Migratory route of Strongyloides venezuelensis in Lewis rats: comparison of histological analyses and PCR.

Strongyloides venezuelensis is a parasitic nematode that has been used as a model to study human and animal strongyloidiasis. In this study, we compared the sensitivity between traditional methodologies and PCR assay to characterize the dynamics of S. venezuelensis infection and its migration route in Lewis rats subcutaneously infected with 4000 L3. The dynamics of the infection was determined by counting the number of eggs and by detecting parasite deoxyribonucleic acid in faeces samples. Both techniques similarly detected the infection at day 6 after larvae inoculation. However, PCR performed with the genus primer showed higher sensitivity during the recovery phase. Histological analysis and PCR assay were then used to follow parasite tissue migration. S. venezuelensis migration route included the muscular fibers below the skin, the pulmonary alveoli and the small intestine vilosities. The sensitivity of these two techniques to detect parasite's presence in these tissues was statistically similar.

Faecal examination and PCR to detect Strongyloides venezuelensis in experimentally infected Lewis rats.

More sensitive methodologies are necessary to improve strongyloidiasis diagnosis. This study compared the sensitivities of the McMaster modified technique and polymerase chain reaction (PCR) assays, both performed in faecal samples. Lewis rats were subcutaneously infected with 4,000, 400 or 40 infective third-stage larvae, considered as high, moderate or low infection, respectively. Seven days later, they were euthanized to count adult nematodes recovered from the small intestine. Stool samples were used to count the number of eggs per gram (EPG) of faeces and to detect parasite DNA by PCR performed with a species and a genus primer pair. The sensitivity of these assays depended upon parasite burden and the primer specificity. All assays presented 100% sensitivity at the highest parasite load. In the moderate infection, EPG and PCR with the genus primer maintained 100% specificity, whereas PCR sensitivity with the species primer decreased to 77.7%. In low infection, the sensitivity was 60% for EPG, 0% for PCR with the species primer and 90% for PCR done with the genus primer. Together, these results suggest that PCR with a genus primer can be a very sensitive methodology to detect Strongyloides venezuelensisin faeces of Lewis rats infected with very low parasite burden.

Is there competition between Haemonchus contortus and Haemonchus placei in a pasture grazed by only sheep?

This study aimed to evaluate the dynamics of Haemonchus contortus and Haemonchus placei infections and hybridization between these species in grazing sheep without contact with cattle. On January 14, 2014, sixteen young sheep were infected with 4000 infective H. placei third-stage larvae L3; 11 days later, another group n = 16 was infected with 4000 H. contortus L3. The establishment rates of H. contortus and H. placei L3 were, on average, 61.6 % and 56.8 %, respectively, in the permanent sheep. After the establishment of patent infections, all permanent sheep were allocated together in the same clean pasture where they grazed for the next 12 months. Euthanasia of a sample of the permanent sheep was performed every three months: in May, August, November and February. Two weeks before the sheep were removed for euthanasia, 2 worm-free tracer sheep were introduced to the pasture to evaluate the larval population in the field. The tracer sheep grazed alongside the permanent sheep for 2 weeks. Then, they were housed indoors for 20 days; at the end of this period, they were euthanized. Parasites were recovered from the permanent and tracer sheep and identified using morphological and molecular techniques. A total of 432 worms (from permanent and tracer animals) were analyzed by PCR using species-specific primer pairs. Of these specimens, only two (0.46 %) male worms were identified as hybrids: one was recovered from a permanent animal euthanized in August and the other from a tracer sheep that grazed in May. The last detection of adult H. placei worms occurred in sheep euthanized in May (approximately 3.5 months after the beginning of the grazing period). The morphological evaluation of the L3 produced in fecal cultures showed that H. placei were progressively replaced by H. contortus populations starting in March. The last trace of H. placei L3 was found in August, when a small percentage (0.5 %) of infective larvae with H. placei morphology was identified in a fecal culture. In conclusion, hybridization between H. contortus and H. placei can occur in the field during coinfection. It was demonstrated that H. placei established successfully in artificially infected worm-free sheep; however, with concomitant natural reinfection with H. contortus, the H. placei population showed a rapid decrease and was eliminated within a few months in an environment without cattle.

A panel of microsatellite markers to discriminate and study interactions between Haemonchus contortus and Haemonchus placei.

Haemonchus contortus and Haemonchus placei are two closely related economically important parasites of ruminants. Their close morphological similarity, common occurrence as co-infections and ability to hybridize makes definitive diagnosis and epidemiological studies in field populations challenging. In this paper, we describe the development of a panel of microsatellite markers that can be used to discriminate and study the genetics of these two parasite species in co-infections and mixed field populations. We have identified two additional microsatellites (Hp52 and Hp53), in addition to three previously reported microsatellites (Hcms3561, Hcms53265 and Hcms36) that have a discrete set of alleles between the two species. Multilocus genotyping of worms with this 5 marker panel from 3 geographically diverse H. placei isolates and 4 geographically diverse H. contortus populations allows unambiguous species assignment of individual worms. This panel of markers should provide a valuable resource in studying the biology and epidemiology of these important ruminant parasite species in the field.

Immune response to Haemonchus contortus and Haemonchus placei in sheep and its role on parasite specificity.

Two trials were conducted to determine the prepatent and the patent period of Haemonchus contortus and Haemonchus placei in Santa Ines crossbred sheep and to determine whether serial infections with both species confer protection against homologous or heterologous challenge. To evaluate the prepatent and patent periods of infection, five lambs received a single infection with 4000 H. contortus-infective larvae (L3), and another five received a single infection with 4000 H. placei L3. H. contortus presented patency earlier than H. placei. Animals infected with both species shed a large number of eggs in the faeces for several months with the highest counts, with means higher than 3000 eggs per gram of faeces (EPG) between 24 and 106 days and between 38 and 73 days post infection with H. contortus and H. placei, respectively. H. contortus eggs were detected in the faeces for a minimum of 302 days and a maximum of 538 days post infection, while the H. placei patent period lasted from 288 to 364 days. In the second trial, one group of lambs (n=12) was serially infected 12 times (three times per week for four weeks) with 500 L3 of H. placei and then challenged with either H. placei (n=6) or with H. contortus (n=6). The lambs in the second group (n=12) were serially infected 12 times with 500 L3 of H. contortus and then challenged with H. contortus (n=6) or with H. placei (n=6), and a third group of lambs was single challenged with H. placei (n=6), H. contortus (n=6), or remained uninfected throughout the trial period (control group, n=6). Animals serially infected with H. placei and then challenged with the same species presented the most intense immune response with the highest levels of anti-parasitic immunoglobulin and number of inflammatory cells in the abomasal mucosa. As a result, this group had the lowest rate of parasite establishment (2.68% of the 4000 L3 given), but this phenomenon did not occur in animals single challenged with H. placei, in which the rate of establishment was relatively high (25.3%), confirming that the protective immune response to H. placei develops only when animals are repeatedly infected with this species. However, when the animals were previously serially infected with H. placei and then challenged with H. contortus, no evidence of significant protection was observed (establishment of 19.18%). The results of the trials showed an important role played by the immune response on parasite-host specificity.

Genetic basis of the resistance to Strongyloides venezuelensis (Nematoda, Rhabdiasidae) infection in mice (Mus musculus)

We investigated the resistance to Strongyloides venezuelensis primary infection of mice strains NIH (resistant) and C57BL/6 (susceptible) and the F1 and F2 offspring of crosses between these strains. The mice were infected with 2000 larvae and seven days later were sacrificed for parasite recovery and counting. There was no statistically significant (p > 0.05) sex effect on resistance. The F1 mice showed an intermediate mean number of parasites as compared to the parental NIH and C57BL6 strains. Out of 400 F2 mice, the 10 percent most resistant mice were infected with 21 to 97 parasites, while the 10 percent most susceptible mice were infected with 1027 to 1433 parasites. We also found that F2 mice with black fur (n = 72), the same color as the C57BL/6 susceptible parental strain, were more susceptible than white (n = 104) or gray furred (n = 224) mice. It is conceivable that some genes determining coat color are located on the same chromosome as where genes controlling helminth resistance.