Acute phase proteins as a tool for differential diagnosis of wasting diseases in growing pigs.

The concentrations of haptoglobin (Hp), C-reactive protein (CRP) and serum amyloid A (SAA) were measured in wasted pigs, first to evaluate their usefulness in the diagnosis of infectious, wasting diseases in pigs, and second, to evaluate whether their concentrations can distinguish the lymphoid depletion score in the lymph tissues of wasted affected pigs. Fifty-three wasted pigs and seven specific pathogen free (SPF) pigs were postmortem examined. Gross lesions were evaluated and samples for histopathological, immunohistochemical, molecular biology and microbiological analysis were taken. Thirty-one pigs were diagnosed as postweaning multisystemic wasting syndrome (PMWS) and 22 as porcine respiratory disease complex (PRDC). Lymphoid depletion degree in lymph tissues of PMWS and PRDC affected pigs was determined. Serum Hp was significantly higher in pigs with PRDC in comparison with the PMWS affected pigs. Serum CRP concentration was significantly lower in pigs with PRDC than in PMWS affected pigs (P<0.001). CRP and SAA levels increased with the lymphoid depletion score, presenting statistical differences between pigs with no depletion and pigs with low, moderate or severe lymphoid depletion (P<0.05, P<0.05 and P<0.001 for CRP and P<0.01, P<0.01 and P<0.01 for SAA, respectively). Hp was higher in pigs with no or low depletion compared with the pigs suffering severe lymphoid depletion (P<0.001 and P<0.05, respectively).

Expression of steroid receptors and calponin in a cervical leiomyoma in a young pig.

Thickening of the uterine cervix and bilateral ovarian cystic change was identified in a 6-month-old pig during routine abattoir inspection. Microscopically, the cervical lesion comprised a non-encapsulated mass of densely packed, large and monomorphic spindle cells within the myometrium. Immunohistochemically, the majority of these neoplastic cells expressed the cytoplasmic terminal smooth muscle differentiation marker calponin, the nuclear oestrogen receptor alpha and the progesterone receptor. The ovarian cysts were classified as follicular cysts. A diagnosis of leiomyoma of the uterine cervix with bilateral ovarian follicular cysts was made. The expression of calponin as a marker of smooth muscle differentiation in tumours of the genital tract of the pig has not previously been reported. The expression of steroid hormone receptors suggests a role for steroid hormones derived from the ovarian follicular cysts in tumourigenesis.

Left perinephric abscess associated with nephrolithiasis and bladder calculi in a bitch.

An eight-year-old, entire female Pekingese cross, weighing 3.8 kg, had been inappetent with fever, depression, abdominal pain, vomiting and diarrhoea for seven days. The radiographic and ultrasonographic findings were consistent with glomerulonephritis, nephrolithiasis in both kidneys, bladder calculi and an accumulation of fluid in the left perinephric space. The clinical signs, together with the results of the diagnostic imaging, suggested that this fluid could be pus. A definitive diagnosis of a subcapsular abscess in the left kidney was established when this kidney was removed surgically. A histopathological examination of the kidney revealed a diffuse suppurative interstitial nephritis, membranous glomerulonephritis and an abscess invading the perinephric adipose tissue from the renal cortex. Twelve months after surgery the dog remains clinically stable, but owing to the disease of its remaining kidney its long-term prognosis is poor.

Endogenous interleukin-12 is not required for resolution of Chlamydophila abortus (Chlamydia psittaci serotype 1) infection in mice.

A Th1 immune response involving gamma interferon (IFN-gamma) production is required to eliminate Chlamydophila abortus infections. In this study, the role of interleukin-12 (IL-12) in protecting against C. abortus infection was investigated using IL-12(-/-) and wild-type (WT) C57BL/6 mice to determine the role of this Th1-promoting cytokine. IL-12(-/-) mice were able to eliminate the C. abortus infection in a primary infection. However, there was a delay in the clearance of bacteria when IL-12(-/-) mice were infected with a sublethal dose of C. abortus, the delay being associated with a lower production of IFN-gamma. The low level of IFN-gamma was essential for survival of IL-12(-/-) infected mice. Both WT and IL-12(-/-) mice developed a Th1 immune response against C. abortus infection, since they both produced IFN-gamma and immunoglobulin G2a antibody isotype. In addition, when mice were given a secondary infectious challenge with C. abortus, a protective host response which resolved the secondary infection was developed by both WT and IL-12(-/-) mice. The lack of IL-12 resulted in few infiltrating CD4(+) T cells in the liver relative to the number in WT mice, although the number of CD8(+) T cells was slightly higher. The more intense Th1 response presented by WT mice may have a pathogenic effect, as the animals showed higher morbidity after the infection. In conclusion, these results suggest that although IL-12 expedites the clearance of C. abortus infection, this cytokine is not essential for the establishment of a protective host response against the infection.

Differences in lymphocyte subpopulations from peripheral blood and lymphoid organs in natural caprine tuberculosis infection.

Although the cell-mediated immune response is known to be a critical factor in host defence against intracellular mycobacterial infection, the different components of the T-cell response are unclear, particularly in caprine infection. In this study we examine the differences in the lymphocyte population of peripheral blood, spleen and mediastinal and superficial lymph nodes in 11 naturally infected goats showing positive reactions in the comparative tuberculine intradermal test. According to the different types of lesion showing, the goats were classified into proliferative or exudative tuberculosis. The results obtained by fflow cytometry analysis indicated that the main differences in peripheral blood were in the CD4 T-cell population, which decreased markedly in goats with exudative tuberculosis, while the CD8 and B cells increased in number. The gamma/delta T cells did not show significant differences in either type of tuberculosis, while interleukin-2 receptor cells decreased slightly in the exudative tuberculosis. The CD4:CD8 ratio was higher than 1 in goats with proliferative tuberculosis and lower than 1 in goats with exudative tuberculosis. In general, the lymphoid organs of the goats with exudative tuberculosis showed a significant increase in the number of CD8 T cells (CD4:CD8 ratio of less than 1) whereas no significant differences were observed in the CD4 T population between either type of tuberculosis.

Arrhythmogenic right ventricular dysplasia/cardiomyopathy in a Siberian husky.

A seven-month-old male Siberian husky was presented with a recent history of anorexia, hindlimb weakness and syncope. Physical examination revealed severe tachycardia, tachypnoea and dyspnoea. Mucous membranes were pale and femoral pulses were weak. An electrocardiogram showed sustained ventricular tachycardia with a left bundle branch block configuration. Thoracic radiographs revealed slight right ventricular enlargement and two-dimensional echocardiography revealed mild right ventricular dilation at the cardiac apex and some hyperechogenic areas on the right side of the interventricular septum. Administration of intravenous lignocaine converted the ventricular tachycardia to sinus rhythm. The maintenance antiarrhythmic therapy consisted of oral procainamide and propranolol. Three weeks later the dog died suddenly. On postmortem examination, the right ventricular free wall was very thin at the apex, infundibulum and caudal aspect of the right ventricular parietal wall, similar to the 'triangle of dysplasia' of human patients. Histopathological examination revealed replacement of several areas of right ventricular free wall myocardium with connective tissue and fat. The right atrium and left ventricle were less severely affected by the same lesions. The clinical and pathological findings are similar to those reported in young people with arrhythmogenic right ventricular dysplasia/cardiomyopathy.

Light and ultrastructural immunocytochemical study of prolactin cells in ovine adenohypophysis. Influence of lactation and weaning.

The influence of lactation and weaning on the number, distribution, and structure of mammotroph cells (PRL cells) in ewes was studied using immunocytochemical procedures for light and electron microscopy, as well as morphometric and stereological techniques. The adenohypophyseal gland of 12 ewes of the Segureña breed at different stages of milk production and weaning were analysed, while the same organ of 3 ewes in anestrus served as control. During lactation, PRL cells increased in number and decreased in size. Ultrastructurally, these cells showed a well-developed rough endoplasmic reticulum (RER) and Golgi complex (GC), and large secretory granules. These findings point to increased synthesis and secretion activities during lactation, and a decrease after weaning. One day after weaning 'storage' cells increased in number, and crinophagy became evident 7 days after weaning, when the first 'inactive' cells were seen (10% of total cells). In anestrus these last cells represented 40% of the total, and showed scarce granules, signs of inactivity, lysosomes and lipid droplets.

Vaccination against swine enzootic pneumonia in field conditions: effect on clinical, pathological, zootechnical and economic parameters.

A field study was carried out to evaluate the effect of vaccination against swine enzootic pneumonia in different production systems (closed or one-site, and open, two- or three-site, systems). The percentage of mortality showed statistically significant differences (p < 0.001) in favour of vaccination in all the production systems examined. Zootechnical and economic parameters also reflected the benefits of vaccination, although only the feed conversion ratio showed statistically significant differences (p < 0.030). A macroscopic study of lungs at slaughter pointed to statistically significant differences (p < 0.001) in favour of vaccination in the one- and two-site production systems. The histopathological and immunocytochemical study showed complete concordance with the macroscopic diagnosis (p < 0.001). Such relationship confirms the suitability of macroscopic diagnosis in field conditions.

Limited role of polymorphonuclear neutrophils in a pregnant mouse model of secondary infection by Chlamydophila abortus (Chlamydia psittaci serotype 1).

The aim of this work was to study the role of polymorphonuclear neutrophils (PMNs) in the clearance of infection, and in the development of specific immunity against Chlamydophila abortus (Chlamydia psittaci serotype 1) secondary infection. A pregnant mouse model depleted of neutrophils by the RB6-8C5 monoclonal antibody was used. No clinical signs were observed in depleted or non-depleted mice after secondary infection and no significant differences were observed in the litter size between the infected and control groups. In PMN-depleted mice C. abortus was not detected in the materno-fetal unit but merely produced low, persistent levels of infection in spleen and liver. In the non-depleted mice the level of infection was significantly lower, being resolved during the first few days post-reinfection. In both infected mice groups the immune response in the liver was quickly established and was seen to be composed mainly of CD4(+)T lymphocytes and macrophages. A Th1 response characterized by the presence of IFN-gamma and TNF-alpha in serum was observed during early infection, with significantly higher levels in the non-depleted animals. Our results suggest that PMNs have little influence on the control of C. abortus secondary infection, although they are a first line of defense and may influence the early production of TNF-alpha and IFN-gamma.

Immunophenotypical characterization of the lymphocyte infiltrate in caprine pulmonary tuberculosis.

A study was made of the immunophenotype of the lymphocytes associated with natural caprine pulmonary tuberculosis at four stages of the disease. Regardless of the stage, CD4(+)and CD8(+)T lymphocytes predominated in lung and mediastinal lymph node lesions, but gamma/delta T and B (IgM(+)) cells were seen only rarely. At the primary complex stage, CD4(+)cells outnumbered CD8(+)cells. At the stage of generalized tuberculosis, however, and still more at the post-primary stage, CD8(+)cells outnumbered CD4(+)cells. At the final stage (tuberculous pneumonia), CD4(+)and CD8(+)cells were present in low but approximately equal numbers.

Light and ultrastructural immunocytochemical study of somatotropic cells (GH cells) in ovine adenohypophysis: lactation and weaning influences.

The influence of lactation period and weaning on the distribution, number, and structure of somatotropic cells (GH cells) in ewes was studied using immunocytochemical procedures for light and electron microscopy, as well as morphometric and stereological techniques. The adenohypophyseal gland of 12 ewes of the Segureña breed in different stages of milk production and weaning was studied, while three ewes in anoestrus served as controls. The size of secretory granules was heterogeneous in all stages, suggesting that this characteristic is not related to functional activity. During lactation and weaning the size of GH cells decreased, while the number of 'synthesis cells' increased. The rough endoplasmic reticulum and Golgi complex appeared more developed and small secretory granules showed lower gold particle labelling. These data suggest that GH cells are more active during lactation, particularly during late lactation.

Characterization and distribution of B cells in the lymphoid organs of goats.

The distribution of B cells in the lymphoid organs of the goat was studied using a panel of 13 monoclonal antibodies (mAbs) developed against different markers for bovine B cells. Samples of mesenteric lymph nodes, jejunal and ileal Peyer's patches, duodenum, jejunum, ileum, colon, caecum and rectum were taken from four 7-month-old male Murciano-granadina goats using the avidin-biotin-peroxidase (ABC) method on frozen sections as described by Hsu et al. (1981). The mAbs against immunoglobulins (Ig) recognized a large number of cells, particularly in the light zones of the germinative centres of the lymphoid follicles, regardless of the isotype against which they were directed. However, the greatest numbers of B cells in the germinative centres and outer coronas of the lymphoid follicles of the lymph node, spleen and Peyer's patches were recognized by mAbs against the L lambda chain of Ig and against IgM. This was also the case in other locations where B cells were abundant, such as the medulla of the lymph node and the dome of the Peyer's patches. These mAbs recognized not only B lymphocytes but also plasma cells, showing an intracytoplasmatic reaction (numerous in the spleen red pulp and the intestinal lamina propria when mAbs were used against the L lambda chain of the Ig, scarce in the intestinal lamina propria when used against IgM and scarce in spleen red pulp and numerous in the intestinal lamina propria when mAbs against IgA were used). The mAbs BAQ44 A, GC65 A and GB25 A are of interest because, besides marking cells in the B areas where lymphocytes show surface Ig, they give a positive reaction in areas where there are Ig-cells (the dark zone of the germinative centre) and do not immunostain plasma cells. Thus, these mAbs recognize a surface marker which is not an Ig.

Postnatal development of lymphocyte subpopulations in the intestinal lymph nodes in goats.

The incidence and location of CD2+, CD4+, CD8+ and gamma/delta T lymphocytes and IgM+ B lymphocytes were studied in the intestinal lymph nodes in 1-week, 1-month, 3-month and 7-month-old goats, using monoclonal antibodies and immunohistochemical methods. The cortical area of the intestinal lymph nodes in 1-week-old animals contains only primary follicles occupied by IgM+ B lymphocytes and some CD2+ CD4+ T lymphocytes. In goats older than 1 month, secondary follicles, that increased in number and size with age, were observed; the light zone of the germinal centre was occupied by IgM+ lymphocytes and some CD2+ and CD4+ T lymphocytes. In the other compartments of the lymph nodes, B lymphocytes were scarce, their number increasing with age in the medulla and diminishing in the paracortex. The numerous CD2+ T lymphocytes in the interfollicular area increased in number in the paracortical area of the 7-month-old goats, simultaneously with an increase in the MHC II+ dendritic cells and the CD4/CD8 ratio, which was greater than 1. The gamma/delta T lymphocytes represented a minor subpopulation scattered through the lymph nodes.

Changes in lymphocyte subsets in the intestine and mesenteric lymph nodes in caprine paratuberculosis.

Changes in the number and distribution of lymphocyte subsets were investigated in the intestinal mucosa and mesenteric lymph nodes of three goats with natural paratuberculosis, comparisons being made with a single uninfected control animal. Lesions in the naturally infected goats varied from small granulomata with scarce epithelioid or multinucleated giant cells, containing few or no bacilli, in the intestine (tuberculoid type) to an extensive, diffuse epithelioid cell infiltrate containing numerous bacilli in the gut and mesenteric lymph nodes (lepromatous type). The number and distribution of lymphocyte subsets in the control were consistent with data reported from other non-infected goats. However, in the goats with paratuberculosis, significant changes were observed in the number and distribution of CD4+ and CD8+ T lymphocytes, the changes being related to the severity of the lesions. In the intestinal mucosa of the goat with tuberculoid lesions no significant changes were observed, but in the cortical area of mesenteric lymph nodes the number of CD4+ T lymphocytes decreased and the number of CD8+ T lymphocytes increased. In the two goats with lepromatous lesions, there was a decrease in the CD4+ T subpopulation and an increase of CD8+ T lymphocytes in the lamina propria of the intestine and particularly in the cortical area of the mesenteric lymph nodes, the CD4:CD8 ratio (< 1) being the opposite of that observed in healthy goats. Because of the small numbers of animals, further studies including additional animals are needed to confirm these preliminary results, which suggest that the progression of paratuberculous lesions may be due to an ineffective host immune response attributable to the CD8+ T lymphocyte subset that "downregulates" the activity of the CD4+ T lymphocytes required for macrophage activation.

Postnatal development of lymphocyte subpopulations in the intestinal mucosa in goat.

The incidence and location of CD2+, CD4+, CD8+ and gamma/delta T-lymphocytes and B-lymphocytes were studied in the intestinal mucosa in goats of one week, one month, three months and seven months old, using monoclonal antibodies and immunohistochemical methods. At all ages CD2+ lymphocytes were the major subpopulation in the intestinal mucosa, more numerous in the small than in the large intestine and in the villi than in the crypt region. In one week-old animals most of CD2+ lymphocytes were scattered in the lamina propria and the number of lymphocytes that expressed CD4 was less than those that expressed CD8, resulting in a CD4/CD8 ratio less than 1. CD2+ lymphocytes increased markedly until one month old, with a higher increase of CD4+ subpopulation than CD8+ with an inversion of the CD4/CD8 ratio. CD4+ lymphocytes were localised in the lamina propria forming clusters, whereas CD8+ lymphocytes were scattered peripherally under the epithelium and in older animals from three month old, CD8+ lymphocytes were also abundant between epithelial cells. The gamma/delta T-lymphocytes recognised with the mAb CACTB6A represented a minor subpopulation located between epithelial cells or in the lamina propria, no evident changes were observed with age. IgM+ cells were a minor subpopulation in all animals (never higher than 1 cell per 1500 microns-2) located in the crypt region of the lamina propria.

Study of lymphocyte subpopulations in peripheral blood and secondary lymphoid organs in the goat using monoclonal antibodies to surface markers of bovine lymphocytes.

Monoclonal antibodies (mAbs) to surface markers of bovine lymphocytes MHC I, MHC II, B-cells, T-cells (CD2, CD4, CD8 and gamma/delta) and interleukin-2 (IL-2) receptor were tested in the goat by flow cytometry and using immunohistochemical methods. Samples from peripheral blood and secondary lymphoid organs (mesenteric lymph nodes, spleen and ileal Peyer's patch) were studied. The percentage of positive cells obtained by flow cytometry and its compartmentalisation in different tissue sections showed that the mAbs against MHC I, MHC II, CD2, CD4, CD8, gamma/delta and IL-2 receptor recognised lymphocyte subpopulations similar to those present in the bovine. However, the mAbs tested on B-cells reacted only partially in the recognition of this subpopulation.

Detection of Mycobacterium paratuberculosis antigen with colloidal immunogold in naturally infected sheep.

The anti-Mycobacterium paratuberculosis polyclonal serum is proved useful for labelling Mycobacterium paratuberculosis in glutaraldehyde-osmium-fixed and epon-embedded intestinal samples from sheep with clinical symptoms of paratuberculosis. M. paratuberculosis marked with antibody-coated colloidal gold stain was seen in macrophages, epithelioid cells, giant cells and neutrophils throughout intestinal mucosa. In large macrophages with a low lysosomal content, a great number of intact mycobacteria was seen within phagosomes. In macrophages with average lysosomal content, very few intact mycobacteria or mycobacterial debris were present and lysosome-phagosome fusions were observed. Mycobacteria within neutrophils were scanty. These results show the usefulness of colloidal immunogold techniques for studies of the pathogenesis of paratuberculosis.