Animals infected with Mycobacterium avium subspecies paratuberculosis show a variety of granulomatous lesions that range from focal forms, seen in the subclinical stages, to diffuse lesions associated with clinical signs. The aim of this study was to phenotypically characterize the macrophages present in the different lesion types using immunohistochemical methods. Lesions from a total of 23 animals with bovine paratuberculosis, natural and experimental, were examined by immunohistochemistry. Antibodies against inducible nitric oxide synthase (iNOS), tumor necrosis factor α (TNF-α), CD163, interleukin 10 (IL-10), transforming growth factor β (TGF-β), major histocompatibility complex (MHC) class II, natural resistance-associated macrophage protein 1 (Nramp-1), calprotectin, Ki-67, CD68, lysozyme, and ionized calcium-binding adaptor molecule 1 (Iba-1) molecules were employed. Samples were scored semiquantitatively using a complete histological score (H-score), reflecting the staining intensity and the percentage of immunolabeled macrophages. Differences in the H-score were seen depending on the lesion type. In focal lesions, with none or few acid-fast bacilli (AFB), macrophages were polarized toward M1 phenotype, with high H-scores for iNOS and TNF-α. Diffuse multibacillary lesions showed M2 differentiation, with high expression of CD163, IL-10, and TGF-β as well as Nramp-1 and MHC class II antigens. Macrophages in diffuse paucibacillary forms showed high H-scores for iNOS but low ones for TNF-α. Diffuse lesions, either multibacillary or paucibacillary, showed high calprotectin and low Ki-67 expression, suggesting a progressive character, while focal forms, with low H-scores for these antigens, would be consistent with latency. Lysozyme and CD68 expression were related to the amount of AFB. H-score for Iba-1 antibody was similar among all types. The findings of this study provide insights into the polarization status of macrophages and lesion development in bovine paratuberculosis.

After oral administration of ewes during mid gestation with 2000 freshly prepared sporulated oocysts of T. gondii isolate M4, abortions occurred between days 7 and 11 in 91.6% of pregnant and infected ewes. Afterwards, a further infection was carried out at late gestation in another group of sheep with 500 sporulated oocysts. Abortions happened again between days 9 and 11 post infection (pi) in 58.3% of the infected ewes. Classically, abortions in natural and experimental ovine toxoplasmosis usually occur one month after infection. Few experimental studies have reported the so-called acute phase abortions as early as 7 to 14 days after oral inoculation of oocysts, and pyrexia was proposed to be responsible for abortion, although the underline mechanism was not elucidated. In the present study, all placentas analysed from ewes suffering acute phase abortions showed infarcts and thrombosis in the caruncullar villi of the placentomes and ischemic lesions (periventricular leukomalacia) in the brain of some foetuses. The parasite was identified by PCR in samples from some placentomes of only one sheep, and no antigen was detected by immunohistochemical labelling. These findings suggest that the vascular lesions found in the placenta, and the consequent hypoxic damage to the foetus, could be associated to the occurrence of acute phase abortions. Although the pathogenesis of these lesions remains to be determined, the infectious dose or virulence of the isolate may play a role in their development.

The relation between gestational age and foetal death risk in ovine toxoplasmosis is already known, but the mechanisms involved are not yet clear. In order to study how the stage of gestation influences these mechanisms, pregnant sheep of the same age and genetic background were orally dosed with 50 oocysts of Toxoplasma gondii (M4 isolate) at days 40 (G1), 90 (G2) and 120 (G3) of gestation. In each group, four animals were culled on the second, third and fourth week post infection (pi) in order to evaluate parasite load and distribution, and lesions in target organs. Ewes from G1 showed a longer period of hyperthermia than the other groups. Abortions occurred in all groups. While in G2 they were more frequent during the acute phase of the disease, in G3 they mainly occurred after day 20 pi. After challenge, parasite and lesions in the placentas and foetuses were detected from day 19 pi in G3 while in G2 or G1 they were only detected at day 26 pi. However, after initial detection at day 19 pi, parasite burden, measured through RT-PCR, in placenta or foetus of G3 did not increase significantly and, at in the third week pi it was lower than that measured in foetal liver or placenta from G1 to G3 respectively. These results show that the period of gestation clearly influences the parasite multiplication and development of lesions in the placenta and foetus and, as a consequence, the clinical course in ovine toxoplasmosis.

The two main genotypes of recognized isolates of Mycobacterium avium subsp. paratuberculosis (Map) are cattle (C) and sheep (S) strains. An experimental infection was conducted to establish the effect of Map strain on the pathogenesis of ovine paratuberculosis. Twenty-four out of thirty 1.5-month-old Assaf lambs were divided into 4 groups of 6 and infected orally with three low passage field isolates, two of S- (22G and the pigmented Ovicap49) and one of C– (764) type, and the reference K-10 strain (C type). The remaining six animals were unchallenged controls. Animals were euthanized at 150 and 390 days post-infection (dpi). Throughout the experiment, the peripheral immune response was assessed and histological and molecular (PCR) studies were conducted on samples of intestine and related lymphoid tissue. Specific antibody and IFN-γ production was significantly higher in animals infected with the C strains, while no consistent IFN- γ responses were observed in the S-type strain infected groups. A positive intradermal skin test response was detected in all infected groups. Lambs infected with S-type strains had granulomatous lesions restricted to the lymphoid tissue with no differences in the lesion intensity over time. In both C–type strain groups, lesions were more severe at 150 dpi while at 390 dpi lesions, characterized by well-demarcated granulomas with fibrosis, decreased in severity. Only infected lambs were positive to PCR. These results suggest that the strain of Map has a strong influence over the immune and pathological responses developed by the host. Lesions induced by C–type strains in lambs show a regressive character and tend to decrease as the infection progresses.