associated with ML resistance and failure to reduce microfilaraemia following ML treatment and to identify a minimal number of SNPs that could be used to confirm ML resistance. In this study, 29 participating veterinary clinics received a total of 148 kits containing supplies for blood collection, dosing and prepaid shipping. Patients recruited after a diagnosis of heartworm infection were treated with a single standard dose of Advantage Multi® and a blood sample taken pre- and approximately 2-4 weeks post-treatment. Each sample was processed by performing a modified Knott's Test followed by isolation of microfilariae, genomic DNA extraction and MiSeq sequencing of regions encompassing 10 SNP sites highly correlated with ML resistance. We observed significant correlation of SNP loci frequencies with the ML microfilaricidal response phenotype. Although all predictive SNP combination models performed well, a 2-SNP model was superior to other models tested. The predictive ability of these markers for ML-resistant heartworms should be further evaluated in clinical and epidemiological contexts.
... Interrogation of the genomes of suspected ML-resistant strains of D. immitis has revealed that these strains are genetically distinct from wildtype strains (Bourguinat et al., 2011b(Bourguinat et al., , 2015(Bourguinat et al., , 2017bGeary et al., 2011;McCall et al., 2014b;Pulaski et al., 2014). We have previously identified a number of single nucleotide polymorphisms (SNPs) that are highly correlated with ML resistance as defined by prophylaxis failure (Bourguinat et al., 2015(Bourguinat et al., , 2017a; although no individual SNP identifies a single gene that accounts for the mechanism of ML resistance, a set of 4-5 SNPs is highly correlated with the phenotype (Bourguinat et al., 2017a). ...
... We have previously identified a number of single nucleotide polymorphisms (SNPs) that are highly correlated with ML resistance as defined by prophylaxis failure (Bourguinat et al., 2015(Bourguinat et al., , 2017a; although no individual SNP identifies a single gene that accounts for the mechanism of ML resistance, a set of 4-5 SNPs is highly correlated with the phenotype (Bourguinat et al., 2017a). A diagnostic test based on SNP markers of ML resistance would be of significant value in distinguishing clinical cases of heartworm disease due to ML resistance from those due to drug compliance failure, as well as for monitoring the extent and spread of resistant genotypes and determining the influence of alternative chemotherapeutic strategies, such as the administration of doxycycline (McCall et al., 2014b), on the spread of ML resistance. However, molecular markers require clinical validation to establish a correlation between genotype and phenotype. ...
... A diagnostic test based on SNP markers of ML resistance could help distinguish clinical cases of heartworm infection due to ML resistance from those due to failure of adequate compliance and for monitoring the extent and spread of resistant isolates in epidemiological surveys. Such a test could also determine the influence of alternative chemotherapeutic strategies on the spread of ML resistance, such as the deployment of doxycycline or melarsomine dihydrochloride (Bowman and Drake, 2017;McCall et al., 2014b). Previously, we identified a set of 10 SNPs that were highly predictive of ML resistance (Bourguinat et al., 2017a). ...
Prophylaxis with macrocyclic lactone (ML) endectocides is the primary strategy for heartworm control. Recent evidence has confirmed that ML-resistant Dirofilaria immitis isolates have evolved. Comparison of genomes of ML-resistant isolates show they are genetically distinct from wild-type populations. Previously, we identified single nucleotide polymorphisms (SNPs) that are correlated with phenotypic ML resistance. Since reliable in vitro assays are not available to detect ML resistance in L3 or microfilarial stages, the failure to reduce microfilaraemia in infected dogs treated with an ML has been proposed as a surrogate clinical assay for this purpose. The goal of our study was to validate the genotype-phenotype correlation between SNPs associated with ML resistance and failure to reduce microfilaraemia following ML treatment and to identify a minimal number of SNPs that could be used to confirm ML resistance. In this study, 29 participating veterinary clinics received a total of 148 kits containing supplies for blood collection, dosing and prepaid shipping. Patients recruited after a diagnosis of heartworm infection were treated with a single standard dose of Advantage Multi® and a blood sample taken pre- and approximately 2-4 weeks post-treatment. Each sample was processed by performing a modified Knott’s Test followed by isolation of microfilariae, genomic DNA extraction and MiSeq sequencing of regions encompassing 10 SNP sites highly correlated with ML resistance. We observed significant correlation of SNP loci frequencies with the ML microfilaricidal response phenotype. Although all predictive SNP combination models performed well, a 2-SNP model was superior to other models tested. The predictive ability of these markers for ML-resistant heartworms should be further evaluated in clinical and epidemiological contexts.
... Pulmonary inflammation following melarsomine is markedly ameliorated by prior treatment with doxycycline and an ML [5][6][7]. Loss of Wolbachia also weakens the worms and decreases their ability to reproduce [8][9][10][11]. The currently recommended 30-day interval between completion of the course of doxycycline and M1 is hypothesized to allow time for the Wolbachia surface protein and other metabolites to be cleared [1]. The delay may also further weaken the worms and render them more susceptible to melarsomine [1]. ...
... There is compelling evidence for the benefits of doxycycline in treatment of HWI [1,[5][6][7][8][9][10][11]. At least some delay prior to M1 is advisable, in order to allow time for doxycycline to take effect. ...
Background
This retrospective study evaluated modified three-dose melarsomine treatment protocols in a shelter setting and compared them to the American Heartworm Society (AHS)-recommended protocol.
Methods
As compared with the AHS protocol, the shelter protocols utilized doxycycline 10 mg/kg once daily (SID) or twice daily (BID), and varied the time from initiation of doxycycline (day 1) to the first melarsomine injection (M1). Dogs were retrospectively grouped based on the shelter’s current protocol (M1 on day 14; Group A) and the AHS protocol (M1 on day 60; Group C), allowing a week on either side of the target M1 day. Treatments that fell outside these ranges formed two additional treatment groups (Groups B and D). Respiratory complications were defined as respiratory signs requiring additional treatment, and were statistically compared for Groups A and C. New respiratory signs and gastrointestinal (GI) signs were compared between dogs receiving SID or BID doxycycline.
Results
One hundred fifty-seven dogs with asymptomatic or mild heartworm disease at presentation were included. All dogs survived to discharge. There was no statistically significant difference between Groups A (n = 79) and C (n = 27) for new respiratory signs post-melarsomine (P = 0.73). The time to M1 for 14 dogs that developed new respiratory signs was a median of 19 days, compared with 22 days for 143 dogs without new respiratory signs (P = 0.2). Respiratory complications post-melarsomine were uncommon. New respiratory signs post-melarsomine occurred in 10/109 (9.2%) dogs receiving SID doxycycline and 4/48 (8.3%) dogs receiving BID doxycycline (P > 0.999). GI signs prior to M1 were recorded for 40/109 (36.7%) dogs receiving SID doxycycline and 25/48 (52.1%) receiving BID doxycycline (P = 0.08). Forty-four follow-up antigen test results were available; all tests performed > 3 months after the third melarsomine injection were negative.
Conclusions
This study provided support for initiating melarsomine after 14 days of doxycycline and for a lower doxycycline dose. Shorter and less expensive treatment protocols can increase lifesaving capacity and improve quality of life for shelter dogs by reducing the duration of exercise restriction and length of stay.
Graphical Abstract
... Pulmonary in ammation following melarsomine is markedly ameliorated by prior treatment with doxycycline and a ML [5][6][7]. Loss of Wolbachia also weakens the worms and decreases their ability to reproduce [8][9][10][11]. The currentlyrecommended 30-day interval between completion of the course of doxycycline and M1 is hypothesized to allow time for Wolbachia surface protein and other metabolites to be cleared [1]. The delay may also further weaken the worms and render them more susceptible to melarsomine [1]. ...
... There is compelling evidence for the bene ts of doxycycline in treatment of HWI [1,[5][6][7][8][9][10][11]. At least some delay prior to M1 is advisable, in order to allow time for doxycycline to take effect. ...
Background
This retrospective study evaluated modified 3-dose melarsomine treatment protocols in a shelter setting and compared them to the American Heartworm Society (AHS) recommended protocol.
Methods
As compared to the AHS protocol, the shelter protocols utilized doxycycline 10 mg/kg once daily (SID) or twice daily (BID), and varied the time from initiation of doxycycline (day 1) to the first melarsomine injection (M1). Dogs were retrospectively grouped based on the shelter’s current protocol (M1 on day 14; Group A) and the AHS protocol (M1 on day 60; Group C), allowing a week on either side of the target M1 day. Treatments that fell outside of these ranges formed two additional treatment groups (Groups B and D). Respiratory complications were defined as respiratory signs requiring additional treatment, and were statistically compared for Groups A and C. New respiratory signs and gastrointestinal (GI) signs were compared between dogs receiving SID or BID doxycycline.
Results
One hundred fifty-seven dogs with asymptomatic or mild heartworm disease at presentation were included. All dogs survived to discharge. There was no statistically significant difference between Groups A (n = 79) and C (n = 27) for new respiratory signs post-melarsomine (P = 0.73). The time to M1 for 14 dogs that developed new respiratory signs was median 19 days, compared with 22 days for 143 dogs without new respiratory signs (P = 0.2). Respiratory complications post-melarsomine were uncommon. New respiratory signs post-melarsomine occurred in 10/109 (9.2%) dogs receiving SID doxycycline and 4/48 (8.3%) dogs receiving BID doxycycline (P > 0.999). GI signs prior to M1 were recorded for 40/109 (36.7%) dogs receiving SID doxycycline and 25/48 (52.1%) receiving BID doxycycline (P = 0.08). Forty-four follow-up antigen test results were available; all tests performed > 3 months after the third melarsomine injection were negative.
Conclusions
This study provided support for initiating melarsomine after 14 days of doxycycline and for a lower doxycycline dose. Shorter and less expensive treatment protocols can increase lifesaving capacity and improve quality of life for shelter dogs by reducing the duration of exercise restriction and length of stay.
... (1). The administration of antibiotics (doxycycline or minocycline) is considered of great value in order to impair the eventual development of the circulating microfilariae to adult worms in a new host, although further confirmation of this effect should be generated [63]. This is the result of removing the filarial endosymbiont Wolbachia pipientis, which is critical for the survival, development, and reproduction of D. immitis [64]. ...
... Treating a dog infected by ML-resistant heartworms with a "slow kill" protocol [63,66], i.e., by repeated doses of MLs that have been shown to slowly kill adult parasites, is not realistic, as both the microfilariae and adult nematodes would not be susceptible to these drugs. Such an approach would only allow the resistance to be transmitted and perhaps increase in intensity. ...
Dirofilaria immitis infection is one of the most severe parasitic diseases in dogs. Prevention is achieved by the administration of drugs containing macrocyclic lactones (MLs). These products are very safe and highly effective, targeting the third and fourth larval stages (L3, L4) of the parasite. Until 2011, claims of the ineffectiveness of MLs, reported as “loss of efficacy” (LOE), were generally attributed to owners’ non-compliance, or other reasons associated with inadequate preventative coverage. There was solid argumentation that a resistance problem is not likely to occur because of (i) the great extent of refugia, (ii) the complexity of resistance development to MLs, and (iii) the possible large number of genes involved in resistance selection. Nevertheless, today, it is unequivocally proven that ML-resistant D. immitis strains exist, at least in the Lower Mississippi region, USA. Accordingly, tools have been developed to evaluate and confirm the susceptibility status of D. immitis strains. A simple, in-clinic, microfilariae suppression test, 14-28 days after ML administration, and a “decision tree” (algorithm), including compliance and preventatives’ purchase history, and testing gaps, may be applied for assessing any resistant nature of the parasite. On the molecular level, specific SNPs may be used as markers of ML resistance, offering a basis for the validation of clinically suspected resistant strains. In Europe, no LOE/resistance claims have been reported so far, and the existing conditions (stray dogs, rich wildlife, majority of owned dogs not on preventive ML treatment) do not favor selection pressure on the parasites. Considering the genetic basis of resistance and the epizootiological characteristics of D. immitis, ML resistance neither establishes easily nor spreads quickly, a fact confirmed by the current known dispersion of the problem, which is limited. Nevertheless, ML resistance may propagate from an initial geographical point, via animal and vector mobility, to other regions, while it can also emerge as an independent evolutionary process in a new area. For these reasons, and considering the current chemoprophylaxis recommendations and increasing use of ML endectoparasiticides as a potential selection pressure, it is important to remain vigilant for the timely detection of any ML LOE/resistance, in all continents where D. immitis is enzootic.
... The administration of antibiotics (doxycycline or minocycline) is considered of great value in order to impair the eventual development of the circulating microfilariae to adult worms in a new host, although further confirmation of this effect should be generated [59]. This is the result of removing the filarial endosymbiont Wolbachia pipientis which is critical for the survival, development and reproduction of D. immitis [60]. ...
... Treating a dog infected by ML resistant heartworms with a "slow kill" protocol [59,62], i.e. by repeated doses of MLs that have been shown to slowly kill adult parasites, is not realistic, as both the microfilariae and adult nematodes would not be susceptible to these drugs. Such an approach would only allow the resistance to be transmitted and perhaps increase in intensity. ...
Dirofilaria immitis infection is one of the most severe parasitic diseases of dogs. Prevention is achieved by the administration of drugs containing macrocyclic lactones (MLs). These products are very safe and highly effective, targeting the third and fourth larval stages (L3, L4) of the parasite. Until 2011, claims of ineffectiveness of MLs, reported as "Lack of Efficacy" (LOE), were generally attributed to owners' non-compliance, or other reason for inadequate preventative coverage. There was solid argumentation that a resistance problem is not likely to occur because of i) the great extent of refugia, ii) the complexity of resistance development to MLs, and iii) the possible big number of genes involved in resistance selection. Nevertheless, today it is unequivocally proven that ML resistant D. immitis strains exist, at least in the Lower Mississippi region, USA. Accordingly, tools have been developed, to evaluate and confirm the susceptibility status of D. immitis strains. A simple, in-clinic, microfilariae suppression test, 14-28 days after ML administration, and a "decision tree" (al-gorithm), including compliance and preventatives' purchase history, and testing gaps, may be applied for assessing any resistant nature of the parasite. On the molecular level, specific SNPs may be used as markers of ML resistance, offering a basis for validation of clinically suspected resistant strains. In Europe, no LOE/resistance claims have been reported so far, and the existing conditions (stray dogs, rich wildlife, majority of owned dogs not on preventive MLs treatment) do not favor selection pressure on the parasites. Considering the genetic basis of resistance and the epizootiolog-ical characteristics of D. immitis, ML resistance neither establishes easily nor spreads quickly, a fact confirmed by the current known dispersion of the problem, which is limited. Nevertheless, ML resistance may propagate from an initial geographical point, via animal and vector mobility, to other regions, while it can also emerge as an independent evolutionary process in a new area. For these reasons and considering the current chemoprophylaxis recommendations and increasing use of ML endectoparasiticides as a potential selection pressure, it is important to remain vigilant for timely detection of any ML LOE/resistance, in all continents where D. immitis is enzootic.
... Additionally, the Wolbachia population from B. malayi contains genes necessary for folate synthesis (Godel et al., 2012). The essential metabolic contributions of Wolbachia renders them a valid drug target to control filariae (Slatko et al., 2010;McCall et al., 2014b;Landmann, 2019;Turner et al., 2020). ...
... The viability of that approach was shown by screening of the predicted Wolbachia proteome of D. immitis which revealed antibiotic drug targets such as Fts proteins essential for cell division (Fts -filamentous temperature-sensitive mutant), and Sec proteins involved in directed transport of proteins across membranes (Secprotein-secreting ATPase complex) (Godel et al., 2012). This was demonstrated experimentally in studies using the tetracycline antibiotic doxycycline, which cleared Wolbachia from D. immitis and led to gradual reduction of microfilariae and adult nematodes (Bandi et al., 1999;McCall et al., 2014b). Doxycycline was also successfully employed in humans as an anti-Wolbachia therapy against O. volvulus in combination with ivermectin (Hoerauf et al., 2003). ...
Dirofilaria immitis, also known as heartworm, is a major parasitic threat for dogs and cats around the world. Because of its impact on the health and welfare of companion animals, heartworm disease is of huge veterinary and economic importance especially in North America, Europe, Asia and Australia. Within the animal health market many different heartworm preventive products are available, all of which contain active components of the same drug class, the macrocyclic lactones. In addition to compliance issues, such as under-dosing or irregular treatment intervals, the occurrence of drug-resistant heartworms within the populations in the Mississippi River areas adds to the failure of preventive treatments. The objective of this review is to provide an overview of the disease, summarize the current disease control measures and highlight potential new avenues and best practices for treatment and prevention.
... So, those two dogs were positive, since they were not yet cured from the adult forms of Dirofilaria. These dogs were treated with a "slow kill" therapy which is based on elimination of larval stages and adults remain live for some time [20]. The possibility of occult infection is about 20 % in dogs and it means infection with adult Dirofilaria immitis in the absence of circulating microfilariae. ...
... For that reason there were more young dogs available for sampling then the older ones. The other reason is that in some shelters (B and partly D) when dogs enter the shelter they are checked for D. immitis infection and if positive, they are in most cases treated with larvicide therapy for some time [20]. ...
Background: Dirofilaria immitis is vector borne parasite of carnivores, with zoonotic potential, endemic in many parts of the world, including Europe. The aim of this study was to determine the prevalence of Dirofilaria immitis infection in dogs from shelters, especially compared to their lifestyle. Dogs living in shelters in Serbia may be at high risk of acquiring vector borne pathogens, mainly because most of them live outside in pens and backyards, in contact with vectors. Also, dogs in shelters are not always regularly treated against ectoparasites, thus, representing an easy feeding source for the vectors. The objective of this study was to determine the prevalence of Dirofilaria immitis infection in dogs from 5 shelters in South Bačka and Central Banat districts, in Autonomous Province of Vojvodina, Northern part of Serbia. Also, the objective was to compare the relation of infection with Dirofiaria immitis with age, sex, type of keeping the animals and preventive treatment in dogs.Materials, Methods & Results: Between May 2017 and October 2019, blood samples were collected from 336 randomly selected dogs from 5 shelters in 2 districts, South Bačka and Central Banat districts, in Autonomous Province of Vojvodina, Northern part of Serbia. The epidemiological survey has been conducted with all of the dogs involved in this research. The survey was designed to collect data about sex, age, lifestyles, food type, treatment against mosquitoes with insecticides and filarioid worms with macrocyclic lactones, regular testing for Dirofilaria infections. The presence of circulating microfilariae was examined using a modified Knott’s test. For the presence of circulating adult female Dirofilaria immitis antigen, serum samples were tested by commercially available enzyme-linked immunosorbent assay, which reacts to antigen of female Dirofilaria. In total, 336 dogs were examined for the presence of Dirofilaria immitis antigen. For that dog population which came from 5 shelters, total prevalence was 25.30%. Most of the positive findings were observed in a shelter where dogs lived exclusively outdoors in fenced yards in big groups and they were partly tested for heartworm infections from time to time. These dogs were not treated with macrocyclic lactones, against mosquitoes with insecticides or filarioid worms. The prevalence in this shelter was 56.36%. On the contrary to that, the lowest positive findings were detected in the shelter, where dogs were allowed to move freely between outside and indoors and they were also provided with accommodation indoors. These dogs have been regularly tested for Dirofilaria infections and treated against mosquitoes with insecticides and filarioid worms with macrocyclic lactones. In this shelter the seroprevalence was 7.69%. Microfilariae of Dirofilaria immitis were detected, by modified Knott’s test, in all of the antigen positive dog samples; except in 2 dogs from one shelter. Discussion: This study shows persistence of cardiopulmonary dirofiariosis in shelter dogs under different maintaining conditions. By comparing the data during the last 17 years, it can be stated that there is a constant increase of prevalence for Dirofilaria immitis in dogs in northern part of Serbia over the years. The results gained in this study are important from the veterinary point of view, but also from the Public Health point of view.
... Endosymbiosis has been defined as the intimate association between two different organisms maintained through generations because host and symbiont equally benefit from the association. Studies have shown that Wolbachia is required for normal parasite development, fertility and long-term survival of different filarial species (Taylor et al., 2013;McCall et al., 2014). Genetic and molecular studies in the last several years have been aimed at defining exactly what these bacteria do for heartworms and other filarial nematodes (Darby et al., 2012, Godel et al., 2012. ...
... However, this study evaluated worm survival at 8 months post-treatment and, as mentioned above, it is likely that death of adult worms takes longer. In a more recent study (McCall et al., 2014), dogs with experimentally-acquired patent infection were treated with doxycycline for 1 month at 20 mg/kg/day. Worm survival was evaluated at 12 and 13 months post-infection (p.i.) and results suggested that treatment had a slow-kill, adulticidal effect, which was more evident at 13, than at 12 months after doxycycline treatment started. ...
Melarsomine dihydrochloride (Immiticide® Merial) is the only approved adulticidal drug for the treatment of canine heartworm disease (HWD). However, in cases where arsenical therapy is not possible or is contraindicated, a monthly heartworm preventive along with doxycycline for a 4-week period, which targets the bacterial endosymbiont Wolbachia, might be considered. There are published reports on the efficacy of ivermectin and doxycycline in both experimentally and naturally infected dogs, but no data on the use of other macrocyclic lactones (MLs) with a similar treatment regime. Preliminary results of studies in dogs show that a topical formulation of moxidectin, the only ML currently registered as a microfilaricide, is also adulticidal when combined with doxycycline. It is not yet known if the efficacy of these combination therapies is due to pharmacokinetic synergism. A recent study showed that serum levels of doxycycline in dogs treated with the combination protocol were not statistically different compared to dogs treated with doxycycline alone. However, lungs from dogs treated with the combination therapy showed a marked reduction in T regulatory cells, indicating that treatment efficacy may be due to a heightened immune response against the parasite. Further studies are necessary to evaluate the long-term clinical outcome of combination protocols and to establish the most efficient treatment for HWD in dogs.
... The benefits of anti-Wolbachia treatment in heartworminfected dogs have been well documented [3][4][5][6][7]. The doxycycline administration protocol for dogs is based on the pivotal work by McCall and his colleagues [3][4][5]. ...
... The benefits of anti-Wolbachia treatment in heartworminfected dogs have been well documented [3][4][5][6][7]. The doxycycline administration protocol for dogs is based on the pivotal work by McCall and his colleagues [3][4][5]. They found that administration of doxycycline to heartworminfected dogs reduces Wolbachia numbers in all stages of heartworms. ...
Background
Doxycycline has been considered the first drug of choice for treating Wolbachia, a member of the Rickettsiaceae, which has a symbiotic relationship with filarial worms, including heartworms. Wolbachia, is susceptible to tetracyclines, which have been used as adjunctive treatments for heartworm disease. Treatment with doxycycline reduces Wolbachia numbers in all stages of heartworms and improves outcomes and decreased microfilaremia in dogs treated for heartworm disease. The American Heartworm Society recommends treatment with doxycycline in dogs diagnosed with heartworm disease at a dose of 10 mg/kg twice daily for 28 days. If doxycycline is not available, minocycline can be considered as a substitute. However, minocycline has not undergone an evaluation in dogs with heartworm disease, nor has an effective dose been established. Minocycline is an attractive option because of the higher cost of doxycycline and new pharmacokinetic information for dogs that provides guidance for appropriate dosage regimens to achieve pharmacokinetic-pharmacodynamic (PK-PD) targets.
Results
Published reports from the Anti-Wolbachia Consortium (A-WOL) indicate superior in vitro activity of minocycline over doxycycline. Studies performed in mouse models to measure anti-Wolbachia activity showed that minocycline was 1.7 times more effective than doxycycline, despite a 3-fold lower pharmacokinetic exposure. To achieve the same exposure as achieved in the mouse infection model, a pharmacokinetic-pharmacodynamic (PK-PD) analysis was conducted to determine optimal dosages for dogs. The analysis showed that an oral minocycline dose of 3.75 to 5 mg/kg administered twice daily would attain similar targets as observed in mice and predicted for human infections.
Conclusions
There are potentially several advantages for use of minocycline in animals. It is well absorbed from oral administration, it has less protein binding than doxycycline (65% vs 92%) allowing for better distribution into tissue, and it is approximately two times more lipophilic than doxycycline, which may result in better intracellular penetration. More work is needed to document efficacy of minocycline for treating canine heartworm disease.
... In the case described here, after the results of the MFST, the dog was treated according to the guidelines of the American Heartworm Society (AHS, 2024) and the European Society of Dirofilariosis and Angiostrongylosis (ESDA, 2024). The administration of doxycycline eliminates the filarial endosymbiont Wolbachia pipientis, thereby reducing its pathological effects following the death of the filariae, but what is relevant in the case of a resistant strain, the elimination of this endosymbiont in microfilariae impairs their development in a new host, ensuring the discontinuation of resistance spreading (McCall et al., 2014). For the same reason, protection against mosquito bites is also important in the management of a suspected or confirmed resistant infection. ...
... In this study, Wolbachia endosymbiont was detected in the DNA extracted from the lungs and was classified as supergroup F. Previous studies have indicated that Wolbachia spp. in filarial nematodes exhibit a stage-specific distribution in their host and play possible critical roles in the synthesis of metabolites and energy production 50,51 , and the depletion of the endosymbionts significantly disrupts its filarial host fitness and development 52 . As used for dirofilariasis and onchocerciasis 53,54 , the use of anti-Wolbachia drugs such as doxycycline and tetracycline might also serve as a control measure for filariasis caused by Onchocercidae sp. characterized in this study. ...
Chickens in free-range environments are at risk of exposure to various pathogens, such as filarioids transmitted via hematophagous vectors. However, the study of filarioids in poultry has been largely neglected compared to the extensive studies focused on viruses, bacteria, and protozoa. Here, we performed histological and molecular investigations of the filarioids detected in domestic chickens from two different flocks in Hiroshima Prefecture, Japan. In the first case, adult worms were present in the pulmonary artery and right ventricle, and microfilariae were present in multiple organs of deceased chickens. In the second case, similar filarioids were detected in the organs and blood of one necropsied layer. Phylogenetic analysis using 18S rRNA gene fragments positioned the filarioid in the same clade as that of Onchocercidae sp., previously identified in a deceased chicken from Chiba Prefecture, Japan, that is located 500 km away from Hiroshima Prefecture. Based on 28S rRNA and mitochondrial COI gene fragments, the filarioid was positioned distinctly from previously reported genera of avian filarioids. These results suggest that the filarioids are potentially associated with the health burden on domestic chickens and belong to the genus Paronchocerca. Furthermore, we developed a nested PCR assay targeting mitochondrial COI and detected the parasite DNA from the biting midge Culicoides arakawae captured near the flock, suggesting that it serves as a vector. Our findings fill the knowledge gap regarding avian filarioids, laying the groundwork for future studies examining the epidemiology, life cycle, and species diversity of this neglected parasite group.
... The concern regarding selection for microfilarial resistance also exists with year-round preventives, and evidence suggests that moxidectin may be effective against resistant strains. Moreover, even if microfilariae do not die, they become non-infective [61,66,67]. In terms of cost, the long duration of the slowkill protocol may raise the total cost of treatment, but it is still less expensive than the full protocol, which is important in the tropical context. ...
Dirofilaria immitis (the canine heartworm) is widespread in the tropics, with prevalence surpassing 30% in high-risk areas. In addition to the suitable climatic conditions that favour mosquito abundance and filarial larva development, there is low compliance with the recommended year-round use of preventives in these transmission hotspots. This represents a major concern, considering that melarsomine (first-line heartworm adulticide) is unavailable in several tropical countries, resulting in the so-called slow-kill protocol being the only available adulticide treatment option. In this article, the members of TroCCAP (Tropical Council for Companion Animal Parasites) review the current distribution of heartworm in the tropics and the availability of melarsomine, and discuss alternatives for the management of heartworm infections in dogs.
Graphical Abstract
... The effectiveness of doxycycline against D. immitis is likely due to its ability to eliminate the intracellular endosymbiont Wolbachia pipientis (Rossi et al., 2010;McCall et al., 2014), and to reduce embryogenesis, with a gradual elimination of the circulating microfilariae (mfs) (Bandi et al., 1999). Wolbachia is found in the lateral chords of male and female worms and in the female reproductive tract (Kramer et al., 2018;Manoj et al., 2021) where it produces enzymes needed for the biosynthesis of heme, purine and pyrimidines (Godel et al., 2012) and is essential for the development, fertility and survival of many filarioid species (Kozek, 2005;Taylor et al., 2005). ...
Bacteria of the genus Wolbachia are endosymbionts of parasitic filarial nematodes, including Dirofilaria immitis, and are a target for the treatment of canine heartworm disease. In the present study, 53 naturally-infected dogs were divided in three groups, based on their positivity to D. immitis by antigen and Knott tests, to assess the efficacy of doxycycline treatment to eliminate Wolbachia from circulating blood. At T0, dogs that scored positive to both tests (G1) or to antigen test only (G2) were submitted to doxycycline (10mg/kg BID PO) treatment and 10% Imidacloprid + 2.5% Moxidectin (Advocate®), while those negative to both tests (G3) received only 10% Imidacloprid + 2.5% Moxidectin (Advocate®). All dogs were followed-up for one year, monthly treated with Advocate® and regularly monitored by antigen and Knott tests. During the whole period, all blood samples were screened for Wolbachia-D. immitis DNA load by quantitative real-time PCR (qPCR). At T0, 88.2% of the microfilariemic dogs were positive for Wolbachia DNA, while none of the dogs from G2 or G3 were positive. Wolbachia DNA was no longer detectable in dogs from G1 following 1 month of doxycycline treatment and microfilariae were cleared at T2. All dogs from the G1 and G2 were negative for D. immitis antigen at 12 months. Results of this study suggest that successful elimination of microfilariae by doxycycline is associated with complete clearance of Wolbachia DNA in D. immitis-naturally infected dogs.
... Thus, current research is aimed at the removal of Wolbachia through antibiotic treatment of infected hosts as an adulticide strategy. Removal of Wolbachia through treatment with doxycycline has adulticidal activity against the parasite in dogs experimentally infected with D. immitis [7,8]. While MLs are highly efficacious against L3 and young fourth stage larvae (L4) of D. immitis, preventive doses have also been reported as having a so-called "slow kill" effect against adult parasites [9]. ...
Due to their marked larvicidal activity, macrocyclic lactones (MLs) are used for the prevention of heartworm disease ( Dirofilaria immitis) in dogs. They have also been shown to eliminate adult parasites after long-term administration, with a so-called “slow-kill” effect. In addition, recent studies have established that a combination of doxycycline, which eliminates the endosymbiont Wolbachia, and MLs has superior adulticide effects when compared to MLs alone. It has been hypothesized that the apparent synergism between doxycycline/MLs may be due to interaction with drug efflux transport proteins. The aim of the present study was to evaluate gene expression of several transport proteins in D. immitis adults treated in vitro either with doxycycline alone, ivermectin alone, moxidectin alone, or a combination of ivermectin or moxidectin with doxycycline for 12 h. Quantitative PCR analysis showed a sex-dependent response to treatments. In female worms, Dim-pgp-10, Dim-haf-1 and Dim-haf-5 were upregulated compared to controls with doxycycline alone and when combined with ivermectin. Moxidectin did not induce any changes in gene expression. In males, moxidectin administered alone induced a slight increase in Dim-pgp-10, Dim-pgp-11and Di-avr-14, while ivermectin in combination with doxycycline produced significant upregulation of the ML receptor Di-avr-14. These results suggest possible synergism between the two drug classes and different susceptibility of males vs. females to adulticide effects.
... Disease presentation will depend on parasite load, normally dogs remain without clinical signs with detectable microfilaria on blood smear, but cough and exercise intolerance can be observed [5]. Alternative slow killing treatment with macrocyclic lactone and doxycycline are reported [3,16]. ...
Background: Canine vector borne diseases (CVBD) are common in tropical countries where the climate favors arthropods abundance. Comorbidity with one or more CVBD are reported as clinical complication and worsen prognostic. Canine visceral leishmaniosis (CanL) is an endemic zoonotic disease in Brazil caused by Leishmania infantum, with several restrictions to canine treatment and suggestion of reservoirs euthanasia for disease control. Heartworm (HW) is a helminthic disease caused by Dirofilaria immitis infection in dogs. It is a chronic heart disease, which can lead to death by congestive heart failure. Canine ehrlichiosis (CE) is caused by Ehrlichia canis bacterial infection with a zoonotic potential and fatal to dogs in acute and chronic presentations. Exposed the above, this study aims to describe a successful treatment and management of a dog with CanL, CE, and HW comorbidity. Case: A 3-year-old male uncastrated black Labrador dog, weighing 35 kg, was admitted to the veterinary clinic due to immunochromatographic CanL positive test performed by municipal zoonosis control center active surveillance. Clinical exam showed a mild shedding, intermittent eye white/yellow discharge and popliteal lymph nodes enlargement. After positive for CanL, veterinary requested more laboratorial exams. IFAT and ELISA were positive for CanL, blood smear showed presence of microfilaria, and bone marrow cytology showed Ehrlichia spp. morulae and microfilaria. Initial treatment prescribed was oral doxycycline, omeprazole, ranitidine, and domperidone for 30 days, and allopurinol and ivermectin until further recommendation. Additionally, repellent collar, repellent spray and vitamin supplement was indicated. After first month, marbofloxacin for 30 days and three doses of immunostimulant drug were administrated. After three months of treatment, dog still positive for heartworm, ehrlichiosis, and CanL. Doxycycline protocol was repeated. Dog became consistently negative for all pathogens one year later with persistent thrombocytopenia but without clinical signs, ergo allopurinol and ivermectin were discontinued. After 4 years of follow up, the animal had an acute pancreatitis and died, with unremarkable total blood count and negative for all pathogens. Discussion: CVBD coinfections are commonly reported as worsen prognostic in endemic regions. The pathogens reported here share a host immunomodulation competence. L. infantum and Ehrlichia spp. downregulates Th1 response, whereas D. immitis increase as Th2 profile. The therapeutic protocol was iniciated by staging CanL. Since the patient had clinical signs, allopurinol was prescribed as a well-established drug for CanL. Marbofloxacin was added due to its high safety drug in clinical improvement of infected dogs with and without renal disease and in vitro effectiveness against L. infantum. Domperidone was used to promote Th1 cytokine profile as INF-γ, IL-2, IL-12, and TNF-α. We used an immunostimulant protocol to favor polarization to the Th1 profile comprised by 30 days of domperidone protocol followed by a vaccine and an immunomodulator. Doxycycline was used successfully for Ehrlichia spp. and HE clearance after 2 treatment courses and 1 year of ivermectin every 15 days. The animal presented intermittent coughing episodes on the first treatment course, but no medical intervention was needed besides exercise restriction. Our report shows the successful management of one dog with CanL, CE and HE comorbidity. This success was possible due to early detection and good therapeutic choice.
... Doxycycline given alone gradually reduces numbers of microfilariae and impairs the development of immature forms of D. immitis resulting in their inability to infect new definitive hosts. This effect is likely to be effective in reducing the rate of selection of D. immitis with genes that confer resistance to MLs [28]. ...
Background:
Dirofilaria immitis is a life-threatening nematode spreading globally. Arsenical treatment is currently recommended for removal of adult worms. However, arsenical treatment is not available in some countries, and there are dogs that cannot tolerate the rapid kill of adult worms; therefore, alternative adulticide slow-kill treatments are needed. Criticisms against the use of these alternative protocols include the potential for allowing disease to progress and for the development of ML-resistant worms.
Methods:
The efficacy of a protocol that includes semi-annual doses (i.e. every 6 months) of commercially available extended-release injectable moxidectin suspension (ProHeart® SR-12) with 30-day oral administration of doxycycline was studied in 20 dogs with naturally occurring D. immitis infections. Each dog received treatment with ProHeart® SR-12 (0.5 mg moxidectin/kg) by subcutaneous injection and oral doxycycline (10 mg/kg/bid × 30 days) every 6 months until two consecutive negative antigen test results were obtained. Pulmonary and cardiac evaluations were performed by radiographic and echocardiographic parameters. Physical examinations, complete blood counts, clinical chemistry profiles, microfilariae and antigen tests were performed periodically.
Results:
At enrollment, all dogs were positive for D. immitis antigen and 18 were microfilaremic. On day 30, microfilaremia counts decreased, and all dogs became amicrofilaremic by day 150. On day 180, 11 dogs were antigen-negative, and 7 more became negative by day 360. The two remaining antigen-positive dogs converted to negative by day 540 or 810. All antigen tests performed 180 days after the first negative test were negative. There was no decline in cardiac performance of the dogs throughout the study. Overall, pulmonary clinical conditions, presence of worms by echocardiography, and enlargement of caudal and main pulmonary arteries improved after treatment. Physical examinations, complete blood count results, and clinical chemistry profiles were within normal reference values. Respiratory conditions were improved, no damage to the heart was observed, and the treatment protocol was well tolerated by the animals.
Conclusions:
This alternative adulticide treatment was efficacious and well tolerated in naturally infected dogs. The injectable formulation provides the advantage of having veterinarians able to administer, monitor, and assess the efficacy and condition of the dog throughout the treatment and post-treatment periods.
... More recent research has focused on the combination of moxidectin and doxycycline based on moxidectin's broad parasiticidal efficacy and the significant impact of doxycycline on all stages of the heartworm life cycle including parasite reproduction through elimination of the parasite's endosymbiotic bacteria, Wolbachia. When doxycycline is used in combination with a ML, their effects are synergistic (Bandi et al., 1999;Bazzocchi et al., 2008;Kramer et al., 2018;McCall et al., 2008McCall et al., , 2014. ...
Canine heartworm disease (CHD) results from infection with Dirofilaria immitis and while it is of global concern, it is most prevalent in tropical climates where conditions support the parasite and vector life cycles. Melarsomine dihydrochloride is the sole treatment for CHD recommended by the American Heartworm Society. However, in cases where cost or access to melarsomine precludes treatment of an infected dog, therapeutic alternatives are warranted. This randomized, controlled field study evaluated the adulticidal efficacy of a combination therapeutic protocol using 10% imidacloprid + 2.5% moxidectin spot-on and a single 28-day course of doxycycline and compared with that of a 2-dose melarsomine dihydrochloride protocol. Of 37 naturally-infected domestic dogs with class 1, 2 or early class 3 CHD enrolled in the study, 30 were evaluated for a minimum of 12 months. Seven dogs were withdrawn due to canine ehrlichiosis, non-compliance, or wrongful inclusion. Dogs were randomly assigned to a control (CP, n = 15) or investigational (IVP, n = 15) treatment group. CP dogs received two injections of melarsomine dihydrochloride (2.5 mg/kg) 24 -hs apart and maintained on monthly ivermectin/pyrantel. IVP dogs were treated with oral doxycycline (10 mg/kg twice daily for 28 days) and topical 10% imidacloprid + 2.5% moxidectin once monthly for 9 months. Dogs were evaluated up to 18 months - monthly for the first 9 months, then every 3 months. Parasiticidal efficacy was based on antigen status using the IDEXX PetChek® 34 Heartworm-PF Antigen test. By month 18, antigen was not detected in any study dog except one from the IVP group. One other IVP dog was persistently antigenemic and treated with melarsomine at month 12 according to the initial study protocol. Mean antigen concentration (based on optical density) decreased more rapidly in the CP group and by month 15 was 0.11 for the IVP and 0.07 for CP groups, with equivalent median concentrations (0.04) in both groups. Conversion following heat-treatment of antigen-negative samples occurred frequently and at similar rates in both treatment groups. Based on the bias of diagnostic tests towards detection of female worms, we conclude that monthly application of 10% imidacloprid + 2. 5% moxidectin for 9 months combined with a course of doxycycline twice daily for 28 days resulted in effective therapy against female adults in CHD. This therapeutic option may be particularly useful in cases where financial constraint or access to melarsomine precludes treatment of an infected individual. This study was supported by Bayer Animal Health.
... Because Wolbachia are members of the Rickettsiaceae family, research has investigated the effect of antibiotics commonly used to treat rickettsial infections on these bacteria. For treatment of Wolbachia in animals and humans with filarial infections, researchers have used doxycycline at doses recommended for other rickettsial infections, which has resulted in a reduction in the number of Wolbachia bacteria present, decreased microfilarial loads, inhibition of the development of larval worms, and female worm infertility (Bandi et al., 1999;McCall et al., 2014). In dogs with heartworm, there have been reports on the clinical benefits of the administration of macrocyclic lactones and doxycycline before administration of melarsomine, the adulticidal drug of choice. ...
The endosymbiont bacteria Wolbachia plays an important role in the pathogenesis and inflammatory immune response to heartworm (Dirofilaria immitis) infection in dogs. Doxycycline is used to reduce Wolbachia from all life stages of heartworm to avoid large releases of the bacteria during the death of the worms. However, the dose and duration currently recommended have been extrapolated from the treatment of other rickettsial infections. Therefore, the aim was to study the dynamics of Wolbachia IgG antibodies in heartworm-infected dogs under adulticide treatment using different dosages of doxycycline. Forty-nine heartworm-infected dogs were recruited. On day 0 (diagnosis), monthly ivermectin (6 μg/kg) was prescribed, as well as daily doxycycline for 30 days, at 10 mg/kg/12 h (n = 13), 5 mg/kg/12 h (n = 19), and 10 mg/kg/24 h (n = 17). Dogs underwent adulticide treatment and blood samples were collected on days 0, 30, 90, and 120. All dogs had antibodies against recombinant Wolbachia surface protein (rWSP), confirming the important role of the bacteria in heartworm. No significant differences were found in anti-rWSP response by presence/absence of microfilariae, or by parasite burden on day 0. In all treated groups, the anti-rWSP antibody response was not significantly different between days 0 and 30 but was significantly lower between days 0 and 120 (p < 0.05). The results of the present study suggest that the administration of a lower dose than currently recommended is sufficient to achieve a significant reduction of Wolbachia in dogs infected by D. immitis.
... Regarding dogs imported from endemic areas, it is more difficult to consider the susceptibility gap, which is defined as the period when adult heartworm disease has been diagnosed, but some stages of D. immitis are not susceptible to treatment with macrocyclic lactones or melarsomine dihydrochloride (Bowman and Drake 2017), as many dogs are presented several months after their entry, that is a long time after exposure to the parasite. Irrespective of the therapy protocol used, administration of macrocyclic lactones and doxycycline one to 2 weeks prior to the first melarsomine injection should be considered in order to minimize side effects induced by the death of macrofilariae (McCall 2005;McCall et al. 2014). ...
A retrospective study based on cases of canine dirofilariosis presented to the University of Veterinary Medicine, Vienna or diagnosed by private practitioners throughout Austria, from 1998 to 2018 was conducted to investigate the long-term development and current state of canine dirofilarial infections in Austria. Included in this study were 146 dogs which were tested positive for D. immitis and/or D. repens. The most commonly used diagnostic methods and the probable geographical origins of the infections were evaluated and the treatment protocols applied were compared with each other and with the literature. The results show that most infections were found due to screening for common travel infections using antigen-ELISA or PCR-testing, or by the incidental finding of microfilariae. Remarkably, only 24.3% of all cases presented showed clinical signs indicating canine dirofilariosis. Regarding the origin and travel history of the dogs, thirteen different countries could be identified. The three treatment protocols used showed a similar outcome after 8 months of treatment and minor side effects, which is consistent with the literature. An alarming increase in reported infections with both D. immitis and D. repens in Austria was noted since 2014. The number of documented cases had almost tripled by 2018, raising severe concerns about the threat of it becoming endemic in Austria. Therefore, the existing recommendations in current guidelines regarding canine dirofilariosis should be widely publicised and more strictly enforced. Prophylactic measures for dogs travelling abroad and diagnostic and therapeutic strategies for dogs imported from endemic countries should be obligatorily established throughout Europe, to reduce the risk of further spread of canine filarial infections to non-endemic regions.
... Wolbachia symbionts can be both facultative (Teixeira et al., 2008;Osborne et al., 2009;Weisman et al., 2009) and obligatory. For example, filarial nematodes could not reproduce without Wolbachia (Rao et al., 2002;Rajan, 2004;McCall et al., 2014), and some treated insects suffer decreasing lifespan and fertility (or even become infertile) (Dedeine et al., 2001;Dong et al., 2007;Hosokawa et al., 2010). ...
Wolbachia are intracellular symbiotic bacteria of terrestrial arthropods and some nematodes. They are found in most insect orders; however, there are insufficient data on symbiont distribution patterns for some taxonomic groups. Here, we examined a collection of Siphonaptera species by conventional and nested polymerase chain reaction. A total of 722 specimens from 30 species were sampled in three regions of Russia: Southern Russia, Ural and the Far East. Wolbachia infection was found in half of the species, which confirmed previous data on the widespread nature of the infection in Siphonaptera. No statistical differences in Wolbachia incidence in flea species from Southern Russia and the Far East were detected, although species lists of these regions completely differed. We did not find totally infected flea species, although high infection rates were detected for Frontopsylla elata botis (64.5%) and Ctenophthalmus wagneri (66%) with sample size exceeding 30 specimens. Our results are in agreement with the data from other regions of the world. Combined data of our study and other authors indicate that Wolbachia symbionts are found in all 11 studied families of Siphonaptera and in 45 out of about 120 studied species in the world.
... Doxycycline has various detrimental effects on developing and adult heartworms, including degeneration of oocytes and early embryonic stages, but its adulticide efficacy is low and slow [10]. Macrocyclic lactones (MLs) are highly efficacious against L3 and young fourthstage larvae (L4) and prevention of disease is based on this activity [11]. ...
Background:
Combination doxycycline/macrocyclic lactone (ML) protocols have been shown to provide a more rapid adulticidal and microfilaricidal effect than either MLs or doxycycline alone, although female worms were reported to have a higher tolerance to treatments compared to male worms. The present study aimed to evaluate how ABC transporters may be involved in the synergic effect of the combination treatment. Adult worms of D. immitis were treated in vitro for 24 hours with doxycycline (DOXY), ivermectin (IVM) and a combination of both, and changes in the modulation of ABC transporter genes were measured. Levels of doxycycline inside different treatment media, post-treatment, were determined through HPLC analysis.
Results:
Quantitative RT-PCR analysis showed the presence of changes in the modulation of ABC transporter genes evaluated in this study. In particular, in female worms, the combination treatment induced a substantial increase in gene expressions, especially of Dim-pgp-10 and Dim-haf-4; whereas in male worms, the greatest increase in gene expression was observed for Dim-pgp-10 and Dim-pgp-11 when treated with DMSO + IVM and DMSO + DOXY/IVM. HPLC analysis of the DOXY concentrations in the media after in vitro treatments of male worms showed a slight difference between the DMSO + DOXY samples and the combination (DMSO + DOXY + IVM), while no difference was observed among females.
Conclusions:
Further studies are required to explain whether the modulation of cellular efflux plays a role, even partially, in the adulticide effect of doxycycline/macrocyclic lactone combinations in heartworm-infected dogs. To the authors' knowledge, this is the first study to evaluate P-gp expression in adult D. immitis.
... The close proximity of the home environment with cognitive development in our model gives it a critical role of protecting against the negative impact of ill-health and adverse SES. This is consistent with recent analysis of 117,000 households by Bornstein et al. (2015), which found that the resources available at home make up three-quarters of the link between SES factors and the child's development, and with findings of earlier studies in Ugandan and Kenyan children that showed no direct relationship between SES and psychomotor development (McCall et al., 2014;Sayed et al., 2005). The focus in this study, children living in poverty and susceptible to frequent and serious illness, accentuates the importance of the home environment when such threats are regular and intense. ...
Background: The home environment is reported to contribute significantly to children’s developing cognitive skills. However, it is not yet evident whether this role prevails in the context of extreme poverty and frequent ill-health. We therefore investigated the role of the home environment in Ugandan children taking into account the frequent infections and extreme poverty in which they lived.
Methods: Cognitive abilities of 163 5-year-old children were assessed. Home environments of these children, their health status and family socioeconomic status (SES) were assessed respectively using the EC-HOME, anthropometry and illnesses, and traditional SES measures. Structural equation analyses compared five models on the influence of the home environment, SES, and child health on the cognitive scores.
Results: The model in which the home environment mediates the combined influence of SES and child health on cognitive performance showed a particularly good fit to the data compared with the four alternative models, i.e. those in which the HOME, SES and health independently influence cognitive performance.
Conclusions: Home environments providing cognitive stimulation can enable children to overcome effects of major adverse life experiences on cognitive development.
... Wolbachia symbionts can be both facultative (Teixeira et al., 2008;Osborne et al., 2009;Weisman et al., 2009) and obligatory. For example, filarial nematodes could not reproduce without Wolbachia (Rao et al., 2002;Rajan, 2004;McCall et al., 2014), and some treated insects suffer decreasing lifespan and fertility (or even become infertile) (Dedeine et al., 2001;Dong et al., 2007;Hosokawa et al., 2010). ...
Wolbachia are intracellular symbiotic bacteria of terrestrial arthropods and some nematodes. They are found in most insect orders; however, there are insufficient data on symbiont distribution patterns for some taxonomic groups. Here we examined a collection of Siphonaptera species by conventional and nested PCR. A total of 722 specimens from 30 species were sampled in three regions of Russia: Southern Russia, the Urals, and the Far East. Wolbachia infection was found in half of the species, which confirmed previous data on widespread of the infection in Siphonaptera. No statistical differences in Wolbachia incidence in flea species from Southern Russia and the Far East were detected, although species lists of these regions completely differed. We did not find totally infected flea species, although high infection rates were detected for Frontopsylla elata botis (64.5%) and Ctenophthalmus wagneri (66%) with sample size exceeding 30 specimens. Our results are in agreement with the data from other regions of the world. Combined data of our study and other authors indicate that Wolbachia symbionts are found in all eleven studied families of Siphonaptera and in 45 out of about 120 studied species in the world. © 2018 Maik Nauka-Interperiodica Publishing. All rights reserved.
... Studies have shown that Wolbachia is required for normal parasite development, fertility and long-term survival of different filarial species. These bacteria have been implicated in adverse effects of drug treatment in filariasis (McCall et al., 2014). Some studies showed a strong interaction of Wolbachia-associated molecules between filarial parasite and the human host WSP (Bandi et al., 2001;Brattig et al., 2004). ...
The present study aimed to evaluate the extent of Dirofilaria immitis and D. repens exposure in humans from eastern and southern areas of Romania and central Moldova by serological methods. The serological screening was performed on a total of 450 serum samples (187 from Romania and 263 from Moldova). The sera were collected using a convenience sampling with the help of physicians from the hospitals of the study areas. All samples were analysed by a non-commercial ELISA test for the detection of IgG antibodies against adult somatic antigens of D. immitis and D. repens. The results showed a total of 49 (10.9%; 95% CI = 8.3-14.1%) individuals from Romania and Moldova with a positive response to IgG antibodies against both adult somatic antigens of D. immitis and D. repens. Specifically, 48 (10.7%; 95% CI = 8.0-14.0%) patients were positive for IgG-antibodies against adult somatic antigens of D. immitis, one (0.2%; 95% CI = 0.4-1.2%) against D. repens antigens, and four (0.9%; 95% CI = 0.4-3.3%). were positive for antigens of both parasites. At country level, out of 187 samples from Romania, 13 (6.9%; 95% CI = 4.1-11.5%) were positive for anti-D. immitis IgG with high exposure in the southern part of the country (Bucharest). Of the 263 people from Moldova, 36 (13.7%; 95% CI = 10.0-18.4%) were positive for D. immitis antigens from which three (1.1%, 95% CI = 0.4-3.3%) were positive for the antibodies against antigens of both parasites. Only one sample was found positive for anti-D. repens IgG. Positive IgG-ELISA results were confirmed by Western blot analysis. In addition, for further confirmation, a complementary ELISA was performed for anti-WSP IgG antibodies against Wolbachia endosymbionts. Our findings showed a noticeable exposure of humans from Romania and Moldova to Dirofilaria parasites. Serology can be useful for indicating exposure to Dirofilaria spp. in a healthy population in order to obtain useful data on the epidemiological scenario of human dirofilariosis in Eastern Europe.
... Studies have shown that heartworm-infected dogs treated with doxycycline to eliminate Wolbachia, before receiving filaricidal therapy, have less severe pulmonary inflammation and thrombosis. Therefore, the administration of doxycycline is an important step during the adulticidal treatment and all official protocols recommend its administration (McCall et al. 2014;Nelson et al. 2017). Doxycycline is given at a dose of 10 mg/kg twice daily for 30 days, which is twice the common oral dose of the doxycycline used in antibacterial treatments in veterinary medicine. ...
Doxycycline is an antibiotic that, in addition to the classic antibacterial use, is also prescribed to fight parasitic diseases, like heartworm disease in dogs. Despite the concern that the overuse of this antibiotic may decrease susceptibility of clinically important bacteria, the consequences of the prolonged doxycycline therapy in heartworm-infected dogs have never been studied before. We have analyzed the impact of this therapy on Staphylococcus aureus and Enterococcus antimicrobial resistance. In this study, 17 heartworm-infected dogs (10 that had completed the doxycycline treatment and 7 dogs that had not yet begun) were included. Twenty-four isolates of Staphylococcus aureus were obtained from two locations of each dog. After treatment, 73.3% of isolates were resistant to at least one antibiotic but only 22.2% of isolates before treatment. Most of doxycycline resistant isolates were obtained from dogs that have received treatment. Erythromycin resistance or intermediate susceptibility was detected in 45.6% of isolates, most of them from dogs after treatment. For Enterococci, 48 isolates were obtained from fecal samples (25 before treatment and 23 after treatment). Before treatment, 32% of isolates were resistant at least to one antibiotic while after, this data increase up to 65%. Comparing isolates before and after treatment, a clear increase in resistance to doxycycline (12% against 21.74%) and erythromycin (20% against 39.13%) was observed. Although the present work is a preliminary research, the results encourages the development of further studies to determinate the effect of prolonged doxycycline therapy on antimicrobial resistance.
... These studies have shown a decrease in gross and microscopic pathology of pulmonary parenchyma in experimental heartworm infections pretreated with doxycycline before melarsomine administration [12,13]. Doxycycline administered at 10 mg/kg twice daily (BID) for 4 weeks has been shown to reduce or eliminate the Wolbachia organisms for up to 12 months [14]. Additional studies have shown that administration of doxycycline in combination with ivermectin provided more rapid adulticidal activity than ivermectin alone [15] as well as more effectively reducing Wolbachia numbers than doxycycline alone [16]. ...
Background
The objective of heartworm treatment is to improve the clinical condition of the patient and to eliminate pre-cardiac, juvenile, and adult worm stages with minimal complications. Pulmonary thromboembolisms are an inevitable consequence of worm death and can result in severe pulmonary reactions and even death of the patient. To minimize these reactions, various treatment protocols involving melarsomine, the only adulticidal drug approved by the US Food and Drug Administrations (FDA), in conjunction with macrocyclic lactone heartworm preventives and glucocorticosteroids have been advocated. The discovery of the bacterial endosymbiont Wolbachia in Dirofilaria immitis has led to several experimental studies examining the effects of administering doxycycline to reduce or eliminate Wolbachia organism. These studies have shown a decrease in gross and microscopic pathology of pulmonary parenchyma in experimental heartworm infections pretreated with doxycycline before melarsomine administration.
Methods
Electronic medical records from a large veterinary practice in northeast Alabama were searched to identify dogs treated for heartworms with melarsomine from January 2005 through December 2012. The search was refined further to select for dogs that met the following criteria: 1) received two or three doses of ivermectin heartworm preventive prior to melarsomine injections, 2) received one injection of melarsomine followed by two injections 4 to 8 weeks later, and 3) were treated with prednisone following melarsomine injections. The dogs were then divided into those that also were treated with doxycycline 10 mg/kg BID for 4 weeks (Group A, n = 47) and those that did not receive doxycycline (Group B, n = 47). The medical notes of all 94 cases were then reviewed for comments concerning coughing, dyspnea, or hemoptysis in the history, physical exam template, or from telephone conversations with clients the week following each visit. Any dog that died within one year of treatment from either cardiovascular or pulmonary problems was noted.
Results
Dogs from Group A receiving doxycycline had fewer respiratory complications (6.52%) and heartworm disease-related deaths (0%) than Group B (19.14% and 4.25%, respectively).
Conclusions
Although there are not enough cases to indicate statistical significance, the results strongly suggest that including doxycycline into canine heartworm treatment protocols decreases post-treatment complications and mortality in naturally infected clinical cases.
... The model was considered successful since the control group was infected at rates (21-64 worms/dog) close to those usually targeted with other experimental infection techniques, such as the use of subcutaneous injection of L3 (up to 37 worms/dog) [20,21], or to natural worm burdens where up to 85 worms/dog were found [22]. This model is expected to be close to the natural exposure of dogs by infected mosquitoes, but higher or lower exposure levels can certainly be achieved because animals can be bitten by uninfected mosquitoes, as well as mosquitoes with variable and unpredictable numbers of L3. ...
Background
This study assessed the influence of a topical ectoparasiticide (dinotefuran-permethrin-pyriproxyfen, DPP, Vectra® 3D, Ceva Animal Health) combined with a macrocyclic lactone (milbemycin oxime, MBO, Interceptor®, Virbac) on transmission of heartworm L3 from mosquitoes to dogs and subsequent development of worms in treated dogs exposed to infected mosquitoes.
Methods
Thirty-two beagle dogs were allocated to four groups of eight: Group 1, untreated controls; Group 2, treated topically with DPP on Day 0; Group 3, treated orally with MBO on Day 51; and Group 4, treated with DPP on Day 0 and MBO on Day 51. Dogs were exposed under sedation for 1 h to Dirofilaria immitis (JYD-34)-infected Aedes aegypti on Days 21 and 28. At the end of each exposure, mosquitoes were classified as live, moribund, or dead and engorged or non-engorged. Live or moribund mosquitoes were incubated for daily survival assessment for 3 days. Mosquitoes were dissected before and after exposure to estimate the number of L3 transmitted to each dog. Dogs were necropsied 148 to 149 days postinfection.
Results
A total of 418 mosquitoes fed on the 16 dogs in Groups 1 and 3, while only 6 fed on the 16 DPP-treated dogs in Groups 2 and 4. Mosquito anti-feeding (repellency) effect in Groups 2 and 4 was 98.1 and 99.1%, respectively. The estimated numbers of L3 transmitted to controls, DPP-treated, MBO-treated and DPP + MBO-treated dogs were 76, 2, 78, and 1, respectively. No heartworms were detected in any of the DPP + MBO-treated dogs (100% efficacy), while 8 out of 8 were infected in the control group (range, 21–66 worms per dog), 8 out of 8 were infected in the MBO-treated group (58% efficacy), and 3 out of 8 were infected in the DPP-treated group (96% efficacy).
Conclusions
DPP repelled and killed most mosquitoes that were capable of transmitting heartworm L3 to dogs. The “Double Defense” protocol of DPP + MBO had better efficacy for protecting dogs against heartworm transmission and infection than MBO alone. This added DPP benefit is more pronounced when macrocyclic lactone-resistant strains of heartworms are involved or lack of compliance in macrocyclic lactone administration is known or suspected.
... Our result of the DOXY treatment regimen, both alone or plus IVM showed a gradual reduction in microfilariae in blood circulation compared to IVM treatment. Similar gradual reductions in D. immitis microfilarial counts were also observed when DOXY was administered orally (McCall et al., 2011(McCall et al., , 2014. Walker et al. (2015) who study the therapeutic efficiency of doxycycline on Onchocerca volvulus stated that "the dynamics of the antifilarial outcomes induced by doxycycline are protracted because of the indirect mode of action of anti-Wolbachia therapy on parasites' vital processes. ...
Lymphatic filariasis (LF) is one of the neglected tropical diseases which causes permanent and long term disability worldwide. LF is caused by filarial nematode parasites, i.e. Wuchereria bancrofti, Brugia malayi, and B. timori. All available antifilarial drugs currently being used have shown a limited adulticidal activity. Discoveries of endosymbiont rickettsia-like bacterium, Wolbachia in filarial nematodes provided a novel approach for antibiotic use in eradication of filarial diseases. The earlier studies revealed the macrofilaricidal efficacy of doxycycline against filarial nematodes. Chemotherapeutic efficiency of doxycycline has been studied against many filarial parasites, but there are still no therapeutic trials of the drug regimens for B. malayi treatment in naturally infected cats. Thus, this study would be the first attempt to study the efficiency of doxycycline (DOXY) alone or in combination with ivermectin (IVM) for treatment of B. malayi in naturally infected cats. A total of 26 B. malayi-infected cats in the endemic areas were recruited and divided into 3 groups, receiving different treatment regimens; a single dose of ivermectin only (IVM), doxycycline only (DOXY) and a combination of ivermectin and doxycycline (DOXY-IVM). The efficacy of each therapatic regimen was evaluated by detecting the presence of microfilaria using parasitological and molecular techniques monthly up to 2 years after starting the treatment. The IVM treated group had a significant rapid reduction of microfilariae in the first month; however, recurrence of microfilaraemia was observed in some cats. By contrast, the DOXY and DOXY-IVM groups showed a better result with a gradual decrease in microfilariae with no recurrence. These 2 groups were not only virtually deprived of infection but also sustained the sterility of infection through the course of study. These results revealed the advantages of using in B. malayi treatment in cats. Doxycycline showed to have both microfilaricidal and adulticidal effects on lymphatic filariae which maintained the long-term response to control of B. malayi infection in cats.
... Известно, что Wolbachia может выступать облигатным симбионтом. Так, филярийные нематоды не способны нормально развиваться и размножаться без Wolbachia (Sungpradit, Nuchprayoon, 2010;McCall et al., 2014). У членистоногих облигатность Wolbachia описана для паразитической осы Asobara tabida (Dedeine et al., 2001;Kremer et al., 2009), сеноеда Liposcelis tricolor (Dong et al., 2007) и клопа Cimex lectularius (Hosokawa et al., 2010). ...
Siberian silk moth (Dendrolimus superans sibiricus) is a very dangerous pest of coniferous trees, in particular, larch and various pine species. Outbreaks of this pest lead to defoliation and forest destruction in a vast area of the Asian part of Russia. Many biological agents, such as viruses, pathogenic microorganisms and parasitoids, prevent the growth of Siberian silk moth population. Here we consider non-pathogen symbiotic Wolbachia bacteria, which are transovarially transmitted between specimens from mother to offspring. This symbiont has an ability to affect biology of its host. In theory, Wolbachia can prevent the growth of population size or induce it, which determines the focus of interest in Wolbachia-host investigation. Two samples from a Siberian silk moth population collected in 2014 and 2016 in Khabarovsk area were studied for Wolbachia infection. We found a high Wolbachia prevalence in the population of Siberian silk moth, in particular, the sample of 2014 was totally infected and the sample of 2016 had 90 % infected specimens. There were at least two distinct Wolbachia strains reveled by analysis of two loci from the MLST protocol, namely f tsZ-36, f bpA-4 and f tsZ-22, f bpA-9. In this study, a possible role of Wolbachia in the symbiotic association with Siberian silk moth and general ways of investigation of this symbiosis are discussed.
... However, this dose may be too low given that a 10 mg/kg PO BID dose is recommended for targeting Wolbachia in dogs infected with D. immitis. 23,24 This concern led to an increase in the dose of doxycycline and minocycline to 10 mg/kg PO BID administered to the dogs that presented later in this series. ...
Objective:
To describe the clinical exam findings, treatment and outcomes of 16 dogs diagnosed with ocular onchocerciasis in New Mexico.
Materials and methods:
Records of dogs diagnosed by the primary author were reviewed (2011-2015). Records that were accessible and included a diagnosis of Onchocerca lupi by histopathologic or molecular identification of the nematode were included.
Results:
Sixteen cases were included. 3/16 dogs were treated with year-round heartworm prophylaxis prior to infection. Clinical exam findings included conjunctival hyperemia and/or episcleral injection (16/16), focal subconjunctival mass(es) (14/16), retinal detachment (7/16), corneal edema (4/16), chemosis (3/16), corneal opacity (2/16), exophthalmia (1/16), glaucoma (1/16), strabismus (1/16), blepharospasm (1/16), and vitreal degeneration (1/16). Ocular involvement was unilateral in 7/16 dogs and bilateral in 9/16 dogs. The diagnosis was confirmed via histologic identification of the nematodes and/or PCR. Treatment consisted of medical management or a combination medical and surgical management. Known or suspected recurrence of disease was documented in 10 dogs.
Conclusions:
Canine ocular onchocerciasis is endemic in New Mexico. Histopathology and molecular identification are useful diagnostic tools. Medical management alone was successful in many cases.
... Studies carried out on some anthroponotic filariae and on Dirofilaria immitis obtained discordant results. However, it seems that only doxycycline may have a possible adulticidal effect [2,11], due to its action on the bacterial endosymbiont Wolbachia, present in most filarial species (D. repens included). It is conceivable that doxycycline had an adulticidal effect and a role in the absence of microfilariae in the bloodstream, but it should be taken in mind that the patient received only a brief course of treatment (14 days), and peripheral blood was tested for microfilariae 3 months after therapy withdrawal. ...
Background:
Human dirofilariosis is still a little known infection even in endemic areas. Dirofilariosis is zoonotic infection usually abortive in humans; instead, we report a very rare case (the 4th in the world), the first in Italy, in which at least two infective larvae became mature adults that mated and produced active microfilariae even though they did not reach peripheral blood.
Case presentation:
A 30-year-old Italian woman presented with a transient oedematous swelling on the left abdominal wall with a creeping eruption followed by the occurrence of a subcutaneous nodular painless mass in the iliac region. One month later, a similar temporary swelling appeared on the contralateral inguinal region associated with intermittent joint discomfort in both knees. The patient had recently travelled abroad, therefore many possible diagnoses were to be ruled out. Routine laboratory investigations revealed eosinophilia. An ultrasound examination of the iliac swelling evidenced a well-defined cyst with a big filamentous formation in continuous movement. A fine-needle aspiration of the lesion was performed for parasitological, cytological and histological exams. The prompt microscopic examination of the aspired material showed the presence of numerous microfilariae that were initially morphologically attributed to Mansonella ozzardi. Subsequently, the revision of the Giemsa stained film and molecular analyses of the biological material, allowed to identify Dirofilaria repens as etiological agent of infection.
Conclusions:
We report of a case in whom microfilariae were detected in fine-needle aspirate of subcutaneous node, without evidence of microfilaraemia, and the infection failed to become fully patent. Therefore we confirm that complete development and fertilization of D. repens worms in human hosts may occur, at variance with what is commonly believed, that Dirofilaria worms cannot fully develop in humans.
... The dynamics of Wolbachia-nematode host interactions have been a topic of active investigation (Taylor and Hoerauf, 1999;Brattig, 2003;Taylor et al., 2005a;Foster et al., 2013;Landmann et al., 2014). Antibiotic depletion of Wolbachia leads to developmental defects in embryogenesis and nematode larvae, as well as killing of adult filarial worms, supporting the essential nature of the Wolbachia-nematode relationship (Hoerauf et al., , 2000Fenn and Blaxter, 2004;Taylor et al., 2001Taylor et al., , 2005bStrubing et al., 2010;Foster et al., 2013;McCall et al., 2014). There is also evidence that Wolbachia spp. ...
The symbiotic relationship of Wolbachia spp. was first observed in insects, and subsequently in many parasitic filarial nematodes. This bacterium is believed to provide metabolic and developmental assistance to filarial parasitic nematodes, although the exact nature of this relationship remains to be fully elucidated. While Wolbachia is present in most filarial nematodes in the family Onchocercidae, it is absent in several disparate species, such as the human parasite Loa loa. All tested members of the genus Acanthocheilonema, such as Acanthocheilonema viteae, have been shown to lack Wolbachia. Consistent with this, we show that Wolbachia is absent from the seal heartworm (Acanthocheilonema spirocauda), but lateral gene transfer (LGT) of DNA sequences between Wolbachia and A. spirocauda has occurred, indicating a past evolutionary association. Seal heartworm is an important pathogen of phocid seals, and understanding its basic biology is essential for conservation of the host. The findings presented here may allow for the development of future treatments or diagnostics for the disease, and also aid in clarification of the complicated nematode-Wolbachia relationship.
Macrocyclic lactones (MLs) are antifilarial drugs. Resistance occurs in Dirofilaria immitis to the MLs, ivermectin (IVM), milbemycin oxime, selamectin, and moxidectin (MOX). To date, resistance in D. immitis has arisen in the southern United States. Factors that may have contributed to selection of ML resistance are (i) high‐intensity local transmission, (ii) a degree of inbreeding of the parasite, the fact that MLs act not only on the developing L3/L4 stages, but (iii) also have microfilaricidal properties and (iv) inhibit fecundity in adult parasites, (v) year‐round monthly treatment, which (vi) may allow periods when drug concentrations are falling and insufficient, against less susceptible parasites to exert these antiparasitic effects. ML heartworm preventives have been used for over 30 years. This long‐term selection pressure coupled with the heartworm life cycle make it not surprising that resistance has arisen in regions where transmission and drug treatments are highest. Tests to confirm ML resistance include a microfilariae reduction test and genetic markers. More research is needed to understand the mechanisms of resistance, factors that could reduce its spread or overcome resistance, as well as monitoring resistance. IVM has been used to treat onchocerciasis for over 30 years, and in sub‐Saharan Africa, to treat lymphatic filariasis (LF) in humans. Recently, MOX was registered for onchocerciasis. IVM and MOX remove microfilariae and suppress fecundity of adult Onchocerca volvulus for several months. There are a number of reports of a decrease in the anti‐fecundity effect of IVM in some countries, and recently evidence of a reduction in the microfilaricidal effect of IVM. These reductions in the anti‐fecundity and microfilaricidal effects of IVM indicate developing IVM resistance in O. volvulus . Diethylcarbamazine (DEC) is widely used for LF. There have been some reports of poor responses to DEC treatment, but no concrete evidence of DEC resistance. Albendazole (ABZ) is used in combination with DEC or IVM, and more recently in a triple combination of these anthelmintics, to enhance the anti‐fecundity effect on LF. There is no evidence of resistance to ABZ in LF. However, single‐nucleotide polymorphisms that confer ABZ resistance in other nematodes have been found in Wuchereria bancrofti . Doxycycline has be en used against filariae in situations in which ML resistance is a concern or where there is a risk of severe adverse events after IVM use in people with high burdens of Loa loa . Doxycycline and related tetracyclines acts on the symbiotic Wolbachia in most filarial parasites, and the elimination of these bacteria impairs the viability of their filarial hosts. Antibiotic resistance to doxycycline can be selected, but so far has not been recorded when it is used as an anti‐filarial treatment.
Chemotherapy options for the prevention of adult Dirofilaria immitis infections and other filarial species in dogs and cats focus primarily on the use of macrocyclic lactones. While macrocyclic lactones are considered safe when used as per their registered product labels, veterinarians should be aware of their basic mechanisms of action and metabolism to understand potential differences in efficacy and safety in the individual animal. With the advent of D. immitis resistance to macrocyclic lactones and the lack of preventatives or treatments for many other filarial species, supportive measures to decrease exposure to the intermediate host are growing in importance. Chemotherapeutic treatment of adult D. immitis infections is only available for dogs and uses melarsomine as the basis. Treatment guidelines differ with regard to the use of antibiotics and treatment regimen but result in similar efficacy and provide veterinarians with options that can be adapted based on the client and patient.
Wolbachia bacteria are endosymbionts of many parasitic filarial nematodes. They are present in human pathogenic filarial species causing lymphatic filariasis ( Wuchereria bancrofti , Brugia malayi , and Brugia timori ), onchocerciasis ( Onchocerca volvulus ), mansonellosis ( Mansonella perstans and Mansonella ozzardi ), but not in filariae causing loiasis ( Loa loa ). Furthermore, they are present in a large number of filariae parasitizing animals, including the canine filariae Dirofilaria immitis and Dirofilaria repens . Wolbachia are proposed to provide the filarial nematodes with essential factors like heme, nucleotides, and riboflavin. The filarial species that contain Wolbachia depend on the endosymbionts for development, embryogenesis, and survival. Due to this crucial role of Wolbachia for filarial transmission and survival, drugs that target the Wolbachia endosymbionts provide an alternative treatment option that is being exploited. Over the past decades, it was shown that the anti‐wolbachial drug doxycycline is a safe treatment that leads to permanent sterilization of the female adult worms and provides a macrofilaricidal effect, i.e. it kills adult worms, in humans suffering from onchocerciasis and lymphatic filariasis. As microfilariae are slowly cleared over several weeks by anti‐wolbachials from the skin or circulation, anti‐wolbachial therapies do not trigger inflammatory responses such as those seen after diethylcarbamazine treatment in onchocerciasis and lymphatic filariasis patients. Furthermore, the lack of Wolbachia endosymbionts in L . loa prevents potential life‐threatening serious adverse events that can occur in highly microfilaremic patients treated with diethylcarbamazine or ivermectin. Similarly, doxycycline is included in the recommended combination treatment with ivermectin and melarsomine for canine heartworm disease and was suggested to lessen treatment‐associated pathology in comparison to melarsomine treatment alone or in combination with ivermectin. Thus, anti‐ Wolbachia compounds have several advantages compared to drugs currently used for human mass drug administration. Nevertheless, contraindications for doxycycline and its treatment regimen of at least four weeks prevent its broader use for human filariasis. Therefore, novel or improved anti‐ Wolbachia therapies are being discovered and developed, including high‐dose rifampicin, moxifloxacin, ABBV‐4083 (Flubentylosin), Corallopyronin A, AWZ‐1066S, and combination therapies with other antibiotics or direct acting drugs to reduce the treatment time required to clear filarial infection or permanently block the transmission of the disease.
Heartworm disease, caused by Dirofilaria immitis, can be lethal in dogs and cats. It is transmitted by mosquitoes, and occurs in many parts of the world. Prevention relies on macrocyclic lactones (MLs). MLs used are ivermectin, selamectin, abamectin, eprinomectin, milbemycin oxime and moxidectin, administered at 30-day intervals during the transmission season. Some moxidectin formulations are long-acting injectables. In the USA, preventives are recommended throughout the year. Loss of efficacy of ML preventives was reported in 2005 and proof of resistance in the USA was published a decade later. Understanding factors which promote resistance is important to maintain control. Factors important for resistance development are discussed. Better, inexpensive tests to confirm resistance are needed. Infection in animals under chemoprophylaxis per se does not imply resistance because lack of compliance in preventive use could be the reason. In vivo confirmation of resistance is expensive, slow and ethically questionable. A microfilariae suppression test can be a surrogate test, but requires a high dose of a ML and repeated blood microfilaria counts 2-4 weeks later. DNA single nucleotide polymorphism (SNP) markers have been successfully used. However, the specific genetic changes which cause resistance are unknown. Surveys to map and follow the extent of resistance are needed. Long acting mosquito repellants and insecticides can play a useful role. High dose rate formulations of moxidectin, coupled with mosquito biting mitigation may reduce transmission of resistant genotypes. Doxycycline, daily for 28 days, as anti-Wolbachia treatment, can reduce transmission and remove adult parasites. However, new classes of heartworm preventives are needed. While any preventive strategy must be highly effective, registration requirements for 100% efficacy may hinder development of useful new classes of preventives. Continued reliance on ML preventives, when they do not work against resistant genotypes, will spread resistance, and allow for more disease.
Heartworm disease is a serious vector‐borne disease that is caused by the mosquito‐borne filarial nematode Dirofilaria immitis. Knowledge of the lifecycle of D. immitis in conjunction with the pharmacology of preventive and adulticidal medications is crucial to understanding prevention and treatment strategies and failures. Because of the limitations of testing in cats and the fact that they do not pose a risk for heartworm transmission to other animals, testing for heartworms in the shelter cat should be reserved for symptomatic cats. Heartworm infections are most commonly detected through the use of a variety of commercial antigen blood tests and screening tests to detect circulating microfilaria. It is important to note that diagnostic test results are not fully predictive of posttreatment complications or success. Many shelter veterinarians successfully treat heartworm disease based on heartworm screening tests and clinical examination without further laboratory or radiographic pre‐treatment evaluation.
Filarial nematodes are tissue-dwelling parasitic worms that can cause a range of disfiguring pathologies in humans and potentially lethal infections of companion animals. The bacterial endosymbiont, Wolbachia, is present within most human and veterinary filarial pathogens, including the causative agent of heartworm disease, Dirofilaria immitis. Doxycycline-mediated drug targeting of Wolbachia leads to sterility, clearance of microfilariae and gradual death of adult filariae. This mode of action is attractive in the treatment of filariasis because it avoids severe host inflammatory adverse reactions invoked by rapid-killing anthelmintic agents. However, doxycycline needs to be taken for four weeks to exert curative activity. In this review, we discuss the evidence that Wolbachia drug targeting is efficacious in blocking filarial larval development as well as in the treatment of chronic filarial disease. We present the current portfolio of next-generation anti-Wolbachia candidates discovered through phenotypic screening of chemical libraries and validated in a range of in vitro and in vivo filarial infection models. Several novel chemotypes have been identified with selected narrow-spectrum anti-Wolbachia specificity and superior time-to-kill kinetics compared with doxycycline. We discuss the opportunities of developing these novel anti-Wolbachia agents as either cures, adjunct therapies or new preventatives for the treatment of veterinary filariasis.
More than 150 million humans in tropical countries are infected by filarial nematodes which harbor intracellular bacterial endosymbionts of the genus Wolbachia (Rickettsiales). These bacteria have been implicated in adverse effects of drug treatment in filariasis. The present study provides evidence that purified major Wolbachia surface protein (rWSP) acts as an inducer of the innate immune system through TLR2 and TLR4: 1) recombinant, stringently purified rWSP elicited the release of TNF-α, IL-12, and IL-8 from cultured blood cells of both Onchocerca volvulus-infected and uninfected people; 2) the inflammatory response to rWSP challenge was TLR2- and TLR4-dependent as demonstrated with TLR-transfected fibroblastoid cells, as well as macrophages and dendritic cells from functional TLR-deficient mice; 3) blood cells of onchocerciasis patients exposed to rWSP also generated down-regulating mediators IL-10 and PGE2 after 6 days of culture; 4) furthermore, rWSP-reactive IgG1 Abs were present in sera of O. volvulus-infected people but not in those of uninfected Europeans. The lack of rWSP-reactive IgE and IgG4 in serum indicated a bias toward a Th1-type adaptive immune response. Abs against rWSP stained endobacteria in living and degenerating adult O. volvulus filariae, tissue microfilariae and host tissue macrophages that apparently had engulfed microfilariae. Thus, filarial helminths, through products of their endobacteria such as WSP, acquire characteristics of a typical microbial pathogen inducing immune responses via TLR2 and TLR4.
The idea that tetracycline may have antifilarial properties was first proposed in 1960. Data were subsequently published on the effects of tetracycline on the development and reproduction of Brugia pahangi in both the mosquito and mammalian hosts. These studies were apparently ignored until recently. In 1998, it was suggested that the antifilarial properties of tetracycline may derive from the presence of the bacterial endosymbiont Wolbachia in fi‐larial worms. In view of this renewed interest, we would like to highlight an old study on the prophylactic activity of tetracycline against three filarial species. Although this work was performed at the begining of the seventies, and presented at a congress in 1973, it has never been published. This study showed that tetracycline had a prophylactic effect against infection of the mammalian host (the gerbil Meriones unguiculatus) with B. pahangi and Lito‐mosoides sigmodontis. On the other hand, no significant prophylactic effect of the drug was observed against Acanthocheilonema viteae in the same host. The new data on the distribution and phy‐logeny of Wolbachia endosymbionts in filarial nematodes, showing that A. viteae is uninfected and that B. pahangi and L. sigmodontis are infected, suggest that the activity of tetracycline on the latter two species is related to the presence of Wolbachia.
The heartworm Dirofilaria immitis is an important parasite of dogs. Transmitted by mosquitoes in warmer climatic zones, it is spreading across southern Europe and the Americas at an alarming pace. There is no vaccine, and chemotherapy is prone to complications. To learn more about this parasite, we have sequenced the genomes of D. immitis and its endosymbiont Wolbachia. We predict 10,179 protein coding genes in the 84.2 Mb of the nuclear genome, and 823 genes in the 0.9-Mb Wolbachia genome. The D. immitis genome harbors neither DNA transposons nor active retrotransposons, and there is very little genetic variation between two sequenced isolates from Europe and the United States. The differential presence of anabolic pathways such as heme and nucleotide biosynthesis hints at the intricate metabolic interrelationship between the heartworm and Wolbachia. Comparing the proteome of D. immitis with other nematodes and with mammalian hosts, we identify families of potential drug targets, immune modulators, and vaccine candidates. This genome sequence will support the development of new tools against dirofilariasis and aid efforts to combat related human pathogens, the causative agents of lymphatic filariasis and river blindness.-Godel, C., Kumar, S., Koutsovoulos, G., Ludin, P., Nilsson, D., Comandatore, F., Wrobel, N., Thompson, M., Schmid, C. D., Goto, S., Bringaud, F., Wolstenholme, A., Bandi, C., Epe, C., Kaminsky, R., Blaxter, M., Mäser, P. The genome of the heartworm, Dirofilaria immitis, reveals drug and vaccine targets.
Lymphatic filarial nematodes are infected with endosymbiotic Wolbachia bacteria. Lipopolysaccharide from these bacteria is the major activator of innate inflammatory responses induced directly by the parasite. Here, we propose a mechanism by which Wolbachia initiates acute inflammatory responses associated with death of parasites, leading to acute filarial lymphangitis and adverse reactions to antifilarial chemotherapy. We also speculate that repeated exposure to acute inflammatory responses and the chronic release of bacteria, results in damage to infected lymphatics and desensitization of the innate immune system. These events will result in an increased susceptibility to opportunistic infections, which cause acute dermatolymphangitis associated with lymphoedema and elephantiasis. The recognition of the contribution of endosymbiotic bacteria to filarial disease could be exploited for clinical intervention by the targeting of bacteria with antibiotics in an attempt to reduce the development of filarial pathology.
Intracellular bacteria have been described in several species of filarial nematodes, but their relationships with, and effects on, their nematode hosts have not previously been elucidated. In this study, intracellular bacteria were observed in tissues of the rodent parasite Litomosoides sigmodontis by transmission electron microscopy and by immunohistochemistry using antiendobacterial heat shock protein-60 antisera. Molecular phylogenetic analysis of the bacterial 16S ribosomal RNA gene, isolated by PCR, showed a close relationship to the rickettsial Wolbachia endobacteria of arthropods and to other filarial intracellular bacteria. The impact of tetracycline therapy of infected rodents on L. sigmodontis development was analyzed in order to understand the role(s) these bacteria might play in filarial biology. Tetracycline therapy, when initiated with L. sigmodontis infection, eliminated the bacteria and resulted in filarial growth retardation and infertility. If initiated after microfilarial development, treatment reduced filarial fertility. Treatment with antibiotics not affecting rickettsial bacteria did not inhibit filarial development. Acanthocheilonema viteae filariae were shown to lack intracellular bacteria and to be insensitive to tetracycline. These results suggest a mutualistic interaction between the intracellular bacteria and the filarial nematode. Investigation of such a mutualism in endobacteria-containing human filariae is warranted for a potential chemotherapeutic exploitation.
Filarial nematodes are important and widespread parasites of animals and humans. We have been using the African bovine parasite Onchocerca ochengi as a chemotherapeutic model for O. volvulus, the causal organism of 'river blindness' in humans, for which there is no safe and effective drug lethal to adult worms. Here we report that the antibiotic, oxytetracycline is macrofilaricidal against O. ochengi. In a controlled trial in Cameroon, all adult worms (as well as microfilariae) were killed, and O. ochengi intradermal nodules resolved, by nine months' post-treatment in cattle treated intermittently for six months. Adult worms removed from concurrent controls remained fully viable and reproductively active. By serial electron-microscopic examination, the macrofilaricidal effects were related to the elimination of intracellular micro-organisms, initially abundant. Analysis of a fragment of the 16S rRNA gene from the O. ochengi micro-organisms confirmed them to be Wolbachia organisms of the order Rickettsiales, and showed that the sequence differed in only one nucleotide in 858 from the homologous sequence of the Wolbachia organisms of O. volvulus. These data are, to our knowledge, the first to show that antibiotic therapy can be lethal to adult filariae. They suggest that tetracycline therapy is likely to be macrofilaricidal against O. volvulus infections in humans and, since similar Wolbachia organisms occur in a number of other filarial nematodes, against those infections too. In that the elimination of Wolbachia preceded the resolution of the filarial infections, they suggest that in O. ochengi at least, the Wolbachia organisms play an essential role in the biology and metabolism of the filarial worm.
Filarial nematodes harbour intracellular endosymbiotic bacteria, which have been assigned to the genus Wolbachia. These bacteria appear to play an important role in the pathogenesis of filarial diseases through their lipopolysaccharides. In view of the presence of Wolbachia endosymbionts in the body of filarial nematodes, one might also expect that proteins from these bacteria play an antigenic role in humans and animals affected by filariases. To test this hypothesis, we produced in recombinant form the surface protein WSP and a portion of the cell-cycle protein FTSZ from the Wolbachia of Dirofilaria immitis. Western immunoblot assays were then performed using cat sera to test the immunogenicity of these proteins. Sera were collected from owners' cats, which were either sero-negative or sero-positive for D. immitis and from cats before and after experimental infection with D. immitis. FTSZ was recognized in Western blots by sera from both positive and negative cats and from both uninfected and experimentally infected cats. WSP was recognized only by sera from positive cats and from cats experimentally infected with D. immitis; this protein was not recognized by sera from negative cats and from cats before experimental infection with D. immitis. The results of Western blot assays on WSP thus support the hypothesis that infection with filarial nematodes induces the production of antibodies against Wolbachia proteins.
The dog parasite Dirofilaria immitis can infect humans. Patients with pulmonary dirofilariasis were tested for immunoglobulin G (IgG) antibodies against the surface
protein of Wolbachia, the bacterial endosymbiont of D. immitis. These patients showed significantly higher IgG titers than healthy individuals from areas in which D. immitis was endemic as well as areas in which it was not endemic. Titration of anti-Wolbachia surface protein IgG could become useful for diagnostic applications.
Intracellular bacteria in filarial nematodes were described as early as the 1970s, yet it was only with the work on Dirofilaria immitis, the agent of canine and feline heartworm disease, that these microorganisms were identified as belonging to Wolbachia, a genus known for encompassing bacteria infecting insects and other arthropods. The implications for the presence of intracellular bacteria in filarial nematodes is now the subject of intense research, particularly regarding their role in the immunology and pathogenesis of disease in infected humans and animals and as a possible target for therapy. Here, the authors report results on the immunohistochemical and immunogold staining of Wolbachia in D. immitis and Brugia pahangi using polyclonal antibodies raised against the recombinant Wolbachia surface protein (WSP). The bacteria were present in the lateral hypodermal chords of both male and female worms and in the reproductive tract of adult females (oocytes, morulae, microfilariae). In D. immitis and B. pahangi from animals treated with tetracycline, positive staining was observed in the lateral chords of adult males and females, but was absent from the oocytes and morulae. These results indicate that Wolbachia endosymbionts can be identified immunohistochemically with anti-WSP polyclonal antibodies, that their distribution matches that already described for Wolbachia of other filarial worms, and that antibiotic treatment may impede the vertical transmission of these bacteria. Unequivocal detection of Wolbachia is essential for the study of this symbiont, in particular to monitor the effects of antibiotic treatment on worms. The use of a specific marker for bacteria in their nematode hosts represents an extremely useful tool in evaluating the pathogenic role and the effect of antibiotic treatment on these potential targets in the control of filarial disease.
Chemotherapy of onchocerciasis by doxycycline, which targets symbiotic Wolbachia endobacteria, has been shown to result in a long-term sterility of adult female worms and corresponding absence of microfilariae. It represents an additional chemotherapeutic approach. The aim of this study was to determine whether a similar regimen would also show efficacy against Wuchereria bancrofti. Ghanaian individuals ( n=93) with lymphatic filariasis and a minimum microfilaremia of 40 microfilariae/ml were included in a treatment study consisting of four arms: (1) doxycycline 200 mg/day for 6 weeks; (2) doxycycline as in (1), followed by a single dose of ivermectin after 4 months; (3) ivermectin only; or (4) no treatment during observation period of 1 year (ivermectin at the end of the study). Doxycycline treatment resulted in a 96% loss of Wolbachia, as determined by real time PCR from microfilariae. After 12 months, doxycycline had led to a 99% reduction of microfilaremia when given alone, and to a complete amicrofilaremia together with ivermectin. In contrast, after ivermectin treatment alone a significant presence of microfilariae remained (9% compared to pretreatment), as known from other studies. This study shows that doxycycline is also effective in depleting Wolbachia from W. bancrofti. It is likely that the mechanism of doxycycline is similar to that in other filarial species, i.e., a predominant blockade of embryogenesis, leading to a decline of microfilariae according to their half-life. This could render doxycycline treatment an additional tool for the treatment of microfilaria-associated diseases in bancroftian filariasis, such as tropical pulmonary eosinophilia and microfiluria.
The symbiosis of filarial nematodes and rickettsial Wolbachia endobacteria has been exploited as a target for antibiotic therapy of filariasis. Depletion of Wolbachia after tetracycline treatment results in filarial sterility because of interruption of embryogenesis and inhibits larval development and adult worm viability. The aim of this study was to investigate if antibiotic intervention of BALB/c mice infected with the rodent filaria Litomosoides sigmodontis with rifampicin or the combination of rifampicin and doxycycline can be used to shorten the treatment period. Both regimens, when given over a period of 14 days initiated with infection, were sufficient to deplete Wolbachia as evidenced by immunohistology and semiquantitative PCR. Worm development and filarial load were significantly reduced in experiments followed up until 63 days p.i. The therapy inhibited embryogenesis and led to filarial sterility. In contrast, treatment with doxycycline alone for 21 days led only to a modest reduction of Wolbachia, filarial growth retardation, worm viability and fertility. In conclusion, the combination of antirickettsial drugs could be used as a suitable tool to explore the minimum duration of therapy required for the depletion of Wolbachia in parasitized hosts subsequent to the onset of patency in human and animal filariasis and the prevention of adverse reactions in human infections.
Symbiotic Wolbachia organisms of filarial nematodes have received much attention as possible chemotherapy targets and disease-causing organisms. In order to further investigate the association between anti-Wolbachia immune responses and chronic filarial disease in humans, antibody responses to Wolbachia surface protein (WSP) were assayed in serum samples collected from 232 individuals living in Leogane, Haiti, an area where Wuchereria bancrofti infection is endemic, and from 67 North Americans with no history of lymphatic filariasis. As opposed to antifilarial antibody responses, which were largely influenced by the patient's infection status, the prevalence and levels of anti-WSP immunoglobulin G (IgG) antibodies among individuals with lymphedema or hydrocele were significantly greater than those in gender- and infection-matched individuals without disease. In at least one case, the anti-WSP IgG response was coincident with the onset of lymphedema development, and among anti-WSP-positive women with lymphedema, anti-WSP IgG levels were negatively correlated with the duration of lymphedema. The presence of anti-WSP IgG was also associated with the severity of inguinal adenopathy among men with hydrocele. In addition to the presence of anti-WSP antibodies among Haitians, 15 of 67 (22%) serum samples collected from individuals from North America, where filariasis is not endemic, were also positive for anti-WSP antibodies. In comparison to those from Haitians, anti-WSP antibodies from North Americans primarily recognized a distinct region of WSP located within the highly conserved second transmembrane domain. The results of this study demonstrate that anti-WSP antibody responses are associated with the presence of chronic filarial morbidity and not filarial infection status in humans and suggest that WSP should be further studied as a potential trigger for the development of filarial disease.
Complete genome DNA sequence and analysis is presented for Wolbachia, the obligate alpha-proteobacterial endosymbiont required for fertility and survival of the human filarial parasitic nematode Brugia malayi. Although, quantitatively, the genome is even more degraded than those of closely related Rickettsia species, Wolbachia has retained more intact metabolic pathways. The ability to provide riboflavin, flavin adenine dinucleotide, heme, and nucleotides is likely to be Wolbachia's principal contribution to the mutualistic relationship, whereas the host nematode likely supplies amino acids required for Wolbachia growth. Genome comparison of the Wolbachia endosymbiont of B. malayi (wBm) with the Wolbachia endosymbiont of Drosophila melanogaster (wMel) shows that they share similar metabolic trends, although their genomes show a high degree of genome shuffling. In contrast to wMel, wBm contains no prophage and has a reduced level of repeated DNA. Both Wolbachia have lost a considerable number of membrane biogenesis genes that apparently make them unable to synthesize lipid A, the usual component of proteobacterial membranes. However, differences in their peptidoglycan structures may reflect the mutualistic lifestyle of wBm in contrast to the parasitic lifestyle of wMel. The smaller genome size of wBm, relative to wMel, may reflect the loss of genes required for infecting host cells and avoiding host defense systems. Analysis of this first sequenced endosymbiont genome from a filarial nematode provides insight into endosymbiont evolution and additionally provides new potential targets for elimination of cutaneous and lymphatic human filarial disease.
Filarial nematodes contain endosymbiotic bacteria of the genus Wolbachia. As described in the April 2005 issue of PLoS Biology, Foster et al. have sequenced the genome of the Wolbachia that lives in the nematode Brugia malayi. What are the clinical implications?
Development of a drug lethal to adult Onchocerca volvulus (i.e., macrofilaricide) is a research priority for the control of human onchocerciasis. Using bovine O. ochengi infections, we investigated the effects of oxytetracycline administered in a short intensive regimen (SIR; 10 mg/kg daily
for 14 days), compared with a prolonged intermittent regimen (PIR; 20 mg/kg monthly for 6 months) or a combination of both
(COM), on the viability of adult worms and their endosymbiotic bacteria (Wolbachia species). The long-term treatments eliminated >80% (COM) or >60% (PIR) of adult female worms (P<.001), and the COM regimen
effected a sustained depletion of Wolbachia organisms. Conversely, SIR was not macrofilaricidal and only transiently depleted Wolbachia densities, which repopulated worm tissues by 24 weeks after treatment. These results unequivocally demonstrate the macrofilaricidal
potential of tetracyclines against Onchocerca infection and suggest that intermittent, protracted administration will be more effective than continuous shorter term treatment
Filarial nematodes are important helminth parasites of the tropics and a leading cause of global disability. They include species responsible for onchocerciasis, lymphatic filariasis and dirofilariasis. A unique feature of these nematodes is their dependency upon a symbiotic intracellular bacterium, Wolbachia, which is essential for normal development and fertility. Advances in our understanding of the symbiosis of Wolbachia bacteria with filarial nematodes have made rapid progress in recent years. Here we summarise our current understanding of the evolution of the symbiotic association together with insights into the functional basis of the interaction derived from genomic analysis. Also we discuss the contribution of Wolbachia to inflammatory-mediated pathogenesis and adverse reactions to anti-filarial drugs and describe the outcome of recent field trials using antibiotics as a promising new tool for the treatment of filarial infection and disease.
Lymphatic filarial nematodes are able to down-regulate parasite-specific and nonspecific responses of lymphocytes and APC. Lymphatic filariae are reliant on Wolbachia endosymbiotic bacteria for development and survival. We tested the hypothesis that repeated exposure to Wolbachia endosymbionts would drive macrophage tolerance in vitro and in vivo. We pre-exposed murine peritoneal-elicited macrophages to soluble extracts of Brugia malayi female worms (BMFE) before restimulating with BMFE or TLR agonists. BMFE tolerized macrophages (in terms of IFN-beta, IL-1beta, IL-6, IL-12p40, and TNF-alpha inflammatory cytokine production) in a dose-dependent manner toward self, LPS, MyD88-dependent TLR2 or TLR9 ligands (peptidoglycan, triacyl lipopeptide, CpG DNA) and the MyD88-independent/TRIF-dependent TLR3 ligand, polyinosinic-polycytidylic acid. This was accompanied with down-regulation in surface expression of TLR4 and up-regulation of CD14, CD40, and TLR2. BMFE tolerance extended to CD40 activation in vitro and systemic inflammation following lethal challenge in an in vivo model of endotoxin shock. The mechanism of BMFE-mediated macrophage tolerance was dependent on MyD88 and TLR2 but not TLR4. Evidence that desensitization was driven by Wolbachia-specific ligands was determined by use of extracts from Wolbachia-depleted B. malayi, aposymbiotic filarial species, and a cell line stably infected with Wolbachia pipientis. Our data promote a role for Wolbachia in contributing toward the dysregulated and tolerized immunological phenotype that accompanies the majority of human filarial infections.
The activity against filarial parasites of the antibiotics rifampicin, oxytetracycline and chloramphenicol was examined. In addition, transmission electron microscopy was used to study the effects of rifampicin and oxytetracycline on filarial tissues and on the endosymbiont bacterium, Wolbachia.
When tested in vitro at a concentration of 50.0 µm, each of the three antibiotics significantly reduced the motility levels of male Onchocerca gutturosa. Rifampicin, however, was the most active, virtually immobilizing the parasite by the end of the 40-day trial and producing an 84% reduction in viability (as measured by formazan-based colorimetry). In tests against O. lienalis microfilariae (mff) in CBA mice, the numbers of mff recovered after treatment with oxytetracycline at 100, 25 or 6.5 mg/kg daily, for 15 days, were 56% (P≤ 0.03), 38% (P>0.05) and 45% (P = 0.05) less than that recovered from the untreated controls, respectively. In another trial in mice, rifampicin (100 mg/kg daily for 15 days) was found to be the most active (causing a 74% reduction in the number of mff recovered—approximately equal to that achieved with the positive control of a single dose of ivermectin at 2 µg/kg), with chloramphenicol also showing significant activity (39% reduction). In further, in-vivo trials, at three dose levels (100, 25 or 6.25 mg/kg daily, for 15 days), all three antibiotics were tested against adult Brugia pahangi in the peritoneal cavities of jirds. None of the antibiotics produced a significant reduction in the numbers of live worms recovered, although a marginal effect was observed in eight of the nine antibiotic-treated groups. A further extended trial with rifampicin and oxytetracycline resulted in 43% and 38% reductions in worm recoveries, respectively (not statistically significant but consistent with a marginal effect); some of these worms appeared less motile and qualitatively in poor condition compared with those recovered from untreated jirds. Ultrastructural studies of these treated worms revealed that virtually all of the endosymbiont bacteria had been cleared from the parasite tissues. The tissues of the adult worms appeared to be largely intact but with a granulomatous response of host cells adhering to some specimens. However, developing uterine forms appeared to be abnormal and extensively damaged, showing an abrogation of embryogenesis. In contrast, worms recovered from control animals contained large numbers of Wolbachia, had no adherent host cells, and showed normal ultrastructure; the female worms exhibited a full range of intra-uterine developing stages from eggs to stretched mff. It is likely that the activity of these antibiotics against the endosymbiont Wolbachia causes the observed antifilarial activity, although some direct effect of each drug on filarial viability cannot be ruled out.
These clinicians think so. Find out how their alternative treatment method may reduce the chances of adverse reactions.
Wolbachia endosymbiotic bacteria have been shown to be widespread among filarial worms and could thus play some role in the biology of these nematodes. Indeed, tetracycline has been shown to inhibit both the development of adult worms from third-stage larvae and the development of the microfilaraemia in jirds infected with Brugia pahangi. The possibility that these effects are related to the bacteriostatic activity of tetracycline on Wolbachia symbionts should be considered. Here we show that tetracycline treatment is very effective in blocking embryo development in two filarial nematodes, B. pahangi and Dirofilaria immitis. Embryo degeneration was documented by TEM, while the inhibition of the transovarial transmission of Wolbachia was documented by PCR. Phylogenetic analysis on the ssrDNA sequence of the Wolbachia of B. pahangi confirms that the phylogeny of the bacterial endosymbionts is consistent with that of the host worms. The possibility that tetracycline inhibition of embryo development in B. pahangi and D. immitis is determined by cytoplasmic incompatibility is discussed.
Summary Endobacteria of the genus Wolbachia in filarial nematodes are related to Rickettsiaceae and can be depleted by tetracycline antibiotics. This depletion blocks female worm development as well as early embryogenesis, in contrast to the currently used microfilaricidal ivermectin which blocks only the last stage of embryogenesis. Since targeting Wolbachia is becoming an area of research for the treatment of human filariases, it was investigated if antibiotics other than tetracyclines are able to deplete Wolbachia from filariae. BALB/c mice infected with the rodent filaria Litomosoides sigmodontis were treated with erythromycin, chloramphenicol or ciprofloxacin. All drugs were well resorbed and resulted in serum levels clearly above breakpoint levels for bacteria susceptible to the respective antibiotic. However, contrary to tetracycline, none of these antibiotics depleted Wolbachia or altered worm development and fertility, as evidenced by immunohistology, immunoelectron microscopy and semiquantitative PCR.
Wolbachia endosymbiotic bacteria have been shown to be widespread among filarial worms and could thus play some role in the biology of these nematodes. Indeed, tetracycline has been shown to inhibit both the development of adult worms from third-stage larvae and the development of the microfilaraemia in jirds infected with Brugia pahangi. The possibility that these effects are related to the bacteriostatic activity of tetracycline on Wolbachia symbionts should be considered. Here we show that tetracycline treatment is very effective in blocking embryo development in two filarial nematodes, B. pahangi and Dirofilaria immitis. Embryo degeneration was documented by TEM, while the inhibition of the transovarial transmission of Wolbachia was documented by PCR. Phylogenetic analysis on the ssrDNA sequence of the Wolbachia of B. pahangi confirms that the phylogeny of the bacterial endosymbionts is consistent with that of the host worms. The possibility that tetracycline inhibition of embryo development in B. pahangi and D. immitis is determined by cytoplasmic incompatibility is discussed.
Unlike in many other helminth infections, neutrophilic granulocytes are major cellular components in the hostˈs immune response against filarial worms. The pathways that drive the immune response involving neutrophils are unclear. This study shows that Wolbachia endobacteria (detectable by polyclonal antibodies against endobacterial heat shock protein 60 and catalase and by polymerase chain reaction being sensitive to doxycycline treatment) are direct and indirect sources of signals accounting for neutrophil accumulation around adult Onchocerca volvulus filariae. Worm nodules from untreated onchocerciasis patients displayed a strong neutrophil infiltrate adjacent to the live adult worms. In contrast, in patients treated with doxycycline to eliminate the endobacteria from O. volvulus and to render the worms sterile, the neutrophil accumulation around live adult filariae was drastically reduced. Neutrophils were absent in worm nodules from the deer filaria Onchocerca flexuosa, a species which does not contain endobacteria. Extracts of O. volvulus extirpated from untreated patients showed neutrophil chemotactic activity and in addition, induced strong TNF-α and IL-8 production in human monocytes, in contrast to filarial extracts obtained after doxycycline treatment. Thus, neutrophil chemotaxis and activation are induced directly by endobacterial products and also indirectly via chemokine induction by monocytes. These results show that the neutrophil response is a characteristic of endobacteria-containing filariae.
The antifilarial effects of tetracycline drugs were first demonstrated when they were found to be highly effective against L(3) and L(4) of Brugia pahangi and Litomosoides sigmodontis in rodent models. Tetracyclines are also now known to have activity against microfilariae and adult Dirofilaria immitis, but assessment of their activity against larval and juvenile heartworms has not been reported previously. This study assessed the effects of doxycycline administered orally at 10mg/kg twice daily for 30-day periods at selected times during the early part of the life cycle of D. immitis in dogs with dual infections of D. immitis and B. pahangi. Twenty beagles were randomly allocated by weight to four groups of five dogs each. On Day 0, each dog was given 50 D. immitis L(3) and 200 B. pahangi L(3) by SC injection. Dogs received doxycycline on Days 0-29 (Group 1); Days 40-69 (Group 2); or Days 65-94 (Group 3). Group 4 served as untreated controls. Blood samples were collected for microfilariae counting and antigen testing. Necropsy for collection of adult heartworms and selected tissues were performed Days 218-222. Heartworms recovered were examined by immunohistology, conventional microscopy/transmission electron microscopy, and molecular biology techniques. No live heartworms were recovered from dogs in Group 1; dogs in Group 2 had 0 to 2 live worms (98.4% efficacy), and dogs in Group 3 had 0-36 live worms (69.6% efficacy). All control dogs had live adult heartworms (25-41). The live worms recovered from dogs in Groups 2 and 3 were less developed and smaller that worms from control dogs. Microfilariae were not detected in any dogs in Groups 1 and 2; one dog in Group 3 had 1 microfilariae/ml at necropsy. All control dogs had microfilariae at necropsy. One dog in Group 1 was antigen positive at one sampling (Day 166). One dog in Group 2 was antigen positive Days 196 and 218-222 and three dogs in Group 3 were antigen positive at one or more samplings All five control dogs were antigen positive at all three sampling times. These findings suggest that doxycycline at 10mg/kg orally twice daily for 30 days has efficacy against migrating tissue-phase larvae and juvenile worms and will delay or restrict microfilarial production.
Canine heartworm disease is caused by infection with Dirofilaria immitis, a filarial nematode that resides in the pulmonary arteries and occasionally in the right heart chambers of infected dogs. Here the authors evaluated the effect of a combination of doxycycline (10 mg/kg/sid for 30 days) and ivermectin–pyrantel(6g/kg of ivermectin+5mg/kg of pyrantel every 15 days for 180 days) on microfilariemia, antigenemia and parasite load at echocardiography in naturally infected dogs from an endemic region of Italy [corrected]. Dogs were examined monthly for 6 months and followed-up 4 months later. One hundred percent of dogs became negative for circulating microfilariae by day 90, while 8/11 (72.7%) of dogs became antigen-negative by day 300. Of the 7 dogs that were positive for visualization of parasites at echocardiography, 6 (85.7%) became negative by day 300. Treatment was well-tolerated by all dogs. These results suggest that a combination of doxycycline and ivermectin is adulticide in dogs with D. immitis.
Since the definitive identification in 1995 of the bacterial endosymbiont Wolbachia that resides in different tissues of the filarial worm Dirofilaria immitis, there has been increasing interest to understand whether and what role it plays in the pathogenesis of and immune response to heartworm infection. The present study evaluated the effects of treatments on lung pathology in 20 beagle dogs experimentally infected with D. immitis. Dogs in Group 1 were treated with doxycycline (10 mg/kg/day) orally from weeks 0-6, 10-12, 16-18, 22-26, and 28-34. Dogs in Group 2 served as infected, non-treated controls. Dogs in Group 3 were given doxycycline as described for Group 1 combined with weekly oral doses of ivermectin (6 mcg/kg) for 34 weeks and intramuscular (IM) melarsomine (2.5 mg/kg) at week 24, followed by two additional melarsomine injections 24h apart 1 month later. Group 4 received only melarsomine as described for Group 3. Lung lesion criteria, scored by two independent blinded pathologists, included perivascular inflammation and endothelial proliferation. Doxycycline treatment alone had no effect on lesion scores, whereas the combination of doxycycline and ivermectin resulted in less severe perivascular inflammation. All lungs were evaluated for positive immunostaining for the Wolbachia surface protein (WSP). Control dogs showed numerous thrombi, intense perivascular and interstitial inflammation and, occasionally, positive staining for WSP. Interestingly, dogs receiving doxycycline/ivermectin/melarsomine showed significantly less severe arterial lesions and the virtual absence of thrombi.
A safer, more effective adulticidal treatment and a safe method for reducing microfilaremia and breaking transmission of heartworm disease early in the treatment are needed. The present study evaluated efficacy of ivermectin (IVM) and doxycycline (DOXY) alone or together (with or without melarsomine [MEL]) in dogs with induced adult heartworm infection and assessed the ability of microfilariae from DOXY-treated dogs to develop to L3 in Aedes aegypti mosquitoes and subsequently to become reproductive adults in dogs. Thirty beagles were each infected with 16 adult heartworms by intravenous transplantation. Six weeks later, dogs were ranked by microfilarial count and randomly allocated to 6 groups of 5 dogs each. Beginning on Day 0, Group 1 received IVM (6 mcg/kg) weekly for 36 weeks. Group 2 received DOXY (10 mcg/(kg day)) orally Weeks 1–6, 10–11, 16–17, 22–25, and 28–33. Groups 3 and 5 received IVM and DOXY according to doses and schedules used for Groups 1 and 2. At Week 24, Groups 3 and 4 received an intramuscular injection of MEL (2.5 mg/kg), followed 1 month later by two injections 24 h apart. Group 6 was not treated. Blood samples were collected for periodic microfilaria counts and antigen (Ag) testing (and later immunologic evaluation and molecular biology procedures). Radiographic and physical examinations, hematology/clinical chemistry testing, and urinalysis were done before infection, before Day 0, and periodically during the treatment period. At 36 weeks, the dogs were euthanized and necropsied for worm recovery, collection of lung, liver, kidney, and spleen samples for examination by immunohistochemistry and conventional histological methods.
Thirty-six Beagles were inoculated with 3rd-stage infective Dirofilaria immitis larvae to determine when 3rd and 4th molts occurred, how long each stage of development persisted in muscle and skin, and the patterns of larval migration from the infection site to the heart. From 22% to 84% of these larvae were recovered when the dogs were euthanatized and necropsied (mean recovery 45%). Larvae were recovered only from the skin and muscle during the first 58 days. The 5th-stage immature adults were first recovered from the heart in dogs killed on postinoculation day (PID) 70. Migration to the heart was essentially completed in dogs killed on PID 120, although 1 immature adult was recovered from subcutaneous tissues of each dog killed on PID 140 and 142. Starting and completion days of molting periods were recorded along with body lengths of male and female worms from the 3rd, 4th, and 5th stages. Microfilariae were first recovered from the peripheral blood in dogs killed on PID 190. Optimal time for drug evaluation against a particular stage of larval development is as follows: 3rd-stage larvae, PID 0 to 2; 4th-stage larvae, PID 15 to 50; and 5th-stage or immature adults, PID 65 to 120.
The ability of oral tetracycline to inhibit the development of third-stage infective larvae (L3) of Brugia pahangi to adult worms in jirds was studied using 2 experimental protocols. Jirds treated with 1.4% tetracycline in drinking water for a period beginning 30 days before inoculation of L3 until 30 days post-inoculation (DPI) had 97% reduction in adult worm recovery compared to untreated controls. Jirds that received 1.2% tetracycline in drinking water beginning 1 day before until either 12 or 26 DPI had adult worm recoveries of 11% and < 1%, respectively. Untreated jirds and those given tetracycline beginning at or later than 13 DPI had similar adult worm recovery (27-29%). Prepatent periods were prolonged, and circulating microfilariae were reduced in jirds given tetracycline from 27 to 54 DPI compared to controls. These data indicate that tetracycline administered to jirds in drinking water inhibits B. pahangi development from L3 to adult worms and suggest that this effect occurs during early larval development. Tetracycline administered to infected jirds prior to and continuing through the onset of patency can also affect development of microfilaremia.
To determine the efficacy of ivermectin (IVM) and milbemycin oxime (MBO) against induced heartworm infection, where monthly treatment is started 3 or 4 months after infection, and to monitor microfilaremia and antigenemia.
21 heartworm-naive Beagles.
Each of 21 dogs was given 50 infective larvae of Dirofilaria immitis by SC inoculation. One group of 5 dogs served as nonmedicated controls, 2 groups of 5 dogs received IVM at 6 micrograms/kg of body weight or MBO at 500 micrograms/kg for 12 months beginning at postinfection (PI) month 4, and 2 groups of 3 dogs received IVM or MBO for 13 months beginning at PI month 3. Blood collected at intervals not > 1 month beginning at PI month 4 was examined for microfilariae and antigen. Dogs were euthanatized at PI month 16.
Adult worm counts, relative to controls, were reduced in the 4-month treatment groups by 95.1 (P < 0.01) and 41.4% for IVM and MBO, respectively. The difference between the IVM and MBO groups was significant (P < 0.01). Live worms were found in all MBO-treated (range, 8 to 27) and control dogs (range, 12 to 39) and in 3 of 5 IVM-treated dogs (range, 2 to 4). In the 3-month treatment groups, worm counts were reduced by 97.7 (P < 0.01) and 96.8% (P < 0.01) for IVM and MBO, respectively. Microfilariae were seen in all control dogs and in only 2 of the 16 treated dogs. The antigen response of MBO-treated dogs in the 4-month treatment group was only slightly weaker than that for control dogs. In all other treated dogs, this response was delayed and weaker.
Ivermectin is highly (> or = 95%) and significantly more effective than MBO against induced heart-worm infection when 1 year of monthly prophylactic dosing is started 4 months after infection.
In some cases of owner compliance failure, monthly administration of IVM gives a high level of protection against young adult heartworms.
The finding that the intracellular bacteria of filarial nematodes are related to the Wolbachia symbionts of arthropods has generated great interest. Here, Mark Taylor and Achim Hoerauf review recent studies by several groups on the structure, distribution and phylogeny of these endosymbionts, and discuss the potential role for these bacteria in filarial disease and as a target for chemotherapy.
The activity against filarial parasites of the antibiotics rifampicin, oxytetracycline and chloramphenicol was examined. In addition, transmission electron microscopy was used to study the effects of rifampicin and oxytetracycline on filarial tissues and on the endosymbiont bacterium, Wolbachia. When tested in vitro at a concentration of 50.0 microM, each of the three antibiotics significantly reduced the motility levels of male Onchocerca gutturosa. Rifampicin, however, was the most active, virtually immobilizing the parasite by the end of the 40-day trial and producing an 84% reduction in viability (as measured by formazan-based colorimetry). In tests against O. lienalis microfilariae (mff) in CBA mice, the numbers of mff recovered after treatment with oxytetracycline at 100, 25 or 6.5 mg/kg daily, for 15 days, were 56% (P < or = 0.03), 38% (P> 0.05) and 45% (P = 0.05) less than that recovered from the untreated controls, respectively. In another trial in mice, rifampicin (100 mg/kg daily for 15 days) was found to be the most active (causing a 74% reduction in the number of mff recovered--approximately equal to that achieved with the positive control of a single dose of ivermectin at 2 microg/kg), with chloramphenicol also showing significant activity (39% reduction). In further, in-vivo trials, at three dose levels (100, 25 or 6.25 mg/kg daily, for 15 days), all three antibiotics were tested against adult Brugia pahangi in the peritoneal cavities of jirds. None of the antibiotics produced a significant reduction in the numbers of live worms recovered, although a marginal effect was observed in eight of the nine antibiotic-treated groups. A further extended trial with rifampicin and oxytetracycline resulted in 43% and 38% reductions in worm recoveries, respectively (not statistically significant but consistent with a marginal effect); some of these worms appeared less motile and qualitatively in poor condition compared with those recovered from untreated jirds. Ultrastructural studies of these treated worms revealed that virtually all of the endosymbiont bacteria had been cleared from the parasite tissues. The tissues of the adult worms appeared to be largely intact but with a granulomatous response of host cells adhering to some specimens. However, developing uterine forms appeared to be abnormal and extensively damaged, showing an abrogation of embryogenesis. In contrast, worms recovered from control animals contained large numbers of Wolbachia, had no adherent host cells, and showed normal ultrastructure; the female worms exhibited a full range of intra-uterine developing stages from eggs to stretched mff. It is likely that the activity of these antibiotics against the endosymbiont Wolbachia causes the observed antifilarial activity, although some direct effect of each drug on filarial viability cannot be ruled out.
Ivermectin is the drug used for mass chemotherapy of onchocerciasis within the WHO African Programme for Onchocerciasis Control. This approach aims to eliminate the disease as a public health problem but using one dose per year may not completely interrupt transmission since it does not suppress microfilaridermia thoroughly enough. Here we show that additional treatment with doxycycline, previously shown to sterilise adult female worms for a few months by depletion of symbiotic wolbachia endobacteria, significantly enhances ivermectin-induced suppression of microfilaridermia, rendering anti-wolbachia treatment a promising basis for blocking transmission by a drug-based approach.
Wolbachia bacteria seem to have evolved as essential endosymbionts of their filarial nematode hosts. Studies in mice have suggested that these bacteria are associated with systemic inflammatory reactions to filarial chemotherapy. We took blood samples from 15 Indonesian patients before and after treatment with diethylcarbamazine for Brugia malayi infection, and recorded the severity of any post-treatment inflammatory reactions. Blood from all three patients with severe adverse reactions and from one of six with moderate reactions was positive for Wolbachia DNA 4-48 h after diethylcarbamazine treatment. We suggest that these severe inflammatory reactions are associated with the release of endosymbionts into the blood after treatment for filariasis.
Wolbachia spp., first reported from filariae nearly 30 years ago, have been suggested to contribute to the pathogenesis associated with human filarial infection. Tetracycline has been used to cure filariae of Wolbachia, as a novel means of chemotherapeutic treatment for both ocular and lymphatic filariasis. Tetracycline treatment of L4 or adult Brugia pahangi in vivo resulted in Wolbachia clearance. Less tetracycline was required to clear Wolbachia when treatment began at the L4 stage, compared with adults. Female worms died earlier than male worms when tetracycline was administered at the L4 stage. In all cases, Wolbachia clearance was closely associated with worm death. Worm recoveries decreased following the L4-L5 molt, suggesting tetracycline does not interrupt molting in this model system. Despite worm death and the assumed release of both bacterial- and worm-derived molecules, differences in inflammatory cell population and T cell cytokine mRNA profiles were negligible between tetracycline-treated and non-treated B. pahangi infected gerbils. These data suggest the contribution of Wolbachia to the in vivo induction of the gerbil immune response to B. pahangi may be small.
Filarial nematodes, including Dirofilaria immitis and D. repens, harbour intracellular bacteria belonging to the genus Wolbachia. These bacteria have been implicated in the pathogenesis of filarial diseases, possibly through their endotoxins. Recent studies have shown that a major surface protein of Wolbachia (WSP) induces a specific IgG response in hosts infected by D. immitis. WSP from the Wolbachia of D. immitis was produced in recombinant form. The purified protein was used in stimulation assays on canine neutrophils. The assays performed using a modified Boyden chamber showed that WSP stimulates neutrophil chemokinesis. In addition, RT-PCR revealed increased production of chemokine IL-8 by cells incubated with this protein. Neutrophils have been shown to play a major role in the pathogenesis of river blindness, and to accumulate in the nodules of onchocerciasis patients. In dogs infected by D. immitis, neutrophils accumulate in kidneys and in the wall of pulmonary arteries. As shown by our studies, Wolbachia could contribute to these inflammatory phenomena through its surface protein WSP, independently from its endotoxin component.
In this review, we describe the pathogenic role of Wolbachia endosymbiotic bacteria in filarial diseases, focusing on the host innate immune responses to filarial and Wolbachia products. A description of the host pathogen recognition and early inflammatory responses including TLR4-mediated signalling, chemokine and cytokine responses and inflammatory cell recruitment is provided from human studies and from animal models of filarial disease. Finally, the impact of the discovery and characterization of Wolbachia on filarial research and treatment programmes is discussed.
The human filarial nematode Brugia malayi contains an endosymbiotic bacterium, Wolbachia. We used real-time quantitative polymerase chain reaction (QPCR) and microscopy to investigate the population dynamics of the bacterium-nematode association. Two Wolbachia (wsp and ftsZ) and one nematode (gst) genes were amplified from all life-cycle stages of B. malayi and results expressed as gene copies per worm and as Wolbachia/nematode ratios. Since the genes were single copy and there was one genome per Wolbachia, the gene copy numbers were equivalent to the numbers of bacteria. These were similar in microfilariae and the mosquito-borne larval stages (L2 and L3), with the lowest ratios of Wolbachia/nematode DNA. However, within 7 days of infection of the mammalian host, bacteria had increased 600-fold and the bacteria/worm ratio was the highest of all life-cycle stages. The rapid multiplication continued throughout L4 development, so that the major period of bacterial population growth occurred within 4 weeks of infection of the definitive host. Microscopy confirmed that there were few bacteria in mosquito-derived L3 but many, in large groups, in L4 collected 9 and 21 days after infection. In adult male worms up to 15 months of age, the bacterial populations were maintained, whilst in females, bacteria numbers increased as the worms matured and as the ovary and embryonic larval stages became infected. These results support the hypothesis that the bacteria are essential for larval development in the mammalian host and for the long-term survival of adult worms.
Sera from three groups of cats under different experimental conditions were studied by ELISA to assess the host's immune response against synthetic peptides derived from Dirofilaria immitis (Dipp) and against the surface protein of its endosymbiont, Wolbachia (WSPr). In experimentally infected cats (Group 1), an increase of IgG antibody against both Dipp and WSPr was observed from 2 months post-infection until the end of the study, 6 months post-infection. In experimentally infected cats, treated against infective larvae (Group 2), anti-Dipp IgG decreased dramatically from 4 months post-infection (3 months post treatment), showing very low values till the end of the study (6.5 months from infection, 5.5 months from treatment), while anti-WSP IgG increased constantly till the end of the study. Of 49 outdoor, asymptomatic cats exposed to a high risk of natural infection (Group 3), 9 were positive for anti-Dipp IgG and for a validated, in-clinic commercial antibody diagnostic kit for cats. Two cats were also found positive for circulating antigens of adult female worm. Anti-WSPr IgG were found in five of nine anti-Dipp IgG-positive sera and from eight ELISADipp-negative sera. Our results confirm the strong IgG response in heartworm infected cats and demonstrate the involvement of the Wolbachia endosymbiont in the immune reaction to the parasite both in experimentally infected cats and in cats exposed to a high risk of natural infection.
Human and animal parasitic filarial nematodes, including the agent of canine and feline heartworm disease Dirofilaria immitis, harbour intracellular bacteria of the genus Wolbachia (Rickettsiaies). It is thought that these bacteria play an important role in the pathogenesis and immune response to filarial infection. Immunoglobulin G (total IgG, IgG1, IgG2) production against and immunohistochemical staining of tissues for the Wolbachia surface protein (WSP) from dogs with natural heartworm infection were evaluated. All infected dogs had significant total anti-WSP IgG levels compared to healthy controls. Interestingly, WSP was recognized by the IgG2 subclass in both microfilariemic dogs and in dogs with no circulating microfilariae (occult infection). However, microfilariemic dogs also produced gG1 antibodies. Positive staining for WSP was observed in lungs, liver and kidneys, in particular in glomerular capillaries of naturally infected dogs who had died from heartworm disease. Our results show for the first time that Wolbachia is recognized specifically by D. immitis--infected dogs and that the bacteria is released into host tissue. Furthermore, microfilariemic status appears to effect immune responses to this endosymbiont.
Wolbachia endosymbionts of filarial nematodes are vital for larval development and adult-worm fertility and viability. This essential dependency on the bacterium for survival of the parasites has provided a new approach to treat filariasis with antibiotics. We used this strategy to investigate the effects of doxycycline treatment on the major cause of lymphatic filariasis, Wuchereria bancrofti.
We undertook a double-blind, randomised, placebo-controlled field trial of doxycycline (200 mg per day) for 8 weeks in 72 individuals infected with W bancrofti from Kimang'a village, Pangani, Tanzania. Participants were randomly assigned by block randomisation to receive capsules of doxycycline (n=34) or placebo (n=38). We assessed treatment efficacy by monitoring microfilaraemia, antigenaemia, and ultrasound detection of adult worms. Follow-up assessments were done at 5, 8, 11, and 14 months after the start of treatment. Analysis was per protocol.
One person from the doxycycline group died from HIV infection. Five (doxycycline) and 11 (placebo) individuals were absent at the time of ultrasound analysis. Doxycycline treatment almost completely eliminated microfilaraemia at 8-14 months' follow-up (for all timepoints p<0.001). Ultrasonography detected adult worms in only six (22%) of 27 individuals treated with doxycycline compared with 24 (88%) of 27 with placebo at 14 months after the start of treatment (p<0.0001). At the same timepoint, filarial antigenaemia in the doxycycline group fell to about half of that before treatment (p=0.015). Adverse events were few and mild.
An 8-week course of doxycycline is a safe and well-tolerated treatment for lymphatic filariasis with significant activity against adult worms and microfilaraemia.
Presence of transovarially-transmitted endosymbiontic Wolbachia bacteria in Dirofilaria immitis, and in other filariae of man and animals, presents a new paradigm for our understanding of pathogenesis, treatment and diagnosis of filarial infections. Many of the basic biological characteristics of Wolbachia have yet to be elucidated, but the results obtained to date suggest that canine or the feline hosts can be exposed to D. immitis Wolbachia when larvae, or adult worms, are killed; when Wolbachia are expulsed, with the deposition of microfilariae, from the uterus of the females; and possibly through the excretory system of both male and female worms. The two organs that have the greatest potential of being affected by the Wolbachial metabolic products/antigens released from the adult worms are the lungs and the kidneys. Population of Wolbachia in D. immitis is polymorphic. The life cycle of Wolbachia is complex and may consist of two reproductive modes: multiplication of the bacillary forms by binary fission and by a more complex mode which resembles the Chlamydia-like cycle that consists of three morphological stages: a small, dense body, an intermediate stage with a dense inclusion, and a bacillary form which represents the final product of development and maturation of the small, dense body. The Chlamydia-like cycle offers a potential survival strategy for the Wolbachia by producing more progeny than multiplication by binary fission, and appears to be more active during growth and development of embryos and of the larvae. The small, dense bodies may be the infectious forms responsible for the spread of Wolbachia through the canalicular system, within the lateral chords of filariae. An amorphous membrane that lines the perienteric surface of the body wall may represent a physical barrier that limits the spread and movement of Wolbachia to the perienteric surface of the lateral chords. Wolbachia in D. immitis may also offer therapeutic and diagnostic possibilities. Elimination of Wolbachia by chemotherapy, and the suppressive effect of aposymbiosis on embryonic development of D. immitis, may have potential application for control (sterilization of female worms) and treatment of dirofilariasis. However, the three stages in the life cycle of Wolbachia may be antigenically different and each stage may have a different susceptibility to therapeutic agents. Persistence of dormant small, dense bodies after treatment would allow the Wolbachia to re-establish once the conditions for development would become favorable. Detection of Wolbachial antigens provides an attractive diagnostic possibility to identify D. immitis early in the infection. Further studies on Wolbachia of filariae, including those of D. immitis, will undoubtedly reveal additional information that can be applied towards treatment, diagnosis, and control of filarial infections.
Recently, experts have warned that mass treatment with ivermectin alone may not interrupt the transmission of Onchocerca. Hence, additional drugs are needed, such as antibiotics acting on symbiotic endobacteria of the filariae, the causative agents of onchocerciasis. Based on animal experiments, human onchocerciasis was treated with doxycycline, and preliminary observations published in 2001 in The Lancet showed sterility in female worms by depletion and marked reduction in symbiotic Wolbachia endobacteria from the filariae. Here, a detailed kinetic analysis of the features of the worms, following administration or not of doxycycline to the patients is reported. Sixty-three onchocerciasis patients in Ghana were treated with 100 mg doxycycline daily for 6 weeks and 2 or 6 months later with ivermectin. Onchocercomas were extirpated 2, 6, 11 and 18 months after the onset of treatment and the filariae were examined by immunohistology and PCR. The analysis showed: (i) progressive depletion of Wolbachia from adult worms and microfilariae by doxycycline over a period of 6 months; (ii) inhibition of embryogenesis by doxycycline after 6 months with respect to all embryo stages followed by decline in microfilariae after 11 months; (iii) reduction in spermatozoa in the female genital tract by doxycycline, whereas spermiogenesis was only partly reduced after 11 and 18 months; (iv) no relevant macro- or microfilaricidal activity; (v) depletion/marked reduction in endobacteria and inhibition of embryogenesis were sustained until 18 months after doxycycline and 12 months after co-administration of ivermectin; (vi) no severe adverse side effects were seen. Due to its long-lasting inhibition of embryogenesis, doxycycline presents an additional strategy for the treatment of onchocerciasis and control of Onchocerca microfilariae transmission. Extension of the existing registration will not require much time or high cost. Treatment of individual patients can be considered immediately.
Dirofilaria immitis is the agent of canine heartworm disease, in which adult worms reside in the pulmonary arteries, producing first stage larvae (microfilariae) that are released into the bloodstream. The present work describes the cytokine and iNOS mRNA expression in the peripheral blood of naturally infected dogs classified as either microfilariemic or amicrofilariemic. Results show that microfilariemic dogs had higher expression of IL-4 and iNOS mRNA than amicrofilariemic dogs. Furthermore, IL-10 mRNA expression was strongly expressed in dogs with circulating microfilariae, compared to only negligible expression in amicrofilariemic dogs. Finally, mf+ status was associated with a predominance in IgG1 production against worm antigens. These results would suggest that circulating mf may stimulate, like in other filarial infections, an immune bias towards unresponsiveness in D. immitis-infected dogs, consenting long-term adult worm survival.
The bacterial endosymbiont Wolbachia of several species of filarial nematodes plays an important role in the inflammatory pathology of filariasis. Nitric oxide (NO) production has also been implicated in the immune response during filarial infections. Here we present data indicating that a recombinant Wolbachia surface protein (rWSP) induces iNOs mRNA expression and NO production, as well as IFN-gamma and a Th1-type antibody response, in inoculated BALB/c mice. This effect is not observed when mice are inoculated with a recombinant heat shock protein from Wolbachia (GroEL).