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Cytokine responses to Cyathostominae larvae in the equine large intestinal wall.

To investigate cytokine responses in cyathostomin infection, we quantified mucosal interleukin-4 (IL-4), interleukin-10 (IL-10), tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma by reverse transcriptase-competitive polymerase chain reaction. The analysis was performed on large intestinal wall samples obtained from six anatomical sites spanning the caecum and colon of 17 naturally exposed horses. The numbers of developing larvae (DL) and early third stage larvae (EL3) were ascertained using transmural illumination and pepsin digestion techniques, respectively. Levels of each cytokine transcript were correlated with local intestinal wall burdens of Cyathostominae larvae. IL-4 and IL-10 levels showed significant correlations with EL3 and DL burdens at several sites. No significant correlations were observed with IFNgamma. A pro-inflammatory response, typified by detection of TNFalpha transcript, was observed at a few sites in some horses with inflammatory enteropathy associated with emerging or emerged larvae. However, this cytokine was measured at an insufficient number of sites to enable statistical analysis. Levels of IL-4, IL-10 and IFNgamma transcript were compared between two groups: one group consisting of horses with low to high mucosal burdens (Group A) and the other, of horses with negative/negligible mucosal burdens (Group B). Significant differences in IL-4 (P<0.001) and IL-10 (P<0.001) transcript levels were observed between the groups, with higher levels observed in Group A. No significant differences in IFNgamma were observed. Taken together, these results indicate that Th2 responses predominate in mucosal Cyathostominae infection prior to larval reactivation.

Animals↗

Efficacy of benzimidazole anthelmintics in goats and sheep in the Philippines using a larval development assay.

The negative effects of nematodes in small ruminants can be reduced by use of dewormers but their effectiveness is increasingly limited by the emergence of anthelmintic resistance. The efficacy of benzimidazole (BZ) anthelmintics in the Philippines was estimated by an in vitro larval development assay using worm eggs recovered from faeces collected from goats and sheep. Two hundred and eighteen farms were selected to represent areas of the country with high goat and sheep populations and the full range of farm sizes, from smallholders with just a few animals to commercial and institutional farms with several hundred. Initial surveys of worm control advisers indicated that BZs have been in continuous widespread use for up to 20 years with little use of other chemical groups. A larval development assay (LDA: DrenchRite) was modified for use with BZs alone to allow up to five samples to be analysed on a single microtitre plate. The assay was validated by comparison with the faecal egg count reduction test (FECRT). The dominant nematode genera were Haemonchus and Trichostrongylus with small numbers of Oesophagostomum. The range of BZ efficacy estimated from the LDA results was 0-100% and the distribution of efficacy levels was continuous, with mean efficacy of 82 and 64% for goats and sheep, respectively. There were significant associations between efficacy and parameters measured to characterize the sampled farms: size of animal management group, FEC of sample, recent importation of stock and no access to common grazing were all correlated with decreased efficacy. Likewise, low efficacy was associated with reported frequency and number of years that BZ drenches had been used. The LDA was found to be highly suited to estimate efficacy in nematode populations from small farms where performance of a FECRT for even one chemical would be impractical. Using a larval development assay, we have demonstrated a wide efficacy range for BZs against nematodes from all sizes of goat and sheep farms in the tropics.

Animal Husbandry↗

Seasonal development of Cyathostominae larvae on pasture in a northern temperate region of the United Kingdom.

A study following the development of Cyathostominae from egg to the infective larval third stage was conducted from April to December 2001 in west central Scotland. Duplicate samples (1 kg) of naturally infected faeces were placed on a 78 cm2 plot each week on a cyathostomin-free pasture. Subsamples of the grass surrounding the faecal plot were collected weekly on four occasions and the number of larvae obtained determined. Few larvae were recovered in the first week of development of individual plots, followed by a rise in the numbers of larvae in second, third and fourth weeks of development of each sample. The climatic conditions were seen to have an effect on the rate of development. Specifically, from multilevel, multivariable linear regression models it was evident that the factors associated with numbers of infective larvae recovered from pasture were the time since the faeces samples were laid down, the average temperature and rainfall during the previous week, as well as the interaction between temperature and rainfall. Conversely, from the model, the number of larvae recovered from pasture was associated with neither the number of eggs within the faeces samples placed on the plots nor with the viability of these eggs.

Animals↗

Resistance of Santa Ines, Suffolk and Ile de France sheep to naturally acquired gastrointestinal nematode infections.

A study was conducted to assess the breed resistance against nematode infections in Santa Ines, Ile de France and Suffolk male lambs over a 9-month period in São Paulo state, Brazil. Lambs were born during the winter (year 2000) and were weaned at 2 months of age. The animals were then housed and treated with anthelmintics to eliminate natural infections by gastrointestinal nematodes. In late October 2000, lambs were placed in a paddock, where they stayed until August of the following year. Fecal and blood samples were taken from each animal every 2 weeks. On the same day, a pasture sample was collected to determine the number of infective larvae on the herbage. To prevent deaths, individual treatment with anthelmintics was provided to lambs with fecal egg counts (FEC) higher than 4000 eggs per gram (EPG) or with a packed cell volume (PCV) lower than 21%. In August 2001, all animals were slaughtered and the worms present in samples of the gastrointestinal contents were identified and counted. Most of the Suffolk and Ile de France sheep received three to six anthelmintic treatments over a period of 7 months, while most of the Santa Ines were not treated. Reductions in PCV and plasma protein values associated with high FEC and worm burdens were recorded, particularly, in Suffolk and Ile de France lambs. Haemonchus contortus and Oesophagostomum columbianum burdens and number of nodular lesions caused in the large intestine by O. columbianum larvae were significantly lower in Santa Ines sheep. All three breeds showed similar Trichostrongylus colubriformis worm burdens. The relative resistance of Santa Ines young male sheep was superior to that of Suffolk and Ile de France sheep.

Abomasum↗

Evaluation of the anthelmintic activity of Khaya senegalensis extract against gastrointestinal nematodes of sheep: in vitro and in vivo studies.

The anthelmintic effect of Khaya senegalensis is described. In vitro and in vivo studies were conducted to determine possible direct anthelmintic effects of ethanolic and aqueous extracts of K. senegalensis towards different ovine gastrointestinal nematode. A larval development assay was used to investigate in vitro, the effect of aqueous and ethanolic extracts towards larvae of strongyles. The LC50 values of the effects of both the aqueous and ethanolic extracts were calculated. Another study was conducted in vivo to evaluate the therapeutic efficacy of the extracts administered orally at a dose rate of 125, 250 and 500 mg/kg of sheep harbouring naturally acquired infection of gastrointestinal nematodes. The presence of K. senegalensis extracts in the cultures decreased the viability of larvae. The LC50 of the aqueous extract (0.69 mg/ml) is not significantly different (P >0.05, t-test) from the ethanolic extract (0.51 mg/ml). The activity of the extract is concentration dependent in vivo. Sheep drenched with 500 mg/kg K. senegalensis ethanolic extract had a mean faecal egg count (FEC) reduction of 88.82%. The extract of K. senegalensis could find application in anthelmintic therapy in veterinary practice.

Animals↗

The influence of flotation solution, sample dilution and the choice of McMaster slide area (volume) on the reliability of the McMaster technique in estimating the faecal egg counts of gastrointestinal strongyles and Dicrocoelium dendriticum in sheep.

The present study was aimed to evaluate the influence of flotation solution, sample dilution, and the choice of McMaster slide area (volume) on the reliability of the McMaster technique in estimating the faecal egg counts of gastrointestinal (GI) strongyles and Dicrocoelium dendriticum in a composite sample of faeces from naturally infected sheep. Fourteen flotation solutions having densities between 1.200 and 1.450, and six sample dilutions, 1:10, 1:15, 1:20, 1:30, 1:40 and 1:50 were used. Each of the six dilutions was divided into 70 aliquots in order to have five replicates of each of the 14 flotation solutions at each of the six dilutions. For each McMaster slide, the GI strongyle and D. dendriticum egg counts were performed under one grid (McM 0.15 ml), two grids (McM 0.3 ml), one chamber (McM 0.5 ml), and both chambers (McM 1.0 ml). Mean eggs per gram (EPG) of faeces of GI strongyles and D. dendriticum were calculated and statistical analyses were performed on the resulting data. The type of flotation solution used significantly influenced the EPG in the GI strongyles and in the D. dendriticum egg counts. All the sucrose-based solutions at density between 1.200 and 1.350 floated more GI strongyle eggs than the others. With respect to D. dendriticum, only six solutions were capable of floating eggs and the potassium iodomercurate solution (density 1.440) floated more eggs than the others. The reliability of the McMaster technique regarding sample dilution was high for both GI strongyle and D. dendriticum EPG at 1:10 and 1:15, and then progressively decreased with increasing dilution. The reliability of the McMaster technique regarding the choice of the McMaster slide area (volume) was high for both GI strongyle and D. dendriticum EPG at the McMaster slide area (volume) of 1.0 ml, i.e. the total area of the McMaster slide. The EPG counts resulting from choosing any of the other three McMaster slide areas (volumes), i.e. McM 0.15 ml, McM 0.3 ml, or McM 0.5 ml, produced unreliable over-estimates. The findings of the present study show that the highest reliability of the McMaster technique for estimating GI strongyle and D. dendriticum egg counts in faeces from pastured sheep is obtained when using flotation solutions based on sucrose for GI strongyles, and potassium iodomercurate for D. dendriticum, dilutions which do not exceed 1:15, and the McMaster slide area (volume) of 1.0 ml.

Animals↗

Effect of repeated benzimidazole treatments with increasing dosages on the phenotype of resistance and the beta-tubulin codon 200 genotype distribution in a benzimidazole-resistant cyathostomin population.

This study was designed to investigate the effect of repeated treatments with increasingly high fenbendazole (FBZ) dosages on the phenotype and genotype of a benzimidazole (BZ)-resistant cyathostomin population. An experimentally infected horse was treated repeatedly with FBZ dose rates between 7.5 and 30.0 mg/kg body weight (bw) over approximately 2 years. Faecal egg counts (FECs) and larval cultures were performed weekly. A total of 45 faecal egg count reduction tests (FECRTs) were analysed, revealing a high variability during the course of experiment with a mean value in faecal egg count reduction (FECR) of -17% (S.D. +/- 78). The FECR was always < 90%, providing the evidence of BZ resistance. Nine egg hatch tests were performed during the course of the experiment and revealed LD(50) values between 0.20 and 0.31 microg/ml thiabendazole (TBZ) and LD(96) values of > 0.36 microg/ml TBZ, confirming the phenotype of resistance. The LD(99) varied between 0.40 and 0.63 microg/ml TBZ. Despite consecutive treatments, no noticeable increase of the LD(50), LD(96) and LD(99) values was detected for the duration of the experiment. The molecular analysis of the codon 200 of 106 third stage larvae (L3) was carried out following repeated treatments with 30 mg FBZ/kg bw. Out of these larvae 32% were homozygous TTC/TTC, 60% showed the heterozygous TTC/TAC genotype, and 8% were homozygous TAC/TAC. The resulting allele frequencies were 62% for TTC and 38% for TAC. These findings suggest that repeated BZ treatments with increasing dosages do not alter significantly the FECRT and EHT characteristics of a BZ-resistant cyathostomin population. Furthermore, it may also be concluded that, in contrast to sheep trichostrongyles, such a selection regime does not result in beta-tubulin codon 200 TAC allele autocracy.

Alleles↗

Aelurostrongylus abstrusus (cat lungworm) infection in five cats from Italy.

Infection by the cat lungworm Aelurostrongylus abstrusus is considered uncommon. Here, the authors report the clinical, diagnostic and therapeutic features of five infections recently observed in Italy. All cats were under 12 months of age. All except one cat had symptomatic infections, with cough, dyspnea, and weight loss with radiographic signs of broncopneumonia. All cats were eosinophilic. Larvae were present in fresh fecal smears and on flotation exam in all cats. Baermann larval recovery permitted definitive identification and, in one case, larvae per gram of feces (lpg) counts. One dose of ivermectin (400 microg/kg) was not effective in one cat, while one dose of selamectin (6 mg/kg) was effective in one of three cases and fenbendazole at 50 mg/kg given daily for 15 days was effective in four of four cases.

Animals↗

Prescription-only anthelmintics--a questionnaire survey of strategies for surveillance and control of equine strongyles in Denmark.

In 1999, legislation in Denmark made anthelmintic drugs available only by prescription, and prohibited their use for routine, prophylactic treatment. A questionnaire survey was conducted in 2004 to determine current strategies for surveillance and control of equine strongyles used in Danish equine veterinary practices. Eighty-seven of 170 (51.2%) registered equine veterinary practices responded. Ninety seven percent of the respondents used faecal egg counts for diagnosis and surveillance, and 41% used larval cultures. Logistic regression revealed that the use of larval cultures was positively correlated with numbers of employees (P = 0.013) and the proportion of equine caseload in the practice (P < 0.000). Performing faecal egg counts and treating horses was most frequent during spring, summer and early autumn. Veterinary practices made treatment decisions based on cut-off values ranging from 20 to 500 eggs per gram (EPG) faeces. Ages of horses, clinical suspicions of parasitic disease, or requests by the owners were the most important factors in the strategies for faecal sampling. Less commonly, sampling strategies included all horses on the premises and random sampling. Ninety five percent of the respondents reported treating certain groups of horses without prior faecal analysis, including horses with clinical signs of parasitic disease (77%), foals (84%), horses less than 3 years of age (52%), and pregnant mares (51%). The respondents regarded the cyathostomin group as the most prevalent cause of parasitic disease and ill-thrift, followed by large strongyles and Parascaris equorum. Sixty seven percent rotated regularly between drugs, while 11% performed routine screening for anthelmintic resistance. Results of this survey suggest that limiting equine anthelmintics to prescription-only availability has increased the level of strongyle surveillance. Veterinary practitioners play a central role in equine parasite management with indications of a lowered intensity of treatment. However, screening for anthelmintic resistance remains uncommon.

Age Factors↗

Molecular diagnosis and equine parasitology.

The future implementation of improved and sustainable control strategies for the major equine parasites will be dependent on a greater insight into their basic biology, pathogenicity and epidemiology together with an enhanced ability for accurate diagnosis. This paper will provide a review of the current molecular methods under development for the detection of equine parasites and their application to current scientific questions. In particular, the strongyles are recognised as important pathogens of horses and recent advances made in the study of this parasitic group at the single species level will be addressed. The ribosomal (r)DNA region of the parasite genome has been employed to distinguish between closely related species. Molecular probes designed to this target region were used in combination with PCR technology to allow the identification of individual species within mixed infections. They have been applied to all parasite stages to look at the role of individual species in natural infection, disease and drug resistance. Similar techniques have been developed to detect other equine parasites and these will also be discussed. Further opportunities for employing existing techniques and the need for new diagnostic tools will be highlighted.

Animals↗

Cyathostomes in horses in Canada resistant to pyrantel salts and effectively removed by moxidectin.

Clinical trials using fecal egg count reduction tests and coproculture were conducted with yearlings and mares on a farm in 1997. Fecal samples were taken from each horse to estimate the number of strongyle eggs/g feces with Cornell-Wisconsin centrifugal flotation and Cornell-McMaster dilution techniques. Eleven of 15 yearlings, which had been on a daily feeding of grain with pyrantel tartrate for 66 d were found with strongyle eggs in feces. This was the first time the in-feed medication had been used on the farm. Nine yearlings were randomised into three groups; continuation of daily pyrantel tartrate or one treatment with pyrantel pamoate or moxidectin. Two of three yearlings given pyrantel tartrate or pamoate had no reduction in the eggs/g feces. These six yearlings were then given moxidectin and in all yearlings the eggs/g feces was reduced to zero. The 66 d of pyrantel tartrate use was an inadequate time for development of resistant cyathostomes and a hypothesis was the resistance was due to extensive use on the farm over many years of pyrantel pamoate at twice the label dose for control of tapeworms. That hypothesis was tested with 12 mares with strongyle eggs in the feces randomised into two treatment groups: pyrantel pamoate at label dose or moxidectin. Five of six mares given pyrantel had <80% reduction in egg/g feces. These mares were then given moxidectin and in all mares the eggs/g feces was reduced to zero. Only cyathostomes were found on culture and apparently there was side resistance among the pyrantel salts.

Animals↗

Cases of reduced cyathostomin egg-reappearance period and failure of Parascaris equorum egg count reduction following ivermectin treatment as well as survey on pyrantel efficacy on German horse farms.

In 2003 and 2004, on a total of 63 different German horse farms, a survey using the faecal egg count reduction (FECR) test was performed to investigate the efficacy of ivermectin (IVM, Ivomec) and pyrantel (PYR, Banminth) treatment against gastro-intestinal nematodes in a total of 767 horses. IVM treatment resulted in 100% reduction of the cyathostomin egg production 14 and 21 days post-treatment (d.p.t.) on 37 farms. On the remaining five farms, the mean faecal egg count reduction ranged between 97.7 and 99.9%. The mean cyathostomin FECR following PYR treatment ranged between 92.2 and 100% on the 25 farms tested. Therefore, based on the 90% FECR threshold suggested for detection of anthelmintic resistance in horses, neither IVM nor PYR anthelmintic resistance was detected. However, if the thresholds recommended for the detection of resistance in small ruminants were applied, on one and four farms signs of reduced IVM and PYR efficacy, respectively, were observed. In 2005, to further investigate these findings, the cyathostomin egg-reappearance period (ERP) following IVM treatment was examined on six selected farms, two of which were found to show less than 99.8% FECR in the previous survey. On these two latter farms, the ERP was less than 5 weeks, while on the other four it was at least 8 weeks. Earlier investigations described IVM cyathostomin ERP of at least 9 weeks. The efficacy of IVM to reduce Parascaris equorum egg excretion was also studied. On one farm in 2 consecutive years, IVM treatment did not lead to a significant reduction in P. equorum faecal egg counts in one and five young horses, respectively.

Animals↗

Evaluation of the specificity of five oligoprobes for identification of cyathostomin species from horses.

Here, we report evaluation of five oligoprobes designed from intergenic spacer (IGS) region sequences for identification of cyathostomin species. Oligoprobes were designed for identification of Cylicocyclus ashworthi, Cylicocyclus nassatus, Cylicostephanus longibursatus, Cylicostephanus goldi and a fifth probe designed to identify all members of this tribe. PCR amplification of IGS DNA from 16 cyathostomin species allowed sequence comparison and identification of four putative species-specific probes. Southern blotting of amplified products from 16 species showed that all probes were species-specific. The fifth probe recognised all 16 cyathostomin species but did not bind to members of the genus Strongylus. Furthermore, these probes were used to identify individual infective L3, eggs and L4 indicating that they will be invaluable to furthering the study of the epidemiology and pathogenesis of these important equine nematodes.

Animals↗

Co-evolutionary relationships between the nematode subfamily Cloacininae and its macropodid marsupial hosts.

Morphologically based phylogenies of the cloacinine genera Cyclostrongylus, Macropostrongylus, Pharyngostrongylus, Popovastrongylus, Rugopharynx, Thallostonema, Wallabinema, and Zoniolaimus were constructed and compared with the phylogeny of their respective macropodid hosts. These comparisons show some evidence of co-speciation. However, there was little consistency among trees of different nematode genera, parasite species were scattered amongst hosts and basal parasite taxa were, in some instances, parasitic in hosts belonging to derived clades. A cladistic analysis, using as characters 208 cloacinine nematode species found in 23 species of host, produced a tree largely resembling that of the host tree but with significant differences explainable by host switching among macropodids occupying similar habitat. Nematodes were moderately host-specific, but some species occurred in three or more distantly related host species indicating a degree of host switching. The results are more consistent with the hypothesis of a colonisation of macropodid hosts by cloacinine nematodes rather than a prolonged period of co-speciation although alternative interpretations of the data are also considered.

Animals↗

Molecular delineation of Cylicocyclus nassatus and C. ashworthi (Nematoda:Strongylidae).

The nucleotide sequences of the first internal transcribed spacer (ITS-1), 5.8S gene and second internal transcribed spacer (ITS-2) of ribosomal DNA have been determined for Cylicocyclus nassatus, C. ashworthi and C. insignis. Pairwise comparisons revealed sequence differences between the taxa ranging from 3.8 to 6.2% for the ITS-2 and 2.2-2.7% for the ITS-1. For the ITS-1, the level of the sequence difference between C. ashworthi and C. nassatus (2.2%) was equivalent to that between C. nassatus and C. insignis (2.2%), indicating that C. ashworthi and C. nassatus represent separate species. Theoretical restriction maps were constructed from the sequence data, and a polymerase chain reaction-linked restriction fragment length polymorphism (PCR-linked RFLP) technique was established to unequivocally distinguish C. ashworthi from C. nassatus.

Animals↗

Phylogenetic relationships of Australian strongyloid nematodes inferred from ribosomal DNA sequence data.

The sequence of the second internal transcribed spacer of the ribosomal DNA was determined for the following strongyloid nematodes: Cylicocyclus insignis, Chabertia ovina, Oesophagostomum venulosum, Cloacina communis, Cloacina hydriformis, Labiostrongylus labiostrongylus, Parazoniolaimus collaris, Macropostrongylus macropostrongylus, Macropostrongylus yorkei, Rugopharynx australis, Rugopharynx rosemariae, Macropostrongyloides baylisi, Oesophagostomoides longispicularis and Paramacropostrongylus toraliformis, and compared with published sequences for species of Strongylus and for Hypodontus macropi. The resultant phylogenetic trees supported current hypotheses based on morphological evidence for the separation of the families Strongylidae and Chabertiidae, but did not support the separation of the endemic Australian genera as a distinctive clade within the Chabertiidae. The implications of this finding for the phylogenetic origins of the Australian strongyloids are discussed.

Animals↗