Clinical signs and hematologic, cytokine, and plasma nitric oxide alterations in response to Strongylus vulgaris infection in helminth-naïve ponies.

The objective of this study was to determine the effect of infection with Strongylus vulgaris on serum cytokines and plasma nitric oxide (NO) concentrations in helminth-naive ponies. Group 1 (n = 21) was given 500 S. vulgaris L3 larvae and group 2 (n = 7) received a saline control. Ponies were monitored daily for clinical signs, and blood was collected for complete blood cell counts and serum cytokines (TNF, IL-1, IL-6) quantification. Group 1 ponies were depressed, anorexic, and febrile for variable periods of time. Plasma NO was increased on day 21 in group 1 and on days 9 and 21 in group 2. Significant increases in total white blood cell counts, fibrinogen, and plasma protein concentrations in group 1 were found. Significant decreases in red blood cell counts and packed cell volume were also noted in group 1. There were no differences in serum cytokines across time in either group of ponies. Despite the lack of proinflammatory cytokine induction with the apparent inflammatory response to S. vulgaris there is evidence of a potential role of NO.

Impact of mixed strongyle infections in foals after one month on pasture.

Twelve Standardbred foals (age 3-6 months), with little previous exposure to parasites, were allocated to 2 groups and put onto pasture with low (Group L) or high (Group H) levels of larval contamination of large strongyles and cyathostomes. After 4 weeks grazing in September, the foals were housed indoors until necropsy 15 weeks later. Foals in Group H became clinically more affected than those of Group L in that they showed loss of vigour, weight gain depression, intermittent soft faeces and inappetence. One foal of Group H had persistent diarrhoea and was subjected to euthanasia 12 weeks after housing. Signs of colic were not observed. Faecal egg counts were significantly higher in Group H than in Group L (P<0.05). At necropsy, the mean number of S. vulgaris and cyathostomes was 20 and 18,000, respectively, in Group L, and 167 and 25,000 in Group H. Routine blood chemistry did not specifically reveal presence of S.vulgaris in pre-patency. A transient neutrophilia and eosinophilia, most prominent in Group H, was seen 2-8 weeks after start of exposure and anaemia was observed later in Group H. Serum albumin and albumin/globulin ratio were reduced, particularly in Group H, and a marked hyperbetaglobulinaemia was observed at 16-20 weeks in Group H. In conclusion, heavy infections with strongyles including S. vulgaris may become established in weaned foals after a brief period on pasture. Infections may be expressed clinically as debilitation, inappetence and intermittent diarrhoea without colic, and the need for control is imperative.

The pathogenic effects of experimental cyathostome infections in ponies.

Nine pony breed foals were reared indoors, then allocated to one of three groups infected with either 3.9 million (Group A) or 3.15 million (Group B) cold-conditioned third stage cyathostome larvae or kept as uninfected controls (Group C). The larvae were administered as a 'trickle' infection of 150000 larvae per dose, three times weekly. Blood biochemical and haematological analyses were performed weekly and faecal worm egg counts bi-weekly. Complete parasitological examinations were performed on all ponies at various times post-initial infection (PI): one infected animal at 9 weeks PI, four animals (three infected, one control) at 20 weeks PI and four animals (two infected, two controls) at 60-62 weeks PI. All ponies in the infected groups experienced a marked reduction in weight gain and two animals developed clinical disease: one pony developed intermittent diarrhoea and colic 8 weeks PI; another pony developed intermittent diarrhoea between 30 and 52 weeks PI. All infected ponies had decreased serum fructosamine concentrations and five had decreased serum albumin, which were first apparent 4-6 weeks PI. Alterations in the composition of serum globulins were detected in all ponies. Transient neutrophilia was observed in five animals from the infected groups 3-9 weeks PI. Serum alkaline phosphatase concentrations were increased in one pony between 30 and 60 weeks PI. During the course of the experiment, faecal samples from all infected animals were negative for worm eggs. At necropsy, cyathostome larvae were present within the mucosa of the large intestine of all infected ponies, however the mucosal larval counts varied considerably between individuals.

Fructosamine measurement in ponies: validation and response following experimental cyathostome infection.

Validation of an assay for measurement of fructosamine in equine serum and plasma utilised blood samples collected from 24 British native breed ponies. The results indicated that fructosamine can be measured easily using an assay which is precise and accurate. Paired plasma and serum fructosamine measurements were highly correlated, however, greater variations were observed within serum compared with those in plasma. A reference range for fructosamine in plasma was calculated to be 256.9 +/- 60.6 mumol litre-1 (mean +/- 2 SD). In order to assess the fructosamine response following experimental cyathostome infection, nine British native breed ponies were allocated to one of three groups: Group 1 (3.9 million third-stage cyathostome larvae (L3) over a nine-week-period), Group 2 (3.15 million L3 over seven weeks) and Group 3 (uninfected controls). From four weeks prior to infection, blood was obtained, once weekly, from all animals for measurement of plasma fructosamine, plasma albumin and serum globulin concentrations. Plasma fructosamine concentrations decreased in all infected ponies post-infection due to enteric protein loss and/or altered protein composition and/or increased protein turnover.

Immune responses of pony foals during repeated infections of Strongylus vulgaris and regular ivermectin treatments.

Ten helminth-free pony foals divided into three groups were used in this study. Eight foals were each experimentally infected per os with 50 Strongylus vulgaris infective larvae weekly for 4 weeks, at which time one foal died of acute verminous arteritis. The remaining seven foals subsequently received 50 S. vulgaris infective larvae every 2 weeks for an additional 20 weeks. Four of the infected foals remained untreated (Group 1) and three of the infected foals were given ivermectin at 8, 16 and 24 weeks post initial infection (Group 2). Two foals served as controls (Group 3). Foals in Group 1 developed eosinophilia, which was sustained throughout the course of infection. A mild eosinophilia also developed in Group 2 foals; however, the eosinophil numbers were markedly reduced for 3 weeks after each ivermectin treatment. Only foals in Group 1 developed significant (P less than 0.05) hyperproteinemia, hyperbetaglobulinemia and a reversal of the albumin/globulin (A/G) ratio 4 weeks after initial infection. Significant (P less than 0.05) IgG anti-S. vulgaris ELISA titers developed in foals in Groups 1 and 2 3 weeks after infection and were sustained for the duration of the experiment. Western blot analysis of soluble somatic antigens of S. vulgaris adult female and male worms probed with sera from foals in Groups 1 and 2 revealed only subtle differences between these animals. The blastogenic reactivity of peripheral blood mononuclear cells (PBMC) to phytohemagglutinin and concanavalin A was not significantly different between groups. Peripheral blood mononuclear cells from foals in Groups 1 and 2 developed significant (P less than 0.05) blastogenic reactivity to S. vulgaris soluble adult somatic antigen when examined at 25 weeks after infection. Mesenteric lymph node cells from foals in Group 2, although not statistically significant, were more reactive to antigen than were the mesenteric lymph node cells from foals in Group 1 when examined at 27 weeks after infection. These results suggest that significant alterations in the immune response of ponies to S. vulgaris does not occur after intravascular killing of larvae by ivermectin treatments.

Plasma thromboxane B2 levels in horses experimentally infected with Strongylus vulgaris.

Plasma thromboxane B2 (TXB2) the stable inactive metabolite of thromboxane A2 (TXA2), was measured daily by specific radioimmunoassay in three groups of animals before and after experimental infection with Strongylus vulgaris. Infection of four 'parasite naive' foals produced a typical acute syndrome with intermittent but statistically insignificant rises in TXB2 levels. Interpretation of results was complicated by the presence of a non-septic peritonitis associated with implantation of the foals with electrodes for recording myoelectrical activity. In two foals of similar age, with some natural exposure to S. vulgaris, there was little or no clinical response to infection and increases in TXB2 were absent. Baseline levels were also much lower, indicating that the peritonitis may have affected the results obtained in the first group of foals. Severe mesenteric arteritis was confirmed at necropsy in all six foals. A third group of yearling horses, all with natural exposure to the parasite, were generally resistant to infection. One animal developed arteritis with clinical signs of diarrhoea and mild colic, and also showed intermittent increases in TXB2. The mean plasma TXB2 level after infection was significantly higher than in the control period, although absolute levels were lower than those recorded in the 'parasite naive' foals. Other animals in this group had low TXB2 levels and minimal arteritis was found at necropsy. These results indicate that although infection appears to have an effect on plasma TXB2, the changes are inconsistent and not reliable indicators of the presence of verminous arteritis. The results also confirm the difficulty in establishing infection and the variability of the response in animals with previous exposure.

Specific serum protein changes associated with primary and secondary Strongylus vulgaris infections in pony yearlings.

The concentrations of haptoglobin, immunoglobin (Ig)G(T) and IgG were measured in the serum of four previously parasite-free pony yearlings following a single dose of 700 (Group H) or 200 (Group L) stage three Strongylus vulgaris larvae (L3) and following a reinfection with the same doses 34 weeks later. The results are compared with an uninfected control pony. The haptoglobin concentration increased during Weeks 1 to 6 and 14 to 17 after infection in the serum of the ponies receiving 200 L3, but in only one pony dosed with 700 L3 (during Weeks 1 to 16). The serum haptoglobin also increased during the first seven weeks after the second infection, in three of the four ponies following the second dose of larvae. The serum IgG(T) concentration started to increase from Week 6 or 9 in the ponies given 700 L3, reaching peaks of 44 and 32 g/litre respectively, eight to nine weeks later, compared with a peak of 16 g/litre 20 to 22 weeks after infection in ponies dosed with 200 L3. The IgG(T) concentration increased to a maximum of 25 g/litre in the serum of only one of the four ponies after the reinfection. The serum IgG concentration in all ponies increased nearly twofold during the first eight weeks after both the primary and secondary dose of larvae. It is concluded that the measurement of specific proteins is more reliable and quicker than the electrophoretic separation and quantitation of protein bands, in tracing changes in serum proteins following the artificial infection of ponies with S vulgaris larvae.

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.

Control of strongylosis in horses by alternate grazing of horses and sheep and some other aspects of the epidemiology of Strongylidae infections.

Alternate grazing of horses and sheep as a control measure for gastrointestinal helminthiasis was studied in three grazing experiments in 1981, 1982 and 1983. Each year a group of three mare yearling Shetland ponies, which were kept on a small pasture from spring to autumn, were compared with a similar group which grazed a similar or the same pasture until July and were subsequently removed to a similar pasture which had been grazed by sheep from April to July. In addition both groups were treated with an anthelmintic when the latter group was removed to the sheep pasture. Pasture larval counts and worm counts and, in 1982 and 1983, faecal egg counts, clinical condition, total protein, albumin and beta-globulin levels demonstrated that the groups removed to sheep pasture acquired considerably lower burdens of nematodes of the subfamilies Cyathostominae and Strongylinae, but considerably higher burdens of Trichostrongylus axei than the groups which were not moved. These T. axei infections resulted in higher serum pepsinogen levels in the former groups compared to the latter in 1981 and 1982. At necropsy an important part of the T. axei burdens and, in 1982 and 1983, the Cyathostominae burdens consisted of inhibited early third stage larvae. A total of 20 species of the subfamily Cyathostominae and 7 species of the Strongylinae were found. Generally the composition of species was in agreement with other observations in western Europe, the most common species being: Cylicostephanus longibursatus, Cylicostephanus minutus, Cylicostephanus calicatus, Cylicostephanus goldi, Cylicostephanus poculatus, Cyathostomum labratum, Cyathostomum coronatum, Cyathostomum catinatum, Cylicocyclus leptostomus, Cylicocyclus nassatus, Cylicocyclus insigne, Strongylus edentatus and Strongylus vulgaris.

Haematological and biochemical values in horses naturally infected with Strongylus vulgaris.

The concentrations of serum proteins (beta 1, beta 2, gamma, alpha 1, alpha 2 globulins and albumin) and absolute numbers of eosinophils, neutrophils and lymphocytes were examined in 64 naturally infected horses and ponies in which the number of larvae of Strongylus vulgaris in the cranial mesenteric artery and the severity of the lesion of verminous arteritis could be determined. The horses were grouped according to the number of larvae found and the severity of the arteritis. The results demonstrated that, although some significant deviation from a random distribution occurred in certain of the values (chi 2 test), there was considerable individual variation in the values obtained for individual animals within groups and overlap of the range of values between groups. Also the number of larvae present in the artery did not necessarily accurately reflect the severity of the arterial lesion. Thus, the parameters examined could not be used reliably to estimate the intensity of infection with S vulgaris in an individual animal.

The elimination of equine strongyles and hematological and pathological consequences following larvicidal doses of thiabendazole.

Twelve horses were divided into three groups and given various doses of a mixed species strongyle inoculum, representing light, moderate, and heavy infections. Three weeks after the larval inoculations, three animals from each group were given larvicidal doses of thiabendazole (TBZ) (440 mg kg-1 on two consecutive days); one animal from each group served as a non-medicated control. Treatment was repeated three weeks later. One treated animal from each group was designated for long-term study; others were necropsied to study adult and larval parasite loads. Six of the twelve animals with strongylosis developed moderate eosinophilia. TBZ given at 440 mg kg-1 on two consecutive days caused depression, lethargy, and anorexia which lasted for five days. Eosinopenia, lymphopenia, and neutrophilia occurred in treated animals, and lasted for three days. During the course of TBZ treatment, one horse died from what appeared to be a mis-dosing or an anaphylactic reaction. At necropsy, active thrombi of the anterior mesenteric artery were seen in parasitized animals, but not in those treated with TBZ. Five out of seven medicated horses were completely free of adult and larval strongyle parasites. One had a few Strongylus edentatus larvae and another had small strongyles. No Strongylus vulgaris larvae or adults were recovered from any horse treated with TBZ.

Effect of diethylcarbamazine on Strongylus vulgaris infection in ponies.

Shetland ponies (n = 4) were given diethylcarbamazine orally at a dose level of 22 mg/kg/day for 1 week before they were inoculated with 800 third-stage larvae of Strongylus vulgaris. Treatment was continued for 86 (1 pony) or 200 days (3 ponies) after the inoculation. As compared with the changes seen in a similarly inoculated group of ponies (group 2) which were not treated, diethylcarbamazine did not prevent the clinical or pathologic changes due to the migrating larvae. Fewer adult parasites were recovered at necropsy from treated ponies than from nontreated (group 2) ponies, even when treatment was discontinued 86 days after inoculation. Treatment appeared to have a detrimental effect on 4th-stage larvae either in the arteries or their intestinal wall, but not until after arterial lesions resulted.

The pathogenesis of single experimental infections with Strongylus vulgaris in foals.

The clinical signs, pathology and clinical pathology associated with single experimental infections of Strongylus vulgaris in worm-free pony foals are described. The major clinical signs which became apparent in the infected foals during the first three weeks were pyrexia, anorexia, dullness and abdominal pain. Within the first two weeks of infection lesions were confined to the intestine and terminal branches of the intestinal arteries and consisted of mucosal, submucosal and serosal haemorrhage together with arteritis of submucosal and serosal arteries and also a marked inflammatory reaction. The main lesion seen three weeks after infection was gross thrombosis of the anterior mesenteric artery or one of its major branches. On section these affected arteries showed marked intimal thickening with infiltration of plasma cells, lymphocytes, macrophages and neutrophils. Between one and four months after infection the gross lesions were predominantly in the arteries and consisted of fibrous thickening of the arterial wall and thrombosis associated with the presence of developing fourth stage larvae. Four months after infection the arterial lesions were still prominent and microscopically there was fibrosis of the wall of the affected artery with wide-spread disruption of the intima. In the adventitia organised thrombi were apparent in the vasa vasorum and resulted in the obliteration of their lumina. The typical lesion associated with the return of fifth stage larvae to the intestine was nodule formation in close proximity to thrombosed terminal intestinal arteries and sections of parasites were seen in the intestinal wall surrounded by neutrophils and necrotic debris. By nine months after infection the arterial lesion had healed, but histologically there was fibrosis of the intima and macrophages containing haemosiderin were seen in the arterial wall. The most significant haematological findings during the experimental period were a marked polymorphonuclear leucocytosis and an increase in the number of circulating eosinophils in the infected animals. Also marked was an increase in the serum globulin levels of the infected foals.

Early development of and pathology associated with Strongylus edentatus.

Pony foals inoculated with infective Strongylus edentatus larvae were monitored for clinical signs and selected blood changes and were examined at necropsy from two to 56 days postinfection. Larvae penetrated the intestine and reached the liver intravenously before 40 hours postinfection. Occasional thrombi and larval tracks associated with the intima of cecal and colic veins suggested aberrant paths. Larvae in the liver doubled in width between seven and 15 days postinfection and a sudden increment in circulating eosinophils occurred between 11 and 15 days. These changes were probably associated with the third molt. At 30 days fourth stage larvae were migrating in the liver; at 42 days they were present in the hepatorenal ligament. White foci were observed in the liver from two to 56 days. They contained mononuclear cells and eosinophils and later necrotic cores of eosinophils. By one month foci were overshadowed by tortuous tracks of migrating larvae. Aberrant larvae in the lungs were confined in granulomas. Massive granulomas in the wall of the cecum and colon contained small larvae which were probably inhibited by antibody associated with the third molt. Severe disruption of omental architecture and adhesions involving the intestine occurred several weeks after infection.