Identification of differentially expressed gene pathways between cytopathogenic and non-cytopathogenic BVDV-1 strains by analysis of the transcriptome of infected primary bovine cells.

The bovine viral diarrhea virus 1 (BVDV-1), belonging to the Pestivirus genus, is characterized by the presence of two biotypes, cytopathogenic (cp) or non-cytopathogenic (ncp). For a better understanding of the host pathogen interactions, we set out to identify transcriptomic signatures of bovine lung primary cells (BPCs) infected with a cp or a ncp strain. For this, we used both a targeted approach by reverse transcription droplet digital PCR and whole genome approach using RNAseq. Data analysis showed 3571 differentially expressed transcripts over time (Fold Change >2) and revealed that the most deregulated pathways for cp strain are signaling pathways involved in responses to viral infection such as inflammatory response or apoptosis pathways. Interestingly, our data analysis revealed a deregulation of Wnt signaling pathway, a pathway described in embryogenesis, that was specifically seen with the BVDV-1 cp but not the ncp suggesting a role of this pathway in viral replication.

Immunogenicity evaluation of recombinant Lactobacillus casei W56 expressing bovine viral diarrhea virus E2 protein in conjunction with cholera toxin B subunit as an adjuvant.

BACKGROUND: Bovine viral diarrhea virus (BVDV) is one of the main causes of infectious diseases in cattle and causes large financial losses to the cattle industry worldwide. In this study, Lactobacillus casei strain W56 (Lc W56) was used as antigen deliver carrier to construct a recombinant Lactobacillus vaccine pPG-E2-ctxB/Lc W56 constitutively expressing BVDV E2 protein fused with cholera toxin B subunit (ctxB) as an adjuvant, and its immunogenicity against BVDV infection in mice model by oral route was explored. RESULTS: Our results suggested that pPG-E2-ctxB/Lc W56 can effectively activate dendritic cells (DCs) in the Peyer's patches, up-regulate the expression of Bcl-6, and promote T-follicular helper (Tfh) cells differentiation, as well as enhance B lymphocyte proliferation and promote them differentiate into specific IgA-secreting plasma cells, secreting anti-E2 mucosal sIgA antibody with BVDV-neutralizing activity. Moreover, significant levels (p < 0.01) of BVDV-neutralizing antigen-specific serum antibodies were induced in the pPG-E2-ctxB/LC W56 group post-vaccination. The recombinant Lactobacillus vaccine can induce cellular immune responses, and significant levels (p < 0.01) of Th1-associated cytokines (IL-2, IL-12, and IFN-γ), Th2-associated cytokines (IL-4, IL-10) and Th17-associated cytokine (IL-17) were determined in the serum of vaccinated mice. Significantly, the recombinant Lactobacillus vaccine provides immune protection against BVDV infection, which can be cleared effectively by the vaccine post-challenge in orally vaccinated animals. CONCLUSIONS: The genetically engineered Lactobacillus vaccine constructed in this study is immunogenic in mice and can induce mucosal, humoral, and cellular immune responses, providing effective anti-BVDV immune protection. It thus represents a promising strategy for vaccine development against BVDV.

Fluorophore labelled BVDV: a novel tool for the analysis of infection dynamics.

Genetic labelling of viruses with a fluorophore allows to study their life cycle in real time, without the need for fixation or staining techniques. Within the family Flaviviridae, options for genetic labelling of non-structural proteins exist. Yet, no system to genetically label structural proteins has been put forward to date. Taking advantage of a previously described site within the structural protein E2, a fluorophore was introduced into a cytopathogenic (cpe) BVDV-1 virus (BVDVE2_fluo). This insertion was well tolerated, resulting in a 2-fold drop in titer compared to the parental virus, and remained stably integrated into the genome for more than 10 passages. The fluorophore E2 fusion protein was readily detectable in purified virus particles by Western blot and fluorescence microscopy and the particle integrity and morphology was confirmed by cryo electron microscopy. The same integration site could also be used to label the related Classical swine fever virus. Also, BVDVE2_fluo particles bound to fluorophore labelled CD46 expressing cells could be resolved in fluorescence microscopy. This underlines the applicability of BVDVE2_fluo as a tool to study the dynamics of the whole life cycle of BVDV in real time.

Detecção do virus 'HoBi'-like (BVDV-3) em bovino no semiárido do Estado da Paraíba / Detection of 'HoBi'-like (BVDV-3) vírus in a cow in the semiarid of Paraíba, Northeastern Brazil

Objetivou-se descrever os aspectos clínicos e anatomopatológicos, e a identificação viral de um caso de infecção pelo vírus 'Hobi'-like (BVDV-3) em bovino do semiárido paraibano, Nordeste do Brasil. Um bovino, fêmea, três meses de idade, foi levado ao Hospital Veterinário da UFCG apresentando salivação, dificuldade de apreensão do teto, falta de apetite, fezes escuras e em pouca quantidade. Diante da piora do quadro clínico optou-se por sua eutanásia in extremis, seguida da realização da necropsia e coleta de material para histopatologia. Histologicamente, nas mucosas do trato digestivo, havia edema, degeneração balonosa, necrose e infiltrado inflamatório, que foi observado na face dorsal da língua e no seu epitélio mais profundo. A imunohistoquímica de amostras de extremidade de pavilhão auricular demonstrou marcação antigênica positiva e pela RT-PCR foi possível detectar RNA viral do BVDV no soro sanguíneo, cujo efeito citopático em células epiteliais de rim bovino da linhagem "Madin Darby bovine kidney" (MDBK) não foi observado. O sequenciamento do gene 5'NCR demonstrou que o vírus isolado estaria mais relacionado ao 'Hobi'-like (BVDV-3). Após a confirmação do diagnóstico foram coletadas amostras de soro dos 23 animais do rebanho para sorologia por ELISA indireto, sendo constatada 69,6% (16/23) de soropositividade. A identificação deste novo caso de infecção por 'Hobi'-like na Paraíba reafirma a necessidade de um monitoramento regular para BVDV na região para detecção precoce da infecção dos rebanhos e adoção de medidas eficazes de prevenção e controle.(AU)

The aim of this study was to describe the clinical and anatomopathological aspects, and the viral identification of a case of 'Hobi'-like (BVDV-3) vírus infection in cattle from the semiarid of Paraiba State, Northeastern Brazil. A female bovine, three months old, was sent to the UFCG's Veterinary Hospital presenting salivation, difficulty of ceiling seizure, lack of appetite, dark feces and in small amounts. Due to worsening of symptoms it was decided to in extremis euthanasia, followed by the necropsy and collection of material for histopathology. Histologically, in the mucous membranes of the digestive tract there were edema, ballooning degeneration, necrosis and inflammatory infiltrate, which was observed on the dorsal surface of the tongue and in its deepest epithelium. The immunohistochemical of skin biopsies of the extremity of the ear (ear notches) showed positive antigenic marking and by RT-PCR it was possible to detect viral RNA of BVDV in the serum, whose cytopathic effect in epithelial cells of bovine kidney lineage "Madin Darby bovine kidney" (MDBK)was not observed. The sequencing of the 5'NCR gene showed that the virus isolated was more related to 'Hobi'-like (BVDV-3). After confirming the diagnosis serum samples were collected from 23 animals for serology by indirect ELISA, and a seropositivity of 69.6% (16/23) was found. The identification of this new case of 'Hobi'-like infection in Paraiba reaffirms the need for regular BVDV monitoring in the region to early detection of infection in the herds and adoption of effective preventive and control measures.(AU)

Bovine Viral Diarrhea Virus Type 2 Impairs Macrophage Responsiveness to Toll-Like Receptor Ligation with the Exception of Toll-Like Receptor 7.

Bovine viral diarrhea virus (BVDV) is a member of the Flaviviridae family. BVDV isolates are classified into two biotypes based on the development of cytopathic (cp) or non-cytopathic (ncp) effects in epithelial cell culture. BVDV isolates are further separated into species, BVDV1 and 2, based on genetic differences. Symptoms of BVDV infection range from subclinical to severe, depending on strain virulence, and may involve multiple organ systems and induction of a generalized immunosuppression. During BVDV-induced immune suppression, macrophages, critical to innate immunity, may have altered pathogen recognition receptor (PRR) signaling, including signaling through toll-like receptors (TLRs). Comparison of BVDV 2 strains with different biotypes and virulence levels is valuable to determining if there are differences in host macrophage cellular responses between viral phenotypes. The current study demonstrates that cytopathic (cp), noncytopathic (ncp), high (hv) or low virulence (lv) BVDV2 infection of bovine monocyte-derived macrophages (MDMΦ) result in differential expression of pro-inflammatory cytokines compared to uninfected MDMΦ. A hallmark of cp BVDV2 infection is IL-6 production. In response to TLR2 or 4 ligation, as might be observed during secondary bacterial infection, cytokine secretion was markedly decreased in BVDV2-infected MDMΦ, compared to non-infected MDMΦ. Macrophages were hyporesponsive to viral TLR3 or TLR8 ligation. However, TLR7 stimulation of BVDV2-infected MDMΦ induced cytokine secretion, unlike results observed for other TLRs. Together, these data suggest that BVDV2 infection modulated mRNA responses and induced a suppression of proinflammatory cytokine protein responses to TLR ligation in MDMΦ with the exception of TLR7 ligation. It is likely that there are distinct differences in TLR pathways modulated following BVDV2 infection, which have implications for macrophage responses to secondary infections.

Experimental infection with cytopathic bovine viral diarrhea virus in mice induces megakaryopoiesis in the spleen and bone marrow.

Here, we infected mice with cytopathic bovine viral diarrhea virus 1 (cp BVDV1) by oral inoculation and investigated the effects of infection by histopathological, immunohistochemical (IHC), hematological methods. Twelve mice were infected, and samples were obtained at day 2, 5, and 9 postinfection (pi). Most of the infected mice exhibited clinical signs of illness such as reduced movement, crouching, loose feces, loss of appetite, and reduced water intake. Blood samples from six mice were positive for BVDV based on reverse transcription polymerase chain reaction (RT-PCR). Blood analysis also revealed thrombocytopenia and lymphopenia. Viral antigens were detected in the spleen (12/12), bone marrow (12/12), and/or mesenteric lymph nodes (4/12) of all infected mice by IHC analysis. The spleens showed significant histopathological changes including (i) substantially increased numbers of megakaryocytes, (ii) lymphocyte depletion, and (iii) hemorrhages. The bone marrow also had an increased number of megakaryocytes, although this increase was not as strong as it was in the spleen. Severe lymphoid depletion was observed in the mesenteric lymph nodes. Viral infections were present in the lymphocytes but not detected in megakaryocytes of the spleen, bone marrow, or mesenteric lymph nodes. These results suggest that the increased numbers of megakaryocytes may be a direct result of BVDV infection. BVDV infection in mice following oral inoculation of cp BVDV1 leads to megakaryopoiesis in the spleen and bone marrow to replenish the platelets.

Development of an APC-targeted multivalent E2-based vaccine against Bovine Viral Diarrhea Virus types 1 and 2.

The aim of this study was to develop and test a multivalent subunit vaccine against Bovine Viral Diarrhea Virus (BVDV) based on the E2 virus glycoprotein belonging to genotypes 1a, 1b and 2a, immunopotentiated by targeting these antigens to antigen-presenting cells. The E2 antigens were expressed in insect cells by a baculovirus vector as fusion proteins with a single chain antibody, named APCH I, which recognizes the ß-chain of the MHC Class II antigen. The three chimeric proteins were evaluated for their immunogenicity in a guinea pig model as well as in colostrum-deprived calves. Once the immune response in experimentally vaccinated calves was evaluated, immunized animals were challenged with type 1b or type 2b BVDV in order to study the protection conferred by the experimental vaccine. The recombinant APCH I-tE21a-1b-2a vaccine was immunogenic both in guinea pigs and calves, inducing neutralizing antibodies. After BVDV type 1b and type 2 challenge of vaccinated calves in a proof of concept, the type 1b virus could not be isolated in any animal; meanwhile it was detected in all challenged non-vaccinated control animals. However, the type 2 BVDV was isolated to a lesser extent compared to unvaccinated animals challenged with type 2 BVDV. Clinical signs associated to BVDV, hyperthermia and leukopenia were reduced with respect to controls in all vaccinated calves. Given these results, this multivalent vaccine holds promise for a safe and effective tool to control BVDV in herds.

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.

Complete genome sequence and pathogenesis of bovine viral diarrhea virus JL-1 isolate from cattle in China.

BACKGROUND: Bovine viral diarrhea virus (BVDV) is a pathogen found worldwide in calves. It can cause significant economic losses in agriculture. Many BVDV strains have been isolated in China. However, the pathogenesis and complete gene characteristics of BVDV isolate have yet not been reported in China. Here, a BVDV isolate was isolated and its pathogenesis and complete genome were studied. RESULTS: A new isolate of bovine viral diarrhea virus, named JL-1, was isolated from the spleen of a sick cow with diarrhea using MDBK cell culture. The complete genome of JL-1 is 12,276 nucleotides and contains a 5'-UTR of 382 nucleotides, a 3'-UTR of 188 nucleotides, and a large ORF encoding a polyprotein consisting of 3,901 amino acids. Genomic comparison and phylogenetic analyses of complete genomic sequence clearly showed that JL-1 fell into the BVDV-1b subtype. The result of pathogenesis of JL-1 strain showed that all infected calves developed clinical signs of elevated rectal temperatures, decreased leucopenia, and viral discharge. Viral antigen was detected in infected animal tissues using immunohistochemistry. Animals in the mock were normal. These results demonstrated that BVDV JL-1 was a virulent strain. CONCLUSIONS: This is the first study to report the pathogenesis and complete gene characterization of the BVDV strain in China. This report may set a good foundation for further study of BVDV in China.

Pathogenetic differences after experimental infection of calves with Korean non-cytopathic BVDV-1 and BVDV-2 isolates.

The purpose of this study was to compare clinical and virological differences between non-cytopathic (ncp) bovine viral diarrhea virus (BVDV)-1 and ncp BVDV-2 isolated from Korean field cases. Each five naïve calves were experimentally infected with Korean ncp BVDV-1 or BVDV-2 isolates. Two additional age-matched animals were used as uninfected controls. Leukocyte, lymphocyte, and platelet counts declined in all infected calves, but were significantly lower and remained decreased longer in calves infected with ncp BVDV-2 isolate. The number of monocytes was greater in calves infected with ncp BVDV-2. Flow cytometric assay showed that lymphocyte apoptosis occurred with an increase of annexin-V positive cells in all infected calves by day 6. Tumor necrosis factor alpha (TNF-α) concentration in all infected calves was lower than in control calves. In ncp BVDV-1 infected calves, interferon gamma (IFN-γ) levels in the serum were increased by day 6 compared to calves infected with ncp BVDV-2. These results demonstrated that the Korean ncp BVDV-2 isolate shows a reduced IFN-γ production, indicating prevention of the antiviral activity, and therefore promotes the development of pathological effects.

Experimental infection of pregnant cows with noncytopathogenic bovine viral diarrhoea virus between days 26 and 50 postbreeding.

The effect of bovine viral diarrhoea virus (BVDV) infection on early pregnant cows between 10 and 24 days after virus inoculation at day 26 of pregnancy was determined. Four cows were inoculated intravenously with either BVDV (treated, n=3) or growth medium (control, n=1). The treated cows were euthanized on either day 10, 17 or 24 post-infection and the control cow was euthanized on day 24 post-infection. The level of serum 2-5A synthetase increased in all of the three treated cows. Progesterone levels decreased to below 1.0 ng/ml between 10 and 22 days after inoculation in two of the three treated cows and the embryos/foetuses of two cows died. Therefore, BVDV may be a cause of early embryonic or feotal loss in early pregnant cows and serum 2-5A synthetase may be useful as an indicator of viral infection in cows.

Distribution of bovine viral diarrhoea virus antigen in persistently infected white-tailed deer (Odocoileus virginianus).

Infection with bovine viral diarrhoea virus (BVDV), analogous to that occurring in cattle, is reported rarely in white-tailed deer (Odocoileus virginianus). This study evaluated the distribution of BVDV antigen in persistently infected (PI) white-tailed deer and compared the findings with those from PI cattle. Six PI fawns (four live-born and two stillborn) from does exposed experimentally to either BVDV-1 or BVDV-2 were evaluated. Distribution and intensity of antigen expression in tissues was evaluated by immunohistochemistry. Data were analyzed in binary fashion with a proportional odds model. Viral antigen was distributed widely and was present in all 11 organ systems. Hepatobiliary, integumentary and reproductive systems were respectively 11.8, 15.4 and 21.6 times more likely to have higher antigen scores than the musculoskeletal system. Pronounced labelling occurred in epithelial tissues, which were 1.9-3.0 times likelier than other tissues to contain BVDV antigen. Antigen was present in >90% of samples of liver and skin, suggesting that skin biopsy samples are appropriate for BVDV diagnosis. Moderate to severe lymphoid depletion was detected and may hamper reliable detection of BVDV in lymphoid organs. Muscle tissue contained little antigen, except for in the cardiovascular system. Antigen was present infrequently in connective tissues. In nervous tissues, antigen expression frequency was 0.3-0.67. In the central nervous system (CNS), antigen was present in neurons and non-neuronal cells, including microglia, emphasizing that the CNS is a primary target for fetal BVDV infection. BVDV antigen distribution in PI white-tailed deer is similar to that in PI cattle.

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.

Bone marrow depletion with haemorrhagic diathesis in calves in Germany: characterization of the disease and preliminary investigations on its aetiology.

Since 2007 a new fatal haemorrhagic diathesis in calves has been observed in all areas of Germany. Analysis of 56 cases submitted for necropsy allowed its characterization. Calves fell ill within the first month of life independent of breed and sex. Only single or a few animals per herd were affected. Petechial and ecchymotic haemorrhages in many organs and tissues, particularly in skin, subcutis and gastrointestinal tract, were major findings in all animals. Microscopically a severe depletion of bone marrow cells was always observed. Lymphocytic depletion (43%) and inflammatory lesions (46%) were less frequently observed. Blood analysis of five animals indicated an aplastic pancytopenia. The resulting thrombocytopenia is regarded as major pathomechanism of this Haemorrhagic Disease Syndrome (HDS). Pedigree analysis gave no indication of hereditary disease. Tests for specific toxins such as S-(1,2-Dichlorovinyl)-L-cysteine (DCVC), furazolidone, or mycotoxins resulting in bone marrow depletion were negative. Bacterial infections, Bovine Viral Diarrhoea Virus, and Bluetongue Virus were ruled out as cause of the disease. HDS shares similarities with a circoviral infection in chickens (chicken infectious anaemia). A broad-spectrum PCR allowed detection of circoviral DNA in 5 of 25 HDS cases and in 1 of 8 non-HDS cases submitted for necropsy. Sequencing of the whole viral genome revealed a high similarity (up to 99%) with Porcine Circovirus type 2b. Single bone marrow cells stained weakly positive for PCV2 antigen by immunohistochemistry in 1 of 8 tested HDS animals. This is the first report of circovirus detection in cattle in Germany. The exact cause of HDS still remains unknown. A multifactorial aetiology involving infection, poisoning, immunopathy, or a genetic predisposition is conceivable. Additional research is necessary to clarify the pathogenesis and the potential role of PCV2 in HDS.

Systemic reactive angioendotheliomatosis-like syndrome in a steer presumed to be persistently infected with bovine viral diarrhea virus.

Unusual proliferative intravascular lesions were seen in multiple organs of a 2-year-old Corriente steer presumed to be persistently infected with bovine viral diarrhea virus (BVDV), based on widespread immunohistochemical detection of BVDV antigen. Proliferations of spindle cells, which were immunohistochemically positive for von Willebrand factor-related antigen, partially-to-completely occluded vessel lumens and were supported by cells that were immunohistochemically positive for smooth muscle actin. Distribution and character of the intraluminal proliferations are strikingly similar to those described in feline systemic reactive angioendotheliomatosis, a rare entity of unknown cause. The presence of occasional intravascular thrombi suggests that the proliferative vasculopathy was associated with an underlying thrombotic process with immunohistochemical similarities to thrombotic thrombocytopenic purpura of humans. Death of the steer was due to hemorrhage from a castration wound, which may indicate thrombocytopenia or platelet dysfunction. The role of persistent BVDV infection in the formation of the intravascular lesions is unknown.

Doença das mucosas associada à dermatite generalizada em bovinos, Mato Grosso do Sul / Mucosal disease associated with generalized dermatitis in cattle, Mato Grosso do Sul, Brazil

São descritos os aspectos epidemiológicos, clínicos, patológicos e diagnósticos de uma forma de dermatite associada à doença das mucosas (DM) em bovinos. Também são abordadas metodologias para a identificação de animais persistentemente infectados (PI) e o impacto nos índices zootécnicos no rebanho afetado. Os casos de dermatite associados com DM ocorreram em dois bovinos Nelore, de 12 e 24 meses de idade, pertencentes a uma fazenda de ciclo completo de bovinos de corte no estado de Mato Grosso do Sul. Os sinais clínicos nesses animais consistiam de emagrecimento lento e progressivo, formação de crostas difusas na pele de todo o corpo, pele ressecada, múltiplas ulcerações nas gengivas e face dorsal da língua, que evoluíram para fendas longitudinais, formação de projeções cornificadas e desprendimento dos cascos. Em um caso, também ocorreu diarréia no estágio final da doença. Na necropsia observaram-se ainda erosões longitudinais no esôfago. O exame histológico revelou focos de necrose de coagulação na mucosa do esôfago e língua, com infiltrado de neutrófilos e linfócitos. As lesões da pele consistiam de necrose de coagulação da epiderme associada com infiltrado de neutrófilos e hiperqueratose. Nos dois casos, a suspeita clínica foi confirmada pelo isolamento viral e identificação dos biótipos citopático e não-citopático do vírus da diarréia viral bovina (BVDV), além da detecção de antígenos virais em tecidos por imunoistoquímica. De um lote de 300 bovinos que tiveram contato com animais afetados, 38 foram testados e apresentaram altos títulos de anticorpos para o BVDV. Amostras de sangue coletadas de 1.025 animais jovens e 40 touros da propriedade foram submetidas a pesquisa de vírus para se identificarem possíveis animais persistentemente infectados (PI). O vírus foi isolado do sangue de três bezerros no teste inicial e, 12 meses depois, em dois deles que permaneceram na propriedade. Imunoistoquímica realizada em biópsia de orelhas identificou...

This paper reports epidemiological, clinical, pathological and laboratory diagnostic aspects of a form of dermatitis associated with mucosal disease (MD) in cattle. It also focuses on the methods used for identifying persistently infected (PI) animals and on the impact of the disease on fertility and weaning rates in the affected herd. Cases of dermatitis associated with MD were diagnosed in two 12 and 24-month-old Nelore calves belonging to a beef cattle farm that operates the full cycle of production (calving, rearing, finishing) in Mato Grosso do Sul, Midwest Brazil. The clinical signs exhibited by affected cattle 0included slow, progressive weight loss; formation of diffuse skin crusts in multiple body areas; skin dryness; multiple ulcerations on the gums and dorsal surface of the tongue which evolved to longitudinal fissures; formation of keratinized projections; and detachment of hoof horn. In addition, diarrhea affected one animal in the late stage of the disease. Necropsies also revealed longitudinal erosions in the esophagus. Histological examination showed coagulation necrosis foci in esophageal and lingual mucosae, with neutrophil and lymphocyte infiltration. Skin lesions consisted of epidermal coagulation necrosis associated with neutrophil infiltration and hyperkeratosis. In both cases, clinical diagnosis was confirmed by the isolation and identification of cytopathic and noncytopathic biotypes of the bovine viral diarrhea virus (BVDV) and immunohistochemical detection of viral antigens in formalin fixed tissues. Out of 300 cattle that had contact with the affected animals, 38 were found to be seropositive - in high neutralizing titers - to BVDV. Blood samples from 1,025 young animals and 40 bulls from the farm were examined for the presence of BVDV to identify potential PI animals. The virus was isolated from blood of three calves in the initial test and, 12 months later, from two of them which had remained...

Immunohistochemical diagnosis of persistent infection with Bovine Viral Diarrhea Virus (BVDV) on skin biopsies.

Detection of persistent infection with BovineViral Diarrhea Virus (BVDV) is essential for both epidemiological and clinical reasons. In addition to the classical virological methods such as virus isolation in tissue culture, ELISA and RT-PCR, immunohistochemistry of skin biopsies has become a useful and reliable tool. Assuming that the presence of BVDV antigen in skin structures is restricted to persistent infection, this method could differentiate from transient infection. In order to answer this question, 6 calves were experimentally infected orally with a non-cytopathic genotype 1 BVDV strain belonging to the subtype k.The calves developed fever, mucopurulent nasal discharge, coughing and leucopenia with relative lymphopenia. Immunohistochemistry of skin biopsies taken daily up to day 13-post infection did not reveal any evidence of BVDV infection. BVDV was, however, isolated from blood samples on cell cultures. Anti-NS3-antibody-ELISA and serum neutralization tests showed that all six calves seroconverted. We conclude that in acute BVDV infections, with genotype 1 and the subtypes found in Switzerland (b, e, h and k) viral antigen is not found in epidermal structures of the skin. In contrast, persistently infected animals test positive for BVD viral antigen by immunohistochemistry of the skin.

Experimental persistent infection with bovine viral diarrhea virus in white-tailed deer.

Bovine viral diarrhea virus (BVDV) infections cause substantial economic losses to the cattle industries. Persistently infected (PI) cattle are the most important reservoir for BVDV. White-tailed deer (Odocoileus virginianus) are the most abundant species of wild ruminants in the United States and contact between cattle and deer is common. If the outcome of fetal infection of white-tailed deer is similar to cattle, PI white-tailed deer may pose a threat to BVDV control programs. The objective of this study was to determine if experimental infection of pregnant white-tailed deer with BVDV would result in the birth of PI offspring. Nine female and one male white-tailed deer were captured and housed at a captive deer isolation facility. After natural mating had occurred, all does were inoculated intranasally at approximately 50 days of pregnancy with 10(6) CCID(50) each of a BVDV 1 (BJ) and BVDV 2 (PA131) strain. Although no clinical signs of BVDV infection were observed or abortions detected, only one pregnancy advanced to term. On day 167 post-inoculation, one doe delivered a live fawn and a mummified fetus. The fawn was translocated to an isolation facility to be hand-raised. The fawn was determined to be PI with BVDV 2 by serial virus isolation from serum and white blood cells, immunohistochemistry on skin biopsy, and RT-PCR. This is the first report of persistent infection of white-tailed deer with BVDV. Further research is needed to assess the impact of PI white-tailed deer on BVDV control programs in cattle.

Activation of cell signaling pathways is dependant on the biotype of bovine viral diarrhea viruses type 2.

Bovine viral diarrhea virus (BVDV), a pestivirus of the Flaviviridae family, is an economically important cattle pathogen with a worldwide distribution. Besides the segregation into two distinct species (BVDV1/BVDV2) two different biotypes, a cytopathic (cp) and a noncytopathic (ncp) biotype, are distinguished based on their behavior in epithelial cell cultures. One of the most serious forms of BVDV infection affecting immunocompetent animals of all ages is severe acute BVD (sa BVD) which is caused by highly virulent ncp BVDV2 strains. Previous studies revealed that these highly virulent ncp viruses cause cell death in a lymphoid cell line (BL3) which is not clearly associated with typical apoptotic changes (e.g. PARP cleavage) observed after infection with cp BVDV. To further characterize the underlying molecular mechanisms, we first analyzed the role of the mitochondria and caspases as key mediators of apoptosis. Compared to infection with cp BVDV2, infection with highly virulent ncp BVDV2 resulted in a delayed and less pronounced disruption of the mitochondrial transmembrane potential (DeltaPsi(m)) and a weaker activation of the caspase cascade. In contrast, infection with low virulence ncp BVDV2 showed no significant differences from the uninfected control cells. Since different pro- and anti-apoptotic cellular signaling pathways may become activated upon virus infection, we compared the effect of different BVDV2 strains on cellular signaling pathways in BL3 cells. Stress-mediated p38 MAPK phosphorylation was detected only in cells infected with cp BVDV2. Interestingly, infection with highly virulent ncp BVDV2 was found to influence the phosphoinositide 3-kinase (PI3K)-Akt pathway. This indicates that BL3 cells respond differently to infection with BVDV depending on virulence and biotype.

Indirect immunohistochemistry on skin biopsy for the detection of persistently infected cattle with bovine viral diarrhoea virus in Italian dairy herds.

Indirect immunohistochemistry (IHC) on skin biopsies for identification of persistently infected (PI) animals has been used as a parallel test to antigen and antibody ELISAs in a bovine viral diarrhoea (BVD) voluntary control program. The aim was to evaluate the reliability and feasibility of IHC on ear skin tissues to detect PI animals in field conditions, including both adult and calves under 6 months of age. In animals over 6 months of age skin biopsy and blood sample were collected at the same time, whereas in young calves blood sampling was performed when animals reached 6 months of age. One hundred and sixty-five animals were tested and immunohistochemical results were compared with those of antigen ELISA. In case of inconclusive results virus isolation and virus neutralization assays were performed. Agreement K value was 0.96. Immunohistochemical staining in positive animals was clearly detectable in the keratinocytes of the epidermis and adnexa.