[Show abstract][Hide abstract] ABSTRACT: Background
Dogs with chronic inflammation, including those with heartworm being managed with macrocyclic lactones and doxycycline (slow kill, SK), may develop immune complexes that block detection of Dirofilaria immitis antigen on commercial tests.Methods
To determine if SK could result in development of false-negative antigen tests, we collected serum samples from dogs that had been diagnosed with heartworm by antigen detection, with or without confirmation by detection of D. immitis microfilariae, placed on monthly macrocyclic lactones and doxycycline, and that later tested negative on an antigen test, and then tested them for antigen of D. immitis before and after treatment to disrupt immune complexes.ResultsSerum samples from a total of 15 dogs managed with SK were negative for antigen prior to heating on commercial assay (DiroCHEK®, Zoetis) by colorimetric detection and spectrophotometry, but after heat treatment, 8/15 (53.3%) samples converted to positive. Review of the medical records of each dog indicated that, after the heartworm diagnosis, only 7/15 (46.7%) dogs appeared to receive preventive monthly as prescribed, including 3 dogs that had detectable antigen after heating the sample and 4 dogs that did not have detectable antigen after heating. Whole blood was available from 9 dogs; microfilariae of D. immitis were detected in 1 sample.Conclusions
These data suggest that immune complex formation in dogs infected with heartworm and managed with SK can induce false negative antigen test results, misleading veterinarians and owners about the efficacy of this approach. Moreover, compliance with preventive administration appears poor, even after a heartworm diagnosis. The presence of persistent microfilaremia in at least one dog has implications for resistance selection.
[Show abstract][Hide abstract] ABSTRACT: Canine serum samples may contain factors which prevent detection of antigen of D. immitis on commercial assays, precluding accurate diagnosis. To determine the degree to which the presence of blocking antibodies or other inhibitors of antigen detection may interfere with our ability to detect circulating antigen in canine samples, archived plasma and serum samples (n = 165) collected from dogs in animal shelters were tested for D. immitis antigen before and after heat treatment. Negative samples were also evaluated for their ability to block detection of D. immitis antigen in a sample from a positive dog. All 165 samples were negative prior to heating, but 11/154 (7.1%) became positive after heat treatment, a conversion that was documented and quantified on spectrophotometric plate assays, and 7/165 (4.2%) samples decreased detection of antigen when mixed with a known positive sample, suggesting some blocking ability was present. An additional 103 plasma and serum samples which tested positive prior to heating also were evaluated; the optical density (OD) of 14/101 (13.9%) increased by ≥ 50%, and one sample by as much as 15-fold, after heat treatment. Our results suggest that canine serum and plasma samples from dogs in the southeastern United States can contain inhibitors of D. immitis antigen detection, and that prevalence estimates of heartworm infection based on these assays would benefit from heat treatment of samples prior to testing.
[Show abstract][Hide abstract] ABSTRACT: Abstract To determine the prevalence of Borrelia burgdorferi and Anaplasma phagocytophilum in a newly established population of Ixodes scapularis in the mountainous region of southwestern Virginia, questing adult ticks were collected and the identity and infection status of each tick was confirmed by PCR and sequencing. A total of 364 adult ticks were tested from three field sites. B. burgdorferi sensu stricto was identified in a total of 32/101 (32%) ticks from site A, 49/154 (32%) ticks from site B, and 36/101 (36%) ticks from site C, for a total prevalence rate of 33% (117/356). In addition, A. phagocytophilum was detected in 3/364 (0.8%) ticks, one from site A and two from site B. The prevalence of both pathogens in ticks at these sites is similar to that reported from established endemic areas. These data document the presence of I. scapularis and the agent of Lyme disease in a newly established area of the Appalachian region, providing further evidence of range expansion of both the tick and public and veterinary health risk it creates.
[Show abstract][Hide abstract] ABSTRACT: Dogs exposed to ticks in the southern US may become infected with multiple species of Ehrlichia. To better define infection risk, blood samples collected from 10 dogs infested with ticks via a natural infestation model were evaluated by blood smear examination, PCR, patient-side ELISAs (SNAP® 4Dx® and SNAP® 4Dx® Plus), IFA, and peptide based ELISA for evidence of infection with Ehrlichiacanis, E. chaffeensis, and/or E. ewingii. Although morulae were rarely identified in blood smears, every dog (10/10) became infected with Ehrlichia spp. as evidenced by nested PCR detection of E. chaffeensis (7/10) and E. ewingii DNA (10/10); real-time PCR detection of E. chaffeensis (0/10) and E. ewingii (9/10); seroconversion on two different patient-side ELISAs (4/10 or 10/10); seroconversion on IFA to E. canis (10/10, maximum inverse titer = 128-4,096, GMTMAX = 548.7) and E. chaffeensis (10/10, maximum inverse titer = 1,024-32,768, GMTMAX= 4,096); and seroconversion on peptide specific ELISA to E. chaffeensis VLPT (7/10) and E. ewingii p28 (9/10). Rickettsemia with E. chaffeensis and E. ewingii, as determined by nested PCR, persisted in dogs for an average of 3.2 or 30.5 days, respectively. Ehrlichia canis was not detected in any dog by any method, and no dogs developed signs of clinical disease. Our data suggest that in areas where ticks are common, dogs are at high risk of infection with Ehrlichia spp., particularly E. ewingii and E. chaffeensis, and can serve as a sentinel for monitoring for the presence of these zoonotic pathogens
[Show abstract][Hide abstract] ABSTRACT: The Companion Animal Parasite Council hosted a meeting to identify quantifiable factors that can influence the prevalence of tick-borne disease agents among dogs in North America. This report summarizes the approach used and the factors identified for further analysis with mathematical models of canine exposure to tick-borne pathogens.
[Show abstract][Hide abstract] ABSTRACT: Abstract Tick infestations and infection with tick-borne agents are commonly recognized in horses in North America, but equine infection with true Ehrlichia spp. has not been described. To determine the degree to which horses in the south-central United States are naturally exposed to and infected with tick-borne disease agents, serum samples were collected at random (n=240) or from horses with active tick infestations (n=73) and tested by immunofluorescence antibody assay (IFA) and/or enzyme-linked immunosorbent assay (ELISA) for evidence of antibodies reactive to Ehrlichia spp., Anaplasma spp., and Borrelia burgdorferi. Positive samples were further evaluated by species-specific serology for antibodies reactive to E. canis and E. chaffeensis, and whole blood samples were tested by PCR for evidence of infection with E. canis, E. chaffeensis, E. ewingii, and an E. ruminantium-like organism referred to as the Panola Mountain Ehrlichia. Antibodies reactive to Ehrlichia spp. were identified in 8.75% (21/240) of the randomly acquired samples and 24.7% (18/73) of the serum samples from tick-infested horses, but species-specific ELISA and PCR failed to confirm exposure to or infection with any known Ehrlichia spp. Antibodies to Anaplasma spp. (5/313; 1.6%) and B. burgdorferi (3/313; 1.0%) were uncommon. These data suggest that horses in the south-central United States are likely exposed to a novel Ehrlichia sp. Further research is needed to identify the etiologic agent responsible for the serologic activity seen and to determine the clinical significance, if any, of this finding.
[Show abstract][Hide abstract] ABSTRACT: Objective-To evaluate the performance of an in-clinic ELISA designed for detection of heartworm antigen and antibodies against 5 tick-borne pathogens. Design-Validation study. Sample-1,601 serum or matched serum, plasma, and blood samples from dogs. Procedures-Samples were tested for Dirofilaria immitis (heartworm) antigen and antibodies against Anaplasma phagocytophilum, Anaplasma platys, Borrelia burgdorferi, Ehrlichia canis, and Ehrlichia ewingii by means of an in-clinic ELISA. Evaluation of assay sensitivity and specificity, agreement of results among sample types, and cross-reactivity of E canis antigens in the assay with anti-Ehrlichia chaffeensis antibodies in stored samples from experimentally infected dogs were performed at a reference laboratory. Field tests of the in-clinic ELISA were performed at 6 veterinary facilities. Results were compared with confirmatory test results. Results-Sensitivity and specificity of the in-clinic ELISA were > 89% for detection of antibodies against A phagocytophilum (93.2% and 99.2%, respectively), A platys (89.2% and 99.2%, respectively), B burgdorferi (96.7% and 98.8%, respectively), E canis (97.8% and 92.3%, respectively), and E ewingii (96.5% and 93.9%, respectively). Sensitivity of the assay for detection of D immitis was 98.9%, with 99.3% specificity. The in-clinic ELISA identified exposure to > 1 vector-borne pathogen in 354 of 1,195 samples. Cross-reactivity of E canis antigens with anti-E chaffeensis antibodies was confirmed. Results of field evaluations confirmed that the in-clinic ELISA could be reliably used under typical clinical conditions to identify dogs exposed to the pathogens of interest. Conclusions and Clinical Relevance-The in-clinic ELISA provided a comprehensive in-house serologic screening test for all vector-borne pathogens evaluated.
Journal of the American Veterinary Medical Association 07/2014; 245(1):80-86. · 1.67 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Diagnosis of Dirofilaria immitis infection in dogs is largely dependent on detection of antigen in canine serum, plasma, or whole blood, but antigen may be bound in immune complexes and thus not detected. To develop a model for antigen blocking, we mixed serum from a microfilaremic, antigen-positive dog with that of a hypergammaglobulinemic dog not currently infected with D. immitis and converted the positive sample to antigen-negative; detection of antigen was restored when the mixed sample was heat-treated, presumably due to disruption of antigen/antibody complexes. A blood sample was also evaluated from a dog that was microfilaremic and for which microfilariae were identified as D. immitis by morphologic examination. Antigen of D. immitis was not detected in this sample prior to heating but the sample was strongly positive after heat treatment of whole blood. Taken together, our results indicate that blood samples from some dogs may contain factors that inhibit detection of antigen of D. immitis, and that heat treatment of these samples prior to testing could improve the sensitivity of these assays in some patients.
[Show abstract][Hide abstract] ABSTRACT: Diagnosis of Dirofilaria immitis infection in cats is complicated by the difficulty associated with reliable detection of antigen in feline blood and serum samples.
To determine if antigen-antibody complex formation may interfere with detection of antigen in feline samples, we evaluated the performance of four different commercially available heartworm tests using serum samples from six cats experimentally infected with D. immitis and confirmed to harbor a low number of adult worms (mean = 2.0). Sera collected 168 (n = 6), 196 (n = 6), and 224 (n = 6) days post infection were tested both directly and following heat treatment.
Antigen was detected in serum samples from 0 or 1 of 6 infected cats using the assays according to manufacturer's directions, but after heat treatment of serum samples, as many as 5 of 6 cats had detectable antigen 6-8 months post infection. Antibodies to D. immitis were detected in all six infected cats by commercial in-clinic assay and at a reference laboratory.
These results indicate that heat treatment of samples prior to testing can improve the sensitivity of antigen assays in feline patients, supporting more accurate diagnosis of this infection in cats. Surveys conducted by antigen testing without prior heat treatment of samples likely underestimate the true prevalence of infection in cats.
[Show abstract][Hide abstract] ABSTRACT: Abstract To determine the risk of canine infection with spotted fever group (SFG) Rickettsia spp. following natural tick exposure, 10 dogs determined to be free of evidence of exposure to or infection with tick-borne disease agents were exposed to ticks via weekly walks in a wooded area in north-central Oklahoma. After each walk, dogs were examined and the number and species of ticks present were recorded. The dogs were then returned to outdoor kennels to allow the infestations and subsequent transmission of any pathogens to proceed. Serum samples and whole blood were collected from each dog twice weekly for 121 days and evaluated via indirect fluorescence antibody (IFA) for antibodies reactive to Rickettsia rickettsii, R. montanensis, and "R. amblyommii," and by PCR for evidence of Rickettsia spp. Dogs became infested with a total of 57-108 ticks over the entire 8-week infestation period (weekly average tick infestation=12.0±4.1). The great majority of the ticks present were Amblyomma americanum (90.5%), with a small number of Dermacentor variabilis and A. maculatum also identified. All (10/10) dogs seroconverted to R. rickettsii, R. montanensis, and "R. amblyommii," with mean maximum inverse titers of 1176, 1448, and 6654, respectively, for all dogs in the study. Maximum inverse titers to "R. amblyommii" ranged from 4096 to 16,384 and were higher in 9/10 dogs than maximum inverse titers to R. rickettsii or R. montanensis. Sequence-confirmed SFG Rickettsia spp. (R. montanensis and "R. amblyommii") were occasionally, but not consistently, identified from whole blood by PCR. Taken together, our data suggest that, in areas where A. americanum is common, antibodies reactive to R. rickettsii in dogs may be due instead to infection with "R. amblyommii" or other, closely related SFG Rickettsia spp.
[Show abstract][Hide abstract] ABSTRACT: Ticks are common on horses, but there is a dearth of contemporary data on infestation prevalence, predominant species, and tick-borne disease agents important in this host. To determine the species of ticks most common on horses and the prevalence of equine exposure to and infection with tick-borne disease agents, ticks and blood samples were collected from 73 horses during May, June, and July of 2010. Adult ticks were identified to species, and antibodies to Ehrlichia spp., Anaplasma spp., and Borrelia burgdorferi were identified using indirect fluorescence antibody assay, a commercial point-of-care enzyme-linked immunosorbent assay, or both. In total, 1,721 ticks were recovered at the majority (85%) of equid examinations. Amblyomma americanum (L.) was the most common tick collected (1,598 out of 1,721; 92.9%) followed by Dermacentor variabilis (Say, 1821) (85 out of 1,721; 4.9%) and Amblyomma maculatum Koch, 1844 (36 out of 1,721; 2.1%); single specimens of Ixodes scapularis Say, 1821 and Dermacentor albipictus (Packard, 1869) were also identified. Antibodies reactive to Ehrlichia spp. were found in 18 out of 73 (24.7%) of horses tested, and were more commonly identified in horses with moderate or high tick infestations than those with low tick infestations (P < 0.001). These data support A. americanum as the most common tick species infesting horses in central Oklahoma from May through July and suggest horses are also commonly exposed to an Ehrlichia sp.
Journal of Medical Entomology 11/2013; 50(6):1330-3. · 1.82 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: In February 2012, 12 farmed mule deer (Odocoileus hemionus) were moved from a facility in southwestern Oklahoma to a facility in southeastern Oklahoma that housed 100 farmed white-tailed deer (Odocoileus virginianus). Between the third and fifth weeks, 9 of the 12 mule deer had died, 4 of which were submitted for necropsy. The deer were heavily infested with Amblyomma americanum (lone star ticks). Hematologic data from 1 deer revealed severe anemia, leukocytosis, and intraerythrocytic hemoparasites consistent with Theileria spp. Microscopically, the liver, lymph nodes, and spleen contained multifocally distributed, enlarged monocytic cells whose cytoplasm was replaced by developing meronts in various stages of merogony. It appears that, upon arrival, the Theileria cervi-naïve mule deer became infested with large numbers of Theileria-infected lone star ticks leading to massive exposure of the mule deer to sporozoites of the protozoan, resulting in an acute hemolytic crisis and fatalities. The merogonic stages of T. cervi are also described. The lack of earlier reports of merogony may be due to the fact that only a single, short-lived, merogonic cycle follows exposure to sporozoites and thus merogonic stages are demonstrable for only a short period. Polymerase chain reaction testing of paraffin-embedded tissue yielded a 507-bp amplicon sequence that was 100% identical with the sequence of T. cervi previously reported from white-tailed deer in Oklahoma and from elk in Wisconsin and Indiana.
Journal of veterinary diagnostic investigation: official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc 09/2013; 25(5):662-5. · 1.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Coyotes (Canis latrans) are commonly infested with ticks, including Amblyomma americanum, the predominant vector of Ehrlichia chaffeensis and Ehrlichia ewingii; Dermacentor variabilis, an important vector of Rickettsia rickettsii; and Amblyomma maculatum, a major vector of Rickettsia parkeri, a spotted fever group (SFG) Rickettsia. To determine the degree to which coyotes are infected with or exposed to tick-borne bacterial disease agents, serum samples collected from coyotes in Oklahoma and Texas were tested for antibodies reactive to R. rickettsii, Ehrlichia canis, E. chaffeensis, E. ewingii, Borrelia burgdorferi, and Anaplasma phagocytophilum by indirect fluorescent antibody (IFA) testing or enzyme-linked immunosorbent assay (ELISA). Of the coyotes tested, 60% (46/77) and 64% (47/74) had antibodies reactive to R. rickettsii and E. chaffeensis, respectively, on IFA. Additionally, 5% (4/77) had antibodies reactive to E. canis, but not B. burgdorferi or A. phagocytophilum, on SNAP(®) 4Dx(®) ELISA; subsequent serologic analysis by plate ELISA using species-specific peptides revealed antibodies to E. ewingii, E. canis, and E. chaffeensis in 46% (23/50), 18% (9/50), and 4% (2/50) of serum samples, respectively. Taken together, these data indicate that coyotes in this region are commonly exposed to SFG Rickettsia and E. ewingii and that further consideration of coyotes as a component of the maintenance cycle for these pathogens may be warranted.
Journal of wildlife diseases 07/2013; 49(3):670-3. · 1.31 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Rickettsial agents, including those in the genera Anaplasma, Ehrlichia, Neorickettsia, and Rickettsia, are important and common vector-borne pathogens of dogs and cats. Disease induced by these organisms ranges from clinically inapparent to severe and potentially fatal. However, laboratory confirmation of a rickettsial etiology can be complicated by a number of factors, including the wide spectrum of disease induced by these organisms, an often low and widely fluctuating level of organism present in infected animals, cross-reactions on serologic and molecular assays, and the presence of co-infections. Correct diagnosis is most likely to be reached when multiple diagnostic strategies, including careful microscopic examination of stained blood films or tissues, both specific and broad serologic tests, and a suite of molecular detection assays, are used in concert. Accurate interpretation of diagnostic tests requires awareness of the likelihood for multiple agents, including novel organisms, to be responsible for the results seen in a given patient. This review provides an overview of current strategies used to diagnose rickettsial infections in dogs and cats.
[Show abstract][Hide abstract] ABSTRACT: "One Health" is a term that encapsulates and underscores the inherent interrelatedness of the health of people, animals, and the environment. Vector-borne infections are central in one health. Many arthropod vectors readily feed on humans and other animals, serving as an ideal conduit to move pathogens between a wide spectrum of potential hosts. As ecological niches flux, opportunities arise for vectors to interact with novel species, allowing infectious agents to broaden both geographic and host ranges. Habitat change has been linked to the emergence of novel human and veterinary disease agents, and can dramatically facilitate expansion opportunities by allowing existing vector populations to flourish and by supporting the establishment of new pathogen maintenance systems. At the same time, control efforts can be hindered by the development of parasiticide and pesticide resistance, foiling efforts to meet these challenges. Using examples drawn from representative diseases important in one health in the Americas, including rickettsial infections, Lyme borreliosis, Chagas disease, and West Nile virus, this paper reviews key aspects of vector-borne disease maintenance cycles that present challenges for one health in the Americas, including emergence of vector-borne disease agents, the impact of habitat change on vector-borne disease transmission, and the complexities faced in developing effective control programs. Novel strategies will be required to effectively combat these infections in the future if we are to succeed in the goal of fostering an environment which supports healthy animals and healthy people.
[Show abstract][Hide abstract] ABSTRACT: Abstract To better define the strains and species of Hepatozoon that infect coyotes in the south-central U.S., whole blood and muscle samples were collected from 44 coyotes from 6 locations in Oklahoma and Texas, and evaluated by a nested PCR using primers amplifying a variable region of the apicomplexan 18S rRNA gene as well as histopathology (muscle only) for presence of tissue cysts. Hepatozoon spp. infections were identified in 79.5% (35/44) of coyotes tested, including 27 of 44 (61.4%) whole blood samples and 17 of 44 (38.6%) muscle samples tested by PCR, and 23 of 44 (52.3%) muscle samples evaluated by histological examination. Analysis revealed 19 distinct sequences comprising 3 major clusters of Hepatozoon spp., i.e., 1 most closely related to H. americanum, another most closely related to H. canis, and the third an intermediate between the 2 groups. The diversity of Hepatozoon spp. in wild canids appears greater than previously recognized and warrants further investigation.
Journal of Parasitology 08/2012; · 1.32 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: There is a lack of knowledge regarding the prevalence of Dirofilaria immitis and Ehrlichia spp. in coyotes in Oklahoma and Texas. Documenting the prevalence of these vector-borne disease agents in coyotes from Oklahoma and Texas underscores the importance of wild canids as reservoir hosts that infect companion animals and humans. To learn more about the sylvatic cycle of D. immitis and Ehrlichia spp. in coyotes from Oklahoma and Texas, we tested for infection with and exposure to, respectively, these disease agents. Coyote carcasses were collected opportunistically from animal control experts and hunters in seven counties in Oklahoma and Texas from January to March, 2010. Serum samples from 77 coyotes were tested with a commercial ELISA test. Five (6.5%) coyotes had D. immitis antigens, and four (5.2%) had antibodies to Ehrlichia spp. The overall prevalence of D. immitis was low relative to studies from the eastern United States. Little is known about the prevalence of Ehrlichia spp. throughout the United States, but coyotes from rural Oklahoma in the current study had a higher exposure rate than those reported from California, and a lower rate than data from an earlier study from Oklahoma.