Comparative evaluation of different molecular methods for DNA extraction from individual Teladorsagia circumcincta nematodes.

BACKGROUND: The purpose of this study was to develop a reliable DNA extraction protocol to use on individual Teladorsagia circumcincta nematode specimens to produce high quality DNA for genome sequencing and phylogenetic analysis. Pooled samples have been critical in providing the groundwork for T. circumcincta genome construction, but there is currently no standard method for extracting high-quality DNA from individual nematodes. 11 extraction kits were compared based on DNA quality, yield, and processing time. RESULTS: 11 extraction protocols were compared, and the concentration and purity of the extracted DNA was quantified. Median DNA concentration among all methods measured on NanoDrop 2000™ ranged between 0.45-11.5 ng/µL, and on Qubit™ ranged between undetectable - 0.962 ng/µL. Median A260/280 ranged between 0.505-3.925, and median A260/230 ranged - 0.005 - 1.545. Larval exsheathment to remove the nematode cuticle negatively impacted DNA concentration and purity. CONCLUSIONS: A Schistosoma sp. DNA extraction method was determined as most suitable for individual T. circumcincta nematode specimens due to its resulting DNA concentration, purity, and relatively fast processing time.

Genetic analyses of the parasitic nematode, Parelaphostrongylus tenuis, in Missouri and Kentucky reveal unexpected levels of diversity and population differentiation.

Wildlife translocations, which involve the introduction of naive hosts into new environments with novel pathogens, invariably pose an increased risk of disease. The meningeal worm Parelaphostrongylus tenuis is a nematode parasite of the white-tailed deer (Odocoileus virginianus), which serves as its primary host and rarely suffers adverse effects from infection. Attempts to restore elk (Cervus canadensis) to the eastern US have been hampered by disease caused by this parasite. Using DNA sequence data from mitochondrial and nuclear genes, we examined the hypothesis that elk translocated within the eastern US could be exposed to novel genetic variants of P. tenuis by detailing the genetic structure among P. tenuis taken from white-tailed deer and elk at a source (Kentucky) and a release site (Missouri). We found high levels of diversity at both mitochondrial and nuclear DNA in Missouri and Kentucky and a high level of differentiation between states. Our results highlight the importance of considering the potential for increased disease risk from exposure to novel strains of parasites in the decision-making process of a reintroduction or restoration.

Range expansion of muskox lungworms track rapid arctic warming: implications for geographic colonization under climate forcing.

Rapid climate warming in the Arctic results in multifaceted disruption of biodiversity, faunal structure, and ecosystem health. Hypotheses have linked range expansion and emergence of parasites and diseases to accelerating warming globally but empirical studies demonstrating causality are rare. Using historical data and recent surveys as baselines, we explored climatological drivers for Arctic warming as determinants of range expansion for two temperature-dependent lungworms, Umingmakstrongylus pallikuukensis and Varestrongylus eleguneniensis, of muskoxen (Ovibos moschatus) and caribou (Rangifer tarandus), in the Canadian Arctic Archipelago from 1980 through 2017. Our field data shows a substantial northward shift of the northern edge of the range for both parasites and increased abundance across the expanded ranges during the last decade. Mechanistic models parameterized with parasites' thermal requirements demonstrated that geographical colonization tracked spatial expansion of permissive environments, with a temporal lag. Subtle differences in life histories, thermal requirements of closely related parasites, climate oscillations and shifting thermal balances across environments influence faunal assembly and biodiversity. Our findings support that persistence of host-parasite assemblages reflects capacities of parasites to utilize host and environmental resources in an ecological arena of fluctuating opportunity (alternating trends in exploration and exploitation) driving shifting boundaries for distribution across spatial and temporal scales.

First report of Davaneidae and Strongylida parasitizing Ramphocelus carbo (Aves: Passeriformes: Thraupidae) in the southwest of the Brazilian Amazon region.

In Brazil, species of the genus Ramphocelus with the presence of various endoparasites have already been reported. Coccidia have been the parasites most frequently found. All species of this genus have similar habitats and ecological niches, which makes parasite transmission easy. The aim of this study was to diagnose the presence of endoparasites in fecal samples from specimens of Ramphocelus carbo that were caught in the Cazumbá-Iracema Extractive Reserve (Cazumbá Resex), in the State of Acre. The specimens (n = 30) were caught in mist nets arranged in different ecosystems of the Reserve. After identification, the bird specimens were placed in cloth bags for a maximum of 30 minutes to collect feces. Among the 22 samples collected, 63.63% (n = 14) were positive for endoparasites. The coccidia were the parasites most frequently. Helminths belonging to Ascaridiidae (Nematoda), Strongylida (Nematoda) and Davaneidae (Cestoda) were recorded for the first time in R. carbo in the State of Acre. Parasites belonging to Strongylida and Davaneidae were recorded for the first time in a species of Ramphocelus. These findings add information on the parasitic fauna of wild birds, since in the Amazon region there are few studies on this subject.

Gastrointestinal helminth parasites of the common wallaroo or euro, Osphranter robustus (Gould) (Marsupialia: Macropodidae) from Australia.

The gastrointestinal helminth parasites of 170 common wallaroos or euros, Osphranter robustus (Gould), collected from all mainland states in which the species occurs as well as the Northern Territory, are presented, including previously published data. A total of 65 species of helminths were encountered, including four species of anoplocephalid cestodes found in the bile ducts and small intestine, and 61 species of strongylid nematodes, all but two of which occurring in the stomach, and with the remainder occurring in the terminal ileum, caecum and colon. Among the mainland subspecies of O. robustus, 52 species of helminths were encountered in O. r. robustus, compared with 30 species in O. r. woodwardi and 35 species in O. r. erubescens. Of the parasite species encountered, only 17 were specific to O. robustus, the remaining being shared with sympatric host species. Host-specific species or species occurring in O. robustus at a high prevalence can be classified as follows: widely distributed; restricted to northern Australia; restricted to the northern wallaroo, O. r. woodwardi; found only in the euro, O. r. erubescens; found essentially along the eastern coast of Australia, primarily in O. r. robustus; and species with highly limited regional distributions. The data currently available suggest that the acquisition of a significant number of parasites is due to co-grazing with other macropodids, while subspeciation in wallaroos as well as climatic variables may have influenced the diversification of the parasite fauna.

Endoparasites Determined by Fecal Examination in Sheep in Erzurum Province

Objective: The aim of the current study was to determine the presence and prevalence of Eimeria and helminth species in sheep raised in Erzurum province by using fecal examination. Methods: Faecal samples were collected from a total of 784 sheep raised in Aziziye, Yakutiye and Palandöken districts between February-March 2019. The samples were examined by Fulleborn's flotation, Benedect sedimentation, and Baermann-Wetzel methods. Results: Eimeria spp. and helminths were found in 49.36% (387/784) and 74.11% (581/784) of the samples, respectively. Identified Eimeria species were as follows: E. parva (59.68%), E. ovina (51.67%), E. faurei (47.80%), E. ahsata (39.27%), E. granulosa (36.62%), E. punctata (28.42%), E. pallida (26.09%), E. ovinoidalis (18.34%), E. crandallis (16.79%), E. intricata (15.76%), E. weybridgensis (11.36%) and E. marsica (6.20%). Helminth species identified at genus/species level were Dicrocoelium spp. (33.91%), Fasciola spp. (5.68%), Paramphistomum spp. (2.58%), Moniezia spp. (5.85%), Trichostrongylid type egg (49.05%), Marshallagia spp. (38.73%), Nematodirus spp. (20.98%), Trichuris spp. (14.46%), Protostrongylus spp. (18.42%), Dictyocaulus filaria (2.41%) and Muellerius capillaris (1.38%). Conclusion: Parasitic diseases cause important economic losses in livestock industry. In following years, it is aimed to plan prevention and control strategies for the parasites detected in this area in line with the data of this study and to share this data with the animal breeders.

Survey of lungworm infection of domestic cats in Hungary.

From 61 settlements of 12 Hungarian counties, 303 domestic cats were included in this survey. Between autumn 2016 and spring 2018, fresh faecal samples were randomly collected and examined by flotation and by the Baermann-Wetzel method for the presence of lungworm infection. No eggs of Eucoleus aerophilus were detected. Morphological identification of first instar larvae (L1) was also carried out. In the faeces of 60 cats (19.8%) from 17 settlements and Budapest, L1 of Aelurostrongylus abstrusus were found. More than half of the cats were from the western part of the country. The average number of larvae per gram of faeces was 190.2 ± 304.88. These results are in line with the former findings on the prevalence of aelurostrongylosis of domestic cats in Hungary. In addition, Oslerus rostratus was also found for the first time in the faecal samples of three cats from the eastern part of the country, infected also with Ae. abstrusus. The average age (2.51 ± 1.26 years) of infected cats indicates that lungworm infection is more common among younger cats. No relationship was found between the lung-worm infection and the sex of cats. Non-neutered cats had a significantly higher proportion of lungworm infections. Two-thirds of the infected cats were apparently healthy, and only 19 individuals showed clinical signs of respiratory disorders.

Development of amplicon sequencing for the analysis of benzimidazole resistance allele frequencies in field populations of gastrointestinal nematodes.

Anthelmintic resistant gastrointestinal helminths have become a major cause of poor health in sheep and goats. Sensitive and specific molecular markers are needed to monitor the genotypic frequency of resistance in field parasite populations. Gastrointestinal nematode resistance to benzimidazole is caused by a mutation in one of three positions within the isotype 1 ß-tubulin gene. In the absence of markers for resistance to the other broad spectrum anthelmintic classes, these provide a relevant study example. Determination of the prevalence of these single nucleotide polymorphisms in field nematode populations can be impractical using conventional molecular methods to examine individual parasites; which can be laborious and lack sensitivity in determining low levels of resistance in parasite populations. Here, we report the development of a novel method based on an Illumina MiSeq deep amplicon sequencing platform to sequence the isotype 1 ß-tubulin locus of the small ruminant gastrointestinal nematode, Teladorsagia circumcincta, and determine the frequency of the benzimidazole resistance mutations. We validated the method by assessing sequence representation bias, comparing the results of Illumina MiSeq and pyrosequencing, and applying the method to populations containing known proportions of resistant and susceptible larvae. We applied the method to field samples collected from ewes and lambs on over a period of one year on three farms, each highlighting different aspects of sheep management and approaches to parasite control. The results show opportunities to build hypotheses with reference to selection pressures leading to differences in resistance allele frequencies between sampling dates, farms and ewes or lambs, and to consider the impact of their genetic fixation or otherwise. This study provides proof of concept of a practical, accurate, sensitive and scalable method to determine frequency of anthelmintic resistance mutations in gastrointestinal nematodes in field studies and as a management tool for livestock farmers.

Impact of a single treatment of injectable doramectin on weight gain post weaning in beef heifers and steers in central Queensland, Australia.

OBJECTIVES: To demonstrate the impact of a single drench with a label dose of injectable doramectin subsequent to weaning on the growth and performance of heifers and steers in central Queensland beef herds. METHODS: Three studies were undertaken on recently-weaned Bos indicus-cross beef calves with ≥ 75% B. indicus content on two farms in central Queensland, just north of the Tropic of Capricorn. Farm 1 was located 50 km north and Farm 2 75 km north-west of Rockhampton. In each study, half of a group of recently-weaned beef calves were treated by random allocation with 0.2 mg/kg of injectable doramectin, and the remainder acting as untreated controls. Study 1 (Farm 1) enrolled 250 heifers, while studies 2 and 3 (Farm 2) both enrolled 200 steers and 200 heifers. The farms involved did not historically use macrocyclic lactone-based drenches on their cattle. There were varying periods of follow-up, with treated and control cattle pastured as one group throughout the study period. Worm burdens were monitored using standard faecal egg counts and larval differentiation procedures. In all studies, the worm genera present were a mix of Cooperia spp., Haemonchus spp. and Oesophagostomum spp. RESULTS: In study 1, conducted on Farm 1 beginning 9 July 2012, doramectin-treated cattle gained an average of 0.27 kg/day while control cattle gained 0.19 kg/day over a monitoring period of 121 days (P < 0.0001). In study 2, conducted on Farm 2 beginning 28 July 2015, doramectin-treated cattle gained an average of 0.15 kg/day versus 0.145 kg/day in the control group (P = 0.44) over a 231-day study period. In study 3, conducted on Farm 2 beginning 4 August 2016, doramectin-treated steers and heifers gained an average of 0.431 and 0.402 kg/day versus 0.342 and 0.311 kg/day in the control group, respectively, over the first 91 days of the study (P < 0.0001 in both cases). The differences in average daily gain (ADG) in subsequent time periods were not statistically significant for steers or heifers. However, overall differences in ADG from day 0 remained statistically significant out to day 258, when the study ended for the heifers. By day 594, when the study ended for the steers, the difference in ADG was no longer significant. CONCLUSION: Treatment with injectable doramectin soon after weaning resulted in improved weight gain in the 3 months after weaning in two of the three studies.

Gastrointestinal parasites in reindeer (Rangifer tarandus tarandus) calves from Fennoscandia: An epidemiological study.

Reindeer (Rangifer tarandus tarandus) host numerous parasites. Although there is a general knowledge about parasite diversity in reindeer, detailed baseline information about parasitic infections is limited. Detailed knowledge of parasite prevalence and diversity provide a pathway for more targeted parasite control, an increasing need expected in the future. The main aim of our cross-sectional study was to estimate the prevalence of gastrointestinal parasites in semidomesticated reindeer calves. The 480 reindeer calves included in our study were aged 6-7 months, originated from 9 reindeer herding cooperatives in Finland and 1 in Norway, and were slaughtered during September-November 2015 in 10 reindeer slaughterhouses. All the reindeer calves passed meat inspection, and the detected parasitic infections were subclinical. As the reindeer included in this study were young animals intended for slaughter, they had never been administrated any antiparasitic treatment. Assessments of gastrointestinal parasitism among these reindeer calves were based on fecal examination and morphological identification of coccidian oocysts or helminth eggs. Individual fecal samples collected from the rectum of each of the reindeer were examined using a modified McMaster method. Most (78.3%) of the reindeer calves had eggs or oocysts of at least one parasite species in their feces, and more than half (53.5%) had a mixed infection. Strongylid eggs were detected in 75.6%, Eimeria sp. oocysts in 50.6%, Moniezia sp. eggs in 28.1%, Nematodirus sp. eggs in 22.1%, Capillaria sp. eggs in 9.4%, and Trichuris sp. eggs in 0.6% of the samples. The prevalence of gastrointestinal parasites was similar or higher relative to previous estimates from the region; the proportion of reindeer calves shedding strongylid eggs and the proportion of reindeer calves shedding Moniezia sp. eggs had increased. Prevalence varied by geographical region, which may reflect different herding practices or environmental parameters. Higher reindeer density was a risk factor for testing positive for Eimeria sp. oocysts, and the odds of testing positive for Nematodirus sp. eggs were higher if a peroral route was used for antiparasitic treatment in the reindeer herding cooperative. The mean proportion of reindeer estimated to receive antiparasitic treatment in Finland was 86% in 2004-2005 and 91% in 2014-2015. During the historical time frames of current management practices, this routine annual antiparasitic treatment of breeding reindeer has not decreased the prevalence of gastrointestinal parasites in reindeer calves, which can be seen as sentinels or indicators of the infection pressure.

Larval survival of Aelurostrongylus abstrusus lungworm in cat litters.

OBJECTIVES: The dehydrating power of cat litters may reduce the vitality of first-stage larvae (L1s) of feline lungworms, limiting copromicroscopical diagnosis. This study assessed the effect of the most commonly used cat litters on Aelurostrongylus abstrusus L1 survival. METHODS: Four types of cat litters were used: clumpling clay (group A); non-clumpling clay (group B); silica crystals (group C); and biodegradable (group D). A control group without litter (group E) was also included. On study day 0 (T0), L1s were obtained by the Baermann-Wetzel technique from the faeces of a naturally infected cat and ~100 larvae were injected in each of the 20 lungworm larvae-free faecal samples (~2 g each). Thereafter, four faecal samples per group were transferred into plastic cups containing the four different types of cat litters, or into empty cups (group E). The survival of L1s was assessed in each group after 3 (T3), 6 (T6), 12 (T12) and 24 (T24) h, using the Baermann-Wetzel technique. RESULTS: A decreasing trend of L1 survival was observed in all groups, with highest significant values at T0 compared with T3, T6, T12 and T24 (P <0.001). However, at T24, a significantly higher number (P <0.05) of L1s was extracted from faeces of the control group compared with the four groups with cat litters. CONCLUSIONS AND RELEVANCE: This study demonstrates how the survival of A abstrusus L1s, and therefore diagnosis, may be negatively influenced by the litter. The effect is time dependent, with a reduction in the number of vital larvae according to the type of litter, over time. False-negative results may be obtained, especially in cases of low parasitic load or when the sample is collected many hours after the emission.

Effects of homogenizing methods on accuracy and precision of equine strongylid egg counts.

Recommendations for control of equine strongylid parasites are based on regular determination of fecal egg counts to identify high strongylid shedders and to evaluate treatment efficacy. The McMaster technique has long been used as the standard egg counting technique in equine veterinary practice in most parts of the world, but recent work has found the Mini-FLOTAC technique to perform with significantly better accuracy and precision. The Mini-FLOTAC system comes with a homogenizing device, termed the Fill-FLOTAC, and it has been hypothesized that this device might have a significant impact on accuracy and precision. The aim of the present study was to investigate the impact of the Fill-FLOTAC homogenizer in comparison with the classical McMaster approach, where samples are suspended in flotation medium by stirring with tongue depressor in a plastic cup. The study compared the McMaster and Mini-FLOTAC techniques, but also included cross-over versions where the Fill-FLOTAC was used with the McMaster chamber, and the tongue depressor and plastic cup homogenizing method was used with the Mini-FLOTAC counting disc. Fecal samples were collected from horses naturally infected with mixed strongylid species. Five samples were included from each of the following egg count levels: 0-500, 501-1000, and >1000 eggs per gram (EPG). Each sample was then analyzed with all four set-ups with three subsamples collected from the same suspension, and three repeated counts determined on each subsample. Both the Fill-FLOTAC homogenizer (p = 0.0098) and the McMaster counting chamber (p = 0.0298) were significantly associated with higher strongylid egg counts, whereas the Mini-FLOTAC chamber was associated with a lower coefficient of variation (p < 0.0001). Precision, however, was not associated with homogenization method (p = 0.9341). Taken together, this study suggests that while the homogenizing method has a positive effect on egg count accuracy, the counting chamber appears to primarily affect precision.

Molecular and morphological characterisation of Pharyngostrongylus kappa Mawson, 1965 (Nematoda: Strongylida) from Australian macropodid marsupials with the description of a new species, P. patriciae n. sp.

BACKGROUND: Pharyngostrongylus kappa Mawson, 1965 is a nematode (Strongyloidea: Cloacininae), endemic to the sacculated forestomachs of Australian macropodid marsupials (kangaroos and wallaroos). A recent study revealed genetic variation within the internal transcribed spacer region of the nuclear ribosomal DNA among P. kappa specimens collected from Macropus giganteus Shaw and Osphranter robustus (Gould). This study aimed to characterise the genetic and morphological diversity within P. kappa from four macropodid host species, including M. giganteus, O. robustus, O. antilopinus (Gould) and O. bernardus (Rothschild). METHODS: Specimens of P. kappa from M. giganteus and Osphranter spp. from various localities across Australia were examined. The first and second internal transcribed spacers (ITS1 and ITS2, respectively) were amplified using polymerase chain reaction and sequenced. Phylogenetic methods were used to determine the interspecific diversification within P. kappa and its evolutionary relationship with other congeners. RESULTS: Morphological examination revealed that P. kappa from M. giganteus, the type-host, can be distinguished from those in Osphranter spp. by the greater length and number of striations on the buccal capsules. DNA sequences showed that P. kappa from M. giganteus was genetically distinct from that in Osphranter spp., thereby supporting the morphological findings. Based on these finding, a new species from Osphranter spp., Pharyngostrongylus patriciae n. sp., is described. CONCLUSION: Pharyngostrongylus patriciae n. sp. from Osphranter spp. is distinguished from P. kappa based on molecular and morphological evidence. The study highlights the importance of combining molecular and morphological techniques for advancing the nematode taxonomy. Although ITS genetic markers have proven to be effective for molecular prospecting as claimed in previous studies, future utilisation of mitochondrial DNA to validate ITS data could further elucidate the extent of speciation among macropodid nematodes.

Troglostrongylus brevior: a feline lungworm of paediatric concern.

The immature immune system of cats in their paediatric age (i.e., first six months of life) favours the establishment of infectious respiratory tract diseases mainly caused by well recognized viruses and bacteria species. Conversely, lungworm infections are less investigated during respiratory disorders in kittens. In the last decade, Troglostrongylus brevior has been found affecting the respiratory tract of cats, along with the better-known Aelurostrongylus abstrusus. Given the scant data available on the epidemiology of feline troglostrongylosis, faecal samples from 575 domestic animals living in three Italian municipalities (i.e., Bari, Messina and Siena) were screened for lungworm infection by Baermann and molecular tecniques. Animals were grouped according to their age as ≤6 months (i.e., paediatric patients), 6-24 months, or >24 months. Paediatric cats were further sub-divided in infant (2-6 weeks), weanling (6-12 weeks) and juvenile (3-6 months). Of the 575 animals tested, 241 (42.0%) were younger than 6 months, 188 (33%) were 6-24-month-old and 146 (25%) were older than 24 months. Lungworm infection was diagnosed in 84 (14.6%) of the examined cats. Of the 49 (20.3%) paediatric animals positive for lungworms, T. brevior was the nematode species most frequently diagnosed (n = 44; 89.8%), followed by A. abstrusus (n = 2; 4.1%), and three cats (6.1%) were co-infected by both species. The diagnosis of T. brevior infection was significantly associated with animals aging ≤6 months (18.2%; P < 0.01) than elder cats. Indeed, the prevalence of infection by T. brevior decreased in animals aging 6-24 months (3.2%) being not detected in cats older than two years. Results of this study indicate that paediatric cats are at higher risk of T. brevior infection compared to adults (P < 0.01).

Molecular identification of parasitic nematodes (Nematoda: Strongylida) in feces of wild ruminants from Tunisia.

In Tunisia and other North African countries, there is a lack of knowledge about parasite biodiversity within threatened wild ruminants and there are not any studies on their gastrointestinal nematodes. Thus the aim of this study was to identify gastrointestinal fauna in the faecal samples of Tunisian wild ruminants. A total of 262 faecal samples were collected from domestic sheep and goat, and wild ruminants (Addax, Barbary sheep, Barbary red deer, Dorcas gazelle, Slender-horned gazelle and Scimitar-horned Oryx) living in protected areas. Samples were examined with floatation (saturated sodium chloride solution), polymerase chain reaction and sequencing of the second internal transcribed spacer region of the rDNA. Microscopic analysis allowed the identification of only Nematodirus genus or molecular tools allowed a first identification of five gastrointestinal nematode species in North African wild ruminants: Chabertia ovina (1.6%), Camelostrongylus mentulatus (1.6%), Marshallagia marshalli (4.7%), Nematodirus helvetianus (62.5%) and Nematodirus spathiger (29.7%). This study reported the first records of C. mentulatus and M. marshalli in Addax and of M. marshalli in Dorcas gazelle and it was the first reported record of N. helvetianus and M. marshalli in Tunisia.

Apex Predators, Rockshelters, and Zoonoses in the Patagonian Holocene.

Carnivorous mammals are a trophic guild with an important role in the dissemination of parasite infective stages (larvae, eggs, cysts, and oocysts). In the present study, new samples of coprolites attributed to carnivorous mammals, obtained from 2 archaeological caves, were analyzed for the presence of parasites with the aim to increase the knowledge about parasites in rockshelters that could have spread to humans and other mammals. To this purpose, fragments of 3 coprolites from Cerro Casa de Piedra, cave 5 and cave 7, were examined. Coprolites were rehydrated in aqueous trisodium phosphate and processed by spontaneous sedimentation. High parasite richness was observed and new parasite species for archaeological contexts were found. The parasitological findings in Puma concolor coprolites associated with caves suggest the importance of these carnivores in the dissemination of parasites in areas with high re-use of space and steady conditions of temperature, humidity, and radiation.

Nematode Species Identification-Current Status, Challenges and Future Perspectives for Cyathostomins.

Human and animal health is globally affected by a variety of parasitic helminths. The impact of co-infections and development of anthelmintic resistance requires improved diagnostic tools, especially for parasitic nematodes e.g., to identify resistant species or attribute pathological effects to individual species or particular species combinations. In horses, co-infection with cyathostomins is rather a rule than an exception with typically 5 to 15 species (out of more than 40 described) per individual host. In cyathostomins, reliable morphological species differentiation is currently limited to adults and requires highly specialized expertize while precise morphological identification of eggs and early stage larvae is impossible. The situation is further complicated by a questionable validity of some cyathostomins while others might actually represent cryptic species complexes. Several molecular methods using different target sequences were established to overcome these limitations. For adult worms, PCR followed by sequencing of mitochondrial genes or external or internal ribosomal RNA spacers is suitable to genetically confirm morphological identifications. The most commonly used method to differentiate eggs or larvae is the reverse-line-blot hybridization assay. However, both methods suffer from the fact that target sequences are not available for many species or even that GenBank® entries are unreliable regarding the cyathostomin species. Recent advances in proteomic tools for identification of metazoans including insects and nematodes of the genus Trichinella will be evaluated for suitability to diagnose cyathostomins. Future research should focus on the comparative analysis of morphological, molecular and proteomic data from the same cyathostomin specimen to optimize tools for species-specific identification.

Infection levels of protostrongylid nematodes in definitive caprine and intermediate gastropod hosts from Uzbekistan.

Morphological analysis of lungworms collected among Caprinae from Uzbekistan resulted in the identification of four species of Protostrongylidae: Protostrongylus rufescens, Protostrongylus hobmaieri, Spiculocaulus leuckarti and Cystocaulus ocreatus. The following species were recorded as definitive hosts: Ovis aries, Ovis ammon, Ovis vignei, Capra hircus, Capra falconeri and Capra sibirica. The prevalence of P. rufescens reached 45.3%, followed by S. leuckarti and C. ocreatus with 31.7% and P. hobmaieri with 16.9%. The sex ratio ranged between 1:3.1 and 1:6.2, with P. hobmaieri showing the strongest predominance of females over males. The prevalence of infection of small ruminants with protostrongylid nematodes increased with the age of the hosts. Protostrongyles use terrestrial gastropods as intermediate hosts, and infective larvae were found in the species Vallonia costata, Gibbulinopsis signata, Pupilla muscorum, Pseudonapaeus albiplicata, Pseudonapaeus sogdiana, Leucozonella ferghanica, Xeropicta candacharica, Candaharia levanderi and Macrochlamys sogdiana. Xeropicta candacharica was the most abundant gastropod and had the highest prevalence of infection with protostrongylids. Adult X. candacharica had a significantly higher infection intensity than juveniles. The epidemiology of protostrongylid infections is dynamic and subject to considerable changes. Further characterization of the interaction of protostrongylid parasites with their terrestrial gastropods as intermediate hosts and Caprinae as definitive hosts is required to understand these processes and to monitor the effects of changing ecological contexts.

The occurrence of the strongylid nematodes Kalicephalus brachycephalus, K. bungari and K. indicus in snake species from Hunan Province, China.

Kalicephalus is a genus of strongylid nematodes infecting snakes and causing serious diseases and even death when it is complicated with secondary bacterial infections. The infection of snakes with Kalicephalus has been reported in many countries in the world. However, little information is available on the prevalence of Kalicephalus in snakes in China. In the present study, the prevalence of Kalicephalus in snakes was investigated. The worms were examined, counted and identified to species according to existing keys and descriptions. Three species of Kalicephalus, namely K. indicus, K. bungari and K. brachycephalus, were found in six species of snakes (Elaphe carinata, Zaocys dhumnade, Naja najaatra, Elaphe taeniura, Bungarus multicinctus and Dinodon rufozonatum). The total prevalence of Kalicephalus in snakes in Hunan Province was 39.7%. The most common species was K. indicus, with the highest prevalence 72.8%, followed by K. bungari (24.0%). The prevalence of K. brachycephalus was 0.9%. This is the first report on the prevalence of Kalicephalus species in snakes in China, and the findings have important implications for the control of Kalicephalus infections in snakes in China.