Optimizing the transport of porcine semen: a proposal for Brazil / Otimizar o transporte do sêmen suíno: uma proposta para o Brasil

Semen from the first 15mL of the ejaculate (P1) obtained from two boars (30mL) was diluted in glycine-egg yolk extender, cooled at 5°C in a special container and rediluted in standard doses of 3x109 mobile spermatozoa after 12h of storage. Semen was also stored up to 24h after redilution. The physical characteristics of the semen were evaluated at different storage periods (fresh, 0h, 12h, rediluted, 24h, and 36h). The reproductive performance of the boars and their fertility regarding the insemination of primiparous sows were also determined. Two treatments were used: T1-15B sows inseminated with semen originated from hyperconcentrated heterospermic doses (15x109 mobile spermatozoa per dose), rediluted after 12h of storage at 5°C for standard doses of 3x109 mobile spermatozoa per dose and stored at 5°C up to 24h after redilution (n=10); T2-3B sows inseminated with standard heterospermic doses (3x109 mobile spermatozoa per dose), stored at 5°C up to 36h after semen collection (n=10). There was no effect (P>0.05) of treatments on the spermatic motility, even though a pronounced decrease (P>0.05) of their values at 12h of storage was recorded. However, they remained higher than 70% until 36h. There was effect of treatments on spermatic vigour at 0h (P<0.05), when T1-15B vigour was higher. There was also effect of the storage period for both treatments with a progressive decrease throughout 36h of storage, although the differences were not always significant. Pregnancy rates (90%) and the number of total farrowed piglets (15, 11-T1-15B; 13, 44- T2-3B) did not differ (P>0.05) between the treatments. It was concluded that the semen hyperconcentration of 15 billion of mobile spermatozoa per dose, stored at 5°C for 12h, did not result in drawbacks considering the physical characteristics of the semen, maintaining the pregnancy rates and prolificacy of the inseminated sows.

Os primeiros 15mL do ejaculado (P1) de dois varrões foram coletados (30mL) e diluídos em diluidor glicina-gema de ovo, resfriados a 5°C em contêiner especial e rediluídos para doses padrão de 3x109 espermatozoides (sptz) móveis, após 12 horas de armazenamento. Além disso, foram armazenados por até 24 horas após a rediluição, sendo as características físicas avaliadas em diferentes períodos de estocagem (fresco, zero hora, 12h, Red12h, 24h e 36h) e a fertilidade avaliada por meio de fêmeas primíparas inseminadas. Foram realizados dois tratamentos: T1-15B: porcas inseminadas com sêmen de doses heterospérmicas hiperconcentradas (15x109 sptz móveis/dose), rediluídas após 12 horas de armazenamento a 5°C para doses padrão de 3x109 sptz móveis/dose, e armazenadas a 5°C por até 24 horas após a rediluição (n=10); T2-3B: porcas inseminadas com doses heterospérmicas padrão (3x109 sptz móveis/dose), armazenadas a 5°C por até 36 horas após coleta. Não houve efeito (P>0.05) dos tratamentos sobre a motilidade espermática e, embora tenha ocorrido queda (P<0.05) às 12 horas, a motilidade foi superior a 70% durante as 36 horas de armazenamento. Houve efeito (P<0.05) dos tratamentos no tempo zero hora quanto ao vigor espermático, sendo E1T1-15B superior. Além disso, houve efeito do período de estocagem para os dois tratamentos, com queda progressiva do vigor ao longo das 36 horas, embora nem sempre as diferenças tenham sido significativas. As taxas de gestação (90%) e o número total de leitões nascidos (15, 11 - T1-15B; 13, 44 - T2-3B) não diferiram (P>0.05) entre os tratamentos. Concluiu-se que a hiperconcentração do sêmen para 15x109 sptz móveis/dose, armazenado a 5°C por 12 horas não resultou em prejuízos quanto à manutenção das características físicas do sêmen e ao desempenho reprodutivo dos varrões, sendo capaz de manter a taxa de gestação e a prolificidade das fêmeas inseminadas.

Detection of Mycoplasma hyopneumoniae in lungs and nasal swabs of pigs by nested PCR / Detecção de Mycoplasma hyopneumoniae em pulmões e suabes nasais de suínos por nested PCR

Fifty-four samples were collected from growing and finishing pigs for the molecular diagnosis of enzootic porcine pneumonia. Nineteen lung fragments were obtained from pigs that showed signs of respiratory disease and 35 nasal swabs were obtained from clinically healthy pigs. For the detection of the bacterial genome in the samples, the nested PCR technique was used to amplify a fragment of 706bp. This fragment was subsequently cloned and sequenced. The sequence of obtained nucleotides was compared with six other sequences of Mycoplasma hyopneumoniae and 11 sequences of other bacteria available in the Genbank. To measure the sensitivity of the nested PCR, serial dilutions (10-1 to 10-15) of cloned fragments were conducted based on the concentration of 300ng. Ten lung fragments and eight nasal swabs showed positive for M. hyopneumoniae and the limit of detection was estimated to be 0.3fg DNA cloned. The sequence of nucleotides obtained showed 99.1 percent homology with the other sequences of M. hyopneumoniae, demonstrating that the nested PCR used in this study may provide an important diagnostic tool for the detection of this agent.

Foram coletadas 54 amostras de animais em fase de crescimento e terminação para o diagnóstico molecular da pneumonia enzoótica suína. Dezenove fragmentos de pulmão foram obtidos de suínos que apresentavam sinais de doença respiratória e 35 suabes nasais foram obtidas de suínos clinicamente saudáveis. Para a detecção do genoma bacteriano nas amostras, foi utilizada a técnica de nested PCR que originou um fragmento de 706pb, o qual foi, posteriormente, clonado e sequenciado. A sequência de nucleotídeos obtida foi comparada com outras seis sequências de Mycoplasma hyopneumoniae e 11 sequências de outras bactérias disponíveis no Genbank. Para medir a sensibilidade da nested PCR, foram realizadas diluições seriadas (10-1 a 10-15) do fragmento clonado, partindo da concentração de 300ng. Dez fragmentos de pulmões e oito suabes nasais apresentaram resultado positivo para M. hyopneumoniae e o limite de detecção foi estimado em 0,3fg de DNA clonado. A sequência de nucleotídeos obtida foi de 99.1 por cento de homologia com as outras sequências de M. hyopneumoniae, demonstrando que a nested PCR utilizada neste estudo pode ser uma importante ferramenta de diagnóstico para a detecção desse agente.

Aplicação da técnica de PCR na detecção de Yersinia enterocolitica em suínos abatidos sem inspeção / PCR technique application in Yersinia enterocolitica detection in non-inspected swine

Avaliou-se a contaminação de carcaças e tonsilas de suínos por Y. enterocolitica em estabelecimentos de abate não inspecionados, comparando a pesquisa microbiológica convencional com a técnica da reação em cadeia de polimerase (PCR) e o tipo de amostra analisada (de tonsila ou de carcaça), como subsídio ao monitoramento microbiano em sistemas de análise de perigos e pontos críticos de controle. Calcularam-se os custos dos dois testes. Não se detectou Y. enterocolitica pela técnica microbiológica convencional, mas, pela técnica de PCR foi possível detectar a bactéria em 40 por cento das carcaças e em 43 por cento das tonsilas, incluindo cepas patogênicas nas tonsilas. Não houve diferença entre os resultados positivos para as amostras de tonsilas e esfregaços de superfície das carcaças. A PCR apresentou-se como uma alternativa na detecção do agente e uma técnica aparentemente mais eficaz, econômica e rápida. Os resultados indicam a PCR como um importante recurso para o controle de qualidade da carne suína.

The contamination of swine carcasses and tonsils by Yersinia enterocolitica in slaughterhouses without inspection was evaluated. The conventional microbiological analysis was compared with the PCR technique of carcass or tonsil swabs, as a subsidy to the microbiological evaluation in the HACCP system. The costs of the two techniques were also calculated. Y. enterocolitica was not detected by the conventional microbiological analysis. Using the PCR, it was possible to detect this bacterium in carcass (40 percent) and tonsil (43 percent) samples. There was no difference between the positive results for the carcass and tonsil samples. The PCR showed to be a more effective, fast and economic alternative for the Y. enterocolitica detection, as compared to the conventional microbiological analysis.

Intrapreputial infection of young bulls with bovine herpesvirus type 1.2 (BHV-1.2): acute balanoposthitis, latent infection and detection of viral DNA in regional neural and non-neural tissues 50 days after experimental reactivation.

Venereal infection of bulls with bovine herpesvirus type 1.2 (BHV-1.2) may result in acute balanoposthitis followed by the establishment of latent infection, presumably in dorsal root nerve ganglia. We herein report the characterization of the acute and latent infection of young bulls with a Brazilian BHV-1.2 isolate and the investigation of neural and non-neural sites in which viral DNA persists during latent infection, i.e. 110 days after inoculation and 50 days after experimental reactivation. Intrapreputial inoculation of BHV-1.2 isolate SV-56/90 (10(6.5)pfu per animal) resulted in severe balanoposthitis, characterized by redness of the penis and preputial mucosa, coalescent vesicles and fibrinous exsudate in all four infected bulls. Virus shedding was detected in preputial secretions and semen up to days 14 and 13 pi, respectively. Dexamethasone administration at day 60 pi led to reactivation of the infection in all animals, resulting in virus shedding in preputial secretions and/or in semen. At day 50 post-reactivation (pr), the animals were euthanized and regional tissues were collected for PCR and virus isolation. Viral DNA was consistently detected in the dorsal root ganglia of nerves genito-femoral (4/4) and obturator (4/4); frequently in the pudendal (3/4), sciatic (3/4) and rectal caudal nerve ganglia (2/3). In addition, viral DNA was detected in the pelvic sympathetic plexus of one bull and in regional lymph nodes (deep inguinal (2/4); sacral (1/4); medial iliac (1/4)) of two bulls. No infectious virus could be recovered from homogenates of DNA positive tissues, indicating the absence of actively replicating virus. These results demonstrate that BHV-1.2 DNA may persist in several sacral nerve ganglia and in regional lymph nodes as well during latent infection, i.e. 50 days after experimental reactivation. These findings may help in understanding the pathogenesis of acute and latent genital infection by BHV-1.2.

Engineering better vaccines for foot-and-mouth disease.

Although efficacious and safe, current vaccines for FMD suffer from drawbacks. Among these are that the immune response to the vaccine interferes with the ability to detect vaccinated animals that have subsequently become infected and could carry and shed the virus, creating an obstacle to re-instating disease-free status to countries/regions that vaccinate to control outbreaks. Multiple diagnostic tests are available to identify animals that have been infected with FMDV by detection of antibodies to viral non-structural proteins (NSP) that are present in low concentration in traditional vaccines and are poorly immunogenic in vaccine preparations. However, these tests are not 100% reliable. To circumvent this problem, we have developed a new generation of vaccines that express the "empty capsid" subunit of the virus, in the absence of one of the most immunogenic NSPs, 3Dpol. Here we describe delivery of the empty capsid subunits by recombinant replication-defective human adenovirus type 5 (Ad5). These Ad5-vectored empty capsid vaccines can protect pigs from FMDV challenge as early as 7 days post-vaccination. A second problem with current FMD vaccines is that they do not induce protective immunity quickly, a drawback that is likely to be shared by our Ad5-vectored empty capsid vaccine. To overcome this problem, we have developed a prophylactic antiviral treatment consisting of an Ad5 encoding porcine interferon alpha (pIFNalpha). Administration of Ad5-pIFNalpha protects swine from FMD as early as one day post-administration. The combination of this antiviral treatment and the empty capsid subunit vaccine should induce rapid and complete protection from FMD, and could overcome current diagnostic problems.

Genome of bovine herpesvirus 5.

Here we present the complete genomic sequence of bovine herpesvirus 5 (BHV-5), an alphaherpesvirus responsible for fatal meningoencephalitis in cattle. The 138390-bp genome encodes 70 putative proteins and resembles the alpha2 subgroup of herpesviruses in genomic organization and gene content. BHV-5 is very similar to BHV-1, the etiological agent of infectious bovine rhinotracheitis, as reflected by the high level of amino acid identity in their protein repertoires (average, 82%). The highest similarity to BHV-1 products (>or=95% amino acid identity) is found in proteins involved in viral DNA replication and processing (UL5, UL15, UL29, and UL39) and in virion proteins (UL14, UL19, UL48, and US6). Among the least conserved (

Early protection against homologous challenge after a single dose of replication-defective human adenovirus type 5 expressing capsid proteins of foot-and-mouth disease virus (FMDV) strain A24.

Previously we demonstrated that two doses of a replication-defective human adenovirus serotype 5 (Ad5) carrying the capsid (P1) and 3C protease coding regions of a laboratory strain of FMDV (A12) completely protected five of six swine challenged with homologous virus. The objective of the current study was to evaluate the efficacy of one dose of an Ad5-vectored vaccine expressing the P1 coding region of an FMDV field strain. A replication-defective Ad5 containing the P1 coding region of FMDV A24 and the 3C coding region of A12 (Ad5A24) was constructed and evaluated for its ability to induce neutralizing antibodies and protect swine against homologous challenge after a single vaccination. Animals were challenged 7, 14 or 42 days after vaccination. Control groups included animals inoculated with commercial vaccine or phosphate-buffered saline. All vaccinated swine were completely protected against homologous challenge at 7, 14 or 42 days after vaccination. Based on these results, we conclude that a single inoculation of Ad5-vectored vaccines could be used as a tool to control FMD in outbreak situations.

pAd5-Blue: direct ligation system for engineering recombinant adenovirus constructs.

We have engineered a new vector that makes use of direct ligation for the generation of replication-defective recombinant adenovirus constructs. In the pAd5-Blue vector, unique yet common restriction endonuclease sites exist, that allow cloning in a directional manner of a gene of interest under control of a cytomegalovirus promoter, upstream of a simian virus 40 polyadenylation signal. The insertion of the new gene replaces the beta-galactosidase alpha gene fragment in the pAd5-Blue vector, allowing the identification of recombinants in bacterial culture by the selection of white colonies. Plasmid DNA from white colonies is digested with PacI and transfected into 293 cells, resulting in the generation of a homogenous population of adenovirus containing the gene of interest. The pAd5-Blue vector system does not rely on recombination either in mammalian or bacterial cells. Furthermore, because of compatible overhangs, the variety of restriction endonucleases that can be used to generate the inserted gene gives flexibility to the process for greater ease of use. The system is quick and straightforward, allowing the generation of recombinant adenoviruses within three weeks of obtaining an appropriate insert. This new vector should greatly enhance the ease and speed with which new recombinant adenovirus constructs can be made.

Levantamento sorológico de parvovírus bovino no rebanho leiteiro do RS, Brasil / Serological survey of bovine parvovirus in dairy herds of Rio Grande do Sul State, Brazil

Para determinaçäo da prevalência de animais com anticorpos contra o parvovírus bovino (BVP), e avaliaçäo do nível de imunidade do rebanho leiteiro do Estado do Rio Grande do Sul (RS), foram testadas 4.096 amostras de soro de bovinos. Os animais eram de diferentes idades e provenientes das diversas bacias leiteiras do Estado. As amostras foram examinadas pela técnica de inibiçäo da hemaglutinaçäo. Do total analisado, 4.000 (97,7 por cento) mostraram anticorpos inibidores da hemaglutinaçäo, sendo 2.715 (66,3 por cento) com títulos de anticorpos entre 1:160 e 1:5120. Näo se observou correlaçäo entre idade e título de anticorpos dos animais. As bacias leiteiras n§ 3 (representada pela regiäo de Bagé) e 6 (representada pelos municípios de Erexim e Tapejara, dentre outros) apresentaram animais com os mais altos títulos de anticorpos e a bacia n§ 7 (representada pelos municípios de Colorado, Espumoso, Selbach e Tapera, dentre outros) com os mais baixos títulos. Concluiu-se que o BVP está amplamente disseminado no rebanho leiteiro do RS e pode ser o agente determinante de diarréia neonatal ou de problemas respiratórios e reprodutivos, apesar da maioria dos animais apresentarem alto grau de imunidade humoral contra o vírus