Tumour necrosis factor-alpha-induced protein 8 (TNFAIP8) expression associated with cell survival and death in cancer cell lines infected with canine distemper virus.

Oncolytic virotherapy is a novel strategy for treatment of cancer in humans and companion animals as well. Canine distemper virus (CDV), a paramyxovirus, has proven to be oncolytic through induction of apoptosis in canine-derived tumour cells, yet the mechanism behind this inhibitory action is poorly understood. In this study, three human mammary tumour cell lines and one canine-derived adenofibrosarcoma cell line were tested regarding to their susceptibility to CDV infection, cell proliferation, apoptosis, mitochondrial membrane potential and expression of tumour necrosis factor-alpha-induced protein 8 (TNFAIP8). CDV replication-induced cytopathic effect, decrease of cell proliferation rates, and >45% of infected cells were considered death and/or under late apoptosis/necrosis. TNFAIP8 and CDVM gene expression were positively correlated in all cell lines. In addition, mitochondrial membrane depolarization was associated with increase in virus titres (p < 0.005). Thus, these results strongly suggest that both human and canine mammary tumour cells are potential candidates for studies concerning CDV-induced cancer therapy.

A three-dimensional cell culture system as an in vitro canine mammary carcinoma model for the expression of connective tissue modulators.

In this study, derived complex carcinoma (CC) and simple carcinoma (SC) cell lines were established and cultured under two-dimensional (2D) and three-dimensional (3D) conditions. The 3D was performed in six-well AlgiMatrix™ (LifeTechnologies®, Carlsbad, CA, USA) scaffolds, resulting in spheroids sized 50-125 µm for CC and 175-200 µm for SC. Cell viability was demonstrated up to 14 days for both models. Epidermal growth factor receptor (EGFR) was expressed in CC and SC in both systems. However, higher mRNA and protein levels were observed in SC 2D and 3D systems when compared with CC (P < 0.005). The connective tissue modulators, metalloproteinases-1, -2, -9 and -13 (MMPs), relaxin receptors 1 and 2 (RXR1 and RXR2) and E-cadherin (CDH1) were quantitated. All were upregulated similarly when canine mammary tumour (CMT)-derived cell lines were cultured under 3D AlgiMatrix, except CDH1 that was downregulated (P < 0.005). These results are promising towards the used of 3D system to increase a high throughput in vitro canine tumour model.

Cellular response markers and cytokine gene expression in the central nervous system of cattle naturally infected with bovine herpesvirus 5.

The present study reports an investigation on the phenotype of inflammatory and immune cells, cytokine and viral gene expression in the brains of cattle naturally infected with bovine herpesvirus 5 (BHV5). Brain sections of 38 affected animals were analysed for the nature and extent of perivascular cuffs in the Virchow-Robin space and parenchyma. Histopathological changes were severe in the olfactory bulbs (Obs), hippocampus, piriform, frontal, temporal and parietal cortices/lobes and were characterized by inflammatory infiltrates in Virchow-Robin spaces. The histopathological changes correlated positively with the distribution of BHV5 antigens (r = 0.947; P < 0.005). Cells of CD3+ phenotype were predominant in areas with severe perivascular cuffs. Viral antigens and genomic viral DNA were detected in the Obs and piriform lobe, simultaneously (r = 0.987; P < 0.005). Similarly, pro-inflammatory cytokine genes INFG, IL2, TNF and LTBR were expressed in the same brain areas (P < 0.005). These results provide important information on the inflammatory and immunological events accompanying BHV5 neurological infections. Our findings provide the first evidence for increased immune activation followed by inflammatory cytokine expression, positively correlated with viral replication in the cranial areas of the brain. Taken together, these results suggest that the host immune response and inflammation play a crucial role in the pathogenesis of acute encephalitis by BHV5 in cattle.

Genome-wide identification and in silico characterisation of microRNAs, their targets and processing pathway genes in Phaseolus vulgaris L.

Common bean (Phaseolus vulgaris L., Fabaceae) is a globally important staple crop, which is an important source of calories, protein and essential micronutrients. At the genomic level little is known regarding the small non-coding RNAs within the common bean genome. One of the most important classes of such small non-coding RNAs is microRNAs (miRNAs), which control mRNA and protein expression levels in many eukaryotes. Computational methods have been applied to identify putative miRNAs in the genomes of different organisms. In this study, our objective was to comprehensively identify and characterise miRNAs from the genome and transcriptome of P. vulgaris, including both mature and precursor miRNA forms. We also sought to identify the putative proteins involved in miRNA processing and the likely target genes of common bean miRNAs. We identified 221 mature miRNAs and 136 precursor miRNAs distributed across 52 different miRNA families in the P. vulgaris genome. Amongst these, we distinguished 129 novel mature miRNAs and 123 miRNA precursors belonging to 24 different miRNA families. We also identified 31 proteins predicted to participate in the miRNA-processing pathway in P. vulgaris. Finally, we also identified 483 predicted miRNA targets, including many which corroborate results from other species, suggesting that miRNA regulatory systems are evolutionarily conserved and important for plant development. Our results expand the study of miRNAs and their target genes in common bean, and provide new opportunities to understand their roles in the biology of this important staple crop.

New Insights on Coffea miRNAs: Features and Evolutionary Conservation.

Small RNAs influence the gene expression at the post-transcriptional level by guiding messenger RNA (mRNA) cleavage, translational repression, and chromatin modifications. In addition to model plants, the microRNAs (miRNAs) have been identified in different crop species. In this work, we developed a specific pipeline to search for coffee miRNA homologs on expressed sequence tags (ESTs) and genome survey sequences (GSS) databases. As a result, 36 microRNAs were identified and a total of 616 and 362 potential targets for Coffea arabica and Coffea canephora, respectively. The evolutionary analyses of these molecules were performed by comparing the primary and secondary structures of precursors and mature miRNAs with their orthologs. Moreover, using a stem-loop RT-PCR assay, we evaluated the accumulation of mature miRNAs in genomes with different ploidy levels, detecting an increase in the miRNAs accumulation according to the ploidy raising. Finally, a 5' RACE (Rapid Amplification of cDNA Ends) assay was performed to verify the regulation of auxin responsive factor 8 (ARF8) by MIR167 in coffee plants. The great variety of target genes indicates the functional plasticity of these molecules and reinforces the importance of understanding the RNAi-dependent regulatory mechanisms. Our results expand the study of miRNAs and their target genes in this crop, providing new challenges to understand the biology of these species.

Bovine herpesvirus type 5 infection regulates Bax/BCL-2 ratio.

Bovine herpesvirus 5 (BoHV-5) is an α-herpesvirus that causes neurological disease in young cattle and is also occasionally involved in reproductive disorders. Although there have been many studies of the apoptotic pathways induced by viruses belonging to the family Herpesviridae, there is little information about the intrinsic programmed cell death pathway in host-BoHV-5 interactions. We found that BoHV-5 is able to replicate in both mesenchymal and epithelial cell lines, provoking cytopathology that is characterized by cellular swelling and cell fusion. Viral antigens were detected in infected cells by immunofluorescence assay at 48 to 96 h post-infection (p.i.). At 48 to 72 h p.i., anti-apoptotic BCL-2 antigens were found at higher levels than Bax antigens; the latter is considered a pro-apoptotic protein. Infected cells had increased BCL-2 phenotype cells from 48 to 96 h p.i., based on flow cytometric analysis. At 48 to 96 h p.i., Bax mRNA was not expressed in any of the infected cell monolayers. In contrast, BCL-2 mRNA was found at high levels at all p.i. in both types of cells. BoHV-5 replication apparently modulates BCL-2 expression and gene transcription, enhancing production of virus progeny.

Resposta humoral de bovinos vacinados contra as toxinas botulínicas tipos c e dem diferentes faixas etárias / Humoral response of vaccinated cattle against toxins of Clostridium botulinum types c and d at different ages

Foi verificada pelo teste de ELISA indireto a resposta humoral contra os toxoides botulínicos C e D em bovinos de diferentes idades. O estudo envolveu 90 animais, que foram divididos em três grupos (n = 30), de acordo com a sua faixa etária; inferior a 2 anos de idade (G1), entre 2 e 5 anos (G2) e superior a 5 anos (G3). Os grupos experimentais foram vacinados com duas doses de vacina antibotulínica bivalente (C e D) comercial, nos dias 0 e 42 após a primo-vacinação (booster). Na avaliação, quando realizada 30 dias após o booster, os animais do G3 apresentaram maior produção de anticorpos (p < 0,05) em relação aos demais grupos. Entre o G1 e G2 não houve diferença significativa na resposta humoral contra a toxina C, no entanto, contra a toxina D, os animais do G1 apresentaram maior produção de anticorpos. Todos os grupos produziram uma resposta significativa de anticorpos contra as toxinas botulínicas após a 2ª dose da vacina bivalente comercial, principalmente contra o tipo D.

Humoral response of vaccinated cattle against toxins of clostridium botulinum types C and D at different ages. Cattle humoral response against type C and D botulinum toxoids (indirect ELISA) was verified in animals of different ages. The animals (n = 90) were divided in three groups (n = 30): group one (G1): less than two years old; group two (G2): from 2 to 5 years old; group three (G3): more than 5 years old. The groups were vaccinated with two doses [0 and 42 days after primary vaccination (booster)] of bivalent (C and D) antibotulinum vaccine. Group three had higher antibody production (p ≤ 0.05) compared to the other groups, 30 days after the booster. There was no difference (G1 and G2; p ≥ 0.05) in the humoral response against C toxin, however, against D toxin, group one had higher antibody production. It was possible to conclude that after two doses of the commercial bivalent vaccine all groups produced a significant antibody response against botulinum toxins, especially against D type.

Detection of the tumour suppressor gene TP53 and expression of p53, Bcl-2 and p63 proteins in canine transmissible venereal tumour.

Canine transmissible venereal tumour (CTVT) is a neoplasm transmitted among healthy dogs by direct contact with injured skin and/or mucous tissue. This study aimed to identify the TP53 gene, messenger RNA (mRNA) as well as the expression of p53, Bcl-2 and p63 proteins in histological sections of 13 CTVT samples at different stages of evolution. The in situ hybridization (ISH) and in situ reverse transcriptase polymerase chain reaction (RT-PCR) assays were used, which showed the DNA homologous to TP53 and its respective mRNA in 92.3% of the samples. We detected p53, p63 and Bcl-2 proteins in most of the cell samples in different grades of intensity. In addition, 46% of the samples were in the progressive and 54% in the regression phase. This is the first description of these proteins and a detailed study of their role in CTVT cells needs to be addressed in or to verify how these cells undergo apoptosis.

Apoptotic and developmental effects of bovine Herpesvirus type-5 infection on in vitro-produced bovine embryos.

Bovine Herpesvirus type-5 (BoHV-5), which is potentially neuropathogenic, was recently described to be related with reproductive disorders in cows. The objective was to elucidate mechanisms involved in propagation of BoHV-5 in embryonic cells. For this purpose, bovine embryos produced in vitro were assayed for apoptotic markers after experimental infection of oocytes, in vitro fertilization, and development. Host DNA fragmentation was detected with a TUNEL assay, expression of annexin-V was measured with indirect immunofluorescence, and viral DNA was detected with in situ hybridization. Infective BoHV-5 virus was recovered from embryos derived from exposed oocytes after two consecutive passages on Madin-Darby bovine kidney (MDBK) cells. The viral DNA corresponding to US9 gene, localized between nucleotides 126243 to 126493, was detected in situ and amplified. There was no significant difference between the ratio of TUNEL stained nuclei and total cells in good quality blastocysts (0.87 ± 0.05, mean ± SD), but there were differences (P < 0.05) between infected (0.18 ± 0.05) and uninfected blastocysts (0.73 ± 0.07). The Annexin-V label was more intense in uninfected embryos (0.79 ± 0.04; P < 0.05). The quality of infected and uninfected embryos was considered equal, with no significant effect on embryonic development. In conclusion, we inferred that BoHV-5 infected bovine oocytes, replicated, and suppressed some apoptotic pathways, without significantly affecting embryonic development.

Pathology and tissue distribution of turkey coronavirus in experimentally infected chicks and turkey poults.

Twenty 1-day-old specific pathogen free chicks and 20 1-day-old commercially derived turkey poults were inoculated with a Brazilian strain of turkey coronavirus (TCoV) to study the pathogenicity and virus distribution up to 14 days post-inoculation by histopathology, immunohistochemistry, reverse transcriptase polymerase chain reaction and sequencing. At 2-14 dpi, TCoV antigens were detected in the paranasal sinus and lachrymal accessory gland (Harderian gland) of infected chicks and in the ileum, ileocaecal junction and caecum of infected poults. Lymphocytic inflammation was present in these tissues. TCoV was re-isolated from pooled tissue suspensions of nasal concha, Harderian gland and paranasal sinus from chicks, as well as from the ileum, ileocaecal junction and caecum of poults, after three consecutive passages in 28-day-old embryonated turkey eggs. Viral RNA corresponding to the spike gene region (1178-2073 genome position) was amplified from the upper respiratory tract of chickens and from the intestinal tract of poults and phylogenetic analysis confirmed the identity as TCoV. This is the first description of TCoV antigens and mRNA in upper respiratory tissues in experimentally infected chickens.

Effects of bovine Herpesvirus Type 5 on development of in vitro-produced bovine embryos.

Bovine (Bos indicus) herpesviruses have been associated with reproductive disease. Type 1, the most studied species, is best known for its reproductive and respiratory effects. Type 5 (BoHV-5) has been detected in bull semen and aborted fetuses but not in oocytes and embryos. This study consisted of three experiments that evaluated (1) BoHV-5-infected oocytes matured in medium with fetal bovine serum (BoHV-FBS) or polyvinyl alcohol (BoHV-PVA) and fertilized by noninfected sperm; (2) noninfected oocytes fertilized by BoHV-5-infected sperm; and (3) infection of presumptive zygotes by BoHV-5. Each treatment involved nine drops of 15 to 20 oocytes. Infection with BoHV-5 was detected by polymerase chain reaction and in situ hybridization assay, and fertilization capacity and embryonic development were assessed using in vitro culture. Experimentally induced infection was obtained in all experiments, and vertical transmission of BoHV-5 by gametes was confirmed. The cleavage rate was reduced (P=0.0201) in BoHV-FBS (80.4+/-8.9%; mean+/-SD) compared with that of noninfected oocytes (89.9+/-6.5%); neither differed from BoHV-PVA (87.3+/-7.1%), and the resulting embryo production rate was not significantly different among groups. Rates of cleavage (87.5+/-7.5% vs. 92.2+/-5.5%, control vs. infected) and development of embryos (41.7+/-9.9% vs. 44.3+/-7.7% to morula/blastocyst/expanded blastocyst [M/B/EB] and 39.6+/-10.3% vs. 40.8+/-9.2% to blastocyst/expanded blastocyst/hatching blastocyst [B/EB/HB] stages) were not compromised by infected sperm (P=0.1462, P=0.5402, and P=0.8074, respectively). However, presumptive zygotes directly infected 1 d after fertilization produced a lower number (P=0.0140 to M/B/EB and P=0.002 to B/EB/HB stages) of in vitro-produced embryos (31.6+/-4.6 vs. 25.0+/-5.5 and 31.6+/-4.6 vs. 20.2+/-5.4; control vs. infected). In conclusion, BoHV-5 infected gametes and was transmissible to the embryo during in vitro development. As zygotes infected 1 d after fertilization had compromised development, BoHV-5 has the potential to be a pathogen with economic consequences.

Neonatal immune response of Brazilian beef cattle to vaccination with clostridium botulinum toxoids types C and D by indirect ELISA

Types C and D strains of Clostridium botulinum are commonly related to avian and mammalian botulism. Although there are numerous vaccine recommendations, little research has been conducted to indicate the real effectiveness of vaccine timing or the ideal immunization protocol for young beef calves. Four commercially available vaccines, two bivalent (Clostridium botulinum types C and D; vaccines 1 and 2) and two polyvalent (all Clostridium spp. including Clostridium botulinum types C and D; vaccines 3 and 4), that are currently used in Brazilian herds, were tested in order to verify the maternal immune response. One hundred cows, divided into four vaccinated groups and one unvaccinated group, were given a two-dose subcutaneous immunization, at day zero, followed by a second dose given at 42 days post-vaccination, which corresponded to 40 days before birth. Serum samples (n = 75) were collected only from healthy neonatal calves at 0, 7, 45 and 90 days post-calving (DPC) and subjected to indirect ELISA using the purified C and D holotoxins as capture antigens. The serological profile showed that all vaccines were able to induce a satisfactory neonatal immune response to both holotoxins at 7 DPC. However, at 45 and 90 DPC, a significant reduction (p < 0.05) was observed in the antibody level against C and D holotoxins in all tested vaccines. Neonatal immunization in calves is compromised by significant levels of maternal antibodies so that the necessity of planning a calf vaccination program involves assessment of disease risks at the production site. Finally, our findings represent the first demonstration of maternal immunity transferred to neonatal beef calves, including immunity levels after vaccination against Clostridium botulinum toxoids C and D.

Epidemiological aspects of astrovirus and coronavirus in poults in the South Eastern Region of Brazil / Aspectos epidemiológicos associados à prevalência de Astrovirus e Coronavirus em lotes de perus da Região Sudeste do Brasil

A survey of Turkey Coronavirus (TCoV) and Astrovirus (TAstV-2) prevalence was carried out from February to December during 2006 year in semiarid region of Brazil, from a turkey producer area, localized in South Eastern of Brazil. To asses the risk factor related to clinical material, climatic condition and type of RT-PCR applied, cloacal swabs (CS), faeces, sera, bursa of Fabricius (BF), thymus (TH) and spleen (SP) and ileum-caeca region were collected from 30-day-old poults suffering of enteritis episode characterized as poult enteritis mortality syndrome (PEMS). The PEMS clinical features were characterized by watery to foamy faeces, light brown-yellow in colour and low mortality rate. Meteorological data (rainfall and relative humidity) observed during along the study presented monthly average temperature ranging from 39.3 and 31.2ºC, precipitation in rainy season from 40 to 270.3 mm/month, and no rain during dry season. Simplex RT-PCR gave odds ratio (OR) values suggesting that ileum-caeca region is at higher chance (OR=1.9; p=0.9741) to have both viral RNA than faeces (OR=1.5; p=0.7319). However, multiplex RT-PCR showed 3.98 (p=0.89982) more chance to give positive results in faeces than CS at dry season. The major risk factors seem to be low rate of humidity and high temperatures at winter, probably responsible for spread, easily, the TCoV and TAstv-2 among the flocks. The positive results of both virus suggested that they can play an important role in enteric disorders, associated to low humidity and high temperatures frequently found in tropical countries.

O presente estudo foi conduzido para avaliar a prevalência do Coronavirus dos perus (TCoV) e Astrovirus tipo 2 (TAstV-2) entre os meses de Fevereiro a Dezembro de 2006, em uma região produtora localizada no semi-árido a Sudeste do Brasil. Os principais fatores de risco associado a prevalência foram material clínico analisado, condições climáticas e tipo de técnica molecular empregada. Os sinais clínicos foram caracterizados como intenso fluido intestinal e baixo crescimento em aves jovens, sendo o material coletado swabs cloacais, fezes, soros, bursa de Fabrícius, segmentos do intestino delgado, timo e baço. Os dados meteorológicos (índice pluviométrico e umidade relativa) desta região, durante o período de estudo, foram de temperatura média mensal variando de 39.3 a 31.2ºC, precipitação na época chuvosa variando de 40 a 270.3mm/mês e ausência de chuva na estação fria e seca. A técnica de simplex RT-PCR resultou em valores de odds ratio (OR) que sugerem que a região do intestino delgado (junção íleo-cecal) possui alta chance (1.9 vezes) de gerar resultados positivos na amplificação de RNA viral que as fezes (1.5 vezes) analisadas. A técnica de multiplex RT-PCR demonstrou ser 3.98 vezes mais eficiente em promover resultados positivos nas fezes que nos swabs cloacais, durante a época de inverno. Os maiores fatores de risco encontrados foram baixa umidade relativa associada a altas temperaturas, durante a estação seca, o que pode permitir uma maior disseminação aérea do ambos os vírus entre os lotes estudados. A alta prevalência detectada para dois vírus sugerem que, no Brasil, estes representam os maiores responsáveis pelos surtos de enterite viral nas regiões semi-áridas, associado a baixas umidades e altas temperaturas típicas de países tropicais.

Epidemiological aspects of astrovirus and coronavirus in poults in the South Eastern Region of Brazil.

A survey of Turkey Coronavirus (TCoV) and Astrovirus (TAstV-2) prevalence was carried out from February to December during 2006 year in semiarid region of Brazil, from a turkey producer area, localized in South Eastern of Brazil. To asses the risk factor related to clinical material, climatic condition and type of RT-PCR applied, cloacal swabs (CS), faeces, sera, bursa of Fabricius (BF), thymus (TH) and spleen (SP) and ileum-caeca region were collected from 30-day-old poults suffering of enteritis episode characterized as poult enteritis mortality syndrome (PEMS). The PEMS clinical features were characterized by watery to foamy faeces, light brown-yellow in colour and low mortality rate. Meteorological data (rainfall and relative humidity) observed during along the study presented monthly average temperature ranging from 39.3 and 31.2ºC, precipitation in rainy season from 40 to 270.3 mm/month, and no rain during dry season. Simplex RT-PCR gave odds ratio (OR) values suggesting that ileum-caeca region is at higher chance (OR=1.9; p=0.9741) to have both viral RNA than faeces (OR=1.5; p=0.7319). However, multiplex RT-PCR showed 3.98 (p=0.89982) more chance to give positive results in faeces than CS at dry season. The major risk factors seem to be low rate of humidity and high temperatures at winter, probably responsible for spread, easily, the TCoV and TAstv-2 among the flocks. The positive results of both virus suggested that they can play an important role in enteric disorders, associated to low humidity and high temperatures frequently found in tropical countries.

Brain and lung cryptococcoma and concurrent corynebacterium pseudotuberculosis infection in a goat: a case report

A four-year-old male goat with a history of neurological disorder was euthanized. It presented uncommon nodules in the brain and lungs associated with multiple abscesses, predominantly in the spleen and liver. Histological examination of brain and lung sections revealed yeast forms confirmed to be Cryptococcus gattii after a combination of isolation and polymerase chain reaction (PCR) procedures. Moreover, Corynebacterium pseudotuberculosis infection was diagnosed by PCR of samples from the lung, spleen and liver. The present report highlights the rare concurrent infection of C. gatti and C. pseudotuberculosis in an adult goat from São Paulo state, Brazil, and indicates the necessity of surveillance in the treatment of goats with atypical pulmonary infections associated with neurological disorders.(AU)

A Bayesian approach to estimate the accuracy of "in-house" ELISA assay to measure rabies antibodies from compulsory vaccinated dogs and cattle

Rabies is a vaccine-preventable disease that causes acute encephalitis in mammals, and it is still a significant public health problem in numerous countries. lnfected dogs represent the main vectors involved in human rabies. Additionally, cattle rearing close to geographic areas where vampire bats are found presents an important connection with rural epidemiology. We applied two "in-house" enzyme-linked immunosorbent assay (ELISA) methodologies, considered alternatives to measure antibodies from vaccinated dogs and cattle, without employing the gold standard approach. The ELISA assays were performed on individual serum samples taken from domestic adult dogs and cows compulsory vaccinated against rabies (147 urban dogs and 64 cows; n equal 211). The sandwich and liquid-phase competitive ELISA (scELlSA and ipcELlSA). considered "in-house" assays. were performed according to previous works. The only statistical methodology that allows this study is the Bayesian approach, developed to replace the conventional Hui-Walter paradigm. For conditional independent Bayesian model (one population, two tests and no gold standard) the prior information for sensitivity and specificity of each test, mode, prevalence and transformed (alpha, beta) were submitted to Bayesian inference. The "in-house" IpcELISA revealed 16 - out of 261 serum samples - negative results, whereas in scELISA all results were positive. The Bayesian approach showed that prior information was specified for all parameters; posterior medians were SescELISA 89%, SpscELISA 88%, SPipcELISA 95% SeipcELISA 98%, and prevalence (pi) of 99%, without the use of gold standard analysis to measure specific anti-rabies antibodies

Validation of an immunohistochemistry assay to detect turkey coronavirus: a rapid and simple screening tool for limited resource settings.

The objective of the present study was to develop and apply the direct immunohistochemistry (D-IHC) assay to search for turkey coronavirus (TCoV) antigens in formalin-fixed embedded-paraffin tissues by the use of biotin-labeled polyclonal antibody. Twenty-eight-day-old embryonated turkey eggs (n = 50) were inoculated with TCoV-purified virus, and 3 d after inoculation, sections from ileum, ileum-cecal junction, and ceca were harvested, fixed in neutral formalin, and embedded in paraffin blocks and used as positive control. In addition, a total of 100 field samples from ileum, ileum-cecal junction, and ceca, collected from 30 to 45-d-old turkeys poults experiencing an outbreak of acute enteritis, were used to search for TCoV by the same D-IHC. All results were compared with those obtained by conventional RT-PCR and indirect fluorescent antibody assay (IFA) for all tested samples. Turkey coronavirus was detected in experimentally infected embryo tissues and also in field samples in 100% of ileum-cecal junction and ceca by the 3 detection procedures. With IFA as a reference assay, sensitivity and specificity of D-IHC were 98 and 58%, whereas sensitivity and specificity of reverse transcription-PCR were 96 and 66%, calculated from the total of tested samples from experimental infection. Each of the examined procedures was highly specific (D-IHC, 93%; RT-PCR, 90%), sensitive (D-IHC, 85%; RT-PCR, 86%), and agreement of both D-IHC and RT-PCR was 99 and 100%, respectively, compared with IFA results obtained from all the field samples. These findings demonstrated the utility of D-IHC for direct detection of TCoV from field samples and considering the sensitivity and specificity found here, can be used as an alternative technique.

Experimental infectious bursal disease in the ostrich (Struthio camelus).

Clinically severe disease was produced in ostriches aged 4 weeks by oral infection with a virulent strain of infectious bursal disease virus (vIBDV), namely strain Faragher 52/70. Four days after infection the birds were humanely killed and tissue samples, including thymus, bursa of Fabricius (BF), brain and kidney were collected for examination. Histopathologically, the thymus and BF showed severe lymphoid depletion and necrosis, while immunolabelling with a polyclonal antibody demonstrated abundant viral antigen.

Detection of bovine Herpesvirus type 5 in formalin-fixed, paraffin-embedded bovine brain by PCR: a useful adjunct to conventional tissue-based diagnostic test of bovine encephalitis.

The aim of this study was to evaluate the application of PCR technique for the detection of BoHV-5 in routinely formalin-fixed, paraffin-embedded brain tissues in 20 naturally infected calves affected by fatal meningoencephalitis. Brains were divided into two halves, one kept fresh for virus isolation and PCR assay, targeting the glycoprotein C gene from BoHV-5 genome. The other half brain, corresponding to posterior cortex region, was submitted to formalin fixation and embedded into paraffin blocks for microscopic evaluation and total DNA isolation. Most of the slides showed severe multifocal non-supurative encephalitis with neuronal degeneration, neurophagia, and no acidophilic intranuclear inclusions could be found in neurons and glial. The 20 fresh samples were confirmed, by virus isolation and PCR assay, as having the BoHV-5 virus and, respective glicoprotein C sequence, while 15 of 20 formalin-fixed, paraffin-embedded samples were considered positive for the same analysis. The results revealed the first description of PCR efficiency, applied to formalin-fixed, paraffin-embedded brain collected from naturally infected calves, improving the detection of BoHV-5 from archival samples in South America.

Chicken embryo related (CER) cell line for quantification of rabies neutralizing antibody by fluorescent focus inhibition test.

Levels of rabies virus neutralization antibody in sera from vaccinated dogs and cattle were either measured by mouse neutralization test (MNT) or by rapid fluorescent focus inhibition test (RFFIT), performed on CER monolayers. The two tests were compared for their ability to detect the 0.5 International Units/ml (I.U.) recommended by the World Health Organization (WHO) as the minimum response for proof of rabies immunization. A significant correlation was found between the two tests (n=211; r=0.9949 in dogs and 0.9307 in cows, p<0.001), good sensitivity (87.5%), specificity (94.7%) and agreement (96.6%) as well. RFFIT method standardized on CER cell system for neutralizing antibodies detection turns the diagnosis easier and less expensive, specially when a great number of samples must be tested from endemic areas as commonly found in Brazil.