Differences in the second internal transcribed spacer (ITS-2) of eight species of gastrointestinal nematodes of ruminants.

Genetic differences in the nucleotide sequence of the second internal transcribed spacers (ITS-2) among Trichostrongylus axei, Trichostrongylus colubriformis, Ostertagia ostertagi, Cooperia oncophora, Cooperia punctata, Nematodirus helvetianus, Nematodirus filicollis, and Haemonchus contortus are described. The ITS-2 sequences of the 8 species ranged between 230 and 241 base pairs in length. Sequence similarities between the different genera varied between 60% and 80%. Identities between the different species within a genus varied between 99% for C. oncophora and C. punctata, 95% for T. axei and T. colubriformis, and 89% for N. helvetianus and N. filicollis. The ITS-2 sequences proved to be useful for species differentiation. Except for the species of Cooperia (2.07% intraspecific variations for C. oncophora and 0.83% for C. punctata) the degree of intraspecific variations (N. filicollis 0.85%, T. colubriformis 1.26%, T. axei 1.27%, H. contortus 2.60%, O. ostertagi, and N. helvetianus no variation) was markedly lower than the interspecific variations allowing a reliable differentiation within the ITS-2 region between single species.     

Observations on the life-cycle and larval morphogenesis of Cooperia fuelleborni (Nematoda: Trichostrongyloidea) parasitic in impala, Aepyceros melampus

The previously unknown life-cycle of Cooperia fuelleborni, parasitic in impala, is described. The morphology of the developmental stages are compared to those of other Cooperia spp. The similarity between C. fuelleborni and C. curticei is discussed as well as the differences between those two species. The morphological studies showed that C. fuelleborni is closely related to C. curticei of sheep, but also has some affinity with the cattle parasite C. punctata and C. oncophora.     

T-cell mediated immune responses in calves primary-infected or re-infected with Cooperia oncophora: similar effector cells but different timing

Cooperia oncophora is the most prevalent intestinal nematode of cattle occurring in Western Europe. Primary infection with 100000 third stage infective larvae (L3) induces acquired immunity in a high proportion of the animals but there is little information on immunity against re-infection. In the current experiment, the contribution of the T-cell mediated immunity in protection against re-infection with C. oncophora was investigated in detail. Priming elicited long-lasting protective immunity that was evidenced by a significantly decreased worm burden and egg excretion in primed animals compared to challenge control animals. Lymphocyte proliferation tests with excretory/secretory products (ESP) of C. oncophora and with three distinct ESP fractions indicated an enhanced reactivity in primed animals and suggested that by fractionating of ESP we selected for proteins involved in protective immunity against re-infection with C. oncophora. Phenotypic analysis of T cell subsets at diverse anatomical locations revealed that the enhanced reactivity of lymphocytes from peripheral blood and lymph nodes of the infected animals coincided with a significantly increased frequency of CD4(+) cells at these locations but a deceased frequency of CD4(+) cells in the lamina propria. These findings were independent of the immune status of the animals but more pronounced in the primed animals than in the challenge control animals. In addition we demonstrated that primary and secondary infections with C. oncophora were associated with two waves of eosinophils and that the kinetics of this cell population differed as a result of priming. Based on the observed correlations we propose that the early increase of eosinophils is T cell independent and merely a consequence of inflammation in the parasitised gut. In contrast, the second wave of eosinophils depends upon CD4(+) cells and correlations with parasitological parameters at this time point support a role of eosinophils as effector cells against adult stages of C. oncophora.     

In vitro interaction of Brazilian strains of the nematode-trapping fungi Arthrobotrys spp. on Panagrellus sp. and Cooperia punctata

In vitro tests were carried out to verify the activity of 26 Brazilian isolates of predatory fungi of the genus Arthrobotrys on a free-living nematode (Panagrellus sp.) and on infective larvae of Cooperia punctata, a parasitic gastrointestinal nematode of cattle. The results showed that the free-living nematode Panagrellus sp. was the most preyed upon, compared to C. punctata, for all the fungal treatments. Also, variable predatory capacity was observed for different fungal isolates belonging to the same genus when applied to different nematode species.     

Differential recoveries from faecal cultures of larvae of some gastro-intestinal nematodes of cattle

Faecal cultures were established using bovine faeces containing known numbers of eggs from either Oesophagostomum radiatum, Haemonchus placei, Cooperia pectinata or a mixture of all three. A substantially greater percentage of larvae was recovered from cultures of O. radiatum and C. pectinata than was recovered from cultures of H. placei. The same pattern was observed in mixed cultures although yields of larvae from all species in mixed cultures were significantly reduced (p less than 0.001). The lower recovery of H. placei was not associated with a lower viability of H. placei eggs. Of the three different methods of harvesting larvae, the jar and mesh recovery technique was the least effective and significantly fewer larvae were recovered with this technique than with the Baermann and inversion techniques (p less than 0.05). The results are discussed with reference to the use of faecal culture and larval differentiation in the diagnosis of mixed species nematode infections of cattle.     

Use of a pre-selected epitope of cathepsin-L1 in a highly specific peptide-based immunoassay for the diagnosis of Fasciola hepatica infections in cattle.

A peptide-based indirect ELISA to detect cattle antibodies against Fasciola hepatica was developed and evaluated for its sensitivity and specificity. An immunogenic antigen released in vitro by F. hepatica was purified. After purification the sequence of the first 20 N-terminal aa of this protein showed considerable homology with cathepsin L-like proteinase. Based on its homology with cathepsin-L1, we further focused on this protein for diagnostic purpose. Predicted B-cell epitopes of cathepsin-L1 were synthesised as single synthetic peptides and tested with respect to their diagnostic potential. An indirect ELISA based on one of these peptides was (i) evaluated further and (ii) compared to the potential of an indirect ELISA with excretion/secretion antigens from adult F. hepatica, or (iii) purified cathepsin-L1. Specificity and sensitivity of the three ELISAs were assessed using sera from calves experimentally infected with pure isolates of Dictyocaulus viviparus, Ostertagia ostertagi, Cooperia oncophora, Nematodirus helvetianus, Schistosoma mattheei, Ascaris suum, Taenia saginata or F. hepatica, respectively, and sera from parasite-naive calves. In addition, sera were analysed from calves naturally infected with F. hepatica. The sensitivities of all three ELISAs were also very high, 98.9% (i), 100% (ii) and 100% (iii). The specificity of the peptide ELISA was very high, 99.8%, whereas specificities of the ES antigens and cathepsin-L1 ELISAs were only 82.8% and 94.6%. In experimentally infected cattle, F. hepatica-specific antibodies were first detected between days 21 and 28 p.i. with all three ELISAs, and the antibody levels persisted in the peptide ELISA until day 183 p.i. All sera from naturally infected calves were positive in the peptide ELISA. These results demonstrate that the peptide-based F. hepatica ELISA is a useful method for detecting antibodies in the sera from cattle infected with F. hepatica. This type of immunodiagnostic will therefore contribute to more accurate diagnosis and to timely curative treatment of animals.     

Titration of subcutaneously administered eprinomectin against mature and immature nematodes in cattle.

Eprinomectin has been approved for use as a topically applied endectocide for beef and dairy cattle. To determine if eprinomectin has utility as an injectable anthelmintic, it was titrated at 0.05, 0.1, and 0.2 mg/kg s.c. against adult (Trial 1) and at 0.05, 0.1, 0.14, and 0.2 mg/kg s.c. against immature (Trial 2) stages of lung and gastrointestinal nematodes in cattle. In Trial 1, every dose of subcutaneously delivered eprinomectin showed maximal or near-maximal (> or = 99%) efficacy against Haemonchus placei, Ostertagia ostertagi, Trichostrongylus axei, T colubriformis, Cooperia punctata, Nematodirus helvetianus, Oesophagostomum radiatum, and Dictyocaulus viviparus. Adult C. oncophora was the only exception. However, even against this species, the lowest dose of 0.05 mg/kg showed 93% efficacy, and the efficacious dose necessary to kill 95% (ED95) of adults was 0.056 mg/kg. In Trial 2, every dose of subcutaneously delivered eprinomectin showed maximal or near-maximal (> or = 99%) efficacy against the immature stages of all of the above species of endoparasites. As a result, ED95 values could not be calculated. Consequently, the exquisite potency against endoparasites through parenteral administration suggests that eprinomectin may also have potential utility as an injectable product for cattle.     

Histopathological changes during experimental infections of calves with Cooperia punctata

Eleven male two-month-old Holstein calves were used to determine the pathological changes induced by a Cooperia punctata infection. After weaning, ten calves received a single oral dose of 45,000 C. punctata infective larvae. One calf remained as a non-infected control. Groups of two calves were killed on days 7, 14, 21, 28 and 35 post-infection (p.i.) for determination of worm burdens and histopathological evaluation. The small intestine was sub-divided into three sections of approximately equal length, and representative samples of mucosa were fixed in 10% formalin, cut, and stained with haematoxylin-eosin. Samples of intestinal contents and mucosal digests were taken and fixed in 10% formalin for an estimation of total worm burdens. An increase in the number of adult parasites and a decrease in the number of larvae were observed with time (P<0.001). A higher concentration of worms was found in the first segment of the small intestine during the five weeks of observation. Histology showed larvae in the intestinal mucosa on day 7 p.i., with a discrete increase in the cellular response. Adult worms and a marked cellular infiltrate with eosinophils and neutrophils were present on day 21 p.i., and these persisted until day 35 p.i. Microcysts resulting from worm destruction were observed from day 21 p.i.     

The comparative efficacy of oxfendazole administered as bolus and suspension to naturally infected sheep in Greece

In a flock of 20 ewes naturally infected with those parasites of sheep most common in Greece, and kept indoors during the whole trial, oxfendazole at the dose rate of approximately 2.9 and 2.8 mg/kg body-weight was tested as a 4 g bolus containing 112 mg active ingredient and a 2.265% suspension. The evaluation of its efficacy was based on the necropsy findings which were also supported by faecal egg counts. No differences in efficacy were noticed between the two formulations of the drug. Both bolus and suspension proved to be 100% effective against Haemonchus contortus, Ostertagia circumcincta, Trichostrongylus axei, T. colubriformis and Chabertia ovina. The efficacy against Cooperia oncophora, Nematodirus spathiger, Bunostomum trigonocephalum, Oesophagostomum columbianum and Moniezia expansa could not be evaluated, because these species, though not found in any of the treated animals, were found in fewer than three controls.     

Field experiments on the ability of Arthrobotrys oligospora (Hyphomycetales) to reduce the number of larvae of Cooperia oncophora (Trichostrongylidae) in cow pats and surrounding grass

In field experiments, conducted on parasite-free grass plots in two consecutive summers, artificially prepared cow pats containing Cooperia oncophora eggs were inoculated with the nematode-trapping fungus Arthrobotrys oligospora. Numbers of infective C. oncophora larvae isolated from the pats as well as from the surrounding herbage were subject to an approximately ten-fold reduction as compared with numbers in fungus-free pats and herbage surrounding these. This reduction was undoubtedly a result of entrapment of the parasite larvae within the faecal pats.     

Interspecific competition between the nematode-trapping fungus, Duddingtonia flagrans, and selected microorganisms and the effect of spore concentration on the efficacy of nematode trapping

The fungus, Duddingtonia flagrans, is able to trap and kill free-living nematode larvae of the cattle parasite Cooperia oncophora when chlamydospores are mixed in cattle faeces. Isolates of Bacillus subtilis (two isolates), Pseudomonas spp. (three isolates) and single isolates of the fungal genera Alternaria, Cladosporium, Fusarium, Trichoderma and Verticillium were isolated from cattle faeces and shown to reduce D. flagrans growth on agar plates. When these isolates were added to cattle faeces containing D. flagrans and nematode larvae of C. oncophora, developing from eggs, none of the isolates reduced nematode mortality attributed to D. flagrans. Similarly, the coprophilic fungus Pilobolus kleinii, which cannot be cultivated on agar, also failed to suppress the ability of D. flagrans to trap and kill developing larvae of C. oncophora. Increasing chlamydospore doses of D. flagrans in faecal cultures resulted in higher nematode mortality. Thus, no evidence of interspecific or intraspecific competition was observed. The consequences of these findings are discussed.     

Field experiment on the ability of earthworms (Lumbricidae) to reduce the transmission of infective larvae of Cooperia oncophora (Trichostrongylidae) from cow pats to grass

In Denmark earthworms play an important role in the disappearance of cow pats on pastures. Field investigations were designed to study the influence of earthworms on the transmission of infective Cooperia oncophora larvae from experimental cow pats to grass. Results from the present experiment showed that cow pats, protected from attack by earthworms, disappeared at a much lower rate than unprotected cow pats. Development in faeces and transmission to grass of C. oncophora larvae were disturbed by the disintegration of cow pats. The rapid disintegration of unprotected cow pats resulted in an approximately 50% reduction of infective C. oncophora larvae on grass in the vicinity of these cow pats, as compared with larval contamination of grass around protected cow pats. How earthworms may cause a reduction in the transmission of infective C. oncophora larvae to grass by creating adverse conditions for the survival of parasite larvae in the cow pats, by eating and killing the parasite larvae, and by depositing parasite larvae in the soil is discussed.     

Cryptosporidium bovis n. sp. (Apicomplexa: Cryptosporidiidae) in cattle (Bos taurus)

A new species of Cryptosporidium, C. bovis, is described. Oocysts of C. bovis, previously identified as Cryptosporidium genotype Bovine B (GenBank AY120911), are morphologically indistinguishable from those of C. parvum. They are excreted fully sporulated and contain 4 sporozoites, but lack sporocysts. Oocysts measure 4.76-5.35 microm (mean = 4.89 microm) x 4.17-4.76 microm (mean = 4.63 microm), with a length-to-width ratio of 1.06 (n = 50). Oocysts were not infectious for neonatal BALB/ c mice, but were infectious for 2 calves that were previously infected with C. parvum. Oocysts were not infectious for 2 experimentally exposed lambs less than 1 wk of age and were not detected in 42 lambs 2-3 mo of age, but were detected in a 2-wk-old lamb. In an earlier study, 79 of 840 calves on 14 dairy farms in 7 states were found infected with the new species. Most calves were 2-7 mo of age and none exhibited signs of diarrhea. This new species has been found in 10 of 162 calves aged 9 to 11 mo on a beef farm in Maryland. Fragments of the 18S rDNA, HSP-70, and actin genes were amplified by PCR, and purified PCR products were sequenced. Multilocus analysis of the 3 unlinked loci demonstrated the new species to be distinct from C. parvum and also demonstrated a lack of recombination, providing further evidence of species status. Based on these biological and molecular data, we consider this highly prevalent Cryptosporidium that infects primarily postweaned calves to be a new species and propose the name Cryptosporidium bovis n. sp. for this parasite.     

Experimental investigations on the effectiveness of fenbendazole in parasitic helminths of the stomach, intestines and lung of cattle (author's transl)]

Helminth-free bulls were injected artificially with Ostertagia ostertagi (120 000 L3), Cooperia oncophora (240000 L3), Haemonchus spec. (1 000 L3) and/or Dictyocaulus viviparus (60 L3/kg) orally or intraruminally. The animals were treated with fenbendazole during the prepatent period or after having reached maturity. The effect of 5 mg fenbendazole/kg p.o. on 3-d, 10-d- and adult stages of Ostertagia ostertagi, Haemonchus spec. and Cooperia oncophora ranges between less than 94% and 100%. Particularly noticeable was the effect on 10-d-old Ostertagia ostertagi with less than 94%. 5 mg/kg fenbendazole orally removed nearly 100% of 6-d-, 13-d-, and adult stages of Dictyocaulus viviparus. Fenbendazole may be administered as a drench or as medicated feed. The safety of the compound permits the administration of excessively high doses without clinical signs.     

Yeasts in the gastrointestinal tract of preweaned calves and possible involvement of Candida glabrata in neonatal calf diarrhea

To examine the possibility of a mycotic involvement in neonatal calf diarrhea (NCD) the presence of fungi was assessed in (a) the intestinal contents of dead calves and fecal samples submitted for routine laboratory examination, (b) fecal specimens, sampled once in winter and once in summer, of calves raised on 2 farms with different management systems, and (c) mucosal scrapings of various segments of the digestive tract of a diarrheic calf, massively shedding Candida glabrata.C. glabrata was the most prevalent fungal species isolated from the routine samples. It was the only fungus which was shed by the calves on the 2 farms, for continuous, more or less prolonged periods, but exclusively in the winter months. Diarrhea and C. glabrata shedding seemed to be associated. C. glabrata colonized the abomasum (the functional equivalent of the monogastric stomach) but not the other segments of the digestive tract of the euthanized calfBased on the findings of this study it seems that while some yeast species may be considered as commensals of the digestive tract of calves, and consequently their isolation from intestinal contents or fecal samples has no clinical significance, others, such as C. glabrata may be involved in enteric pathogenic processes. Moreover, characteristics of the culture, previous chemotherapeutic treatments, the animal's age and possibly climatic conditions should be taken into account before deciding on the fungal isolate's clinical relevance. Determination of mycotic involvement in NCD by routine mycological examination of intestinal contents and fecal samples of diarrheic calves may be useful to avoid unnecessary and potentially harmful antibacterial therapy.This revised version was published online in October 2005 with corrections to the Cover Date.     

B cells and antibody response in calves primary-infected or re-infected with Cooperia oncophora: influence of priming dose and host responder types

We investigated whether the generation of protective memory humoral immunity in Cooperia oncophora infected calves occurs in a dose-dependent way and whether it depends on the animal responder types. To this end, serum and mucus antibody responses were measured in animals primary-infected with 30000 or 100000 L3, treated with anthelmintics and subsequently challenged with 100000 L3. A detailed phenotypic and functional analysis of B cells was done in animals infected once or twice with 100,000 L3. Based on the similarity in parasitological variables of animals primed with 30000 or 100000 L3, we concluded that with these doses priming conferred protection in a dose-independent way. Upon challenge significant increases in Cooperia-specific serum and mucus IgG1 and IgA and total serum IgE titres were induced in primed animals in a dose-independent way. In contrast, intermediate and low responders differed in the onset of the production of Cooperia-specific serum IgG1. Furthermore, not only the onset but also the level of total serum IgE significantly differed between intermediate and low responders. Phenotypic and functional analysis of B lymphocytes revealed that (i). priming induced the generation of memory B cells which upon challenge readily differentiated into antibody secreting cells; (ii). sensitised B cells were more efficiently recruited to the intestinal effector sites; (iii). based on the expression of CD62L and CD86 two distinct B cell subpopulation could be differentiated. CD62L(+)CD86(-) B cells that were likely lymphocytes not yet activated and with an enhanced recirculation capacity, and CD62L(-)CD86(+) B cells that were activated B cells with a reduced recirculation ability; and finally (iv). the increased expression of CD86 and subsequent correlations with parameters of the T helper 2 immune response induced by C. oncophora, suggested that CD86- interactions are involved in the generation of protective immunity against Cooperia.     

Coccidia of the domestic goat (Capra hircus) in Saudi Arabia.

Faeces of 228 domestic goats (Capra hircus) from the central region of Saudi Arabia were examined for the presence of coccidian oocysts. Ten species of coccidia were identified and described. A total of 90.3% of the specimens were positive, most of them contained 100-1000 oocysts per g of faecal sample. Kids less than 1 year old had higher oocyst counts than goatlings or adult goats. Mixed infections with three to five species were found in 69.7% of the specimens and six to eight species were found in 10.1%. Eimeria arloingi and E. hirci were most prevalent. E. alijevi, E. ninakohlyakimovae, E. caprina, E. christenseni and E. apsheronica were less common. E. jolchijevi, E. caprovina and E. punctata were relatively rare.     

Helminths of roe deer (Capreolus capreolus) in the Middle Black Sea Region of Turkey

Fifteen roe deer were examined at necropsy from Northern Turkey in the period 2006-2010 for the helminth infections. Totally 6470 helminth specimens were collected and identified by morphological criteria. Twenty-five helminth species were identified (1 of the Class Trematoda, 1 of Cestoda and 23 of Nematoda). Dicrocoelium dendriticum (Prevalence 20%) was found in liver. Cysticercus tenuicollis (6.6%) was found in mesentery. Haemonchus contortus (53.3%), Ostertagia leptospicularis (73.3%), O. leptospicularis (minor morph: kolchida) (53.3%), Ostertagia ostertagi (26.6%), Spiculopteragia spiculoptera (66.6%), S. spiculoptera (minor morph: mathevossiani) (6.6%), Teladorsagia circumcincta (40.0%), T. circumcincta (minor morph: davtiani) (6.6%), T. circumcincta (minor morph: trifurcata) (6.6%), Trichostrongylus axei (66.6%) were found in abomasum. Trichostrongylus andreevi (6.6%), T. colubriformis (6.6%), T. longispicularis (26.6%), T. vitrinus (40.0%), T. capricola (6.6%), Cooperia oncophora (26.6%), C. punctata (6.6%), Nematodirus filicollis (66.6%), and Capillaria bovis (26.6%) were found in small intestine. Oesophagostomum venulosum (46.6%), Chabertia ovina (26.6%), and Trichuris ovis (13.3%) were found in large intestine. Dictyocaulus capreolus (6.6%) was found in lungs.     

The influence of weather and egg contamination on the development of third-stage larvae of Cooperia oncophora on pasture.

The influence of weather and egg contamination on the dynamics of herbage contamination with infective larave of Cooperia oncophora was investigated on artificially contaminated grass plots and in a grazing experiment with 24 first-year grazing calves from May to October 1987 in Lower Saxony, Germany. On the experimental plots the larval translation was highest at the beginning of July and in the second part of September, following high mean weekly temperatures. Between July and September peak recovery of larvae from herbage occurred 4 weeks after contamination. A seasonal pattern of larval translation similar to that on the experimental plots could be demonstrated on the grazed pastures when the number of larvae per m2 of pasture had been adjusted to the previous egg output by means of a contamination index. The resulting 'relative larval density' is regarded as a good indicator for larval development on pasture. From July to September the larval population on pasture resulted mainly from the egg contamination 2-3 weeks earlier. The short persistence of the infective larvae on herbage was probably due to the frequent and heavy rainfall throughout the season, causing a passive washout of larvae into the soil. On single pastures the larval density started to increase within 1 week after the calves had first contact with these fields. The impact of the calves on the distribution of larvae is discussed.