Comparison of slot blot nucleic acid hybridization, immunofluorescence, and virus isolation techniques to detect bluetongue virus in blood mononuclear cells from cattle with experimentally induced infection.

A slot blot hybridization technique was applied for detection of bluetongue virus (BTV) in blood mononuclear cells (BMNC) obtained from cattle with experimentally induced infection. This technique lacked sensitivity to detect the viral nucleic acid directly in clinical specimens. When aliquots of mononuclear cells from these cattle were cultivated in vitro for 10 days to amplify virus titer, only 33.3% of the samples collected during viremia gave a positive signal in the slot blot hybridization format. By contrast, results for 34.3% of noncultured and 63.3% of cultured mononuclear cell samples collected during viremia were positive by immunofluorescence. The average number of infected cells, as detected by immunofluorescence in the noncultured mononuclear cell samples, was 1 to 5/300,000, and was usually > 10/300,000 in the cultured cell samples. Virus was isolated from all postinoculation blood samples obtained from 4 heifers that were seronegative at the time of inoculation, but was not isolated from any of the preinoculation samples, or from any of the postinoculation samples obtained from 2 heifers that were seropositive at the time of inoculation. When virus isolation was attempted from separated mononuclear cells in 2 heifers, 43.7% of the noncultured and 87.5% of the cultured samples had positive results.     

Cyclooxygenase ‐2 Expression in Mammary Tumors in Dogs and its Correlation to Histologic and Biologic Behavior

Introduction:  Cyclooxygenase‐2 (Cox‐2) is the inducible form and the rate‐limiting enzyme, for conversion of arachidonic acid to prostaglandins. Cox‐2 overexpression, common in carcinomas, is associated with increased growth rate, resistance to apoptosis, angiogenesis, and overall, both local and distant aggressive behavior. Cox‐2 overexpression has been detected in human and canine mammary tumors (MTs). Histopathology of canine MT is not always predictive of biologic behavior, and anecdotally, only 50% of the malignant MTs are expected to metastasize. We hypothesize that Cox‐2 expression correlates with aggressive behavior.
Methods:  This retrospective study evaluated 48 bitches, presented for excision of MT between 2000 and 2003 at FMVZ de Botucatu‐UNESP, Brasil. Follow‐up varied from 18 months to 24 months and included physical examination and thoracic radiographs. Histopathologic examination was performed in all tumors, as well as in metastatic lesions when detected in the follow‐up period. Immunohistochemistry was used to detect expression of Cox‐2 in paraffin blocks (Rabbit polyclonal anti‐PGHS‐2. Oxford Biomedical). 10 adenomas, 10 carcinomas, 10 benign mixed tumors, 10 malignant mixed tumors and 8 cases of primary carcinomas and their metastatic lesions.
Results:  Expression of Cox‐2 varied among groups. Adenomas (32.1%), mixed benign tumors (38%), carcinomas (60.3%), malignant mixed tumors (65.8%), and metastatic carcinomas (81.25%) and their metastatic lesions (84.35%). Statistically significant differences (p < 0.05) were observed between the benign and malignant counterparts and between carcinomas and metastatic carcinomas.
Conclusions:  Cox‐2 expression correlates with both histologic and biologic behavior in mammary carcinomas, and may serve as a predictor of metastatic potential.     

全日制放牧BMY牛的活体采卵-体外胚胎生产-胚胎移植研究

为建立BMY牛的活体采卵(OPU)-体外胚胎生产(IVP)-胚胎移植(ET)快速扩繁技术体系,从人工草地全放牧牛群中选择健康的经产空怀BMY牛作为供体(n=14)集中OPU一次;卵母细胞经TCM-199体外成熟培养(IVM)、Fert-TALP液体外受精(IVF),受精卵用合成输卵管液(SOF)体外培养(IVC)生产胚胎;BMY牛受体(n=81)用CUE-MATETM孕酮阴道栓+EB+FSH+PG方法同期发情处理,对黄体合格的移植OPU-IVP来源鲜胚。研究结果表明:①OPU头均获卵母细胞23.50枚,获10枚以上卵母细胞的供体占85.7%;IVP的平均囊胚数和可用胚数分别为5.21和4.00枚;OPU前8～9d卵巢上有黄体(CL)的供体,头均获卵母细胞数及IVP的囊胚数、可移植胚数、囊胚率和可移植胚率分别比对照组高7.28枚(P<0.05)、7.29枚(P<0.05)、6.00枚(P<0.05)、27.3%(P<0.01)和23.2%(P<0.01);②受体黄体合格率69.1%(56/81),胚胎移植妊娠率37.5%(21/56),产犊率35.7%(20/56);③受体在胚胎移植时注射GnRH,其妊娠率比对照组高16.2%(P>0.05)。结果显示,BMY母牛具有良好的OPU潜力,但个体间差异较大;可通过严格供体选择及改善IVP技术等措施,实现OPU-IVP效率最大化。CUE-MATETM+EB+FSH+PG同期发情处理方法适于在牛规模化胚胎移植(ET)和人工授精(AI)中应用。综合应用OPU、IVP、同期发情和ET技术是加快优良BMY牛扩繁的重要途径之一。     

Serobiotypes and virulence genes of Escherichia coli strains isolated from diarrheic and healthy rabbits in Brazil

A total of 178 Escherichia coli isolates from diarrheic and healthy rabbits in the S?o Paulo State (Brazil) were serobiotyped and investigated by PCR for the presence of virulence genes. Among the 90 (50.6%) isolates which possessed the eae gene, 74 were from diarrheic animals and all but one encoded intimin beta. Sixty five (72.2%) of the eae+ isolates had insertion of the locus of enterocyte effacement locus in the pheU locus, 11 (12.2%) in the selC and 14 (15.6%) did not insert in either of these loci. All isolates were negative for genes of the E. coli enterotoxins, Stx1, Stx2, CNF1, CNF2 and EHEC hemolysin. The O132:H2 serotype was dominant, being present in 63 isolates (70%) of the 90 eae+ isolates, and 57 of the 63 isolates of this serotype belonged to biotype 30. PCR detected the gene for AF/R2 fimbriae in 75 (83.3%) of the 90 eae+ isolates. Adherence to HeLa cells was best detected following 6h incubation and a positive fluorescence actin staining (FAS) test was given by 52 isolates. These data show that isolates of E. coli associated with diarrhea in rabbits in Brazil possess the genotype and phenotype typically associated with rabbit enteropathogenic E. coli (EPEC). We conclude that EPEC that possess the eae gene are a common cause of diarrhea in Brazilian rabbit farms and that the pathogenic eae+ AF/R2+ isolates of O132:H2:B30 serobiotype are especially predominant.     

Prevalence of anti-Neospora caninum antibodies in cattle and dogs from Western Amazon, Brazil, in association with some possible risk factors

For evaluation of the prevalence of anti-Neospora caninum antibodies and its associated risk factors, serum samples from 2109 cattle (11 beef, 50 dairy and 25 mixed farms) and 174 dogs were examined in the State of Rond?nia, Western Amazon, Brazil. An inquiry was applied in each farm. Sera were examined by the Indirect Fluorescence Antibody Test (IFAT) using cut off dilution of 1:25 for cattle and 1:50 for dogs. Statistical association between the serologic status and several variables were analyzed by linear and logistic regression. The overall herd prevalence of anti-N. caninum antibodies for 86 farms was 72% (61.3-81.2%). Prevalence values were 100, 70 and 64% in beef, dairy and mixed herds, respectively. Herd prevalence in beef herds was significantly different (P<0.05) from dairy and mixed herds. The overall animal prevalence of N. caninum in cattle was 8.8%. Prevalence values by animal were similar in different production types (P>0.05), with values of 9.5, 11.2 and 9.7% for beef, dairy or mixed cattle, respectively. Antibodies were found in 12.6% of the 174 examined dogs. Sixteen (22.8%) out of 70 farms with dogs had at least one dog with anti-N. caninum antibodies. The occurrence of antibodies in cattle was statistically associated with farms having more than 25 cows (OR 9.7, 95% IC 2.9-32.2; P=0.0002). There was no significant association between the presence of the dogs, jungle contact or reproductive variables with the occurrence of antibodies in cattle.     

Detection of enzootic nasal tumor virus (ENTV) in a sheep flock in southern Brazil

Enzootic nasal tumor (ENT) is a contagious neoplasm associated with enzootic nasal tumor virus (ENTV), which may induce disease in sheep (ENTV-1) and goats (ENTV-2). This study aimed to describe the occurrence of ENT in two Texel sheep (Ovis aries) from a 75-sheep flock, located in the city of Gravataí, southern Brazil. Animals used to be purchased from different origins, and no specific tests for disease monitoring or quarantine procedure were performed. Affected animals presented respiratory distress, anorexia with severe weight loss, and mucopurulent unilateral nasal discharge. Necropsy was performed in both animals and nasal cavity masses were observed. Histopathological analysis demonstrated an epithelial neoplasm compatible with nasal adenocarcinoma. PCR using a protocol that amplifies a 591 bp sequence of 5’LTR-gag region of ENTV1 was performed followed by DNA sequencing. Both samples were positive, and the sequences obtained presented highest identity (97%) with ENTV strain TN28 (GenBank accession number MH899613) detected in a Texel sheep from Scotland. This is the first report of ENTV-1 leading to enzootic nasal tumor in sheep in Latin America, which confirms the presence of the retrovirus in sheep flocks in the Brazilian territory.

     

Necrotic enteritis challenge regulates peroxisome proliferator-1 activated receptors signaling and β-oxidation pathways in broiler chickens

Necrotic enteritis (NE) is an important enteric disease in poultry and has become a major concern in poultry production in the post-antibiotic era. The infection with NE can damage the intestinal mucosa of the birds leading to impaired health and, thus, productivity. To gain a better understanding of how NE impacts the gut function of infected broilers, global mRNA sequencing (RNA-seq) was performed in the jejunum tissue of NE challenged and non-challenged broilers to identify the pathways and genes affected by this disease. Briefly, to induce NE, birds in the challenge group were inoculated with 1 mL of Eimeria species on day 9 followed by 1 mL of approximately 10⁸ CFU/mL of a NetB producing Clostridium perfringens on days 14 and 15. On day 16, 2 birds in each treatment were randomly selected and euthanized and the whole intestinal tract was evaluated for lesion scores. Duodenum tissue samples from one of the euthanized birds of each replicate (n = 4) was used for histology, and the jejunum tissue for RNA extraction. RNA-seq analysis was performed with an Illumina RNA HiSeq 2000 sequencer. The differentially expressed genes (DEG) were identified and functional analysis was performed in DAVID to find protein–protein interactions (PPI). At a false discovery rate threshold <0.05, a total of 377 DEG (207 upregulated and 170 downregulated) DEG were identified. Pathway enrichment analysis revealed that DEG were considerably enriched in peroxisome proliferator-activated receptors (PPAR) signaling (P < 0.01) and β-oxidation pathways (P < 0.05). The DEG were mostly related to fatty acid metabolism and degradation (cluster of differentiation 36 [CD36], acyl-CoA synthetase bubblegum family member-1 [ACSBG1], fatty acid-binding protein-1 and -2 [FABP1] and [FABP2]; and acyl-coenzyme A synthetase-1 [ACSL1]), bile acid production and transportation (acyl-CoA oxidase-2 [ACOX2], apical sodium–bile acid transporter [ASBT]) and essential genes in the immune system (interferon-, [IFN-γ], LCK proto-oncogene, Src family tyrosine kinase [LCK], zeta chain of T cell receptor associated protein kinase 70 kDa [ZAP70], and aconitate decarboxylase 1 [ACOD1]). Our data revealed that pathways related to fatty acid digestion were significantly compromised which thereby could have affected metabolic and immune responses in NE infected birds.     

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.