Antibody responses of ponies to initial and challenge infections of Strongylus vulgaris

An indirect fluorescent antibody assay (IFA) was developed using Strongylus vulgaris third stage larvae (L3) as antigens. Observations using the IFA indicate that a species-specific antibody response to S. vulgaris L3 develops in S. vulgaris-infected ponies and that some surface L3 antigens are shared by adult worms. Sequential antibody levels against S. vulgaris were measured in strongyle-naive and in immune ponies following initial and challenge infections using the IFA and an indirect hemagglutination assay (IHA). Antibody levels measured by IFA increased faster following initial infections than did levels measured by IHA. Antibody levels appear to increase following challenge infections of immune ponies when measured with the IFA, but not with the IHA. Significant differences in antibody titers were not seen between ponies which developed colic following challenge infections and those that did not develop colic. Antibodies were not detectable in ponies unexposed to larval migrations, but which received surgical implantation of S. vulgaris adults into the cecum.     

Purification and analyses of the specificity of two putative diagnostic antigens for larval cyathostomin infection in horses

Cyathostomins are important equine gastrointestinal parasites. Mass emergence of mucosal stage larvae causes a potentially fatal colitis. Mucosal stages are undetectable non-invasively. An assay that would estimate mucosal larval stage infection would greatly assist in treatment, control and prognosis. Previously, we identified two putative diagnostic antigens (20 and 25 kDa) in somatic larval preparations. Here, we describe their purification and antigen-specific IgG(T) responses to them. Western blots confirmed the purity of the antigens and showed that epitopes in the 20 kDa complex were specific to larval cyathostomins. No cross-reactive antigens appeared to be present in Parascaris equorum or Strongyloides westeri species. Low levels of cross-reactivity were observed in Strongylus edentatus and Strongylus vulgaris species. Use of purified antigens greatly reduced background binding in equine sera. These results indicate that both antigen complexes may be of use in a diagnostic assay.     

Critical anthelmintic trials in ponies with oxfendazole and caviphos and concomitant studies on the spontaneous elimination of small strongylids.

The efficacy of the benzimidazole, oxfendazole, and the organophosphate, caviphos, against gastrointestinal parasites of ponies was evaluated by the critcial test method. Oxfendazole (10 mg/kg of body weight) given in single oral doses was 100% effective against adult large strongylids (Strongylus vulgaris, Strongylus edentatus, and Strongylus equinus), 99% effective against adult small strongylids, and 97% effective against 4th-stage small strongylids (genera identified in order of frequency: Cylicostephanus, Cyathostomum, Cylicocyclus, Triodontophorus, Poteriostomum, Oesophagodontus, Cylicodontophorus, Gyalocephalus, and Craterostomum). Caviphos (40 mg/kg of body weight) admixed in the grain ration (horse crunch) was 89% effective against adult large strongylids (S vulgaris and S edentatus) and 99% effective against adult small strongylids (genera identified earlier in order of frequency above), but only 35% effective against 4th-stage small strongylids. Both drugs were effective (100%) against adult and immature pinworms (Oxyuris equi) but ineffective against Habronema spp and Draschia megastoma. Oxfendazole was only 11% effective against stomach bots (Gasterophilus spp); caviphos was 73% effective against these species. During a three-day pretreatment interval, about 34% of the total population of small strongylids was lost spontaneously from the 29 ponies.     

Serine protease activity in excretory-secretory products of Oestrus ovis (Diptera: Oestridae) larvae

The sheep bot fly, Oestrus ovis, is a very common myiasis of nasal and sinus cavities of sheep and goats causing severe welfare and production implications. As the viability of O. ovis adult flies strictly depends on larval abilities to assimilate and to stock nutrients from the host, it was necessary to investigate proteolytic activities in larval excretory/secretory products (ESP). ESP of O. ovis larvae degrade mucosal and plasmatic components such as mucin, albumin or immunoglobulin G. A preliminary biochemical characterization, using substrate gel analysis and inhibitor sensitivity, demonstrated the presence of at least six major serine proteases (molecular weights from 20 to 100 kDa), mainly trypsin-like, secreted in the digestive tube of larvae. Their involvement in larval trophic activity and evasion from the host immune response is further discussed as O. ovis excretory/secretory serine proteases could represent potential vaccinal targets.     

Assessing the agreement between Ostertagia ostertagi ELISA tests performed using the crude adult antigen and the adult and larval stage 4 excretory/secretory antigens

The objective of this study was to determine the agreement between ELISA tests conducted using three O. ostertagia antigens: crude adult worm, larval stage 4 (L4) excretory/secretory (ES) and adult ES. This study was carried out on 289 Holstein cows from five herds in Prince Edward Island and one herd in Nova Scotia. Composite milk samples of these cows were collected (between May and September 2002) from the respective provincial laboratories and sent to the Atlantic Veterinary College where each sample was tested for antibodies to O. Ostertagi using an indirect microtitre ELISA test. Results were expressed as optical density ratio (ODR) values. Each milk sample was tested with three ELISA tests, with each test using a different O. ostertagi antigen. There was a slight rise in ODR values of both adult antigens, between May and August, with higher values obtained using the adult ES antigen. L4 ES ODR values were generally higher than those for both adult antigens during the study period, except for May. There was a more dramatic rise in L4 ES ODR values between May and August. Rises in ODR in May and end of July coincided with periods of mass maturation of L4 to adult worms. The results of the study showed that the concordance correlation coefficient (CCC) between tests performed using both ES and the crude antigens were low (crude adult versus adult ES=0.31, crude adult versus L4 ES=0.30). The highest CCC was observed between tests done using both ES antigens (CCC=0.56). Generally, the study results suggest that the antibody response (detectable by the ELISA) is mainly directed against ES antigens (especially L4) than the crude adult worm antigen.     

An evaluation of the efficacy of oxfendazole against the common nematode parasites of the horse.

In a controlled trial in naturally-infected young ponies, oxfendazole administered orally at dose-rates of 10 mg per kg and 50 mg per kg resulted in complete elimination of Trichostrongylus axei, Parascaris equorum, Oxyuris equi and adult Strongylus vulgaris. Also, all migrating Strongylus edentatus larvae recovered from the subperitoneal tissues of the flank were found to be dead. Minimum efficiencies of 99.8 per cent and 99.1 per cent were obtained against adult small strongyles (Trichonema spp) and 97.6 per cent and 100 per cent of developing small strongyle larvae at dose-rates of 10 mg per kg and 50 mg per kg respectively. Although the arterial lesions caused by migrating S vulgaris larvae were less severe in the treated compared with the untreated animals, reductions in mean larval numbers over controls were only in the region of 49 to 59 per cent.     

Serum protein changes in ponies on different parasite control programmes

Serum protein responses were examined in 52 ponies divided into five groups and subjected to various control strategies that resulted in pasture infectivity ranging from 706 to 18,486 infective third stage, cyathostome and Trichostrongylus axei larvae per kilogram of herbage (L3/kg) by 17 September 1984. Major protein changes occurred only in young ponies (Groups 4 and 5) and were observed before exposure to maximum numbers of pasture larvae (Group 4; 10,210 L3/kg, Group 5: 10,042 L3/kg) on 17 September. It appeared that a primary infection of T axei was a greater stimulus to serum beta-globulin and immunoglobulin (Ig)G(T) responses that provided by continued infection with cyathostome (small strongyle) worms. The large strongyles (Strongylus vulgaris, S edentatus and S equinus) were not detected in any larval cultures or on pastures grazed by the young ponies. A fall in beta-globulin and IgG(T) concentrations of Group 5 ponies one month after treatment with ivermectin indicated a larvicidal action against T axei and/or the cyathostomes. A subsequent rise in serum albumin concentrations of Group 5 ponies suggested that a protein-losing gastroenteropathy had been alleviated by the larvicidal action of ivermectin. Mature control ponies (Group 1) showed little beta-globulin response and only a modest IgG(T) response in six of the 10 ponies after exposure to heavily infected lawns (18,486 L3/kg) in September 1984. It was concluded that serum protein and IgG(T) responses were of limited value as an aid to diagnosis of parasitism because of numerous difficulties of interpretation.     

The activity of closantel as an equine antiparasitic agent

Eighteen pony foals were experimentally infected with 500 third stage larvae of Strongylus vulgaris at 2 weeks, and at 2, 4, 6 and 8 months after birth. For the duration of the study, all foals were kept in the same pasture with their mothers to allow natural infection with other parasites by exposure to a contaminated environment. Twelve of the foals were utilized in groups of 3 and treated orally five times at two month intervals starting at one month of age with closantel at doses of 5, 10, 20 or 40 mg kg-1. Ten months after birth the foals were necropsied to determine the parasitic burdens in the gastrointestinal tracts and the cranial mesenteric arteries. The results indicate a high antiparasitic activity of closantel against larval stages of Gasterophilus intestinalis and S. vulgaris, as well as against adult S. vulgaris, S. edentatus, Anoplocephala perfoliata and Triodontophorus spp., when used at doses of 20 or 40 mg kg-1.     

The efficacy of fenbendazole in the control of immature strongyle infections in ponies

The efficacy of fenbendazole against immature stages of Trichonema spp., Strongylus vulgaris and Strongylus edentatus was evaluated. Naturally infected 6 to 12 month old ponies were given single, oral doses of 0, 15, 30 and 60 mg/kg of body weight. A dose response relationship was noted between increasing dose levels and efficiency against larval trichonemes and migrating stages of S. vulgaris and S. edentatus. Dose levels of 30 mg/kg and higher removed 93 per cent of mucosal stages of Trichonema spp., while doses of 60 mg/kg removed 83 per cent and 89 per cent of the migrating larvae of S. vulgaris and S. edentatus respectively.     

Precipitin response of the mitogen produced by Strongylus vulgaris arterial larvae.

The precipitin response of the mitogen produced by Strongylus vulgaris arterial larvae was investigated. IgG (T) from the sera of horses naturally infected with S. vulgaris adults and arterial larvae recognised the presence of two antigenic components of the mitogenic fractions. The results obtained seem to confirm that these antigens are immunogenic in stimulating the production of increased levels of IgG(T) in infected animals, and showed that the procedures could be used as immunological tools in the diagnosis of S. vulgaris infection.     

Detection of specific antibodies in dogs infected with Angiostrongylus vasorum

Canine angiostrongylosis, caused by the nematode Angiostrongylus vasorum, is an emerging cardiopulmonary disease in Europe which can be fatal if left untreated. We determined the diagnostic value of the specific detection of antibodies against A. vasorum adult somatic antigen, adult excretory/secretory (E/S) antigen and first stage larvae (L1) somatic antigen in ELISAs. Also, A. vasorum adult somatic antigen purified by monoclonal antibodies (mAb) was evaluated in a sandwich-ELISA. Among the crude antigens, the best sensitivities when testing 21 naturally infected dogs were obtained using adult E/S and somatic antigen (85.7% and 76.2%, respectively), which were comparable with the results of the sandwich-ELISA based on mAb-purified antigens (81%). The ELISA performed with L1 antigen had the lowest sensitivity (42.9%). In experimentally inoculated dogs, the sensitivities ranged from 97.7% to 100% with all test settings. The specificity was 98.8% (92.5-99.9%, 95% CI) with all ELISAs using sera of 82 randomly selected dogs. Cross-reactions using adult somatic, adult E/S and L1 somatic antigen were observed in sera of dogs infected with Crenosoma vulpis, Dirofilaria immitis, Dirofilaria repens, and Eucoleus aerophilus. In contrast, using the mAb-purified antigens, the cross-reactions were minimal. Depending on the antigens used, specific antibodies were detected starting between 13 and 21 days post experimental inoculation (dpi), and at latest between 35 and 48 dpi, thus before or around the onset of patency. The serological follow-up of four A. vasorum-infected dogs after anthelmintic treatment at 88 dpi showed a decrease of antibody levels after drug administration, and the animals became seronegative 2-9 weeks later. Two untreated dogs remained seropositive. In four dogs treated 4 dpi, virtually no antibody-reaction was detectable, with the exception of the ELISA performed with L1 antigen. The early detection of specific antibodies against A. vasorum by ELISA represents a valid alternative for a reliable diagnosis and for follow-up investigations after anthelmintic treatment.     

Strongylus vulgaris in donkeys (Equus asinus) from the highveld of Zimbabwe

Strongylus vulgaris populations in the cranial mesenteric arteries, caecum and colon were studied in 14 donkeys obtained from a communal area of the Zimbabwean highveld during July and November, 1986, and January and April 1987. Adult parasites were present in all animals and larvae in the cranial mesenteric arteries of 12 animals. Aged animals had high worm burdens. The number of adult parasites varied from 63 to 1255 (mean 382) and of larvae in the arteries from 0 to 181 (mean 69). The mean adult worm burdens were highest in July (400) and November (488), and lowest in April (107). The mean arterial larval burden was highest in July (130) and lowest in November (21). These observations indicate that infection with S. vulgaris takes place during the rainy season resulting in the heavy arterial larval population from January onwards and the heavy adult population during the dry season.     

Humoral immune response of turkeys to strain S6 and a variant Mycoplasma gallisepticum studied by immunoblotting.

Two putative variant Mycoplasma gallisepticum (MG) strains (M876 and M35), originally isolated from commercial turkeys, were compared with eight well-characterized MG strains by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE protein profiles indicated that the variant strains were correctly classified as MG based on homologous patterns in species-specific regions of the electrophoretic profiles. However, differences in protein profiles also indicated that variant strains M876 and M35 were different from each other and the other MG strains tested. Immunoblotting was used to assess the humoral immune response of turkeys to infection with the S6 reference strain or M876 variant strain of MG. Immunoblots using antisera to M876 showed that seroconversion to this isolate was slower, and to fewer MG proteins when compared with immunoblots using antisera to S6. Immunoblot analyses further indicated that pooled antisera from turkeys inoculated with either S6 or M876 reacted with each of 10 MG strains tested. However, pooled S6 antisera reacted with greater intensity and with more MG proteins than did pooled M876 antisera. The species-specific immunodominant proteins with the greatest potential for use as antigens in serologic tests appeared to be those of 64 (p64) and 56 (p56) kilodaltons molecular mass.     

Separation of a Soluble Antigen and Infectious Particles of Bovine Viral Diarrhea Viruses and their Relationship to Hog Cholera

A soluble antigen present in infectious tissue culture fluids was separated from the infective virus particle by ultracentrifugation of two serologically related strains of bovine viral diarrhea viruses, NADL-MD and Oregon C24V.

Neutralizing antibodies against the two viruses were absent in four hog cholera antisera, but present in significant titer in the commercially prepared antiserum. Precipitin tests utilizing the agar double diffusion technique formed a single line of identity between the concentrated soluble antigen of both viruses and NADL-MD and hog cholera antisera. No lines were observed using concentrated virus pellet and noninfected BEK cell antigens or control SPF calf and swine sera.

     

Effect of early season ivermectin and pyrantel treatments on strongylid infections in young Shetland ponies in The Netherlands

Two groups of three ponies were used to study the effect of three ivermectin or pyrantel treatments given at intervals of 5 weeks at the beginning of the grazing season. Although each pyrantel treatment resulted in a greater than 95% reduction in faecal egg counts during the first 3 weeks, high pasture larval counts were seen from the beginning of August onwards and substantial cyathostomine burdens were found at necropsy in December. The ivermectin treatments resulted in an even more pronounced reduction in faecal egg output, and the pasture larval counts and cyathostomine burdens at necropsy were considerably lower than in the pyrantel group. The proportion of inhibited early L3 of the cyathostomines was lower in the ivermectin than in the pyrantel group. Faecal egg output of the large strongyles was completely suppressed in the ivermectin group. Nevertheless, Strongylus vulgaris larvae were found in the arteries of all three ponies, possibly as a result of overwintering of infective larvae on pasture. In the pyrantel group, the egg output of Strongylus edentatus and, to a much lesser extent, Strongylus vulgaris, was not completely suppressed.     

Effects of repeated Strongylus vulgaris inoculations and concurrent ivermectin treatments on mesenteric arterial lesions in pony foals

Eight of 10 pony foals reared under helminth-free conditions were inoculated PO with 50 Strongylus vulgaris infective larvae/week for 4 weeks, at which time 1 foal died of acute verminous arteritis. Inoculation of 7 remaining foals continued at 2-week intervals for 20 weeks. Of the 7 foals, 3 were treated with ivermectin (0.2 mg/kg of body weight) in an oral paste formulation at experiment weeks 8, 16, 24; 4 foals were not treated. Two foals were not inoculated or treated and served as controls. After the first ivermectin treatment, ivermectin-treated foals had fewer days (12 +/- 2.9) with rectal temperatures greater than 38.6 C than did nontreated foals (23.3 +/- 3.8). Mean baseline rectal temperatures were 38 +/- 0.2 C. Adverse clinical reactions to ivermectin treatment were not observed in foals. Foals were euthanatized and necropsied 3 weeks after the last ivermectin treatment (week 24). Ivermectin was effective in reducing S vulgaris arterial larval and intestinal adult parasite numbers by 100% in 3 treated foals. Strongylus vulgaris arterial larvae and/or adults were recovered from all 4 nontreated inoculated foals. One nontreated inoculated foal lacked arterial larvae or active arterial lesions, indicating that protective resistance had developed in this individual. Marked gross and histopathologic lesions typical of chronic S vulgaris infection were observed in the 3 nontreated inoculated foals with arterial larvae. Repeated killing of intra-arterial S vulgaris fourth-stage larvae in ivermectin-treated foals did not exacerbate lesions associated with verminous arteritis or induce unique lesions associated with repeated destruction of arterial larvae.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterisation of humoral immune responses in dogs vaccinated with irradiated Ancylostoma caninum

Infection with Ancylostoma caninum, an intestinal hookworm of dogs, can cause debilitating and potentially life-threatening disease. In the current study, protective immunity to hookworm infection was induced in dogs following vaccination with irradiation-attenuated third-stage larvae (L3) with significant reductions in both worm (P<0.03) and faecal egg counts (P<0.0004) following a challenge infection. Vaccination with irradiated L3 and challenge with infective L3 stimulated a dominant antibody response to antigens of less than 20 kDa in an excretory/secretory extract from adult parasites. Immunoscreening of an adult A. caninum cDNA library with antisera from the vaccine trial identified a number of clones. The three clones with the strongest immunoreactivity proved to be identical and encoded a peptide with similarity to the N-terminal domain of the tissue matrix metalloproteinase inhibitor (TIMP)-2 mammalian tissue metalloproteinase inhibitor family.     

Antigenic similarity between squamous cell carcinoma of horn (horn cancer) and normal bovine foetal tissues.

Normal bovine foetal (liver and skin) and horn cancer tissue antigens were examined using double diffusion agar gel precipitation and immuno-electrophoretic tests to detect any cross reactivity among them. Rabbit horn cancer antisera absorbed with normal bovine liver, skin and horn core epithelium antigens, when tested with foetal skin and liver (4 to 6 months of gestation), revealed the presence of 2 foetal antigens in horn cancer. Immuno-chemically 2 of the horn cancer antigens were found to be identical to the bovine foetal antigens.     

Isolation and partial characterization of excretory/secretory antigens of Gastrothylax crumenifer

The present study was carried out to identify the excretory/secretory (E/S) antigens of the rumen infecting digenetic trematode Gastrothylax crumenifer that may be useful for the immunodiagnosis of rumen amphistomosis particularly during the pre-monsoon season during which this rumen parasite stops shedding eggs. The in vitro released E/S proteins were purified on a Sephadex G-200 column. The gel filtration profile revealed three distinct fractions F1-F3 where F1 and F3 appeared as sharp peaks while the F2 fraction was dispersed. The antibody titre against each of the purified E/S fractions was determined by ELISA using anti-whole E/S polyclonal antibodies raised in rabbit. Among the three fractions, the antibody titre against F1 was highest (1:12,800) whereas IgG titre was very low (1:50) for fraction F2 and F3 (1:100). Of the total polypeptides resolved on gradient SDS-PAGE, only a few antigenic polypeptides were detected in each fraction with hyperimmune anti-serum as revealed by Western Blot analysis. However, a 33 kDa antigen detected in each fraction appeared to be immunodominant which could be exploited for the diagnosis of the pouched amphistome.     

Internal parasites of horses on mixed grassveld and bushveld in Transvaal, Republic of South Africa

Between 1980 and 1982, the gastrointestinal tracts of 17 horses which had been grazing on mixed grassveld at Potchefstroom and bushveld at Onderstepoort in the province of Transvaal, Republic of South Africa, were examined at necropsy and processed for parasite recovery. The large strongyles and their prevalences were as follows: Strongylus vulgaris and associated lesions (88-94%), Strongylus edentatus (24%), Strongylus equinus (30%), Triodontophorus nipponicus (35%) and Craterostomum acuticaudatum (18%). The seven most prevalent and abundant cyathostomes collected were Cylicostephanus longibursatus, Cylicostephanus goldi, Cylicostephanus calicatus, Cylicocyclus nassatus, Cyathostomum catinatum, Cylicostephanus minutus and Cyathostomum coronatum. Gasterophilus intestinalis was the most prevalent and abundant botfly larva recovered. Most of the cyathostome larvae and adults were present during all seasons except winter. The distribution of cyathostome species in the large intestine of the host is discussed.