Subclinical paratuberculosis in goats following experimental infection. An immunological and microbiological study.

An experimental oral infection of goats with a caprine isolate of Mycobacterium a. subsp. paratuberculosis was used to investigate immunological and bacteriological events during the subclinical phase of infection. Seven goats at 5-8 weeks of age were given a bacterial suspension in milk-replacement three times weekly for 9 weeks. Six animals were kept as controls. Cellular recall responses against M. a. paratuberculosis were analysed by means of a lymphocyte proliferation test, an IFN-gamma assay and an IL-2 receptor assay. All inoculated animals had detectable CMI responses from 9 weeks post-inoculation and through the 2 years of study, although the responses were highest during the first year. Antibodies against M. a. paratuberculosis could be detected from weeks 15-20 in four of the seven animals, and one additional animal became antibody positive at week 35, while two inoculated animals did not produce significant antibody titres during the experiment. At about 1-year post-inoculation, two animals became faecal shedders, while two others started to excrete bacteria into faeces about 2 years post-inoculation. The appearance of M. a. paratuberculosis in faeces was not associated with a decline in cellular responses as far as could be assessed using the current methods for measuring CMI. Pathological lesions due to M. a. paratuberculosis infection and presence of bacteria were recorded in the intestine and/or mesenteric lymph nodes of five animals while lymph node changes suggestive of paratuberculosis were observed in one animal. Only the two animals with no signs of an active infection at necropsy showed a considerable decline in the cellular parameters during the last year of the study, particularly in the IFN-gamma assay. The two animals with the highest levels of M. a. paratuberculosis responsive CD8+ lymphocytes in the circulation about 1-year post-inoculation had no detectable lesions in the distal ileum and colon at necropsy, while high numbers of gammadelta T-cells responsive to M. a. paratuberculosis in the circulation were associated with disseminated lesions in the distal ileum and colon.

Lesions in subclinical paratuberculosis of goats are associated with persistent gut-associated lymphoid tissue.

The organized gut-associated lymphoid tissue (the Peyer's patches [PPs] of domestic ruminants) is an important site of lesions caused by Mycobacterium avium subsp. paratuberculosis. To investigate the association between PP morphology and the lesions of paratuberculosis in goats, two experiments were performed. Five healthy kids aged 4-5 weeks were examined and the morphology of organized lymphoid tissue in the small intestine was described. Morphological similarities were observed between the ileocaecal-valve PP (ICVPP) and the jejunal PPs (JPPs), with pear-shaped follicles, large submucosal interfollicular T-cell areas, and many intraepithelial leucocytes in the follicle-associated epithelium. The ileal PP (IPP) consisted of elongated follicles, small T-cell areas and few intraepithelial leucocytes. The association between these three locations of PPs and lesions of paratuberculosis was then studied in seven goats inoculated with M. a. paratuberculosis at 5-8 weeks of age and killed 2 years later, while in the subclinical phase of infection. Gross lesions were recorded in five animals and microscopic lesions were observed in the intestine and mesenteric lymph nodes of six animals. The lesions in the small intestine were mainly located in the PPs of the mid-jejunum (JPPs) and ICVPP. Lesions were not present in the intestinal segments that had contained IPP, which had undergone involution during the first 12-18 months of life. These observations indicate that the persistent organized lymphoid tissue in the JPPs and ICVPP, but not the involuted IPP, sustains the development of granulomatous inflammation due to paratuberculosis during the subclinical phase of infection.

Eperythrozoon ovis infection in a commercial flock of sheep.

A flock of sheep consisting of 60-70 winterfed ewes was examined. Blood was collected from approximately 30% of the sheep four times from 1991 to 1995. Eperythrozoon ovis (Ep. ovis) organisms were not detected in blood smears stained with Wright's stain in 1991 and 1993. In 1995, examination of blood smears stained with acridine orange revealed Ep. ovis organisms in 11 of 26 sheep, whereas examination of smears stained with Wright's stain revealed organisms in only one ewe with a high degree of infection. The prevalence of antibodies detected by IFAT varied from 58-100%. The antibody titres varied from 1:20 to 1:12,000. This investigation indicates that Ep. ovis infection once present in a flock of sheep may spread easily among the sheep, and that the infection seems to persist within the flock.

Experimental infection with Ehrlichia phagocytophila in cattle.

Twenty Norwegian Red cattle, aged 8-10 months were used. Ten animals were inoculated intravenously with 3 ml of a whole blood dimethyl sulphoxide stabilate of a bovine strain of Ehrlichia (Cytoecetes) phagocytophila. The animals were observed on a daily basis for clinical symptoms and rectal temperatures were recorded daily for 36 days post inoculation. Blood and serum samples were collected regularly during the observation period. All infected animals developed fever of 2-4 days duration within a week. Six out of 10 animals had secondary temperature rises of 1-2 days duration. Three animals showed swellings in the hind limbs and a stiff gait. Compared with the control group, there was a 10% decrease in the mean haematocrit and a leukopenia that initially was due to a lymphopenia and later to a neutropenia. A monocytosis occurred after the subsidence of the primary fever period. A thrombocytopenia occurred during the early phase of infection. Granulocytic inclusions were detected for 18-32 days. All infected animals developed antibodies > or = 1:4096 to E. phagocytophila within 14 days post infection.

Experimental infection with Ehrlichia phagocytophila and Babesia divergens in cattle.

Nine Norwegian Red Cattle, 9-12 months old, were inoculated simultaneously with Ehrlichia (Cytoecetes) phagocytophila and Babesia divergens. Ten cattle of the same breed and age were kept as controls. Clinical evaluation was done on a daily basis and rectal temperatures were recorded every morning for 4 weeks post inoculation. Blood and serum samples were collected regularly during the observation period. Sera were examined for the presence of antibodies to E. phagocytophila and B. divergens by an indirect immunofluorescence antibody test. All infected animals showed two periods of fever that corresponded to the proliferation of B. divergens and E. phagocytophila in the peripheral blood. The simultaneous infection did not induce more severe clinical symptoms than infection with either organism alone. Compared with the control group, there was a 25% decrease in the mean haematocrit. The leukogram showed a biphasic pattern and corresponded to the changes seen in infection with either organism alone. The simultaneous infection did not suppress antibody formation towards E. phagocytophila or B. divergens.

Experimental infection with Babesia divergens in cattle persistently infected with bovine virus diarrhoea virus.

Nine Norwegian Red cattle, aged 7-14 months, persistently infected with bovine virus diarrhoea virus (BVDV) were inoculated with a Swedish strain of Babesia divergens. Six persistently infected cattle of the same age and breed were kept as controls. Blood and serum samples were collected regularly during the observation period. Rectal temperatures were recorded every morning for 25 days post infection, and the animals were examined clinically on a daily basis. Sera were examined for antibodies to B. divergens by indirect immunofluorescence antibody test (IFAT). Eight of the infected animals developed fever of 2-5 days duration. Babesia divergens organisms appeared in the erythrocytes of all infected animals on the day after inoculation. The parasitaemia lasted for 4-11 days. One animal had a transient haemoglobinuria. Compared with the control group, there was a 20% decrease in the haematocrit. There was a transient lymphopenia and thrombocytopenia during the period of fever. There were no differences in mean numbers of neutrophils between the two persistently infected groups. Compared with cattle free of BVDV, the persistently infected cattle had consistently lower total leucocyte count that was mainly due to decreased mean numbers of neutrophils and monocytes. All infected animals develop antibodies > or = 1:1280 between day 7 and 10 post infection. The magnitude of the antibody response was considerably lower than that of BVDV-free animals inoculated with the same strain and dosage of B.divergens.