Strongylid Nematodes Research Articles

Characterization of a Caenorhabditis elegans glc seven-like phosphatase ( gsp) orthologue from Haemonchus contortus (Nematoda)

A full-length complementary DNA (cDNA; designated Hc-stp-1) encoding a serine/threonine phosphatase ( Hc-STP-1) was isolated from Haemonchus contortus, a strongylid nematode parasite of small ruminants. Hc-stp-1 was shown to be transcribed in males of both adults and fourth-stage larvae, but not in females, early larval stages or eggs. The full-length gene (2854 bp) contained ten exons and nine introns, and encoded a cDNA of 951 bp. Comparisons of the conceptually translated protein (316 amino acids, estimated at ∼35 kDa) with serine/threonine phosphatases (STPs) from other organisms revealed the presence of the conserved motif LRGNHE. Structural analysis, by comparative modelling, confirmed strict conservation of residues and features involved in catalytic activity, and variation in the ligand-binding interface. Phylogenetic analysis of amino acid sequence data revealed that Hc-STP-1 clustered with STPs from other nematodes (including Caenorhabditis elegans, Trichostrongylus vitrinus, Oesophagostomum dentatum, Ascaris suum and Brugia malayi) to the exclusion of STPs from other organisms. The protein was inferred to be most closely related to the PP1 class of STPs of C. elegans, within a group containing STPs encoded, amongst others, by the genes gsp-3 and gsp-4 in this free-living nematode. The functions of proteins GSP-3 and GSP-4 are known to be central to spermatogenesis and other male-specific processes in C. elegans. The findings from the present and previous studies support the proposal that Hc-stp-1 and its product play a significant role in reproductive and/or developmental processes in maturing or adult male H. contortus.

Intraspecific Epitopic Variation in a Carbohydrate Antigen Exposed on the Surface of Trichostrongylus colubriformis Infective L3 Larvae

The carbohydrate larval antigen, CarLA, is present on the exposed surface of all strongylid nematode infective L3 larvae tested, and antibodies against CarLA can promote rapid immune rejection of incoming Trichostrongylus colubriformis larvae in sheep. A library of ovine recombinant single chain Fv (scFv) antibody fragments, displayed on phage, was prepared from B cell mRNA of field-immune sheep. Phage displaying scFvs that bind to the surface of living exsheathed T. colubriformis L3 larvae were identified, and the majority of worm-binding scFvs recognized CarLA. Characterization of greater than 500 worm surface binding phage resulted in the identification of nine different anti-CarLA scFvs that recognized three distinct T. colubriformis CarLA epitopes based on blocking and additive ELISA. All anti-CarLA scFvs were specific to the T. colubriformis species of nematode. Each of the three scFv epitope classes displayed identical Western blot recognition patterns and recognized the exposed surface of living T. colubriformis exsheathed L3 larvae. Surprisingly, each of the anti-CarLA scFvs was able to bind to only a subset of worms. Double-labelling indirect immunofluorescence revealed that the three classes of anti-CarLA scFvs recognize distinct, non-overlapping, T. colubriformis sub-populations. These results demonstrate that individual T. colubriformis L3 larvae display only one of at least three distinct antigenic forms of CarLA on their surface at any given time, and suggest that antigenic variation within CarLA is likely a mechanism of immune evasion in strongylid nematodes.

Modelling parasite density in ruminants

Models of parasite density in ruminants usually involve three nonlinear, coupled, ordinary differential equations for larval density, adult parasite density and host resistance. We deal with the inverse problem of objectively determining the nonlinear coefficients within the equations using available data. Experiments are suggested to allow efficient calculation of these coefficients. An alternative model is also proposed which reproduces key features of experimental results and shows the importance of including time delay equations in parasite modelling. References Anderson, R. M. and May, R. M. Herd immunity to helminth infection and implications for parasite control. Nature 315, 493--496, 1985. doi:10.1038/315493a0 Gordon, H. M. The epidemiology of parasitic diseases, with special reference to studies with nematode parasites of sheep. Australian Veterinary Journal 24, 17--45, 1948. doi:10.1111/j.1751-0813.1948.tb01569.x Hansen, J. and Perry, B. The epidemiology, diagnosis and control of helminth parasites of ruminants. International Laboratory for Research on Animal Diseases, 1994. Kao, R. R., Leathwick, D. M., Roberts, M. G. and Sutherland, I. A. Nematode parasites of sheep: a survey of epidemiological parameters and their application in a simple model. Parasitology 121, 85--103, 2000. http://journals.cambridge.org/action/displayIssue?jid=PAR&volumeId=121&issueId=01&iid=53786 Louie, K., Vlassoff, A. and Mackay, A. Nematode parasites of sheep: extension of a simple model to include host variability. Parasitology 130, 437--446, 2005. doi:10.1017/S003118200400678X Roberts, M. G. and Grenfell, B. T. The population dynamics of nematode infections of ruminants: periodic perturbations as a model for management. IMA Journal of Mathematics Applied in Medicine and Biology 8, 83--93, 1991. doi:10.1093/imammb/8.2.83 Roberts. M. G. and Heesterbeek, J. A. P. A simple parasite model with complicated dynamics. J. Math. Biol., 37: 272--290, 1998. doi:10.1007/s002850050129 Sargison, N. D, Wilson, D. J, Bartley, D. J, Penny, C. D and Jackson, F. Haemonchosis and teladosagiosis in a Scottish sheep flock putatively associated with the overwintering of hypobiotic fourth stage larvae, Veterinary Parasitology 147, 326--331, 2007. doi:10.1016/j.vetpar.2007.04.011 Vlassoff, A., Leathwick, D. M. and Heath, A. C. G. The epidemiology of nematode infections of sheep. New Zealand Veterinary Journal 49, 213--221, 2001. http://www.sciquest.org.nz/default.asp?pageid=69&pub=2&vol=49&iss=6 Waller, P. J., Dobson, R. J., Donald, A. D. and Thomas, R. J. Populations of strongylid nematode infective stages in sheep pastures: comparison between direct pasture sampling and tracer lambs as estimators of larval abundance. International Journal of Parasitology 11, 359--367, 1981. doi:10.1016/0020-7519(81)90006-0

Genetics of Mating and Sex Determination in the Parasitic Nematode Haemonchus contortus

Genetic analysis of parasitic nematodes has been a neglected area of research and the basic genetics of this important group of pathogens are poorly understood. Haemonchus contortus is one of the most economically significant livestock parasites worldwide and is a key experimental model for the strongylid nematode group that includes many important human and animal pathogens. We have undertaken a study of the genetics and the mode of mating of this parasite using microsatellite markers. Inheritance studies with autosomal markers demonstrated obligate dioecious sexual reproduction and polyandrous mating that are reported here for the first time in a parasitic helminth and provide the parasite with a mechanism of increasing genetic diversity. The karyotype of the H. contortus, MHco3(ISE) isolate was determined as 2n = 11 or 12. We have developed a panel of microsatellite markers that are tightly linked on the X chromosome and have used them to determine the sex chromosomal karyotype as XO male and XX female. Haplotype analysis using the X-chromosomal markers also demonstrated polyandry, independent of the autosomal marker analysis, and enabled a more direct estimate of the number of male parental genotypes contributing to each brood. This work provides a basis for future forward genetic analysis on H. contortus and related parasitic nematodes.

Antibodies to surface epitopes of the carbohydrate larval antigen CarLA are associated with passive protection in strongylid nematode challenge infections

Sheep were immunized by multiple truncated infections with the gastrointestinal nematodes Trichostrongylus colubriformis, Haemonchus contortus and Teladorsagia circumcincta. Three infections with T. colubriformis of 14 days plus five booster doses of L3 stimulated highly effective protection against challenge (99%). Three infections of 14 days plus three booster doses with H. contortus also resulted in significant protection against challenge infection (87%), but the same procedure was not effective for T. circumcincta. Antibodies derived from gastrointestinal mucus of these immunized sheep were tested for their ability to reduce worm burden following injection of antibody-coated exsheathed larvae into the abomasum (H. contortus and T. circumcincta) or duodenum (T. colubriformis) of nematode-naïve sheep in a passive immunity test. The IgG fraction from the mucus of immunized sheep reduced worm burdens by 62%, 76% and 91% in three tests with T. colubriformis but was not effective for either of the abomasal dwelling nematodes H. contortus and T. circumcincta. Antibodies in immune mucus predominantly recognized two larval surface antigens on immunoblots of L3 extract, a high MW surface glycoprotein and the carbohydrate larval antigen (CarLA). Antibodies raised against purified T. colubriformis glycoprotein Tc-120 and CarLA were tested in the passive immunity model and it was found that only the antibody against CarLA resulted in a significant reduction of infection (87%). The protective anti-CarLA antibodies strongly recognized the surface of living T. colubriformis L3. Antibodies from abomasal mucus of sheep immunized by H. contortus and T. circumcincta infections reacted weakly with CarLA and the larval surface and did not reduce worm counts in a passive immunity test. The results provide further evidence that the larval surface carbohydrate antigen CarLA has potential as a mucosal immunogen for a strongylid nematode vaccine.

The house fly ( Musca domestica) as a potential vector of metazoan parasites caught in a pig-pen in Germany

In the present study a total of 224 specimens of the synanthropic house fly ( Musca domestica) were caught in a pig-pen of an organic farmer in Dormagen (Germany). The flies were examined for their potential as a carrier of metazoan parasites. On the exoskeletons and in the intestines of the flies the eggs and/or larvae of four endoparasite nematode species of domestic pigs ( Ascaris suum, Strongyloides ransomi, Metastrongylus sp., undetermined Strongylida) were isolated. Also one ectoparasite species, the hog louse ( Haematopinus suis), was detected on the exoskeleton of one fly. The analysis of the pig faeces as potential source revealed many eggs and larvae of nematodes. A high number of A. suum eggs (62.0% of all found nematode eggs), many eggs of strongylid nematodes (21.0%), some eggs of S. ransomi and few eggs of Trichuris suis were detected. However Metastrongylus sp. could not be identified in the faeces. Further laboratory experiments verified the potential of the house fly as a transmitter of the pig parasites A. suum and T. suis. In the intestines of 59 flies (49.2%) from 120 experimentally used house flies, nematode eggs of both nematode species were detected. The present study clearly demonstrates the potential of the house fly as a vector of metazoan pig parasites.

Illustrated identification keys to strongylid parasites (strongylidae: Nematoda) of horses, zebras and asses (Equidae)

The Equidae (the horse, Equus caballus, the ass, Equus asinus, zebras and their hybrids) are hosts to a great variety of nematode parasites, some of which can cause significant morbidity or mortality if individual hosts are untreated. Worldwide the nematode parasites of horses belong to 7 suborders, 12 families, 29 genera and 83 species. The great majority (19 of 29 genera and 64 of 83 species) are members of the family Strongylidae, which includes the most common and pathogenic nematode parasites of horses. Only the Strongylidae are included in this treatise. The Strongylidae (common name strongylids) of horses – nematodes with a well-developed buccal capsule, a mouth collar with two leaf-crowns, and a strongyloid (common name of superfamily Strongyloidea) copulatory bursa – can be separated into two subfamilies: Strongylinae (common name strongylins), usually large or medium-sized with a globular or funnel-shaped buccal capsule; and Cyathostominae (common name cyathostomins), usually small to medium-sized with a cylindrical buccal capsule. The increased attention to strongylid nematode parasites of horses has resulted in the need for updated diagnostic keys to these parasites using readily recognizable characters and the most recent literature on their systematics. Because the cyathostomins have been historically difficult to identify, and because they have emerged as the most significant nematode pathogens of horses, we provide a brief nomenclatural and taxonomic history and an introduction to the morphology of this group. This treatise is intended to serve as a basic working tool—providing easy identifications to genus and species of adult strongylid nematodes of equids. All strongylid nematodes normally parasitic in horses, the ass (and their hybrids), and zebras are included. The keys are illustrated with line drawings and halftone photomicrographs of each species. A short discussion of the systematics of the genus and species is provided for each genus following the species descriptions. Species diagnoses and a synonymy of each species is provided. Geographic distribution, prevalence, and location in host are also given for each species.

Analysis of the transthyretin-like (TTL) gene family in Ostertagia ostertagi – Comparison with other strongylid nematodes and Caenorhabditis elegans

The transthyretin-like ( ttl) gene family is one of the largest conserved nematode-specific gene families, coding for a group of proteins with significant sequence similarity to transthyretins (TTR) and transthyretin-related proteins (TRP). In the present study, we investigated the ttl family in Ostertagia ostertagi (a nematode of the abomasum of cattle). Mining of expressed sequence tag (EST) databases revealed the presence of at least 18 ttl genes in O. ostertagi ( Oo-ttl), most of which are constitutively transcribed from the free-living, third larval stage onwards. The full-length cDNA of one of these genes ( Oo-ttl-1) was amplified and cloned for recombinant expression. Western blot analysis using a specific antiserum showed that the native protein Oo-TTL-1 was highly present in the excretory–secretory (ES) products of adults of O. ostertagi. The protein was immunolocalized to the pseudocoelomic fluid of adult worms. A phylogenetic–bioinformatic analysis of all amino acid sequence data for TTL proteins from a range of strongylid nematodes showed that they could be divided into at least five different classes. This classification was based on conserved amino acids in the first TTL signature domain and the number and location of cysteine residues. The biological role(s) of the TTLs in nematode biology is still unclear. A theoretical three-dimensional model of Oo-TTL-1 indicated that it had a similar structure to TTRs (i.e., containing β-sheets, arranged in a β-sandwich). In contrast to TTRs, competitive binding studies using recombinant Oo-TTL-1 indicated that the protein was devoid of any hydrophobic ligand- or thyroid hormone-binding properties. Finally, combinatorial analysis by double-stranded RNA interference of five ttl genes in the free-living nematode Caenorhabditis elegans did not reveal any visible phenotypes. More information on the transcription profile and tissue distribution of TTLs in nematodes is needed to provide new insights into the biological role of this gene family.

Rodents as reservoirs of parasites in India

We monitored the prevalence of endoparasitic infections of rodents in Punjab State, India, between January 2004 and December 2005. Three species of wild rodents, namely the house rat, Rattus rattus (n= 42), the lesser bandicoot rat, Bandicota bengalensis (n= 34) and the Indian gerbil, Tatera indica (n= 15), were live-captured from houses and crop fields. Examination of various organs revealed that the highest rates of endoparasitic infection occurred in R. rattus (40.5%), followed by B. bengalensis (35.3 %) and then T. indica (20.0%), with an overall infection rate of 35.2%. All three rodent species were found naturally infected with one or more species of helminths. Metacestodes (1-6) of Cysticercus fasciolaris (larvae of Taenia taeniaeformis) were found in all three rodent species (in the liver). In one male T. indica, numerous robust T. taeniaeformis metacestodes were found in oval sacs attached to the mesentery and the abdominal wall, an unusual site. The cauda epididymal fluid of the same gerbil was also found to be infected with a very rare species of strongylid nematode, which could not be identified to genus or species level. It is possible that this nematode is transmitted sexually and thus may affect the reproductive potential of gerbils. This appears to be the first report of this phenomenon. In one B. bengalensis individual, the intestine was found to be obstructed with an acanthocephalan, Moniliformis moniliformis, with concurrent infection with C. fasciolaris in the form of multiple cysts in the liver. Although no natural protozoan infection was found in these field rodents, experimental Trypanosoma evansi infection has been established in all three species with high pathogenicity, and the possibility of sexual transmission was supported by the presence of T. evansi in the cauda epididymal fluid of male rats.

The Community of Strongylids (Nematoda, Strongylida) of Working Donkeys (Equus Asinus) in Ukraine

The Community of Strongylids (Nematoda, Strongylida) of Working Donkeys (Equus Asinus) in UkraineThe aim of our work was to study the species composition of the strongylid community of donkeys and to explore the influence of anthelmintic treatments on the community structure. Strongylid nematodes were collected by the diagnostic deworming technique from 33 donkeys from the riding school "Chudo-oslik" (Crimea) (25), from Kharkiv (3) and Kyiv zoos (2) and Kyiv riding schools (3). Seventeen species were found in donkeys studied: 16 species of Cyathostominae and 1 of Strongylinae. Between 2 and 7 species were found per donkey (average of 4.2 ± 2.8). Cyathostomum tetracanthum, C. catinatum, Cylicocyclus nassatus, Cylicostephanus goldi and C. longibursatus) dominated in the community; they were found in 80-100% animals studied and comprised 91.7% of the total number of strongylids collected. Two species C. tetracanthum and Cylicocyclus auriculatus were found to be specific for donkeys. The results obtained showed a reduction of the species richness of the strongylid community in donkeys from riding schools and zoos caused by lack of grazing and by regular anthelmintic treatments.

Trichostrongylus vitrinus (Nematoda: Strongylida): Molecular characterization and transcriptional analysis of Tv-stp-1, a serine/threonine phosphatase gene

A full-length cDNA ( Tv-stp-1) encoding a serine/threonine protein phosphatase ( Tv-STP-1) was isolated from Trichostrongylus vitrinus (order Strongylida), an economically important parasitic nematode of small ruminants. The uninterrupted open reading frame (ORF) of 951 nucleotides encoded a predicted protein of 316 amino acids (aa), containing the characteristic motif [LIVMN]-[KR]-G-N-H-E. Comparison with other sequences in non-redundant databases showed that Tv-STP-1 had significant identities/similarities to those from a range of metazoans and protists. Sequence similarity was most pronounced in the central region of the protein, in which the catalytic activity is inferred to be modulated by eight conserved residues (Asp 61, His 63, Asp 92, Asp 95, Asn 121, His 171, His 246 and Tyr 270), known to coordinate the binding of two metal ions (Mn 2+ and Fe 2+) in various organisms. Phylogenetic analyses of selected amino acid sequence data using the neighbor-joining and maximum parsimony methods revealed Tv-STP-1 to be most closely related to the glc seven-like phosphatases inferred for genes from the free-living nematode Caenorhabditis elegans and the parasitic nematode Oesophagostomum dentatum (order Strongylida). Comparison of the genomic organization of the full-length Tv-stp-1 gene with related molecules from other nematodes revealed substantial variation in the lengths and numbers of the exons and introns. The entire genes Tv-stp-1 (5041–5362 bp; 10 exons and 9 introns) and Od-mpp-1 (10,271 bp; 8 exons and 9 introns) from the parasitic nematodes T. vitrinus and O. dentatum were considerably longer than the C. elegans genes (1222–1603 bp; 3–7 exons and 2–6 introns). Transcriptional analysis by reverse transcription polymerase chain reaction (RT-PCR) showed that Tv-stp-1 was transcribed in adult males of T. vitrinus, but not in the adult female or in any larval stages of this species. In spite of considerable variation at the genomic level, the findings of the present study suggest that there is relative conservation in features and function of the serine/threonine protein phosphatase characterized among T. vitrinus, O. dentatum and C. elegans, which should have implications for exploring molecular reproductive and developmental processes in strongylid nematodes of socio-economic importance.

Three surface antigens dominate the mucosal antibody response to gastrointestinal L3-stage strongylid nematodes in field immune sheep

Although gastrointestinal nematode parasites are a major human and veterinary health problem, little is known about how the host is sometimes able to mount an effective immune rejection response. In previous work, we identified a carbohydrate larval surface antigen (CarLA) as the target of mucosal antibodies that can elicit rejection of Trichostrongylus colubriformis L3s in sheep. Here we characterise the natural mucosal antibody responses to L3s from three major strongylid gastrointestinal parasites of sheep, Trichostrongylus colubriformis, Haemonchus contortus and Teladorsagia circumcincta. The mucosal antibody repertoire of naturally field-immune sheep was displayed on bacteriophage as single-chain antibodies (scFvs) and phage were selected for the ability to bind to the surface of living L3s of the three nematode species. All nematode-binding scFvs were found to recognize one of three different antigen classes that are each found in the three strongylid species. These three antigen classes appear to represent all of the major antigens recognized on Western blots by pooled mucosal antibodies from field-immune sheep. One of the antigen classes is a heterogeneous, high molecular weight molecule that is protease-sensitive. The scFvs recognizing this surface antigen also recognize a similar antigen in all strongylids tested. A second antigen class is a protease-insensitive, low molecular weight antigen found only in sheaths and scFvs recognizing this antigen cross-react with a similar molecule found in all strongylids tested. The third surface antigen class is CarLA and all of the anti-CarLA scFvs obtained from the field-immune sheep repertoire were specific to L3s of only one species and often recognized only a subset of the worms. Thus three different L3-stage surface antigens, two that lack a protein component, dominate the natural mucosal antibody response to L3-stage gastrointestinal strongylid nematodes in sheep.

Anthelminthic efficacy of aqueous extracts of <i>Garcina kola</i> seed and stem bark against strongylid nematodes of small ruminants in Nigeria

The anthelminthic efficacy of the crude aqueous extracts of the seed and stem of Garcina kola against strongylid nematodes of small ruminants was investigated using the invitro egg hatch assay method. Compared to the water (control0 cultures, the extracts demonstrated significant (P

Diversity of the infracommunities of strongylid nematodes in the ventral colon of Equus caballus from Rio de Janeiro state, Brazil

Nematodes from the ventral colon of 31 adult horses, 24 males and 9 females, in the metropolitan region of the state of Rio de Janeiro were analysed. There were 53,444 (86.4%) adults of the total recovered strongylid nematodes. They belonged to 21 species of Cyathostominae and seven of Strongylinae. Larval forms made up 13.6% (8407) of the total recovered, and 49% of the strongylid nematodes were observed in ventral colon. The most prevalent and abundant species were Cyathostomum tetracanthum, Cylicocyclus nassatus, Cylicostephanus minutus, Cylicostephanus longibursatus, Cylicostephanus leptostomus, Cylicostephanus calicatus and Cylicostephanus goldi, corresponding to 93.2% of the total adult population; these species were classified as central species, except C. goldi; and other species were classified as secondary ( n = 8) and satellite ( n = 14). The low Green's aggregation index, presented to central species, indicated high dispersion because they were found in a greater number of infracommunities and were proven by the greater prevalence. Only 12.1% of the horses were uninfected or infected by a single species; the other infections were multiple, ranging from 3 to 23 species.

The occurrence of strongylid nematodes in the epididymides of wood mice

The recent discovery of a larval nematode in the epididymides of free-living wood mice (Apodemus sylvaticus) suggests a sexual transmission of these parasites. They have been placed within the bursate nematodes (order Strongylida) through 18S rDNA analysis, suggesting that they are undetermined metastrongyloid nematodes. The possibility that these parasites are transmitted sexually opens an intriguing field of research because sexually transmitted metazoan parasites are known to occur mainly in invertebrates, whereas in vertebrates sexually transmitted parasites are usually microparasites such as viruses, bacteria and protozoa.

Semi-nested PCR for the specific detection of Habronema microstoma or Habronema muscae DNA in horse faeces

Habronema microstoma and Habronema muscae (Spirurida: Habronematidae) are parasitic nematodes which infect the stomach and/or skin of equids. The accurate diagnosis of gastric habronemosis is central to studying its epidemiology, but data on its distribution and prevalence are lacking, mainly due to the limitations of clinical and coprological diagnosis in live horses. To overcome this constraint, a two-step, semi-nested PCR-based assay was validated (utilizing genetic markers in the nuclear ribosomal DNA) for the specific amplification of H. microstoma or H. muscae DNA from the faeces from horses (n = 46) whose gastrointestinal parasite status had been determined at autopsy and whose faeces were examined previously using a conventional parasitological approach. Of these horses examined at autopsy, some harboured adults of either H. microstoma (n= 19) or H. muscae (n =4), and others (n = 7) harboured both species. Most of them were also infected with other parasites, including strongylid nematodes (subfamilies Cyathostominae and Strongylinae), bots and/or cestodes; there was no evidence of metazoan parasites in 2 horses. Larvated spirurid eggs were detected in the faeces of 1 of the 30 horses (3.3 %) shown to be infected with Habronema at autopsy. For this set of 46 samples, the PCR assay achieved a diagnostic specificity of 100 % and a sensitivity of approximately 97 % (being able to specifically detect as little as approximately 0.02 fg of Habronema DNA). The specificity of the assay was also tested using a panel of control DNA samples representing horse, the gastric spirurid Draschia megastoma and 26 other species of parasites from the alimentary tract of the horse. H. microstoma, H. muscae and D. megastoma could be readily differentiated from one another based on the sizes of their specific amplicons in the PCR. The results of this study showed that the performance of the PCR for the diagnosis of gastric habronemosis was similar to that of autopsy but substantially better than the traditional coprological examination procedure used. The ability to specifically diagnose gastric habronemosis in equids should have important implications for investigating the epidemiology and ecology of H. microstoma and H. muscae.

Comparison of two techniques used for the recovery of third-stage strongylid nematode larvae from herbage

A laboratory trial to determine the efficacy of two methods in recovering known numbers of third-stage (L 3) strongylid nematode larvae from herbage was carried out. Herbage samples consisting almost entirely of star grass ( Cynodon aethiopicus) that had no L 3 nematode parasitic larvae were collected at Onderstepoort, South Africa. Two hundred grams samples were placed in fibreglass fly gauze bags and seeded with third-stage strongylid nematode larvae at 11 different levels of herbage infectivity ranging from 50 to 8000 L 3/kg. Eight replicates were prepared for each of the 11 levels of herbage infectivity. Four of these were processed using a modified automatic Speed Queen heavy-duty washing machine at a regular normal cycle, followed by isolation of larvae through centrifugation–flotation in saturated sugar solution. Larvae in the other four samples were recovered after soaking the herbage in water overnight and the larvae isolated with the Baermann technique of the washing. There was a strong correlation between the number of larvae recovered using both methods and the number of larvae in the seeded samples, indicating that the two methods give a good indication of changes in the numbers of larvae on pasture if applied in epidemiological studies. The washing machine method recovered higher numbers of larvae than the soaking and Baermann method at all levels of pasture seeding, probably because the machine washed the samples more thoroughly and a sugar centrifugation–flotation step was used. Larval suspensions obtained using the washing machine method were therefore cleaner and thus easier to examine under the microscope. In contrast, the soaking and Baermann method may be more suitable in field-work, especially in places where resources and equipment are scarce, as it is less costly in equipment and less labour intensive. Neither method recovered all the larvae from the seeded samples. The recovery rates for the washing machine method ranged from 18 to 41% while those for the soaking and Baermann method ranged from 0 to 27%. Practical application of the two methods to estimate the number of nematode larvae on pastures without applying a correction factor would therefore result in a significant underestimation. This study provides a model, which can be applied in various laboratories to determine the larval recovery rates for techniques being used and the application of a correction factor when estimating the actual numbers of larvae on pasture.

Sexual transmission of a nematode parasite of Wood Mice (Apodemus sylvaticus)?

Sexual transmission occurs commonly in microparasites such as viruses and bacteria, but this is an unusual transmission route for macroparasites. Here we present evidence which suggests that a nematode parasite of Wood Mice (Apodemus sylvaticus) may be sexually transmitted and we have classified the nematode using molecular data. Wood Mice were collected annually in the course of work on their reproductive physiology. Larval nematodes were found in the epididymides of 19.6% of males. It seems likely that they would be transmitted to females at ejaculation. To identify these larval nematodes, which we were unable to do using morphological features, we sequenced the 18S rDNA. Sequence comparisons with the molecular phylogeny of Blaxter et al. (1998) demonstrated that they were bursate nematodes (Order Strongylida). The relationships between strongylid taxa were poorly resolved by 18S rDNA. However, both distance and parsimony analyses grouped the nematode with the superfamily Metastrongylidea in a clade containing Filaroides and Angiostrongylus sp. Importantly, the sequences were distinct from those of Heligmosomoides polygyrus and Angiostrongylus dujardini, two common strongylid nematodes of Apodemus. We were therefore unable positively to identify these worms by matching their sequences with those from morphologically identifiable adult strongylid nematodes infecting Apodemus. These results demonstrate that an as yet unidentified strongylid is quite commonly found in large numbers in the male reproductive tract of Wood Mice. Further work is required to understand the biology and transmission dynamics of this interesting system.

Host population density and body mass as determinants of species richness in parasite communities: comparative analyses of directly transmitted nematodes of mammals

Epidemiological theory predicts positive correlations between host population density or body mass and species richness among parasite communities. Here I test these predictions by a comparative study of communities of directly transmitted mammalian parasites, gastrointestinal strongylid nematodes. I use data from 45 species of mammals, representing examination of 17 200 individual hosts. The variable studied was the average number of gastrointestinal strongylid nematode species per host population, and three different methods were used to obtain estimates of parasite species richness that are unbiased by number of host individuals examined. Analyses were done using the phylogenetically independent contrast method. Host population density and parasite species richness were strongly positively correlated when the effects of host body weight had been controlled for. Controlling for other variables did not change this, and the relationship was found regardless of method used to correct for uneven sampling effort among host species. A positive relationship between parasite species richness and host body weight was also found, but the effect of host densities had to be controlled for to see this. These relationships between host traits and species richness of directly transmitted parasites are stronger than patterns found using data on indirectly transmitted mammalian parasites, and suggests that links between host traits and parasite species richness are stronger than previously suggested. The results are consistent with parasite species richness being positively linked to pathogen transmission rates and reductions in transmission rates possibly increasing extinction probabilities in parasite populations. The results also suggest that parasites may exert a cost of increases in rate of population energy usage, and thus show that pathogens may be important in generating independence between body mass and rate of population energy usage among host species.

Single-strand conformation polymorphism analysis of genetic variation in Labiostrongylus longispicularis from kangaroos.

Single-strand conformation polymorphism (SSCP) analysis was employed to screen for sequence heterogeneity in the second internal transcribed spacer (ITS-2) of ribosomal (r) DNA of Labiostrongylus longispicularis, a parasitic strongylid nematode occuring in some species of kangaroo in different geographical regions of Australia. The results showed that most of the nematodes screened had different SSCP profiles, which were subsequently shown to correspond to polymorphisms and/or an indel in the ITS-2 sequence. These variable sites related mainly to unpaired regions of the predicted secondary structure of the precursor rRNA molecule. SSCP profiles could be used to distinguish L. longispicularis in Macropus robustus robustus (New South Wales) from L. longispicularis in Macropus robustus erubescens and Macropus rufus (South Australia). This difference corresponded to a transversional change in the ITS-2 sequence at alignment position 82. The study demonstrated clearly the effectiveness of SSCP analysis for future large-scale population genetic studies of L. longispicularis in order to test the hypothesis that L. longispicularis from different geographical regions represents multiple sibling species.