The LEC rat is reported to exhibit hypersensitivity to X-irradiation, deficiency in DNA double-strand break repair, and radio-resistant DNA synthesis. This character of the LEC rat has been thought to be due to abnormal G1 arrest in cells after X-irradiation. In this report, we re-investigated the effect of X-irradiation on the cell cycle in primary-cultured fibroblasts. Primary-cultured fibroblasts derived from LEC and BN rats were exposed to 4 Gy of X-ray and their cell cycle analysis was performed with a flow cytometer. Fibroblasts derived from both rats showed normal response of the cell cycle, indicating the arrest at both G1- and G2/M-phase and no difference in the cell cycle population between fibroblasts derived from both rats. In contrast, when the same analysis was performed using the cell line, L7 and W8, which had been established from the lung fibroblasts of LEC and control WKAH rats, respectively, by immortalizing with SV40 T-antigen, L7 cells but not W8 cells showed impaired G1 arrest and abnormal cell cycle. These results suggest that fibroblasts derived from LEC rats possess the normal cell cycle response after X-irradiation, if they are kept naive as not immortalized with SV40 T-antigen.

The major cause of infection in animal prion diseases is thought to be consumption of prion-contaminated stuff. There is evidence that the enteric nerve system (ENS) and gut-associated lymphoid tissues (GATL) are involved in the establishment of prion infection through alimentary tract. To elucidate the initial entry port for prion, we inoculated prion to alymphoplasia (aly) mice showing a deficiency in systemic lymph nodes and Peyer's patches. The aly/aly mice were susceptible to prion infection by intra-cranial inoculation and there were no differences in incubation periods between aly/aly mice and wild-type C57BL/6J mice. Incubation periods in aly/aly mice were about 20 days longer than those in C57BL/6J mice with the intra-peritoneal inoculation. The aly/aly mice were completely resistant to prion infection by per os administration, while C57BL/6J mice were sensitive as they entered the terminal stage of disease around 300 days post inoculation. PrPsup(sc) were detected in the intestine and spleen of C57BL/6J mice inoculated with prion intra-peritoneally or orally; however PrPsup(sc) was not detected in the spleen and intestine of aly/aly mice. Prion infectivity was detected in the intestines and spleens of prion-inoculated C57BL/6J mice, even after the early stages of ex-posure, while no infectivity was detected in these tissues of prion-inoculated aly/aly mice. No apparent differences were observed in the organization of the enteric nerve system between wild-type and aly/aly mice. These results indicate that GALT rather than ENS acts as the primary entry port for prion after oral exposure.

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.

Serological analysis was performed to detect morbillivirus infection in Kuril harbor seals in Hokkaido, Japan. Serum samples were collected from the seals at Nosappu (231 sera), Akkeshi (16), and Erimo (75) between 1998 and 2005. Antibodies to phocine distemper virus (PDV) were detected by ELISA in seals from Nosappu and Erimo. Antibodies to PDV were found in 56% (5/9) of the sampled seals from Nosappu in 1998, versus only 5% (3/66) for 2003, 1% (1/79) for 2004, and 1% (1/77) for 2005. These suggest epidemic caused by the virus in or before 1998. As antibody-positive seals included juvenile seals in 2003 and 2005, sporadic infections of the virus are thought to have occurred in recent years. In Erimo, antibodies to PDV were found in 50% (14/28) of sampled seals in 2004, versus only 13% (1/8) for 1999, 7% (1/15) for 2003, and 0% (0/24) for 2005. These suggest sporadic infection by the virus before 2003 and the epizootic between after autumn in 2003, when samples of 2003 were collected, and 2004. Since antibodies to canine distemper virus (CDV) were detected in one adult seal from Nosappu in each year from 2003 to 2005, sporadic infections of the virus, were suggested. There were no difference in incidence of seals with antibodies to the viruses between males and females and between juveniles and adults.

The study determined the types of pesticide/herbicide pollutants in water, sediment and fish from the Kafue River. A preliminary investigation of the oxidative stress from these pesticides/herbicides was also assessed by measurement of catalase activity. Water, sediment and fish samples were collected upstream, midstream and downstream the Kafue river in Chingola, Kitwe, Kafue National Park and Kafue Town. Water, sediment and fish muscle were sampled and analysed for pesticides using Gas chromatography. For catalase activity fish liver samples only were examined. The pesticides/herbicides detected in all samples collectively included: Heptachlor, pp'-DDE, Cypermethrin, Chlordane, Toxaphene, Terbufos, Kelthane, Endosulfan, Dieldrin, pp'-DDD, pp'-DDT, Atrazine, Disulfoton, d-trans-Allethrin and Endrin. On the other hand, catalase activity was detected in all fish liver samples from all sites. Its levels increased significantly from Chingola upstream to sites down-stream with highest being in Kafue town. This study therefore, demonstrates that there is widespread contamination of the Kafue River with pesticides/herbicides. It also demonstrates that organochlorides are found throughout the river especially in fish samples. The spectrum of pesticides/herbicides was much wider in fish probably due to bioaccumulation. It was also observed that fish are subjected to oxidative stress as determined by catalase levels. The stress is more pronounced downstream where the catalase levels were significantly higher than Chingola. The observation that more pesticide varieties are also found downstream may suggest a likely causative effect of the pesticides on oxidative stress although this needs further investigation. This study further tentatively highlights the potential dangers of these agro-related substances to dependants of the Kafue River water body and the need to carry out risk assessments and thereafter institute corrective measures to help reduce contamination and adverse effects.

Anthrax is endemic in Western and North-western Provinces of Zambia. The disease occurs throughout the year and impacts negatively on the economy of the livestock industry and public health in Zambia. During 1989-1995, there were 1,626 suspected cases of anthrax in cattle in Western province and of these 51 were confirmed. There were 220 cases of human anthrax cases in 1990 alone and 248 cases during 1991-1998 with 19.1% and 7.7% case fatality rates, respectively. Interplay of the ecology of affected areas and anthropogenic factors seem to trigger anthrax epidemics. Anthrax has drawn considerable attention in recent years due to its potential use as a biological weapon. In this paper, the history, current status and approaches towards the control of the disease in Zambia are discussed. Quarantine measures restrict trade of livestock and exchange of animals for draught power resulting in poor food security at household levels. Challenges of anthrax control are complex and comprise of socio-political, economical, environmental and cultural factors. Inadequate funding, lack of innovative disease control strategies and lack of cooperation from stakeholders are the major constraints to the control of the disease.

A manipulação nutricional da capacidade de absorção de ácidos graxos voláteis pela parede do rúmen pode ser uma estratégia para controlar acidose em vacas leiteiras. Objetivando induzir variação morfológica da parede do rúmen através da dieta e estabelecer marcadores morfológicos eficientes para epitélio ruminal, dois experimentos foram relizados. No experimento um, sete vacas com cânula ruminal foram alimentadas com concentrado uma ou quatro vezes ao dia por 19 dias seguidos por 72 horas de jejum. Papilas ruminais foram coletadas nos dias zero, quatro, 12 e 19 do período de tratamento e 24, 48 e 72 horas após o início do período de jejum. Baixa freqüência de alimentação concentrada foi associada a um aumento de insulina plasmática através do tempo (P=0,02) e a um maior (P=0,03) índice mitótico (IM), mas não afetou outros parâmetros morfológicos. No experimento dois, foram realizados dois ensaios não-simultâneos com três ovinos canulados no rúmen, os quais foram submetidos abruptamente a 72 horas de jejum. Papilas ruminais foram coletadas no final do período de alimentação e no final do jejum. O IM foi mais alto no período de alimentação do que no período de jejum (P<0,01), mas outros parâmetros morfológicos não foram capazes de responder à variação nutricional. Entres os marcadores morfológicos estudados o IM parece ser a melhor variável para avaliação da resposta morfológica do epitélio ao plano alimentar. A freqüência de alimentação concentrada pode ser usada para regular a morfologia das papilas ruminais.