Investigation into the Pathology of Idiopathic Systemic Amyloidosis in Four Captive Badgers (Meles meles).

Systemic idiopathic amyloidosis was described in four captive badgers (Meles meles). Two animals (B1 and B2) were not enrolled in any trial, while animals B3 and B4 took part in a vaccine efficacy study and had been challenged with Mycobacterium bovis. A full set of tissues was collected and processed routinely for histopathological, immunohistochemical and ultrastructural studies. Splenomegaly was found in three out of four animals. Histopathological evaluation revealed congophilic, permanganate-resistant systemic amyloid deposits in the tissues of all badgers. Animals B2 and B4 displayed a marked granulomatous response to amyloid within the spleen. Animals B1 and B2 also displayed clinicopathological findings suggestive of chronic kidney disease. Ultrastructural examination identified peculiar star-shaped arrays of amyloid. Immunohistochemical studies were unrewarding. Systemic amyloidosis should be considered among the differentials of wasting in captive badgers.

Comparative analysis of cellular immune responses and cytokine levels in sheep experimentally infected with bluetongue virus serotype 1 and 8.

Protective immunity in sheep with bluetongue virus (BTV) infection as well as the role of BTV-induced cytokines during immune response remains unclear. Understanding the basis immunological mechanisms in sheep experimentally infected with serotypes 1 and 8 (BTV-1 and -8) was the aim of this study. A time-course study was carried out in order to evaluate cell-mediated immune response and serum concentrations of cytokines (IL-1ß, TNFα, IL-12, IFNγ, IL-4 and IL-10) with inflammatory and immunological functions. Depletion of T cell subsets (mainly CD4(+), γδ and CD25(+)) together with the absence of cytokines (IFNγ and IL-12) involved in the regulation of cell-mediated antiviral immunity at the first stage of the disease suggested that both BTV-1 and BTV-8 might impair host's capability against primary infections which would favor viral replication and spreading. However, cellular immune response and cytokines elicited an immune response in sheep that efficiently reduced viremia in the final stage of the experiment. Recovery of T cell subsets (CD4(+) and CD25(+)) together with a significant increase of CD8(+) T lymphocytes in both infected groups were observed in parallel with the decrease of viremia. Additionally, the recovery of CD4(+) T lymphocytes together with the significant increase of IL-4 serum levels at the final stage of the experiment might contribute to humoral immune response activation and neutralizing antibodies production against BTV previously described in the course of this experiment. These results suggested that both cellular and humoral immune response may contribute to protective immunity against BTV-1 and BTV-8 in sheep. The possible role played by IL-10 and CD25(+) cells in controlling inflammatory and immune response in the final stage of the experiment has also been suggested.

Immunohistochemical detection of dendritic cell markers in cattle.

Dendritic cells (DCs) are "professional" antigen-presenting cells with a critical role in the regulation of innate and adaptive immune responses and thus have been considered of great interest in the study of a variety of infectious diseases. The objective of this investigation was to characterize the in vivo distribution of DCs in bovine tissues by using potential DC markers to establish a basis for the study of DCs in diseased tissues. Markers evaluated included MHCII, CD208, CD1b, CD205, CNA.42, and S100 protein, the latter 2 being expressed by follicular dendritic cells whose origin and role are different from the rest of hematopoietic DCs. Paraffin wax-embedded tissues from 6 healthy Friesian calves were subjected to the avidin-biotin-peroxidase method, and the most appropriate fixatives, dilutions, and antigen retrieval pretreatments were studied for each of the primary antibodies. The most significant results included the localization of CD208-positive cells not only in the T zone of lymphoid organs but also within lymphoid follicles; CD1b-positive cells were mainly found in thymus and interfollicular areas of some lymph nodes; cells stained with anti-CD205 antibody were scarce, and their location was mainly in nonlymphoid tissues; and CNA.42- and S100 protein-positive cells localized in primary lymphoid follicles and light zones of germinal centers, although showing differences in the staining pattern. Furthermore, MHCII was established as one of the most sensitive markers for any DC of hematopoietic origin. These results increase our understanding of DC immunolabeling and will help in future DC studies of both healthy and diseased tissues.

Pathology of African swine fever: the role of monocyte-macrophage.

African swine fever (ASF) is a viral hemorrhagic disease with different clinical and lesional changes depending of virulence of strains/isolates and immunological status of pigs. In acute and subacute forms of ASF, severe vascular changes are present, with hemorrhages in different organs (mainly melena, epistaxis, erythema, renal petechiaes and diffuse hemorrhages in lymph nodes), pulmonary edema, disseminate intravascular coagulation and thrombocytopenia. Lymphopenia and monocytopenia are developed during acute and subacute ASF. Lymphopenia is associated with lymphoid depletion in primary and secondary lymphoid organs, which is caused by apoptosis. All these lesions are not related to viral replication in endothelial cells or lymphocytes. Monocytes-macrophages show viral replication and cytophatic effect, including hemadsorption. The more significant changes in these cells are increased number and secretory activation (increased levels of proinflammatory cytokines) in targets organs. Proinflammatory activation is the initial cause of clinical and lesional pictures in ASF, including fever and changes in levels of acute phase proteins. Levels of IFN-ß and -γ are increased from initial phase of acute ASF. Anti-inflammatory response, represented by increased level of IL-10, is observed also, although in the final phase of acute ASF only.

Potential role of proinflammatory cytokines in the pathogenetic mechanisms of vascular lesions in goats naturally infected with bluetongue virus serotype 1.

In vitro studies have demonstrated that bluetongue virus (BTV)-induced vasoactive mediators could contribute to the endothelial cells dysfunction and increased vascular permeability responsible of lesions characteristic of bluetongue (BT) like oedema, haemorrhages and ischaemic necrosis in different tissues. However, few in vivo studies have been carried out to clarify the causes of these lesions. The aim of this study was to elucidate in vivo the pathogenetic mechanisms involved in the appearance of vascular lesions in different organs during BT. For this purpose, tissue samples from goats naturally infected with bluetongue virus serotype 1 (BTV-1) were taken for histopathological and immunohistochemical studies to determine the potential role of proinflammatory cytokines (tumour necrosis factor alpha, TNFα and interleukin one alpha, IL-1α) in the increased vascular permeability and their relationship with the presence of virus. Gross and histopathological examination revealed the presence of vascular damage leading to generalized oedema and haemorrhages. Immunohistochemical studies displayed that endothelial injury may have been due to the direct pathogenic effect of BTV infection on endothelial cells or may be a response to inflammatory mediators released by virus-infected endothelial cells and, possibly, other cell types such as monocytes/macrophages. These preliminary results of what appears to be the first in vivo study of tissue damage in small BT-infected ruminants suggest a direct link between the appearance of vascular changes and the presence of BTV-induced vasoactive cytokines.

Characterization of apoptosis pathways (intrinsic and extrinsic) in lymphoid tissues of calves inoculated with non-cytopathic bovine viral diarrhoea virus genotype-1.

Previous studies have shown that activation of effector caspase-3 is associated with the apoptosis of lymphocytes occurring during infection with bovine viral diarrhoea virus (BVDV); however, the regulation of the apoptosis pathways that induce cell death via activation of effector caspase-3 has not yet been clarified. The aim of this study was to examine immunohistochemically the expression of cleaved caspase (CCasp)-8 (initiator caspase of the extrinsic pathway), CCasp9 (initiator caspase of the intrinsic pathway) and Bcl-2 (an anti-apoptotic marker) in gut-associated lymphoid tissue (GALT) of the ileum from calves inoculated with a non-cytopathic strain of BVDV genotype-1. CCasp8 had similar expression to that of CCasp3. In interfollicular T-cell areas there was moderate apoptosis and evidence of moderate activation of initiator caspase-8. In B-cell follicles there was marked lymphocyte apoptosis and evidence of intense caspase-8 activation, highlighting the potentially major role of the extrinsic pathway in lymphocyte apoptosis in the GALT during BVDV infection. Additionally, there was a significant decrease in the number of CCasp9(+) cells from the start of the experiment and this was linked to inactivation of caspase-9. Therefore, the intrinsic pathway may play only a minor role in the induction of lymphocyte apoptosis. Finally, the observed overexpression of Bcl-2 protein could play a major role in protecting lymphocytes in the T-cell areas against apoptosis, while low levels of Bcl-2 expression could be associated with the follicular lymphocyte apoptosis occurring during BVDV infection.

Virus distribution and role of thymic macrophages during experimental infection with noncytopathogenic bovine viral diarrhea virus type 1.

Thymic depletion, presence of viral antigen, and changes in distribution and cytokine production of thymic macrophages were investigated in calves experimentally infected with a noncytopathogenic bovine viral diarrhea virus type (BVDV) 1 strain. Ten clinically healthy colostrum-deprived calves were used. Eight calves were inoculated with the virus and two were used as uninfected controls. Calves were sedated and euthanized in batches between 3 and 14 days postinoculation. At necropsy, thymus samples were collected for structural, immunohistochemical, and ultrastructural study and TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling). From 6 days postinoculation, the thymic cortex was multifocally depleted with increased frequency of pyknosis and karyorrhexis, suggestive of apoptosis and confirmed by the TUNEL technique. Although the onset of lymphoid depletion was coincident with the detection of viral antigen by immunohistochemistry, the number of infected lymphocytes was very low through the experiment. There was an increase in number of macrophages in cortex and medulla, accompanied by ultrastructural changes indicative of phagocyte activation, and a decrease in cells expressing tumor necrosis factor-alpha (TNF-α) and IL-1α. These results suggest that the increase in number of these cells could be related to phagocytosis of cell debris and apoptotic lymphocytes. Furthermore, the results imply that, in contrast to the situation with classical swine fever virus, the lymphocyte apoptosis resulting from bovine viral diarrhea virus infection is not mediated by TNF-α or interleukin-1 alpha (IL-1α) production by virus-infected macrophages. This is the first study that describes this decrease in the number of thymic cells expressing TNF-α and IL-1α in cattle experimentally infected with bovine viral diarrhea virus type 1.

Response of proinflammatory and anti-inflammatory cytokines in calves with subclinical bovine viral diarrhea challenged with bovine herpesvirus-1.

The aim of this work was to investigate the susceptibility of calves infected with bovine viral diarrhea virus (BVDV) against secondary infections. For this purpose, the profile of cytokines implicated in the immune response of calves experimentally infected with a non-cytopathic strain of BVDV type-1 and challenged with bovine herpesvirus 1.1 (BHV-1.1) was evaluated in comparison with healthy animals challenged only with BHV-1.1. The immune response was measured by serum concentrations of cytokines (IL-1ß, TNFα, IFNγ, IL-12, IL-4 and IL-10), acute phase proteins (haptoglobin, serum amyloid A and fibrinogen) and BVDV and BHV-1.1 specific antibodies. BVDV-infected calves displayed a great secretion of TNFα and reduced production of IL-10 following BHV-1 infection, leading to an exacerbation of the inflammatory response and to the development of more intense clinical symptoms and lesions than those observed in healthy animals BHV-1-inoculated. A Th1 immune response, based on IFNγ production and on the absence of significant changes in IL-4 production, was observed in both groups of BHV-1-infected calves. However, whereas the animals inoculated only with BHV-1 presented an IFNγ response from the start of the study and high expression of IL-12, the BVDV-infected calves showed a delay in the IFNγ production and low levels of IL-12. This alteration in the kinetic and magnitude of these cytokines, involved in cytotoxic mechanisms responsible for limiting the spread of secondary pathogens, facilitated the dissemination of BHV-1.1 in BVDV-infected calves.

Hepatic immune response in calves during acute subclinical infection with bovine viral diarrhoea virus type 1.

Eight colostrum-deprived calves aged 8-12 weeks were inoculated intranasally with a non-cytopathic strain of bovine viral diarrhoea virus (BVDV) genotype-1 and the effects on the hepatic immune response were studied. Two calves were sacrificed at each of 3, 6, 9 and 14 days post-inoculation (dpi) and two uninoculated animals were used as negative controls. BVDV was detected in hepatic macrophages and monocytes from 3 to 14dpi and in Küpffer cells (KCs) from 6 to 14dpi. Increases in the numbers of MAC387(+) KCs and monocytes, but not interstitial macrophages, differentiated by morphological features, were evident in the liver following inoculation with BVDV. There was a substantial increase in the number of monocytes positive for tumour necrosis factor (TNF)-α, but only small increases in the numbers of TNF-α(+) KCs and interstitial macrophages and interleukin (IL)-6(+) monocytes, KCs and interstitial macrophages. There was an increase in the number of interstitial CD3(+) T lymphocytes in the liver, but no substantial changes in the numbers of circulating CD3(+) T lymphocytes, interstitial or circulating CD4(+) or CD8(+) T lymphocytes, or CD79αcy(+) B lymphocytes. Serum haptoglobin and serum amyloid A increased transiently at 12dpi. Upregulation of some pro-inflammatory cytokines by hepatic macrophages is evident in subclinical acute BVDV type 1 infection in calves.

Cytokine expression in paraffin wax-embedded tissues from conventional calves.

The cross-reactivity of antibodies against human tumour necrosis factor (TNF)alpha, interleukin (IL)-1alpha, IL-1beta and porcine IL-6, and the distribution of immunolabelled cells were evaluated on paraffin wax-embedded tissues from five healthy calves. The tissues were fixed in 10% buffered formalin or Bouin's solution and processed for structural studies and immunohistochemical studies by the avidin-biotin-peroxidase technique. Bouin's solution proved to be the more suitable fixative and Tween 20 the most effective antigen unmasking technique for increasing detectable antigenicity. Constitutive expression of TNFalpha, IL-1alpha, IL-1beta and IL-6 by different cell populations, mainly macrophage-like cells, was detected. Lymphoid organs displayed a higher presence of immunolabelled cells than did lung, liver or kidney. TNFalpha and IL-1alpha appeared as the predominant cytokines, especially in the gut-associated lymphoid tissue of the ileum and in the regional mesenteric lymph nodes. The results will facilitate investigation of the role of these cytokine-producing cells in inflammatory disease processes in calves.

Atoxoplasma spp. infection in captive canaries (Serinus canaria).

Clinical signs, histopathological and ultrastructural findings associated with Atoxoplasma spp. natural infection in captive canaries (Serinus canaria) are described. Intracytoplasmic Atoxoplasma-like protozoa were found in the liver and lung. In the liver, protozoa were found in hepatocytes and Kupffer's cells and were associated with granulomatous hepatitis and a marked bile duct hyperplasia. An usual finding was the presence of infected mononuclear cells adhered to the endothelium of the blood vessels in lung. The diagnosis was confirmed by ultrastructural examination of reprocessed paraffin-embedded tissues.

An immunohistochemical study of the tonsils in pigs with acute African swine fever virus infection.

An immunohistochemical study of the tonsils was carried out to gain further insight in the pathogenesis of acute African swine fever (ASF). Twenty-one pigs were inoculated by intramuscular route with a highly virulent isolate of ASF virus and painlessly killed at 1-7dpi. Viral antigen was highly distributed in the tonsil from 3 to 4dpi and an increase in the number of monocyte-macrophages was very evident at the same days post inoculation. This phenomenon was observed together with an increase of the expression of proinflammatory cytokines (Tumour necrosis factor alpha and Interleukin-1 alpha) and the apoptosis of lymphocytes studied by the terminal deoxynucleotidyltransferase-mediated dUTP nick end labelling (TUNEL) technique and haemorrhages. With these results, we can conclude that the tonsil is suffering similar lesions than those observed in other lymphoid organs in acute African swine fever, even when the route of inoculation is the intramuscular and not oral-nasal.

Lymphocyte apoptosis and thrombocytopenia in spleen during classical swine fever: role of macrophages and cytokines.

Thirty-two Large White x Landrace pigs, 4 months old, were inoculated with the classical swine fever (CSF) or hog cholera virus strain "Alfort" in order to identify the mechanism responsible for the lymphopenia and thrombocytopenia observed in the spleen during the experimental induction of disease, by immunohistochemical and ultrastructural techniques. Results showed a progressive depletion of splenic lymphoid structures and evidence of platelet aggregation processes. Lymphoid depletion was due to lymphocyte apoptosis, which could not be ascribed to the direct action of the virus on these cells; direct virus action could play only a secondary role in the death of these cells. Absence of severe tissue and endothelial damage, together with moderate procoagulant cytokine levels in the serum, suggest that these pathologies can be ruled out as the cause of platelet aggregation and thrombocytopenia in CSF. Monocyte/macrophages were the main target cells for the CSF virus, and they exhibited phagocytic and secretory activation leading to the synthesis and release of tumor necrosis factor alpha, which proved to be the chief mediator, followed by IL-6, IL-1alpha, and C1q complement component. In view of their characteristics, TNF-alpha and, to a lesser extent, IL-1alpha and IL-6 appear to be the major cytokines involved in the pathogenesis of lymphocytopenia and thrombocytopenia; a clear spatial and temporal relationship was observed between these two phenomena.

Proinflammatory cytokines induce lymphocyte apoptosis in acute African swine fever infection.

Twenty-one pigs inoculated with a highly virulent isolate (E70) of African swine fever (ASF) virus were killed 1-7 days later; a further three animals served as uninfected controls. An early increase in TNF-alpha, IL-1alpha, IL-1beta and IL-6 expression was detected in lymphoid organs from infected animals, together with an increase in the serum concentrations of TNF-alpha and IL-1beta. These changes were accompanied by increased apoptosis of lymphocytes, and the presence of infected and uninfected macrophages showing changes indicative of secretory and phagocytic activation. The present study demonstrated an increase in the number of macrophages expressing TNF-alpha, IL-1 and IL-6 in proximity to lymphocytes undergoing apoptosis, supporting previous suggestions that in acute ASF proinflammatory cytokines induce lymphocyte apoptosis.

Immunohistochemical detection of the expression of pro-inflammatory cytokines by ovine pulmonary macrophages.

The aim of this study was to determine the expression of three proinflammatory cytokines by pulmonary macrophages of sheep in paraffin wax-embedded tissue. Samples of lung from seven healthy sheep were fixed by immersion in either 10% neutral buffered formalin, acetic formalin, paraformaldehyde-lysine-periodate or Bouin's solution and processed for structural and immunohistochemical studies. The expression of interleukin (IL)-1alpha, IL-6 and tumour necrosis factor (TNF)-alpha by pulmonary intravascular macrophages (PIMs) and alveolar macrophages (AMs) was detected by the avidin-biotin-peroxidase (ABC) technique. Bouin's solution proved to be the most suitable fixative and Tween 20 the most effective pretreatment for increasing permeability. Constitutive expression of IL-1alpha, IL-6 and TNF-alpha by both macrophage populations was detected. The number of PIMs expressing IL-1alpha (the predominant cytokine in ovine lung) was higher than that of AMs, while the expression of IL-6 was greater in AMs. No differences between PIMS and AMs were found in respect of TNF-alpha expression. The evaluation of cytokine expression represents a valuable tool for studying the pathogenesis of disease in the ovine lung.

Apoptosis of thymocytes in experimental African Swine Fever virus infection.

This paper report on the lesions occurred in the thymus in experimental acute African swine fever (ASF). Twenty-one pigs were inoculated with the highly virulent ASF virus (ASFV) isolate Spain-70. Animals were slaughtered from 1 to 7 days post infection (dpi). Three animals with similar features were used as controls. Thymus samples were fixed in 10% buffered formalin solution for histological and immunohistochemical study and in 2.5% glutaraldehyde for ultrastructural examination. For immunohistochemical study, the avidin-biotin-peroxidase complex (ABC) technique was used to demonstrate viral protein 73 and porcine myeloid-histiocyte antigen SWC3 using specific monoclonal antibodies. Cell apoptosis was evaluated by the TUNEL assay. Blood samples were taken daily from all pigs and were used for leukocyte counts. The results of this study show a severe thymocyte apoptosis not related to the direct action of ASFV on these cells, but probably to a quantitative increase in macrophages in the thymus and their activation. A decrease in the percentage of blood lymphocytes was observed at the same time No significant vascular changes were observed in the study. With these results we suggest that ASFV infection of the thymus does not seem to play a critical role in the acute disease. Although severe apoptosis was observed, animals died because of the severe lesions found in the other organs.

Classical Swine Fever: pathology of bone marrow.

Twenty pigs were inoculated with a virulent isolate (Quillota strain) of classical swine fever (CSF) virus to determine the chronological development of lesions in bone marrow. Histopathologic, ultrastructural and immunohistochemical (detection of viral antigen gp55, myeloid-histiocyte antigen, CD3 antigen, and FVIII-rag), and morphometric techniques were employed. Viral antigen was detected from 2 days postinfection (dpi) in stromal and haematopoitic cells, and severe atrophy related to apoptosis of haematopoitic cells was observed. Megakaryocytes (MKs) did not show significant changes in number, but there were important qualitative changes including 1) increased numbers of cloud-nuclei MKs, microMKs, apoptotic MKs, and atypical nucleated MKs and 2) decreased number of typical nucleated MKs. Morphometric study of these cells showed a decrease in cytoplasmic area. MK infection was detected from 2 dpi, but in a small percentage of cells. Myeloid cells showed quantitative changes, with an increase in granulocyte numbers. Apoptosis of lymphocytes and viral infection of erythroblasts were also observed. The main changes in stroma were depletion of T lymphocytes in the middle phase of the experiment and macrophages. Viral infection was also observed in these cells. MK lesions suggest dysmegakaryocytopoiesis, which would aggravate the thrombocytopenia already present and could be responsible for it. Granulocyte changes would lead to the appearance of circulating immature forms, whereas lymphocyte apoptosis in bone marrow would contribute to lymphopenia.

Apoptosis of thymocytes related to cytokine expression in experimental classical swine fever.

Atrophy of the thymic cortex and loss of thymocytes were studied in 32 pigs inoculated with the virulent strain "Alfort" of classical swine fever (CSF) virus and killed at intervals from 2 to 15 days after infection. Immunohistochemical, ultrastructural, ELISA and TUNEL methods were used. The results suggested that direct action of CSF virus on thymocytes played no more than a minor role. The massive lymphoid depletion observed in the thymus, may, however, have been associated with the numerical increase in monocytes-macrophages in this organ, and their secretory activation, leading to synthesis and release of tumour necrosis factor (TNF)-alpha, interleukin (IL)-1alpha and C1q complement component as main chemical mediators, and IL-1beta and IL-6 as minor mediators. These cytokines (TNF-alpha and IL-1alpha) may have played a role in the apoptosis of thymocytes, demonstrated by TUNEL and ultrastructural methods. The pathogenetic mechanism outlined may contribute to the lymphoid depletion observed in others organs in CSF and may explain the lymphopenia characteristic of the disease.

Changes in macrophages in spleen and lymph nodes during acute African swine fever: expression of cytokines.

To gain further insight into the pathogenesis of African swine fever (ASF), the cytokine expression by macrophages in spleen and lymph nodes were examined. Twenty-one piglets were inoculated with the highly virulent isolate Spain-70 of ASF virus and killed in groups at 1-7 post-inoculation days (pid). An increase in the immunohistochemical detection of proinflammatory monokines in spleen and renal and gastrohepatic lymph nodes is reported, along with an increase in the serum levels of TNF-alpha and IL-1 beta. The expression of these cytokines is detected simultaneously in time and space with the viral protein 73 (vp 73) of the ASF virus detection. Our results demonstrate that mononuclear phagocyte system cell activation results in the release of several cytokines that could induce apoptosis of lymphocytes and haemodynamic changes.

Glomerulonephritis associated with simultaneous canine adenovirus-1 and Dirofilaria immitis infection in a dog.

This article describes a case of glomerulonephritis and immunocomplex (IgM, IgG and C3c) deposition in the mesangium and basement membranes of a 2-year-old dog with canine viral hepatitis and dirofilariasis. The deposits observed in the mesangium were in the vicinity of cells with viral replication. However, no clear relationship was found between viral replication and the deposition of immunocomplexes in the glomerular capillary basement membranes, which may be the reason why these deposits have only been tentatively related to the concomitant infestation by Dirofilaria immitis.