Detection of hepatitis E virus RNA in saliva for diagnosis of acute infection.

Diagnosis of acute hepatitis E virus (HEV) infection is established by detection of anti-HEV IgM antibodies by ELISA or by amplification of serum viral RNA. Here, we evaluate the diagnostic value of testing HEV RNA in saliva to identify patients with acute HEV infection. Prospective proof-of-concept study including patients with acute hepatitis. Whole blood and neat saliva samples were obtained from all patients. Saliva samples were processed and analysed for HEV RNA by RT-PCR within 2 hr after collection. A total of 34 patients with acute hepatitis and 12 healthy donors were included in the study. HEV RNA in serum was confirmed by RT-PCR in eight of these patients (23.5%; 95% CI: 12.2%-40.2%). HEV was isolated in the saliva of eight of 34 patients (23.5%; 95% CI: 12.2%-40.2%). All patients with HEV RNA amplified in saliva had detectable HEV RNA in serum. HEV was isolated neither in the saliva of any of the 26 patients without detectable HEV RNA in serum nor in healthy donors. Our study suggests that acute HEV infection could be diagnosed by assessing viral load in saliva.

Familial Hepatitis E Outbreak Linked to Wild Boar Meat Consumption.

An HIV-infected patient was diagnosed with acute hepatitis E infection in our hospital. An epidemiological inquiry was performed to collect demographic, food and animal exposure variables in order to identify the potential route of transmission. The patient reported that his family traditionally hunted wild boar for food. All family members were analysed for hepatitis E virus infection. Additionally, route of transmission by wild boar meat consumption and prevalence of HEV infection among wild boar from the same hunting area were investigated. In all-family members (n = 8), HEV-RNA was amplified. Two wild boar meat slices consumed was analysed, showing the presence of HEV. The virus isolated was consistent with genotype 3, revealing 100% homology between family members and meat. Additionally, we tested nine wild boar hunted in the same hunting area. All of them were RNA-HEV positive, isolating the same HEV genotype 3 viral strain. We demonstrated by phylogenetic analysis zoonotic transmission of HEV by wild boar meat consumption. The prevalence of HEV infection among wild boar found in our study suggests that this species is an important route of transmission to human.

Immunopathologic Changes in the Thymus of Calves Pre-infected with BVDV and Challenged with BHV-1.

The aim of this work was to investigate the effect of pre-infection with bovine viral diarrhoea virus (BVDV) on thymus immune cells from calves challenged with bovine herpesvirus 1 (BHV-1). Twelve Friesian calves, aged 8 to 9 months, were inoculated with non-cytopathic BVDV-1. Ten of them were subsequently challenged with BHV-1 and euthanized in batches of two at 1, 2, 4, 7 or 14 dpi with BHV-1. The other two calves were euthanized prior to the second inoculation and were used as BVDV-infected controls. A further 10 calves were inoculated solely with BHV-1 and euthanized at the same time points. Two calves were not inoculated with any agent and were used as negative controls. Quantitative changes in immune cells were evaluated with immunohistochemical methods to compare coinfected calves and calves challenged only with BHV-1. The results of this study pointed out BVDV as responsible for the thymic lesions observed in the experiment as well as for the majority of immunopathologic changes, including a downregulation of Foxp3 lymphocytes and TGFß, which reverted as BVDV was cleared, and an overexpression of medullary CD8+ T cells. However, despite not inducing evident lesions in the thymus, BHV-1 seemed to prompt some immune alterations. Collectively, these data contribute to the knowledge on the immunopathologic alterations of the thymus during BVDV infections, and its importance in the development of secondary infections.

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.

Characterization of thymus atrophy in calves with subclinical BVD challenged with BHV-1.

Since the thymus is a target organ for the bovine viral diarrhea virus (BVDV), our experiment aimed to understand its relationship with the immunosuppressive effect by studying the consequences of a previous infection with BVDV on the thymus of calves challenged with bovine herpesvirus 1.1 (BHV-1). For this purpose, 12 animals were inoculated intranasally with non-cytopathic BVDV-1; 12 days later, 10 of them were coinfected intranasally with BHV-1. These animals were euthanized in batches of two at 0, 1, 2, 4, 7 or 14 dpi with BHV-1. Another 10 calves were inoculated solely with BHV-1 and euthanized in batches of two at 1, 2, 4, 7 or 14 dpi with BHV-1; two uninoculated calves were used as negative controls. Thymus samples from these animals were processed for viral detection and histopathological, immunohistochemical, and ultrastructural studies focused on BVDV/BHV-1 antigens, cortex:medulla ratio, apoptosis (TUNEL and caspase-3), collagen deposition, and factor VIII endothelial detection. Our study revealed the immunohistochemical presence of BVDV antigen in all animals in the BVDV-infected group, unlike BHV-1 detection, which was observed in animals in both infection groups only by molecular techniques. BVDV-preinfected animals showed severe atrophic changes associated with reduced cortex:medulla ratio, higher presence of cortical apoptosis, and increased collagen deposition and vascularization. However, calves solely infected with BHV-1 did not show atrophic changes. These findings could affect not only the numbers of circulating and local mature T cells but also the T cell-mediated immunity, which seems to be impaired during infections with this virus, thus favoring pathogenic effects during secondary infections.

An update on the epidemiology and pathology of African swine fever.

African swine fever (ASF) is one of the most important infectious diseases of swine and has major negative consequences for affected countries. ASF is present in many sub-Saharan countries, Sardinia and several countries of eastern and central Europe, where its continuous spread has the swine industry on heightened alert. ASF is a complex disease for which no vaccine or treatment is available, so its control is based on early detection and rapid control of spread. For a robust and reliable early detection programme it is essential to be able to recognize the clinical signs and pathological changes of ASF, keeping in mind that in most cases the first introductions don't show high mortality nor characteristic clinical signs or lesions, but fever and some hemorrhagic lymph nodes. Knowledge of the main characteristics of this infection, including its current distribution and routes of transmission, is also essential for preventing and controlling ASF. This review addresses each of these topics and aims to update knowledge of the disease in order to improve early detection of ASF in the field and allow implementation of public health programmes.

Effects of Preinfection With Bovine Viral Diarrhea Virus on Immune Cells From the Lungs of Calves Inoculated With Bovine Herpesvirus 1.1.

The aim of this work was to study the interstitial aggregates of immune cells observed in pulmonary parenchyma of calves preinfected with bovine viral diarrhea virus and challenged later with bovine herpesvirus 1. In addition, the intent of this research was to clarify the role of bovine viral diarrhea virus in local cell-mediated immunity and potentially in predisposing animals to bovine respiratory disease complex. Twelve Friesian calves, aged 8 to 9 months, were inoculated with noncytopathic bovine viral diarrhea virus genotype 1. Ten were subsequently challenged with bovine herpesvirus 1 and euthanized at 1, 2, 4, 7, or 14 days postinoculation. The other 2 calves were euthanized prior to the second inoculation. Another cohort of 10 calves was inoculated only with bovine herpesvirus 1 and then were euthanized at the same time points. Two calves were not inoculated with any agent and were used as negative controls. Pulmonary lesions were evaluated in all animals, while quantitative and biosynthetic changes in immune cells were concurrently examined immunohistochemically to compare coinfected calves and calves challenged only with bovine herpesvirus 1. Calves preinfected with bovine viral diarrhea virus demonstrated moderate respiratory clinical signs and histopathologic evidence of interstitial pneumonia with aggregates of mononuclear cells, which predominated at 4 days postinoculation. Furthermore, this group of animals was noted to have a suppression of interleukin-10 and associated alterations in the Th1-driven cytokine response in the lungs, as well as inhibition of the response of CD8+ and CD4+ T lymphocytes against bovine herpesvirus 1. These findings suggest that bovine viral diarrhea virus preinfection could affect the regulation of the immune response as modulated by regulatory T cells, as well as impair local cell-mediated immunity to secondary respiratory pathogens.

Role of thymic epithelial cells in lymphoid depletion after experimental infection with the noncytopathogenic BVDV1 strain 7443.

Thymic epithelial cells could play an important role in lymphoid depletion during bovine viral diarrhea virus (BVDV) infection. To evaluate this hypothesis, we examined proliferation of lymphocytes, expression of cytokeratins by thymic epithelial cells, and ultrastructural features at sequential time points after experimental infection of colostrum-deprived calves with the noncytopathogenic BVDV1 strain 7443. Ten clinically healthy Friesian calves were used. Eight were inoculated with the virus, and 2 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. Thymic lymphoid depletion was accompanied by a decrease in lymphocyte proliferation and immunohistochemical and ultrastructural changes in thymic epithelial cells. Immunohistochemical and ultrastructural results reflect a disturbance of the thymic epithelial cell network, which may explain the decrease in lymphocyte proliferation by defective thymocyte-epithelial cell interactions.

Cell-mediated immune response during experimental acute infection with bovine viral diarrhoea virus: evaluation of blood parameters.

Acute infections with bovine viral diarrhoea virus (BVDV), a major pathogen of cattle, are often asymptomatic or produce only mild clinical symptoms. However, they may play an important role in the bovine respiratory disease complex by exerting a marked immunosuppressive effect, as a result of the death of the immunocompetent cell populations involved in controlling innate and adaptive immune responses, together with a marked reduction of both cytokine expression and co-stimulatory molecule synthesis. Although experimental research and field studies have shown that acute BVDV infection enhances susceptibility to secondary infection, the precise mechanism involved in BVDV-induced immunosuppression remains unclear. The present study is aimed at measuring a range of blood parameters in a single group of fourteen calves infected with non-cytopathic BVDV-1. Focus has been put on those related to the cell-mediated immune response just as leucocyte populations and lymphocyte subpopulations, serum concentrations of cytokines (IL-1ß, TNF-α, IFN-γ, IL-12, IL-4 and IL-10) and acute phase proteins [haptoglobin, serum amyloid A (SAA), fibrinogen and albumin], as well as BVDV-specific antibodies and viremia. After non-cytopathic BVDV-1 infection, clinical signs intensity was never more than moderate coinciding with the presence of viremia and leucocyte and lymphocyte depletion. An early increase in TNF-α, IFN-γ and IL-12 levels in contrast to IL-1ß was observed in line with a raise in haptoglobin and SAA levels on the latest days of the study. As regards IL-4 levels, no evidence was found of any changes. However, a slight increase in IL-10 was observed, matching up the TNF-α decline during the acute phase response. These findings would help to increase our knowledge of the immune mechanisms involved in acute infection with non-cytopathic BVDV-1 strains, suggesting the existence of a clear tendency towards a type 1 immune response, thereby enhancing resistance against viral infections.

Comparative study of clinical courses, gross lesions, acute phase response and coagulation disorders in sheep inoculated with bluetongue virus serotype 1 and 8.

Bluetongue virus serotypes 1 (BTV-1) and 8 (BTV-8) have been described as the most prevalent in Europe during recent outbreaks displaying intense virulence, sheep being among the most severely affected livestock species. However, BTV pathogenesis is still unclear. This study sought to elucidate differences in the pathogenetic mechanisms of BTV-1 and -8 in sheep. For this purpose, a time-course study was carried out, with sequential sacrifices in order to relate pathological lesions to changes in a range of virological and serological parameters. A greater virulence of BTV-1 was probed. BTV-1 infected sheep showed a longer clinical course, with a significant increase of clinical signs and more severe gross lesions than BTV-8 infected sheep. These differences appear not to be attributable to greater virus replication, suggesting viral loads did not influence in the pathogenicity of these serotypes. While both groups displayed an early, intense antibody response, they still developed clinical signs and lesions characteristic of bluetongue, indicating a lack of correlation between antibody levels and protection against the disease. Both acute phase response (APR) and thrombocytopenia induced by BTV-1 in sheep were more intense. Furthermore, an association between acute phase proteins (APPs) concentrations and the evolution of clinical signs and gross lesions was also observed, suggesting the existence of a direct link between the pathogenicity of BTV serotypes, the severity of vascular lesions and the serum concentrations of APPs. To our knowledge, this is the first verification of a measurable APR in sheep with both experimental and naturally occurring bluetongue.

Effect of infection with BHV-1 on peripheral blood leukocytes and lymphocyte subpopulations in calves with subclinical BVD.

Bovine viral diarrhea virus (BVDV) and bovine herpesvirus-1 (BHV-1) are important cattle pathogens that induce a broad immunosuppression on cell-mediated immune response on its own participating in the bovine respiratory disease complex (BRDC). The aim of our study was to evaluate the quantitative changes in immunocompetent cells in healthy calves and calves with subclinical bovine viral diarrhea (BVD), both inoculated with BHV-1. Total leukocyte counts exhibited changes mainly in neutrophils and lymphocytes that can contribute to the BVDV immunosuppression, thus accounting for some of the intergroup differences. Monocytes did not display numerical changes in either group. Regarding lymphocyte subpopulations, even though CD4+ T lymphocytes and B cells were depleted around 4 dpi in both infected groups, the main difference observed between both groups was in CD8+ T cells which displayed an earlier depletion in BVDV inoculated calves that can promote a greater BHV-1 dissemination, thus aggravating the course of the disease.

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.

Quantification and determination of spread mechanisms of bovine viral diarrhoea virus in blood and tissues from colostrum-deprived calves during an experimental acute infection induced by a non-cytopathic genotype 1 strain.

To detect and monitor the sequential changes in virus levels, a reverse transcription quantitative real-time polymerase chain reaction assay using a TaqMan probe was carried out on frozen blood and tissues samples collected from calves experimentally infected with a non-cytopathic Bovine viral diarrhoea virus (BVDV) genotype 1 strain. Blood samples were collected among days 1-14 post-inoculation (p.i). On day 3 p.i, viral RNA was detected in blood samples from six of the eight inoculated animals. Viral RNA was detected in all remaining inoculated animals between 5 and 12 days p.i. The levels of viral RNA increased along the experiment, with a maximal peak between 6 and 9 days p.i. Analysis of virus load in tissues collected from calves euthanized on days 3, 6, 9 and 14 p.i displayed that BVDV was detected on day 3 p.i, being especially abundant in tonsils and ileocaecal valve, highlighting the role of tonsils as the main earliest viral replication sites as well as the principal source for virus spread to other lymphoid tissues and visceral organs. Coinciding with the highest viraemia levels, the highest viral loads were recorded at 9 days p.i. in tonsils, ileal lymph nodes, distal ileum and spleen, showing the main role of these secondary lymphoid organs in the pathogenic mechanisms of BVDV. However, virus levels in the liver and lung increased only towards the end of the infection. This fact could influence in the appearance of bovine respiratory diseases because of the capacity of BVDV for enhancing susceptibility to secondary infections.

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.

Immunohistochemical detection of bluetongue virus in fixed tissue.

The VP7 structural protein is the most abundant of the major core proteins and is highly conserved in all serotypes of bluetongue virus (BTV). The aim of this study was to develop immunohistochemical techniques for the detection of BTV VP7 in Bouin's- and formalin-fixed and paraffin wax-embedded tissues from small ruminants (sheep and goats) naturally infected with BTV. Tissue samples were taken from animals in which BTV infection had been confirmed by reverse transcriptase polymerase chain reaction. Optimal results were obtained by incubation of monoclonal antibody 2E9 on samples fixed with Bouin's solution or neutral buffered formalin. Optimum antigen retrieval for Bouin's-fixed samples was by microwave heating (6 min) of tissue samples in citrate buffer (pH 6.0, 0.01 M), while for formalin-fixed samples a 30 min heating period in pH 9.0 buffer was required. In both species, BTV was mainly detected in the spleen, lymph nodes and lungs; specifically within the arteriolar and capillary endothelial cells, together with macrophages and lymphocytes. The immunohistochemical method described will be a useful tool for future research.