In genome sequencing project, we encounter the DNA regions that often contain stable secondary structure with high GC content. These regions are difficult to not only amplify by PCR for template preparations, but also deter mine the DNA sequences using standard Cycle sequencing (CS) method. Transcriptional sequencing (TS) is a unique DNA sequencing method using RNA polymerase, and is based on the principles of the chain-termination method, which is a powerful method to analyze GC-rich sequences. In this study, we examined the multiple displacement amplification (MDA) to overcome low efficiency of PCR amplification in GC-rich regions and subjected to TS reaction. Combination of MDA and TS (MDA-TS) was extremely successful with GC content ranging from 65 % to 85%, which are difficult to analyze with PCR and CS. We also report plasmid vector, pTS1, which has the stronger T7 and T3 promoters than those of conventional vectors, and the sequence that decreases transcriptional efficiency was removed from its multiple cloning sites. pTS1 resulted in the improved sequencing accuracy and reduced reaction time up to 5 min. These results showed that MDA-TS is a rapid and accurate method for the analysis of GC-rich templates.

We examined whether Bovine leukemia virus (BLV) was transmitted by rectal palpation using a common sleeve between a BLV-infected cow and BLV- negative steers. Three of four steers developed antibodies against BLN as determined by agar-gel immunodiffusion (AGID) test between 7 to 10 weeks after the first rectal palpation using common sleeves from BLV-infected cow. In the steers, BLV proviral DNA were detected by PCR 1 to 5 weeks earlier than detection of the antibodies by the AGID test. Our experiments demonstrated that rectal palpation is a potential cause of BLV spread in herds and that detection of BLV proviral DNA in cattle by PCR is useful screening test for early diagnosis of BLV infection.

To develop ELISA to detect antibodies specific to Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis), the carboxyl termini of the 34 kDa proteins of M. paratuberculosis and Mycobacterium avium subsp. avium (M. avium) were expressed in Escherichia coli expression system. Antibodies specific to M. paratuberculosis were detected with the truncated 34 kDa protein of M. paratuberculosis in ELISA after pre-absorption of serum samples with the truncated 34 kDa protein of M. avium. All the serum samples from cattle confirmed to be infected with M. paratuberculosis were positive and those from healthy cattle were negative in the present ELISA system. These results indicate that the established ELISA detects antibodies specific to M. paratuberculosis with high specificity and sensitivity and is an useful tool for the screening of Johne's disease.

The mitochondrial DNA control region of the sun bear (Helarctos malayanus) was sequenced using 21 DNA samples collected from confiscated sun bears to identify conservation units, such as evolutionarily significant units and management units, in Sarawak, Borneo Island. A total of 10 haplotypes were observed, indicating the presence of at least two lineages in the sun bear population in Sarawak. Presumably, these two lineages could represent evolutionarily significant units. However, the geographical distributions of the two lineages remained unknown due to the lack of information regarding the exact capture locations of the confiscated sun bears. It is essential to elucidate the geographical distributions of these lineages in order to create a proper conservation plan for the sun bears in Sarawak. Therefore, further studies examining the haplotype distributions using DNA samples from known localities are essential.

Eco-tourism depending on wildlife is becoming increasingly profitable and landowners are beginning to favor game farming and ecotourism. In these areas, large-scale translocation of wildlife involves a diversity of species and large populations. The African buffalo (Syncerus caffer) is one of the major tourist attractions in Zambia. It accounts for 8.7% and 12.4% of the total animal species hunted in the Game Management Areas and the total hunting revenue earned in Zambia, respectively. It is ecologically an important animal species essential for the purpose of habitat control and facilitating the provision of suitable grazing pastures. However, the rearing of the African buffalo on game ranches has been hampered by its carrier state of the Southern Africa Terroritory (SAT) serotypes of foot and mouth disease virus (FMD). The African buffalo is also known to be a carrier of Theileria parva lawrencei, the causative agent of corridor disease (CD) that continues to have devastating effects on the livestock industry in Zambia. In addition, the importation of buffaloes from countries with populations endemic to bovine tuberculosis is highly restricted. Veterinary regulations in Zambia, strongly advocate against the translocation of buffaloes from protected areas to private ranches for disease control purposes thereby mounting a considerable constraint on the economic and ecological viability of the industry. It is hoped that this review will motivate the relevant government authorities in exploiting ways in which this animal species play a central role in eco-tourism.

Genotypes and subgenotypes of bovine viral diarrhea virus (BVDV) field isolates from Japan, Germany and the United States of America (USA) were identified, and the prevalent pattern of BVDV in individual countries was estimated genetically. Subgenotypes were determined based on phylogenetic analyses of nucleotide sequences of a part of the E2-coding gene of BVDV. Forty-five, 61 and 56 BVDV strains were isolated from naturally infected cattle in Japan, Germany and USA, respectively, between 1980 and 2003. The most prevalent BVDV in these three countries was BVDV - 1b. The second most prevalent BVDV strains were 1a, 1d and BVDV - 2 in Japan, Germany and USA, respectively. The most prevalent subgenotype 1b in each country constructed individual small clusters in the subgenotype 1b branch in the phylogenetic tree. Although cattle and/or cattle products were moving among the three countries as part of international trade, the distribution of BVDV in the field in each country showed long-standing individual patterns.

Infecção das estruturas umbilicais pode resultar em bacteremia, septicemia e morte em neonatos com falha na imunidade passiva sendo os microrganismos usuais de onfalites isolados freqüentemente em animais com bacteremia. Um estudo foi desenvolvido entre setembro 2002 e setembro 2003 utilizando-se 44 bezerros, visando verificar a freqüência de bacteremia em bezerros neonatos e a correlação entre os agentes isolados a partir de amostras de sangue de estruturas umbilicais. Amostras de sangue foram obtidas por punção da jugular e submetidas à cultura para isolamento de agentes microbianos, sendo 24 obtidas entre 48 e 72 horas e 20 entre o terceiro e o quinto dia após o nascimento. Através de "Swabs" procedeu-se à coleta de material das estruturas umbilicais para a mesma finalidade. Obteve-se crescimento microbiano em 17 (38,67%) amostras de sangue e 100% das amostras de estruturas umbilicais. Os microorganismos mais freqüentes em amostras de sangue foram Staphylococcus sp., E. coli, Bacillus spp, Pseudomonas sp. e Streptococcus. Todos os animais com bacteremia apresentaram manifestações de enfermidade focal ou sistêmica. O sinal clínico mais freqüentemente relacionado com cultura de sangue positiva foi o espessamento das estruturas umbilicais. Bacillus spp, Enterobacter spp, Micrococcus spp. e E. coli foram os microrganismos isolados das estruturas umbilicais. Os dados confirmam uma flora bacteriana mista nos casos de onfalite e sugerem uma prevalência alta de bacteremia em bezerros neonatos, sobretudo aqueles com onfalite, evidenciando a importância de boa transmissão de imunoglobulinas através do colostro. | Umbilical structures infections can be followed by bacteremic, septicemic infections and death in newborns with passive immunity deficiency. Microorganisms isolated in omphalitis have been also isolated from animals with bacteremia. From september/2002 to september/2003, a research was developed using 44 calves, in order to evaluate bacteremia frequency in newborns and to correlate microorganisms isolated from blood samples and umbilical structures. Blood samples were collected from jugular vein for microorganisms isolation. Twenty four samples were collected in a period of 24 to 48 hours, the other 20 samples from thirty to fifty days after birth. Umbilical structures materials were collected though swabs. Microbial growth occurred in 17 (38,67%) blood samples and in 100% of umbilical samples. Staphylococcus spp., E. coli, Bacillus spp, Pseudomonas spp. and Streptococcus spp were the major isolated microorganisms from blood. All bacteremic animals presented systemic or localized clinical manifestations. The most reported clinical sign was thickness of umbilical structures. Bacillus spp, Enterobacter spp, Micrococcus spp. and E. coli were isolated from umbilical structures. Data confirm a mixed bacterial environment in omphalitis, and suggest a high prevalence of bacteremic infections in newborns calves, pointing out the need of passive immunity transfer through colostrum.

Em fêmeas bovinas a utilização da técnica de inseminação artificial em tempo fixo (IATF) é possibilitada pelo emprego de fármacos que tem como objetivos sincronizar a emergência das ondas foliculares, os estros e a ovulação. Em rebanhos comerciais, o custo de tais fármacos deve estabelecer uma vantajosa relação com os benefícios. O presente estudo, objetivou comparar as taxas de prenhez em vacas Nelore (Bos taurus indicus) inseminadas em tempo fixo, tratadas com implantes auriculares contendo 3mg de norgestomet (Crestar®), novos (IN) ou utilizados uma vez (IR; reutilizados), associados a administração de norgestomet (NG) e valerato de estradiol (VE) ou progesterona (P4) e benzoato de estradiol (BE). Vacas Nelore PO (n=241), amamentando, com o bezerro ao pé, receberam um dos quatro tratamentos: Crestar®novo durante 10 dias associado a administração de 3mg de NG e 5mg de VE (grupo IN/NG+VE; n=61); Crestar®novo inserido durante 8 dias associado a 50mg de P4 e 2mg de BE (grupo IN/P4+BE; n=61); Crestar®reutilizado inserido durante 10 dias associado a administração de 3mg de NG e 5mg de VE (grupo IR/NG+VE; n=58) ou Crestar®reutilizado inserido durante 8 dias associado a e 50mg de P4 e 2mg de BE (grupo IR/P4+BE; n=61). No dia da remoção dos implantes os animais receberam 7,5mg de Luprostiol e 24 horas após a remoção 1mg de BE. A IATF foi realizada 54 a 56 horas após a retirada dos implantes. Após a IATF, as vacas tiveram os estros observados durante um período de 49 dias e em estro foram reinseminadas 12 horas após a observação. O diagnóstico de gestação foi realizado por ultra-sonografia 35 dias após a IATF e após o final da estação de monta. Foram avaliadas as taxas de prenhez na IATF (TP IATF) e no final da estação de monta (TP EM). Não houve interação entre as características dos implantes (novos e reutilizados) e os tratamentos administrados no dia da inserção do implante (NG+VE e P4+BE). A utilização do CIDR®novo ou reutilizado não teve efeito nas TP IATF (48,3% vs 48,7%) e nas TP EM (85,2 vs 86,5%). Os tratamentos com NG+VE e P4+BE não tiveram efeito nas TP IATF (49,5 vs 47,5%) e TP EM (86,5 vs 85,2%). Houve efeito do número de parições (primíparas e multíparas) nas TP IATF (35% vs. 52,7%; P<0,01) e nas TP EM (71,9% vs. 90,2%; P<0,01). Conclui-se que implantes de norgestomet novos e reutilizados, quando associados ao NG+VE ou P4+BE promovem taxas de prenhez bastante satisfatórias em vacas Nelore e que melhores taxas são obtidas em fêmeas multíparas. | The use of fixed-time artificial insemination (IATF) in cows is possible by the use of drugs that aim at the synchronization of the folicular wave pool, the estrus and ovulation. In trading cattle the costs of these drugs must stablish a profitable relation with the benefits. The present study meant to compare the pregnancy rates in Nelore (Bos taurus indicus) cows inseminated at fixed-time, treated with new (IN) or once used (IR; reused) auricular releasing devices with 3mg of norgestomet (Crestar®), associated with the administration of norgestomet (NG) and estradiol valerate (VE) or progesterone (P4) and estradiol benzoate (BE). Pure breed Nelore cows (n=241), on lactation with calf received one of the four treatments: new Crestar®during 10 days administrated with 3mg of NG and 5 mg of VE (group IN/NG+VE; n=61); new Crestar®inserted during 8 days with 50mg of P4 and 2mg of BE (group IN/P4+BE; n=61); reused Crestar®inserted during 10 days in association with 3 mg of norgestomet and 5 mg of VE (group IR/NG+VE; n=58) or reused Crestar®inserted during 8 days in association with 50mg of P4 and 2mg of BE (group IR/P4+BE; n=61). On the day the releasing devices were removed the animals received 7,5mg of Luprostiol and after 24 hours 1mg of BE. A fixed-time artificial insemination was done 54 to 56 hours after the removal of the releasing devices. Cows detected in estrus after the insemination at fixed-time were observed during a period of 49 days and reinseminated. The pregnancy diagnosis was done by ultrassonography 35 days after the IATF and after the end of the breeding season. The pregnancy rates of IATF (TP IATF) and at the end of the breeding season (TP EM) were estimated. There was no interaction between the characteristics of the (new or reused) releasing devices and the treatment given at the day the releasing devices were inserted (NG+VE e P4+BE). The use of new or used CIDR®had no effect on TP IATF (48,3% vs 48,7%) and on TP EM (85,2% vs 86,5%). The treatments with NG+VE e P4+BE did not have effect on TP IATF (49,5% vs 47,5%) and TP EM (86,5 vs 85,2%). There was a effect on the number of parturition (primipara and multipara) on the TP IATF (35% vs 52,7%; P<0,01) and on TP EM (71,9% vs 90,2%; P<0,01). It can be concluded that the new or once used releasing devices with norgestomet when associated with NG+VE or P4+BE promote highly satisfatory pregnancy rates in Nelore cows, being the better rates obtained in multipara females.

Para descrever a anatomia ultra-sonográfica foram examinados os ligamentos patelares (medial, intermédio e lateral) e as estruturas relacionadas de 20 articulações fêmuro-tibio-patelares de cavalos adultos. Tanto a ecogenicidade como o alinhamento das fibras de colágeno, a espessura, a forma e relação com as estruturas vizinhas foram avaliadas. Os três ligamentos apresentaram ecogenicidade uniforme e paralelismo das fibras colágenas. Em secção longitudinal os ligamentos patelares medial (LPM) e intermédio (LPI) apresentaram maior espessura do segmento distal (p<0,01) quando comparado com o proximal. Comparando-se o mesmo segmento entre os três ligamentos, o LPI apresentou espessura maior (p<0,01) em relação ao LPM e ligamento patelar lateral (LPL). Em secção transversal (cm²) o LPL apresentou a maior área (p <0,01), seguido pelo LPI e LPM. Em secção transversal do segmento médio, o LPM apresentou forma triangular, o LPI em forma triangular ou circular, e o LPL formato achatado. As estruturas adjacentes identificadas durante o exame dos ligamentos foram cristas femorais, cartilagem articular, tecido gorduroso periligamentar, patela e tíbia proximal. | In order to describe the ultrasonographic anatomy of patellar ligaments (medial, middle, lateral) and associated structures, 20 femorotibiopatellar joints of adult horses were investigated. Echogenicity, collagen fibers alignment, size, shape and surrounding structures were evaluated. All ligaments presented uniform echogenicity and collagen fibers parallelism. The longitudinal section of the medial and middle patellar ligaments presented a significant larger thickness (p<0,01) of the distal region when compared to the proximal region. When comparing the same site between the three ligaments, LPI was thicker (p<0,01) than lateral patellar ligament (LPL) and LPM. The LPL transversal section (cm²) presented a significant larger area (p<0,01), followed by the LPI and LPM. The transversal section of the medium region revealed a LPM with triangular shape, triangular or circular LPI, and flattened LPL. The surrounding structures observed during the evaluation were: femoral groove, articular cartilages, periligamentar fat tissue, patella and proximal tibia.

Claw lameness can be associated to biomechanical factors caused by unbalanced pressure distribution under the hooves when cows are confined in modern dairy operations with hard concrete flooring. In the present study, an original claw subdivision4 was slightly modified to differentiate between the anterior (typical sole lesion spot) and posterior portions of the medial sole, and to emphasize the maximum pressures applied only on the area of contact without including the total area within these regions during midstance. The results, obtained showed significance (p < 0.044) for the interaction among Group, Leg and region (G*L*R). It was observed that the rear portion of the claws (heels) on the hind limb of untrimmed cows, are more stressed than the heel region on trimmed cows (23 % versus 16.72% of total pressure applied on the claw for untrimmed and trimmed respectively). The typical sole lesion spot pressures were increased slightly on trimmed cows as compared to untrimmed (20.20% versus 15.9%). The front feet presented differences in pressure concentration on the lateral sole between both groups (29% versus 23.25% for untrimmed versus trimmed respectively). It was concluded that, although the differences were small (5%) changes in pressure concentration, untrimmed cows stress more the sole lateral as compared to trimmed on the front feet, and on the rear feet, they stress more the heel region whereas trimmed cows tend to have a slight better balance among regions. Conversely, when cows are trimmed, the typical sole lesion spot concentrates more pressure than the heel itself (20.20% versus 16.72% respectively) and may favor the occurrence of sole ulcers. | Laminite (manqueira) pode ser associado a fatores mecânicos, causados por falta de balanceamento na distribuição de pressão na sola dos cascos de vacas confinadas em instalações modernas, que utilizam pisos de concreto. No presente estudo, a subdivisão original dos cascos de vacas leiteiras foi modificada para diferenciar-se entre a porção anterior (local típico de lesão) e posterior da sola medial dos cascos, e para enfatizar as pressões máximas aplicadas somente na área de contato não levando em consideração a área total da sola. Os resultados mostraram significância estatística (p < 0.044) para a interação entre Grupo, Pata e Região (G*L*R). Foi observado que a porção posterior (calcanhar) das patas traseiras de vacas não-casqueadas foram estressadas mais intensamente que de vacas casqueadas (23 % versus 16.72% da pressão total aplicada nas patas em não-casqueadas e casqueadas respectivamente). As pressões na região do local típico de lesão aumentaram em animais casqueados comparado com não-casqueados (20.20% versus 15.9%). As patas da frente apresentaram diferenças na concentração de pressão da sola lateral (29% versus 23.25% em não-casqueadas versus casqueadas, respectivamente). Foi concluído que, apesar das diferenças serem pequenas (5%) mudanças nas concentrações de pressão, vacas não-casqueadas estressaram mais a porção da sola lateral, comparado a vacas casqueadas nas patas da frente, enquanto nas traseiras elas estressam mais a região do calcanhar, e as vacas casqueadas tendem a ter uma distribuição melhor de pressão entre as regiões. No entanto, quando as vacas são casqueads, a região típica de lesão tende a concentrar mais pressão do que o próprio calcanhar (20.20% versus 16.72% respectivamente) podendo favorecer a incidência de úlcera de sola.