Mônica Regina Vendrame Amarante

Haemonchus contortusandHaemonchus placeiare among the major parasites of small ruminants and cattle. Although infection with these nematodes is host-specific, withH. placeipredominating in cattle andH. contortusin sheep, cross-infections are observed in areas where both parasites are sympatric, and hybrid offspring can occur. Therefore, a fast and precise method is required for differentiating the parasites. Identification based on spicule morphometry is the most common technique for differentiatingHaemonchusspecies. However, because these measurements overlap between species, morphological analysis is insufficient for differentiating between helminth species. In this work, we present a reliable, conventional polymerase chain reaction (PCR)-based method that uses two species-specific primer pairs to differentiate betweenH. contortusandH. placeispecimens and their hybrids. Each primer pair produces one single and distinct amplification band for each species, which enables the detection of hybrid specimens. These primer pairs were validated by testing eight different populations ofH. contortus, H. placeiand hybrids.

Molecular and morphological methods were evaluated to distinguish between Haemonchus contortus and Haemonchus placei species. A total of 141 H. contortus and 89 H. placei male adult specimens collected from artificially infected lambs were identified individually by PCR analysis, using a species-specific primer pair. These PCR results were used as gold standard for Haemonchus spp. identification. Haemonchus placei presented higher mean spicule and barb lengths than H. contortus (PH. contortus and one H. placei specimen. The sheath tail length of the third stage larvae (L3), which comprises the distance between the tip of the larval tail and the end of the sheath tail, were measured. Only three of the 485 H. placei larvae (0.619%) had a sheath tail shorter than 85 µm, while only four of the 500 H. contortus larvae (0.8%) presented a sheath tail longer than 85 µm. The results indicated that 6.09% of the male adult specimens would be misclassified based on the discriminate function, while only 0.71% of infective larvae would be misclassified. Therefore, identification of L3 can be used as the first method to indicate the presence of H. placei and/or H. contortus in a population of domestic ruminants.

To better evaluate the usefulness of female Haemonchus specimens for specific identification, we undertook a detailed analysis of the morphology of a collection of worms obtained from cattle and sheep in shared pastures. Based on the results, we also more precisely evaluated the host-specificity of Haemonchus contortus, H. placei and H. similis occurring sympatrically in a farm located in the western region of São Paulo State, Brazil. A synlophe analysis was employed to identify the three species of Haemonchus. In cattle, the predominant species was H. similis (90.9%), followed by H. placei (9.1%). With the exception of one H. placei specimen, only H. contortus was found in sheep. The longest body length was found for H. placei specimens, followed by H. contortus and then H. similis. It was possible to distinguish H. similis females from H. contortus and H. placei on the basis of vulval structure. The synlophe analysis proved to be very useful for identification of H. contortus, H. placei and H. similis in epidemiological studies involving different species of ruminants in the same pastures. The finding that H. placei and H. similis were adapted to cattle and that H. contortus was adapted to sheep also confirmed the high host-specificity of the three nematodes species.

Agricultural ruminants usually harbour mixed infections of gastrointestinal nematodes. A specific diagnosis is important because distinct species can differ significantly in their fecundity and pathogenicity. Haemonchus spp. and Cooperia spp. are the most important gastrointestinal nematodes infecting ruminants in subtropical/tropical environments. In Brazil, C. punctata is more adapted to cattle than sheep. Additionally, C. spatulata appears to be more adapted to cattle, whereas C. curticei is more adapted to sheep. However, infection of sheep with C. punctata is common when cattle and sheep share the same pasture. Although morphological analyses have been widely used to identify nematodes, molecular methods can overcome technical limitations and help improve species-specific diagnoses. Genetic markers in the first and second internal transcribed spacers (ITS-1 and ITS-2, respectively) of nuclear ribosomal DNA (rDNA) have been used successfully to detect helminths. In the present study, the ITS-1 region was analysed and used to design a species-specific oligonucleotide primer pair to identify C. curticei. The polymerase chain reaction (PCR) product was sequenced and showed 97% similarity to C. oncophora partial ITS-1 clones and 99% similarity to the C. curticei sequence JF680982. The specificity of this primer pair was corroborated by the analysis of 17 species of helminths, including C. curticei, C. punctata and C. spatulata. Species-specific diagnosis, which has implications for rapid and reliable identification, can support studies on the biology, ecology and epidemiology of trichostrongylid nematodes in a particular geographical location.

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

In this study, we investigated the characteristics of the infection and subsequent immunity induced by Strongyloides venezuelensis in Lewis rats. Animals were infected with 4000 L3 of S. venezuelensis and number of eggs per gram of faeces indicated an acute phase around day 8 and a recovery phase around day 32 after infection. A strong Th2 polarization during recovery phase was ascertained by a significant increase in IgG1 and IgE compared with that in the acute period. A shift in the cytokine profile confirmed these findings. A predominant production of IFN‐γ during the acute phase was followed by IL‐10 production during recovery. Together these findings show that experimental infection of Lewis rats with S. venezuelensis presents a kinetics of parasite establishment and immunity similar to that described in other models of helminthic infection.

Radiation hybrid maps were constructed for river buffalo and cattle Y chromosomes. A total of 41 cattle-derived Y-chromosome molecular markers were selected and tested with 2 previously described 5,000-rad whole-genome radiation hybrid (RH) panels (river buffalo – BBURH5000 and cattle – BTARH5000) for generation of maps. Among the initial 41 selected markers, a subset of 26 markers generated PCR products suitable for scoring with the BBURH5000 panel. Of these, 19 markers (73%) were distributed in 1 linkage group spanning 341.3 cR. Retention frequencies (RF) for individual markers ranged from 17.8% for SMCY to 56.7% for BTY1, with an average RF of 37.6%. From the selected markers, 37 generated reliable scores using the BTARH5000 panel. The newly constructed BTAY RH map contains 28 markers distributed within 1 linkage group. Twenty-four of these markers had been previously mapped on BTAY using a 7,000-rad cattle-hamster WG-RH panel and 4 markers were mapped for the first time (ZFY, SeqRep, RepSeqS4 and BTY1). The length of the BTAY RH map was estimated to be 602.4 cR. Retention frequencies for individual mapped markers ranged from 10% (INRA126) to 63.3% (SeqRep), with an average RF of 35.3%. RH marker positions along the Y chromosome were compared between BBUY and BTAY, which revealed differences in the order of some of the markers. The BBUY pseudoautosomal region (PAR) is delineated by 3 BTAY PAR markers (MAF45, TGLA325 and UMN2008). These markers are telomeric in both species but are not found in the same order. Here we have demonstrated the effective use of bovine Y chromosome markers for the development of the first BBUY RH map. Likewise, these set of markers can be used for comparative assessment of Y chromosomes in other members of the Bovidae family.

The aim of the present study is to report morphological data from parasitic female, rhabditoid and filarioid larvae, free-living female worms and eggs of Strongyloides ophidiae (Nematoda, Strongyloididae). In addition, a molecular DNA analysis was carried out using a pool of eight S. ophidiae parasitic females. Samples were obtained from the small intestine of Oxyrhopus guibei (Serpentes, Colubridae) collected in the municipality of Lençóis Paulista, State of São Paulo, Brazil. DNA amplification by polymerase chain reaction (PCR) resulted in a 350 bp band for samples containing S. ophidiae and Strongyloides venezuelensis DNA. Strongyloides ophidiae nucleotide sequence analysis showed 98% similarity with Strongyloides procyonis and 97% with Strongyloides cebus, Strongyloides stercoralis, Strongyloides fuelleborni and Strongyloides sp. from snakes.