Bovine herpesvirus-5 infection in a rabbit experimental model: Immunohistochemical study of the cellular response in the CNS

Since little information is available regarding cellular antigen mapping and the involvement of non-neuronal cells in the pathogenesis of bovine herpesvirus type 5 (BHV-5) infection, it were determined the BHV-5 distribution, the astrocytic reactivity, the involvement of lymphocytes and the presence of matrix metalloproteinase (MMP)-9 in the brain of rabbits experimentally infected with BHV-5. Twelve New Zealand rabbits that were seronegative for BHV-5 were used for virus inoculation, and five rabbits were used as mock-infected controls. The rabbits were kept in separate areas and were inoculated intranasally with 500 μl of virus suspension (EVI 88 Brazilian isolate) into each nostril (virus titer, 107.5 TCID50). Control rabbits were inoculated with the same volume of minimum essential medium. Five days before virus inoculation, the rabbits were submitted to daily administration of dexamethasone. After virus inoculation, the rabbits were monitored clinically on a daily basis. Seven rabbits showed respiratory symptoms and four animals exhibited neurological symptoms. Tissue sections were collected for histological examination and immunohistochemistry to examine BHV-5 antigens, astrocytes, T and B lymphocytes and MMP-9. By means of immunohistochemical and PCR methods, BHV-5 was detected in the entire brain of the animals which presented with neurological symptoms, especially in the trigeminal ganglion and cerebral cortices. Furthermore, BHV-5 antigens were detected in neurons and/or other non-neural cells. In addition to the neurons, most infiltrating CD3 T lymphocytes observed in these areas were positive for MMP-9 and also for BHV-5 antigen. These infected cells might contribute to the spread of the virus to the rabbit brain along the trigeminal ganglia and olfactory nerve pathways. © 2013 Elsevier Ltd.

Vitrification of immature feline oocytes with a commercial kit for bovine embryo vitrification

The aim of this study was to evaluate the suitability of a commercial kit for bovine embryo vitrification for cryopreserving cat oocytes and to evaluate comparatively the effects of its use with slow freezing procedure on cryotolerance in terms of morphology and oocyte resumption of meiosis. Germinal vesicle stage oocytes isolated from cat ovaries were either vitrified (n=72) using a vitrification kit for bovine embryo or slow frozen (n=69) by exposing oocyte to ethylene glycol solution before being transferred to a programmable embryo freezer. After thawing and warming, oocytes were cultured for 48h and then were examined for meiosis resumption using bisbenzimide fluorescent staining (Hoechst 33342). Fresh immature oocytes (n=92) were used as the control group. The proportion of oocytes recovered in a morphologically normal state after thawing/warming was significantly higher in frozen oocytes (94.5%) than in the vitrified ones (75%, p<0.01). Morphological integrity after culture was similar in vitrified (73.6%) and slow frozen oocytes (76.8%); however, only 37.5% of the morphologically normal oocytes resumed meiosis after vitrification compared to 60.9% of those submitted to slow freezing procedure (p<0.01). Fresh oocytes showed higher morphological integrity (91.3%) and meiosis resumption rates (82.6%, p<0.002) than cryopreserved oocytes, irrespective of the procedure used. These results suggest that immature cat oocytes vitrified with a kit for bovine embryos retain their capacity to resume meiosis after warming and culture, albeit at lower rates than slow frozen oocytes. Vitrification and slow freezing methods show similar proportions of oocytes with normal morphology after culture, which demonstrate that thawed and warmed oocytes that resist to cryodamage have the same chances to maintain their integrity after 48h of culture. © 2012 Blackwell Verlag GmbH.

Deproteinized bovine bone mineral particles and osseointegration of implants without primary bone contact: An experimental study in dogs

Objectives: To evaluate the influence on osseointegration of Deproteinized bovine bone mineral (DBBM) particles used to fill defects of at least 1 mm around implants having no primary contact with bone. Material and methods: Premolars and first molars were extracted bilaterally from the mandible of six Labrador dogs. After 3 months of healing, mucoperiosteal full-thickness flaps were elevated, and one recipient site was prepared in the molar region of each hemi-mandible to place implants. These were installed with a deliberate circumferential and periapical space to the bone walls of 1.2 mm. All implants were stabilized with passive fixation plates to maintain the implants in situ and without any contact with the implant bed. The control sites were left to be filled with coagulum, while at the test sites, the residual gap was filled with DBBM. After 3 months of submerged healing, the animals were sacrificed. Ground sections were prepared and analyzed histomorphometrically. Results: Mineralized bone-to-implant contact was 4.0% and 3.9% for control and test sites, respectively. The width of the residual defects was 0.48 mm and 0.88 mm at the control and test sites, respectively. The percentage of implant surface covered by a layer of dense connective tissue of 0.12 mm of width on average was 84.9% and 88.5% at the control and test sites, respectively. Conclusion: A minor and not predictable degree of contact or distance osteogenesis was obtained on the implant surface when primary contact of the implant surface with the implant bed had deliberately been avoided. DBBM grafting of the artificial gap did not favor osseointegration. Neither did it enhance the ability to bridge the gap with newly formed bone in an artificial defect wider than 1 mm. © 2013 John Wiley & Sons A/S.

Inefficacy of albendazole sulphoxide and ivermectin for the treatment of bovine parasitic otitis caused by rhabditiform nematodes

O objetivo deste estudo foi avaliar a eficácia do sulfóxido de albendazol administrado oralmente e da ivermectina "pour-on" no tratamento da otite parasitária bovina causada por nematóides rhabditiformes. Dezoito vacas Gir apresentando otite clínica foram divididas em três grupos de seis animais cada. O primeiro não recebeu tratamento (grupo controle). O segundo foi tratado com ivermectina "pour-on" a 0,05% na dose de 500µg/kg de peso vivo. O terceiro grupo foi tratado com sulfóxido de albendazol oral a 6% na dose 6,0mg/kg. Os condutos auditivos de todos os animais foram reexaminados nos dias 7 e 21 pós-tratamento. Os animais do grupo controle permaneceram infectados nos dias de observação. O tratamento com ivermectina não demonstrou eficácia alguma para os dias 7 e 21 pós-tratamento. O tratamento com sulfóxido de albendazol obteve 16,7 e 25% de eficácia nos dias 7 e 21, respectivamente. Mais estudos são necessários para determinação de tratamentos eficazes para tal doença parasitária, especialmente através de vias alternativas de administração, por causa de seu significante impacto na criação de Bos taurus indicus nas Regiões Tropical e Subtropical.

Cryopreservation of bovine spermatozoa from the epididymal tail using conventional and automated methods

Cryopreservation of sperm is important to preserve the gerrnplasm from animals of genetic value, which can die unexpectedly. This study compares conventional and automated methods of cryopreservation of spermatozoa obtained from the epididymis of bulls post-mortem. Twenty-two epididymides were obtained from a commercial slaughterhouse. Spermatozoa were collected from the tail of the epididymis using the retrograde flow technique. Thus, the samples, which were diluted in 10 ml of extender without glycerol (Botubov (R) I, Botupharma, Botucatu, SP, Brazil), were evaluated on motility, sperm vigor, structural and functional (swelling hypoosmotic test) membrane integrity, mitochondrial activity, sperm viability and ADN fragmentation. The samples were divided into two aliquots and diluted in extender with glycerol (Botubov (R) II, Botupharma, Botucatu, SP, Brazil) at a concentration of 50x10(6) motile sperm/0.5 French straws. One sample was frozen by the conventional method (4 hours at 5 degrees C, in a refrigerator and 20 min in nitrogen vapor) and the other by the automated method (Cryogen (R) Dualflex, Neovet, Uberaba, MG, Brazil). The parameters were higher in all the tests of fresh sperm samples, with the exception of the swelling hypoosmotic test, which showed no significant difference when the results were compared with sperm frozen by the conventional method. The average motility of fresh spermatozoa was 74%, and conventional and automated averages were 29 and 25%, respectively. Therefore, although cryopreservation techniques reduce sperm quality parameters, the viability of the sperm is maintained, and these methods can be used to preserve sperm.

Artificial activation of bovine and equine oocytes with cycloheximide, roscovitine, strontium, or 6-dimethylaminopurine in low or high calcium concentrations

Knowledge on parthenogenetic activation of oocytes is important to improve the efficiency of nuclear transfer (NT) and intracytoplasmic sperm injection (ICSI) because artificial activation of oocyte (AOA) is an essential step to achieve embryo production. Although different procedures for AOA have been established, the efficiency of in vitro production of embryos remains low, especially in equines and Bos taurus bovines. In an attempt to improve the techniques of NT and ICSI in bovine and equine species, we tested different combinations of drugs that had different mechanisms of action for the parthenogenetic activation of oocytes in these animals. The oocytes were collected, in vitro matured for 24 to 30 h and activated artificially, in the presence of low or high concentrations of calcium, with combinations of calcium ionophore (ionomycin) with cycloheximide, roscovitine, strontium, or 6-dimethylaminopurine (6-DMAP). For assessment of activation rates, oocytes were stained with Hoechst 33342 and observed under an inverted microscope. We showed that all combinations of drugs were equally efficient in activating bovine oocytes, with the best results obtained when high concentrations of calcium were adopted. For equine oocytes, high concentrations of calcium were not beneficial for the parthenogenetic activation and the combination of ionomycin with either 6-DMAP or roscovitine was effective in inducing artificial activation of oocytes. We believe that our preliminary findings provide some clues for the development of a better AOA protocol to be used with these species.

Commercial Bovine Proteoglycan Is Highly Arthritogenic and Can Be Used as an Alternative Antigen Source for PGIA Model

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Deproteinized Bovine Bone Mineral or Autologous Bone at Dehiscence Type Defects at Implants Installed Immediately into Extraction Sockets: An Experimental Study in Dogs

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Outbreak control and clinical, pathological, and epidemiological aspects and molecular characterization of a bovine herpesvirus type 5 on a feedlot farm in S(a)over-tildeo Paulo State

This paper describes the control, epidemiological, pathological, and molecular aspects of an outbreak of meningoencephalitis in calves due to bovine herpesvirus 5 at a feedlot with 540 animals in Sa (a) over tildeo Paulo State, Brazil. The introduction of new animals and contact between the resident animals and the introduced ones were most likely responsible for virus transmission. Bovine herpesvirus 1 vaccine was used, resulting in the efficacy of the outbreak control, although two bovine herpesvirus 1 positive animals, vaccinated and revaccinated, presented meningoencephalitis, thereby characterizing vaccinal failure.

Bioanalytical methods for the metalloproteomics study of bovine longissimus thoracis muscle tissue with different grades of meat tenderness in the Nellore breed (Bos indicus)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of oestrogen deficiency and 17 beta-estradiol therapy on bone healing in calvarial critical size defects treated with bovine bone graft

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effects of FGF10 on bovine oocyte meiosis progression, apoptosis, embryo development and relative abundance of developmentally important genes in vitro

Contents Fibroblast growth factor (FGF10) acts at the cumulus oocyte complex, increasing the expression of cumulus cell expansion-related genes and oocyte competency genes. We tested the hypothesis that addition of FGF10 to the maturation medium improves oocyte maturation, decreases the percentage of apoptotic oocytes and increases development to the blastocyst stage while increasing the relative abundance of developmentally important genes (COX2, CDX2 and PLAC8). In all experiments, oocytes were matured for 22h in TCM-199 supplemented with 0, 2.5, 10 or 50ng/ml FGF10. In Experiment 1, after maturation, oocytes were stained with Hoechst to evaluate meiosis progression (metaphase I, intermediary phases and extrusion of the first polar body) and submitted to the TUNEL assay to evaluate apoptosis. In Experiment 2, oocytes were fertilized and cultured to the blastocyst stage. Blastocysts were frozen for analysis of COX2, CDX2 and PLAC8 relative abundance. In Experiment 1, 2.5ng/ml FGF10 increased (p<0.05) the percentage of oocytes with extrusion of the first polar body (35%) compared to 0, 10 and 50ng/ml FGF10 (21, 14 and 12%, respectively) and FGF10 decreased the percentage of oocytes that were TUNEL positive in all doses studied. In Experiment 2, there was no difference in the percentage of oocytes becoming blastocysts between treatments and control. Real-time RT-PCR showed a tendency of 50ng/ml FGF10 to increase the relative abundance of COX2 and PLAC8 and of 10ng/ml FGF10 to increase CDX2. In conclusion, the addition of FGF10 to the oocyte maturation medium improves oocyte maturation in vitro, decreases the percentage of apoptotic oocytes and tends to increase the relative abundance of developmentally important genes.

Bovine cysticercosis in slaughtered cattle as an indicator of Good Agricultural Practices (GAP) and epidemiological risk factors

This study focused on estimating the economic losses resulting from cysticercosis at beef cattle farms that supply an export slaughterhouse located in the state of Sao Paulo, Brazil, and to identify the epidemiological risks factors involved in the disease to ascertain if these farms adopt Good Agricultural Practices (GAP). To this, we used data recorded in 2012 by Brazil's Federal Inspection Service (SIP) on the daily occurrence of the disease, according to the farm from which the animals originated. In addition, the associated risk factors were determined based on a case-control study at 48 farms. Cysticercosis was detected in 2.26% (95% CI 2.2-2.33) of the 190,903 bovines supplied by 556 farms in the following four states: 2.92% (95% CI 2.83-3.03) in Sao Paulo, 1.81% (95% CI 1.71-1.93) in Minas Gerais, 0.71% (95% CI 0.6-0.82) in Goias and 1.11% (95% CI 0.79-1.57) in Mato Grosso do Sul, with significant differences in the epidemiological indices of these states. Cysticercosis was detected at 58.45% (95% CI 54.36-62.55) of the farms of this study, representing estimated economic losses of US$312,194.52 for the farmers. Lower prevalence of this disease were found at the farms qualified for exports to the European Union, indicating a statistically significant difference from those not qualified to export to Europe. The access of cattle to non-controlled water sources, as well as sport fishing activities near the farms, was identified as risk factors. Cysticercosis causes considerable losses in Brazil's beef supply chain, with lower prevalence appearing only at farms qualified to export to the European Union. As for the access of cattle to non-controlled water sources, this is an indication that GAP are not implemented by some farms, demonstrating the violation of international agreements by the industry and the farms. (C) 2015 Elsevier B.V. All rights reserved.

Molecular detection and phylogenetic analysis of bovine astrovirus in Brazil

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)