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Molecular differentiation of sheep and cattle isolates of Fasciola hepatica using RAPD-PCR
Abstract
Understanding genetic structure and status of genetic variation of Fasciola hepaticaisolates from different hosts, has important implications on epidemiology and effective control of fasciolosis. Random amplified polymorphic DNA (RAPD-PCR) was used to study the genetic variation of F. hepatica in sheep and cattle. DNA was extracted from adult helminthes removed from livers of each infected animal in slaughterhouse at East-Azerbaijan province, North-West of Iran. DNA template amplified by the polymerase chain reaction, usingthree oligonucleotide decamers with arbitrary DNA sequences as primers. RAPD patterns showed the specific but different pattern DNA patterns for each primer. The intraspecific similarity coefficient within two isolates of F. hepatica was ranged between 69 to 100%. Present findings showed that the interspecific genetic distance was higher than intraspecific genetic distances (19-47% compares to 0-19%). Pair wise similarity matrices generated from each isolates-primer combination were totaled and the similarity coefficient betweenstrains were calculated both manually (Nei and Li method) and software analysis (Free-Tree-Freeware program). The inferred phylogenetic tree on the fingerprinting of these isolates clearly demonstrated the existence of population genetic diversity sub structuring within F. hepatica of sheep and cattle of Iran, raising interesting questions on the host specificity, epidemiology (e.g., zoonotic transmission) and ecology of this fluke. RAPD-PCRis useful for both individual identification and epidemiological investigations in endemic regions.
... On the basis of genetic distance data (Table 4), liver flukes used in this study showed great variation with a range of 0.0690 -0.6109 as compared to the findings by other authors in their study on the genetic variation of Fasciola hepatica isolates from sheep, cattle and buffalo, they found the interspecific genetic distance to range between 0.22 and 0.50 and intraspecific genetic distance ranged between 0 and 0.22 [23]. In a study on the Molecular differentiation of sheep and cattle isolates of Fasciola hepatica using RAPD-PCR the interspecific genetic distance had a range of 0.19-0.47 and intraspecific genetic distances had a range of 0-0.19 [24]. RAPD variability and genetic diversity for F. hepatica in particular have been examined in cattle populations of liver fluke from Ukraine and Armenia [25]. ...
... RAPD variability and genetic diversity for F. hepatica in particular have been examined in cattle populations of liver fluke from Ukraine and Armenia [25]. Studies [16,24,25] suggested multiple genetically different parasites but did not provide adequate information about genetic diversity, distribution and population structure of studied liver fluke isolates. Specific RAPD assays have been developed for differentiation of fasciolid species in UK, Peru, Ghana and Sudan [26]; and the technique enabled differentiation of F. hepatica and F. gigantica from cattle and sheep hosts from countries mentioned above. ...
... All liver flukes have been correctly identified to species level. Some investigations do not give detailed and complete information for genetic structure and diversity of liver fluke intrapopulations and do not established intra-and interpopulation relationships in different geographic regions [16,24,26]. ...
The study sought to characterize Fasciola gigantica isolates from cattle in different localities using RAPD-PCR. Adult flukes morphologically identified as F. gigantica were collected from slaughtered infected animals during meat hygiene inspections. DNA was extracted from single flukes and subjected to RAPD-PCR analysis. In the RAPD-PCR analysis, genomic DNA isolated from the conical anterior end of the worms was amplified by the polymerase chain reaction using 10 random oligonucleotide primers. Depending upon the Fasciola gigantica isolate-primer combination, 1-13 DNA fragments in the range of 75-2000bp were amplified. It was observed that all the 10 primers directing amplification of DNA were of potential interest in the generation of polymorphic DNA. The percentage polymorphic loci ranged from 33.33-100%. Polymorphic bands were scored and used to calculate Nei's 1978 genetic distance. The genetic distance values ranged between 0.0690 (isolate 5 and 6 from Gwanda and 0.6109 (isolate 6 from Gwanda and isolate 14 from Matopo). The mean Nei's gene diversity was 0.2839. The study showed the variability of Fasciola gigantica isolates from the same host, using RAPD markers could be applied as a low cost way of identification.
... Therefore, people infected with this parasite can be present in the community for a long time as carriers of the parasite without a diagnosis, which will have adverse and destructive effects on public health. Fasciola can act as an erratic parasite and be seen as an ectopic infection in other organs of the human body, such as subcutaneous tissue and other organs inside and outside the abdomen, which has been reported by Taghipour et al., but this issue is not widespread in endemic areas of this parasite [13,41]. Human infection with the fasciola parasite is associated with short-term and long-term symptoms. ...
... Bithionol is the first known adenylyl cyclase solution inhibitor to act through a bicarbonate junction by a predominantly allosteric mechanism [39,40]. LD50 of Bithionol in rats at the oral dose is 2100 mg/kg [39,41,42]. ...
Background
Fascioliasis is a worldwide parasitic infection caused by a food-borne trematode called Fasciola, and Fasciola infection has been reported in more than 80 countries. Recently, the WHO has presented a roadmap for overlooked diseases from 2021 to 2030, which aims to increase the prevention and control of overlooked different diseases such as Fascioliasis.
Methods
Our main objective was to conduct a systematic review aiming to summarize recent knowledge on the Antiparasitic Compounds against Human Fascioliasis. A keyword search was performed in PubMed, Web of Science, to gather relevant literature published between the 17th of April 1992 and the 23rd October 2022. A total of 329 records were initially retrieved, with 28 full-text articles retained for the qualitative synthesis.
Results
Up to now, various antiparasitic drugs have been used to treat human fascioliasis, the most important of which are: Triclabendazole, Albendazole and Bithionol, Praziquantel, Emetine and Dehydroemetine, Mebendazole in combination with Metronidazole and Nitazoxanide, Chloroquine, Hexylresorcinol. From the past to the present, natural herbal medicines have traditionally been used in most countries to treat various parasitic diseases in humans and animals so that these are known as active anthelmintic phytochemicals such as Artemisinin, Mirazid, Plumbagin, Lycium chinense.
Conclusion
Although Triclabendazole is an effective and useful drug of choice for the treatment of human fascioliasis, but due to the gradual resistance of fasciolas to Triclabendazole, further research is needed to find new drugs. Despite many advances in antiparasitic compounds used against human fascioliasis, a number of integrated control measures should be implemented as strong management strategies for fascioliasis.
... Other diagnostic methods for fascioliasis include ultrasound, CT scan, and PCR [6]. But in practice, often only the first option is done. ...
... But in practice, often only the first option is done. Control methods are expensive and will become more difficult with the development of resistance to flukicide drugs [5,6]. ...
The World Health Organization (WHO) includes fascioliasis in the list of the NTDs (Neglected Tropical Diseases), among the group of food-borne trematodiases.
... However, a survey carried out in Kassala, eastern Sudan, near the Ethiopian border (560 km from Khartoum) reported an eastern kdr mutation. The strong association between the presence of the kdr allele and susceptibility/resistance phenotype is consistent with, while others, in contrast, revealed the absence or weak association of the kdr with DDT and pyrethroid resistance (31). Our kdr frequency aligns with the study conducted by others which found no significant difference in kdr frequency between urban and suburban areas, except for the presence of DDT. ...
... However, a survey carried out in Kassala, eastern Sudan, near the Ethiopian border (560 km from Khartoum) reported an eastern kdr mutation. The strong association between the presence of the kdr allele and susceptibility/resistance phenotype is consistent with, while others, in contrast, revealed the absence or weak association of the kdr with DDT and pyrethroid resistance (31). Our kdr frequency aligns with the study conducted by others which found no significant difference in kdr frequency between urban and suburban areas, except for the presence of DDT. ...
Abstract
Introduction: Insecticide resistance is one of the major challenges in vector control programs around the globe. This study investigated the insecticide resistance and kdr mutation in the Anopheles arabiensis malaria vector in Khartoum State, Sudan.
Methods: Entomological cross-sectional surveys were carried out at four urban and suburban sites in Khartoum State. Four insecticides were tested for World Health Organization (WHO) susceptibility, and kdr frequencies were estimated using two allele-specific PCR assays.
Results: WHO bioassay tests revealed that DDT, malathion, and permethrin showed high resistance in both urban and suburban sites. There is no significant difference in mortality rates between urban and suburban sites (P > 0.05), with the exception of DDT, where mosquitoes from urban sites showed more susceptibility [64 (51.23-76.77)] than those from suburban areas [53.5 (69.73-95.27)]. In general, all populations from the four sites showed faster KDT50% to bendiocarb and permethrin than to malathion and DDT insecticides. Generalized linear model analysis revealed that insecticide type, site type, and their interaction were determinant factors in mortality rate. A high to moderate frequency of the West African kdr mutation (L1014F) was observed in urban and suburban sites, and the association between the presence of the kdr mutation and resistance phenotype was strong for permethrin and DDT (OR > 7 in the allelic test).
Conclusion: This study showed the susceptibility status of the malaria vector A. arabiensis to four insecticides belonging to different classes in urban and suburban sites. This provides important knowledge that helps vector surveillance and control programs. Additionally, more research is necessary to explore the impact of pyrethroid resistance, particularly in bednets, and other resistance mechanisms in this malaria vector.
Keywords: Anopheles arabiensis, kdr mutation, Insecticide resistance
... Trypanocidal Drug Use Trypanocides are the main method of controlling bovine trypanosomosis in most sub-Saharan African nations where tsetse and trypanosomosis are present (48)(49)(50)(51). Historically, for the past 50 years, the three trypanocide compounds -isometamidium chloride, homidium (bromide and chloride), and diminazene aceturatehave been marketed. ...
Most vector-borne human diseases worldwide are spread by arthropod disease vectors, including mosquitoes, ticks, tsetse flies, and sandflies, which are greatly impacted by environmental factors. The tsetse-transmitted animal Trypanosomiasis severely hampers animal production and agricultural development in Ethiopia. The southern portion of the Rift Valley, the southwest corner of the nation, the western lowlands and escarpments, and the Blue Nile are the only areas in Ethiopia where tsetse flies are found. These areas are limited to longitudes 33° and 38° E and latitudes 5° and 12° N. The three Trypanosoma species that have the most impact on cattle in Ethiopia are Trypanosoma brucei, Trypanosoma vivax, and Trypanosoma congolense. Particularly in the “tsetse belt,” which includes the Omo, Borena, and Metekel zones of the Benishangul Gumuz region, the illness is extensively spread throughout the western and southwest regions of the nation. Ethiopia’s main river systems, including
the Abay/Didessa, Omo/Gibe, Baro/Akobo, and southern Rift Valley, are linked to the prevalence of tsetse fly infestations and
the Trypanosomiasis they cause. Mixed livestock and crop production is the major farming style in Ethiopia’s highlands and
plays a crucial role in agricultural activity. The bioclimatic threshold for tsetse flies in Ethiopia has not been met. Since it has
long been typical for the peasantry in general and livestock owners, in particular, to avoid tsetse-infested areas to reduce their chance of contracting Trypanosomiasis, the impact of tsetse-borne Trypanosomiasis on development efforts in rural Ethiopia is becoming increasingly significant. This review examines the economic impact and epidemiology of tsetse-transmitted bovine
trypanosomiasis in Ethiopia.
Keywords: Bovine, Distribution, Epidemiology, Trypanosomiasis, Tsetse fly�
... In any case, regarding recombinant worm vaccines, a group believes that the effectiveness of such vaccines cannot be very reliable and it is necessary to conduct more investigations for their effective effectiveness (12). The vaccine prepared against Boophilus micropolus ticks is a type of recombinant vaccine prepared from the outer surface proteins of the intestinal wall of the tick (13). ...
Although anti-parasitic drugs in humans and animals have been used for many years to control parasitic diseases in developed, developing and low-income countries. However, the continued use of anti-parasitic drugs is associated with two basic problems, which include the gradual resistance of the parasite to the drug and its residue in animal products such as meat and milk, and the pollution of the environment.Therefore, according to the mentioned points, researchers tried to use vaccines to solve the problems caused by drug consumption, although conventional vaccines and recombinant vaccines have been associated with effective results in preventing the spread of bacterial and viral diseases in humans and animals, and in the case of vaccines Recombination related to parasitic diseases brilliant research activities have been carried out so far. But no significant results have been obtained. Despite this, a limited number of protozoan vaccines such as Theilerian, Babesian, Coccidial, Toxoplasmic and Leishmanian vaccines have been prepared and produced in the form of attenuated vaccines, but the use of such vaccines is accompanied by problems and obstacles.In the case of molecular parasitic vaccines, vaccines with high reproductive immunity have not yet been prepared and investigations are ongoing in this regard, and in the current situation, only anti-mite vaccines such as Tick Gard in Australia and Gavac in Cuba and others. It is used in South American countries, but it should be noted that the success rate of such vaccines is not more than 30-35%, and it is necessary to conduct more studies to prepare anti-mite molecular vaccines with an efficiency of more than 35%.
... These mushrooms demonstrate antibacterial, anticancer, and antihypertensive, immune system modulator, antioxidant, and antiandrogenic properties. Currently, there are very few studies and reports on the anti-parasitic ability of Cantharellus and Ganoderma mushrooms (13). ...
Abstract
Fasciola hepatica and Fasciola gigantica are among the most interesting and at the same time important subjects of parasitology. The relative ability of these zoonoses in performing laboratory operations and their high prevalence in animals have made fascioliasis a suitable research subject. Today, consistent and continuous control measures are widely recognized as planned management strategies for fascioliasis. These programs may include scheduled grazing of animals and killing of snails and the use of antiparasitic drugs. In the past, various medicinal plants have been tested against fascioliasis. Usually, mushrooms are known as valuable food and contain various active chemical substances with nutritional and therapeutic properties. Ganoderma applanatum and Cantharellus cibarius are species of macrofungi that have great medicinal and therapeutic value. These mushrooms show a wide range of medicinal properties (antibacterial, anticancer, antihypertensive, immune system modulator, antioxidant, and antiandrogenic properties). In the biological control method of various Fasciola species, macrofungi and predatory fungi are mostly used, and due to the naturalness of their compounds, they have extremely less side effects and risks compared to chemical synthetic compounds, and because of this alternative biological control, they will be suitable for other fascioliasis control methods in the future.
Keywords: Fasciola spp., Biological control, macrofungi, Ganoderma applanatum, Cantharellus cibarius
... This might be due to environmental factors causing diversity. The percentage of similarity between isolates and standard subspecies was used to evaluate the similarity between isolates [34]. ...
Escherichia coli (E. coli) is shocked by various temperature processes in milk, which forces the organism to make proteins as a result of changes in the synthesis of enzymes that might give the strain special characteristics. The purpose of this study was to investigate the effects of the heat shock factor on changing the results of biochemical and molecular tests among E. coli strains obtained from ice cream and non-pasteurized milk when compared to a reference strain from the American-type culture collection (ATCC) in order to determine the phenotypic variation caused by the temperature conditions of the manufacturing process. Furthermore, isolates with characteristics similar to E. coli were discovered, but they were not E. coli and caused some ambiguity. To test the E. coli contamination of traditional and industrial ice cream, 82 samples were chosen at random. SDS-PAGE and 16S rDNA sequencing were carried out, as well as phenotypic testing. Isolated strains did not exactly match the reference strain. The results of biochemical testing and protein analysis revealed that the isolates were diverse. Samples E. coli phenons were classified. In the electrophoresis, the ice cream strain had two protein bands in the 20.75 and 23.59 kDa ranges that were distinct from the reference strain. These isolates appear to experience alterations in enzyme characteristics and structural proteins as a result of being exposed to various temperature conditions, such as pasteurization and frigidity. When compared to the reference strain, the calculated similarity percentage of the elicited isolate varied from 60 to 70%. The electrophoretic patterns of E. coli isolated elicited from milk samples differed from E. coli isolated obtained from the ice cream. The distinctions were in the intensity or position of the bands. The results also revealed that when isolates are subjected to thermal stresses, they exhibit a pattern similar to that of ice cream isolates. These considerations are made because a change in protein composition might result in a change in biochemical features, resulting in uncertainty in its identification. Sequences revealed that the sequences were related to E. coli 16S rDNA, despite differences in phenotypic and electrophoretic features between the isolated bacteria and the reference strain E. coli ATCC 25922. Our findings revealed that 16S rDNA could potentially be used to instantly implement an appropriate preventive measure for the purpose of identifying this type of bacteria and avoid some ambiguity.
... However, this study investigated eggs of Fasciola species of living ruminants in Iran. Khakpour and Garedaghi (2012) examined the molecular differentiation of sheep and cattle isolates of F. hepatica using RAPD-PCR and found that RAPD-PCR can efficiently detect F. hepatica, which is in line with the findings of the present study. Ayaz et al. (2014) investigated F. hepatica in many buffaloes and cattle using PCR and microscopic examination and reported that the microscopic method could detect 3.58% of infected samples, which is similar to the results of this study. ...
Fascioliasis is an emerging and important food and water-borne disease in human communities which has become one of the most important health challenges in countries, like Iran. It causes weight loss, a decrease in feed conversion ratio as well as milk and meat production, and also reduces fertility in animals the prevalence of fasciolosis is increasing in some regions of the world due to various factors. Different methods have been used for the detection of Fasciola hepatica in animals. This study is the first to detect F. hepatica in Lori sheep using polymerase chain reaction (PCR) and conventional diagnostic methods in Western Iran. During three months, 195 fecal samples were collected from sheep in Lorestan province, Iran, using the stratified random sampling method. The conventional diagnostic methods, including wet mount microscopic examination and concentration assays, as well as the PCR technique targeting the intergenic spacer gene of F. hepatica, were used for the detection of the parasite in sheep. In total, 4 (2.1%) out of 195 examined stool samples were positive for F. hepatica based on the conventional assays. The PCR test was positive for F. hepatica in7 (3.6%) samples of 195 studied specimens. Statistical analyses of the data revealed that there is a significant difference between the results of diagnostic methods for F. hepatica detection (P=0.0421). Finally, the results showed that PCR has more diagnostic sensitivity, compared to conventional diagnostic methods, including the concentration techniques and microscopic examination. Hence, it can be advised to use PCR for the detection of F. hepatica in sheep.
... The developed detection methods of heterophyid trematodes have so far contributed to fast and accurate identification methods. Most have been involved with coprodiagnosis and the simplex PCR technique, such as sequence characterized amplified regions marker [25], high annealing temperature random amplified polymorphic DNA profile [26] and PCR-restriction fragment length polymorphism [11,12] , which are considered both time-consuming and expensive. Accordingly, a recent report that was published in 2008 concluded that multiplex PCR was the most sensitive and accurate method for specific diagnosis and could easily be applied to achieve results as a low cost technique. ...
... The developed detection methods of heterophyid trematodes have so far contributed to fast and accurate identification methods. Most have been involved with coprodiagnosis and the simplex PCR technique, such as sequence characterized amplified regions marker [25], high annealing temperature random amplified polymorphic DNA M A N U S C R I P T A C C E P T E D ACCEPTED MANUSCRIPT profile [26] and PCR-restriction fragment length polymorphism [11,12], which are considered both time-consuming and expensive. Accordingly, a recent report that was published in 2008 concluded that multiplex PCR was the most sensitive and accurate method for specific diagnosis and could easily be applied to achieve results as a low cost technique. ...
Objective: To develop the multiplex PCR method based on the internal transcribed spacer 2 to discriminate the intestinal trematodes, Centrocestus caninus (C. caninus), and Stellantchasmus falcatus (S. falcatus).
Methods: Four species of heterophyid trematodes including C. caninus, S. falcatus, Haplorchis taichui and Haplorchoides sp. were amplified and the specific primer was designed based on the internal transcribed spacer 2 region. Two specific primers were used to validate the optimized PCR conditions: the specificity test and the sensitivity test.
Results: Both of these specific primers confirmed the specificity through multiplex PCR reaction which generated both PCR products (231 and 137 bp) in the mixed DNA template of C. caninus and S. falcatus with no cross-reaction with other heterophyid trematodes. The optimum annealing temperature of both primers was 54–59 °C. The sensitivity test used the two-fold serial dilution DNA template, which was concentrated between 10 and 0.3125 ng/μL. The lowest concentration of the DNA template of this multiplex PCR was 2.5 ng/μL.
Conclusions: The technique described here proved to be a species-specific technique and was found to be a rapid method for the diagnosis of C. caninus and S. falcatus in terms of the larval and adult stages in intermediate and/or definitive hosts in the endemic area.
For elucidation of the taxonomic status of the Japanese Fasciola species, whole mitochondrial DNA of Fasciola hepatica from Australia, F. gigantica from Malaysia, and Fasciola sp. from Japan was digested with three four-base-cutting endonucleases: HinfI, MspI, and RsaI. The resulting digestion patterns showed that for each enzyme there were some bands specific for each geographical isolate and that the Japanese Fasciola sp. shared more bands with F. gigantica than with F. hepatica. Nucleotide sequences of two regions, the second internal transcribed spacer (ITS2) of the nuclear ribosomal RNA cluster and mitochondrial cytochrome c oxidase subunit I (COI), were also compared among them. The ITS2 sequence was highly conserved among the three isolates. F. gigantica and the Japanese Fasciola sp. were identical, but they differed from the Australian F. hepatica at six sites, one of which was a deletion. The COI sequence was less conserved but implied a similar relationship between the isolates. There seems no reason to regard the Japanese Fasciola sp. as anything other than a strain of F. gigantica.
We have generated a 3' cDNA pool from the RNA of only 1000 or fewer cells by reverse transcription (RT) from an extended oligo(dT) primer with a 3' degenerate base and a second strand primer with four degenerate 3' bases, followed by PCR. Reproducible differential displays (DD) can be made from this essentially inexhaustible source of DNA. The method produced DD patterns that are comparable but not identical in band number and size distribution with those obtained by the original RT-DD technique. Northern blots performed with the excised bands verified altered gene expressions. The data indicate that this 3'-end cDNA pool can supplement current PCR-based methods of expression genetics. This pool of cDNA sequences also provides a reliable source for primer-specific gene amplifications.
Random amplified polymorphic DNA (RAPD) was used to study genetic variation within Oesophagostomum bifurcum in Ghana. Four different decamer primers were used for the amplification of DNA from individual O. bifurcum adults (n = 41) from humans and non-human primates (including the Mona monkey, Patas monkey and Olive baboon) from different geographic regions. Analysis of the amplicons from all 41 nematodes by high resolution, denaturing polyacrylamide gel electrophoresis defined a total of 326 informative RAPD bands. Cluster analysis of the RAPD data (based on pairwise comparison of banding profiles) showed that O. bifurcum from humans was genetically distinct from O. bifurcum from the Mona and Patas monkeys, and from the Olive baboon. These findings clearly demonstrate the existence of population genetic substructuring within O. bifurcum from different primate hosts in Ghana, and raise interesting questions about host specificity, epidemiology (e.g., zoonotic transmission), and ecology of the different genotypes of O. bifurcum.
Random amplified polymorphic DNA (RAPD) markers were used to quantify the genetic diversity and its distribution in liver fluke, Fasciola hepatica recovered from seven definitive hosts belonging to Ukrainian and Armenian cattle populations. Five oligonucleotide primers produced complex and highly variable patterns of amplified DNA in F. hepatica. Intra- and interpopulation genetic variability of adult parasites was analyzed on the base of polymorphic and monomorphic reproducible bands. Genotypic diversity calculated by Shannon's indices showed that the majority of variance occurred within, rather then between hosts and was also greater within than between populations. Analysis of molecular variance was used to test the significance of genetic differentiation within and between populations of worms. Of total gene diversity, 86.54% were partitioned within hosts, 8.17% of variability accounted by the differences among hosts/within populations and 5.3% of variation by the differences between populations. These results suggest that each individual cow is infected by numerous genetically different parasites. Migration of definitive hosts and other factors influencing the distribution of liver fluke RAPD variability are discussed.
Rates at which AP-PCR patterns diverge among isolated taxa were examined to test whether they exhibit clocklike regularity.
The results showed that rates of divergence differed significantly among the groups examined (primates, antelopes, and Hawaiian
Drosophila grimshawi). Therefore, AP-PCR divergence rates cannot be used as a "universal clock" with an invariant rate in
all animals. Nevertheless, within each group, a strong relationship existed between degree of AP-PCR pattern divergence and
time since separation of isolated taxa. Thus, AP-PCR divergence may prove useful for dating evolutionary events if calibrated
within a more limited taxon.
Various protein separation techniques have been used as alternative means of differentiating species and strains of parasites. Of these techniques, isoelectric focusing (IEF) has proved to be specific and reproducible with cestodes, protozoa and trematodes. As with the traditional morphological approach, there is, however, the potential for host influence. For a particular separation technique to be useful, it is important that banding profiles differentiating parasites be consistent, regardless of host species. To test for host influence, Fasciola hepatica were maintained in four definitive hosts. Soluble proteins of flukes from different hosts and their respective excretory-secretory product (ESP) were then subjected to IEF together with respective host tissues, and the resulting banding patterns were compared. Protein banding profiles of fluke body protein from llama, rat and mouse were very similar, while those of flukes from calves were distinct. Banding patterns of fluke ESP from different hosts were dissimilar. Overall banding patterns of body protein and ESP of flukes from different hosts were distinct from those of respective host tissues. The unique banding patterns of the body protein of flukes from calves indicate that there was host influence in their protein banding patterns, and the uniqueness of the calf fluke is discussed relative to host's resistance to the fluke.
A cDNA clone that encodes the large subunit of mitochondrial ribosomal RNA (LSU rRNA) from the liver fluke F. hepatica was isolated and characterized. This RNA molecule is polyadenylated at the 3′ end and represents 10% of the poly A+RNA in adult F. hepatica. Fluke LSU rRNA has significant sequence homology to mosquito mitochondria LSU rRNA and is more closely related to the mitochondrial
rRNA of hermaphroditic than dioecious trematodes. Mitochondrial DNA constitutes approximately 10% of the total cellular DNA
of adult flukes. This percentage is lower in non-embryonated eggs as are the levels of LSU rRNA indicating eggs have lower
metabolic activity. Analysis of transcription and the number of mitochondrial genomes in S.mansoni shows that the LSU rRNA is more abundant in females than in males. Restriction endonuclease analysis of the fluke mitochondrial
LSU rRNA genes suggests the presence of heterogeneous repeated copies in the mitochondrial genome or heterogeneity among individual
genomes of mitochondria.
The reproducibility of the generation of random amplified polymorphic DNA fragments from three commonly used thermal cyclers was determined using identical assay conditions. In all cases, different results were obtained from the three instruments. Variation in the length of the primer (20 nt or 10 nt) did not have any effect on the reproducibility of the assays from the three machines tested. A DNA concentration of 1 ng generated poorly staining DNA fragments whereas concentrations between 10 ng and 100 ng gave similar banding patterns when using the same thermal cycler. Low concentrations of primer (0.05 microM) did not produce any detectable DNA fragments. Increased primer concentrations of 0.25 microM or higher generated intensely staining DNA fragments, and concentrations above 0.5 microM did not improve the clarity of the banding patterns but did direct the synthesis of increasing amounts of very short DNA fragments. Surprisingly, the 20 nt-long primer was able to direct the synthesis of more DNA fragments than a primer of only 10 nt long.
Genetic variability in Entamoeba histolytica was analyzed by random amplified polymorphic DNA (RAPD) using ten arbitrary primers. Due to intrinsic characteristics of the RAPD technique only axenic samples were analyzed. since the presence of any microorganism in the cultures interfered in the DNA profile by generating RAPDs not pertaining to E. histolytica. The RAPD profiles of E. histolytica samples isolated from patients with different clinical manifestations from different regions of the Americas shared about 70% of the bands produced. These profiles were compared to those obtained for E. moshkorskii, and E. invadens. The combined data for the ten primers were used in the phenetic analysis of all the isolates studied by using the Dice similarity coefficient as the genetic distance measure between the samples. Three distinct groups could be separated by phenon line: one including E. moshkovskii samples, which shared > 90% of the RAPDs produced by the different primers; one consisting solely of E. invadens; and a third comprising samples of E. histolytica, which showed considerable intraspecific variability.
Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease common in the tropics. Melioidosis is most prevalent in the northeastern part of Thailand. The diseases has diverse clinical manifestations ranging from mild localized to fatal septicemic forms. The bacterial genetic factors contributing to the severity of melioidosis have not been completely identified. We have developed a genotyping method based upon randomly amplified polymorphic DNA (RAPD) analysis. Eighteen deca-oligo nucleotide primers with 70% GC content, eight previously published 60%GC RAPD primers, and four random deca oligomers were tested on nine strains of B. pseudomallei isolated from five patients with localized and four with septicemic melioidosis. The RAPD patterns were analyzed by polyacrylamide gel electrophoresis using a laser based automated fragment analyzer, GS2000. Based upon the pattern complexity, seven pairs consisting of eight primers were chosen for further analysis. Six hundred and thirty-two samples, including duplicates/triplicates, of B. pseudomallei isolated from melioidosis patients and the environment were analyzed. Two controls were included in each run of the test samples. All the samples were tested and patterns analyzed by blinded technical staff. Apparently, the method is reproducible. This is indicated by the RAPD patterns of the two controls of between run assay. Interestingly, some RAPD patterns were more prevalent in the clinical isolates than the environmental specimens and vice versa. For example, Q162KKU4-0 and Q162KKU1-0 were found 3. 5 and 3.3 times more often in the clinical specimens (P<0.025). Likewise, Q162KKU1-1 and Q162KKU4-1 were found 18 and 37 times more often in the environment (P<0.0000001). In addition, there was a bias in the distribution of arabinose positive strains and particular RAPD patterns; RAPD patterns of B. pseudomallei that were found frequently in septicemic patients were less likely to be arabinose positive. The data suggest the existence of bacterial genetic differences between the clinical and environmental isolates of B. pseudomallei. Further analysis of the RAPD patterns searching for common polymorphic DNA fragments and systemic comparative genomic analysis of B. pseudomallei in accordance with the clinical data should reveal genetic factors involved in severity and bacterial pathogenesis of B. pseudomallei in melioidosis.
The present study shows the molecular characterization of Fasciola gigantica and F. hepatica isolates collected from cows and sheep, using the random amplified polymorphic DNA fragments-polymerase chain reaction (RAPDs-PCR) technique. Optimal standardization of amplification conditions and thermocyclation were made, using genetic markers. The methodology used compared the genetic pattern between the two species (inter-species) and inside each species (intra-species) between cow and sheep and the amplification fragments were between 135 and 741 base pairs of marker. The results showed genetic variations (polymorphisms) of Fasciola gigantica and F. hepa-tica with amplification fragment based on a 500-400 base pair (bp). Inside each species, there were genetic variations in bovine and ovine and the amplification fragments were between 600 and 400 base pairs (bp). This assay is useful for both individual diagnosis and epidemiological surveys in endemic regions.
Random amplified polymorphic DNA (RAPD) markers were assayed in an attempt to discriminate among five species of Paragonimus. Genomic DNAs of two strains of Paragonimus heterotremus from two provinces in Thailand, Saraburi and Phitsanulok, as well as of P. siamensis, P. harinasutai, P. westermani and P. bangkokensis were extracted and amplified by an arbitrary primer, namely P2 (5-GTTTCGCTCC-3). RAPD patterns showed that those five species were genetically distinct, although they shared genomic DNA to some extent. This primer could also distinguish between two strains of P. heterotremus. The polymorphism observed allowed to construct a relationship dendrogram. The phylogenetic dendrogram showed that the P. heterotremus strains were closest to P. harinasutai, followed by P. siamensis, P. bangkokensis and P. westermani.