Development of a biosecurity assessment tool and the assessment of biosecurity levels by this tool on Japanese commercial swine farms.

It is well known that infectious diseases such as porcine reproductive and respiratory syndrome (PRRS) and porcine epidemic diarrhea (PED) decrease herd productivity and lead to economic loss. It is believed that biosecurity practices are effective for the prevention and control of such infectious diseases. Therefore, the objective of the present study was to investigate whether or not an association between biosecurity level and herd productivity, as well as disease status exists on Japanese commercial swine farms. The present study was conducted on 141 farms. Biosecurity in each farm was assessed by a biosecurity assessment tool named BioAsseT. BioAsseT has a full score of 100 and consists of three sections (external biosecurity, internal biosecurity and diagnostic monitoring). Production data for number of pigs weaned per sow per year (PWSY) and post-weaning mortality per year (PWM) were collected for data analysis. Regarding PRRS status, the farms were categorized into two groups: unknown or unstable and stable or negative. In addition, these farms were categorized based on their PED status, either positive or negative. The total BioAsseT score was associated with herd productivity: as total score increased by 1, PWSY increased by 0.104 pigs and PWM decreased by 0.051 % (P < 0.05). Herd productivity was associated with the score of external and internal biosecurity (P < 0.05), but did not correlate with the score of diagnostic monitoring. Regarding PRRS status, farms with an unknown or unstable status had lower total score than those with stable or negative status (P < 0.05). Similarly, PED positive farms had a lower total score compared to PED negative farms (P < 0.05). In conclusion, the present study provides evidence for the association between high biosecurity levels and increased herd productivity as well as a decreased risk for novel introductions of infectious diseases such as PED.

Does decrease of the thoracic kyphosis influence decrease knee adduction moment during gait? A preliminary study of a healthy population.

[Purpose] The purpose of this study was to investigate the influence of a decrease in thoracic kyphosis angle on the knee adduction moment during gait in healthy young individuals. [Subjects and Methods] Twenty-nine healthy adults, consisting of 15 males and 14 females (21.6 ± 1.1 years old), participated. The draw-in maneuver was used to decrease thoracic kyphosis, and thoracic kyphosis was measured using a SpinalMouse during normal standing and standing with the draw-in maneuver. The participants were required to maintain the draw-in maneuver during gait. A 3-D motion analysis system and a force plate were used to obtain knee adduction moment. [Results] Thoracic kyphosis angles during the draw-in maneuver (41.0 ± 7.4 degrees) were significantly decreased compared with the angles during normal standing (43.0 ± 7.9 degrees). Although the knee adduction moment during gait with the draw-in maneuver was not significantly decreased compared with that during level gait, in the 20 subjects who had decreased kyphosis due to the draw-in maneuver, the 1st peak knee adduction moment (55.7 ± 24.3 × 10(-3)) with the draw-in maneuver was significantly decreased compared with the knee adduction moment (57.0 ± 16.3 × 10(-3)) during level gait. [Conclusion] Knee adduction moment in the case of a decreased thoracic kyphosis angle due to the draw-in maneuver was decreased compared with that during level gait.

Expression analysis of programmed death ligand 2 in tumors caused by the avian oncovirus Marek's disease virus.

PD-L2 is a ligand of the immunoinhibitory receptor PD-1. Here, we report functional and expression analyses of PD-L2 in tumor lesions and spleens from chickens infected with gallid herpesvirus 2 (GaHV-2, Marek's disease virus), which induces malignant lymphomas in chickens. We show that the expression of IFN-γ protein was decreased in PBMCs and splenocytes co-cultured with PD-L2-expressing cells and that the expression of PD-L2 mRNA was significantly higher in the spleens of infected chickens in the latent phase and in tumor lesions caused by GaHV-2. These results suggest that chicken PD-L2 has an immunoinhibitory function and is involved in the establishment of latency and tumor formation by GaHV-2.

Molecular characterization of immunoinhibitory factors PD-1/PD-L1 in chickens infected with Marek's disease virus.

BACKGROUND: An immunoinhibitory receptor, programmed death-1 (PD-1), and its ligand, programmed death-ligand 1 (PD-L1), are involved in immune evasion mechanisms for several pathogens causing chronic infections and for neoplastic diseases. However, little has been reported for the functions of these molecules in chickens. Thus, in this study, their expressions and roles were analyzed in chickens infected with Marek's disease virus (MDV), which induces immunosuppression in infected chickens. RESULTS: A chicken T cell line, Lee1, which constitutively produces IFN-γ was co-cultured with DF-1 cells, which is a spontaneously immortalized chicken fibroblast cell line, transiently expressing PD-L1, and the IFN-γ expression level was analyzed in the cell line by real-time RT-PCR. The IFN-γ expression was significantly decreased in Lee1 cells co-cultured with DF-1 cells expressing PD-L1. The expression level of PD-1 was increased in chickens at the early cytolytic phase of the MDV infection, while the PD-L1 expression level was increased at the latent phase. In addition, the expression levels of PD-1 and PD-L1 were increased at tumor lesions found in MDV-challenged chickens. The expressions levels of PD-1 and PD-L1 were also increased in the spleens and tumors derived from MDV-infected chickens in the field. CONCLUSIONS: We demonstrated that the chicken PD-1/PD-L1 pathway has immunoinhibitory functions, and PD-1 may be involved in MD pathogenesis at the early cytolytic phase of the MDV infection, whereas PD-L1 could contribute to the establishment and maintenance of MDV latency. We also observed the increased expressions of PD-1 and PD-L1 in tumors from MDV-infected chickens, suggesting that tumor cells transformed by MDV highly express PD-1 and PD-L1 and thereby could evade from immune responses of the host.

Analysis of transcriptional activities of the Meq proteins present in highly virulent Marek's disease virus strains, RB1B and Md5.

Marek's disease virus (MDV) is an oncogenic herpesvirus that causes malignant lymphomas in chickens. Recent field isolates of MDV have tended to exhibit increasing virulence, and MDV strains are currently classified into four categories based on their relative virulence. Meq, a putative MDV oncoprotein, resembles the Jun/Fos family of basic leucine zipper (bZIP) transcription factors and can regulate the expression of viral and cellular genes as a homodimer or as a heterodimer with a variety of bZIP family proteins. MDV isolates display distinct diversity and point mutations in Meq, which may contribute to changes in the transcriptional activities of Meq and subsequently, to observed increases in MDV oncogenicity. In this study, we introduced mutations into the meq gene and used dual luciferase reporter assays to analyze the transcriptional activities of the resulting Meq proteins to determine whether distinct mutations in Meq could be responsible for differences in transcriptional activity among MDV strains. A proline-to-alanine substitution at position 217, the second position of one of the proline direct repeats in the transactivation domain, enhanced the transactivation activity of Meq. In addition, we found that two substitutions at positions 283 and 320 affected transactivation activity. These results suggest that the distinct diversity of and point mutations in the Meq proteins are responsible for differences in transactivation activity among MDV strains.

Cytokine profiles in chickens infected with virulent and avirulent Marek's disease viruses: interferon-gamma is a key factor in the protection of Marek's disease by vaccination.

Marek's disease has been controlled byvaccination with avirulent strains of MDV. However, the protection mechanism following vaccination is not fully understood. In this study immune responses of PBMC and splenocytes derived from vaccinated chickens challenged with virulent MDV were examined using real-time PCR and ELISA. Higher levels of IFN-gamma induction were observed in chickens vaccinated during the latent phase of infection with virulent MDV than in similarly challenged, unvaccinated chickens. Furthermore, the mean expression of IFNGR2 and IFN regulatory factor-3 mRNAs was significantly higher in vaccinated than in unvaccinated chickens. These results show that IFN-gamma could be one of the important factors in prevention of MD by vaccination and is effective during the latent phase of the infection.

Microarray analysis of host immune responses to Marek's disease virus infection in vaccinated chickens.

Marek's disease (MD) is a commercially important disease of chickens caused by MD virus (MDV). Although avirulent MDV strains have been used for vaccination to prevent MD outbreaks, the protective mechanism of the vaccine has not been elucidated. In this study, a comprehensive transcriptional analysis using microarray was conducted in MDV-infected chickens with and without vaccination at 7 and 21 days post-infection (dpi). The data suggested that the expression of T cell receptor (TCR) 1-related genes was up-regulated in vaccinated-challenged compared to unvaccinated-challenged chickens during the latent phase of infection. Consistently, this induction was confirmed by quantitative PCR. Flow cytometric analysis revealed that most of TCR1(+) cells expressed CD8alpha chain brightly. The number of this subpopulation was significantly and specifically increased in vaccinated-challenged chickens at 21 dpi compared to unvaccinated-challenged chickens, though it was not the major population in spleen of chickens. The number of CD8alpha(high) TCR2(+) cells, the major subpopulation of chicken CD8alpha(high) cells, was increased in vaccinated chickens with or without challenge compared to unvaccinated control chickens. These data suggested that both CD8alpha(high) TCR1(+) and CD8alpha(high) TCR2(+) cells could be induced by the vaccination. It is also possible that CD8alpha(high) TCR1(+) cells might be primed by the vaccination and specifically induced by the challenge with virulent strain of MDV during the latent phase of infection. Thus, CD8alpha(high) TCR1(+) cell population is probably one of the key factors involved in the protective mechanism induced by a vaccine strain, CVI988.

Prevalence and source of trypanosome infections in field-captured vector flies (Glossina pallidipes) in southeastern Zambia.

The prevalence of trypanosome infections in tsetse flies, Glossina pallidipes, collected from Chiawa and Chakwenga in Zambia with endemic trypanosomosis was assessed by polymerase chain reaction (PCR). Out of the 550 G. pallidipes, 58 (10.5%) flies were found to harbor trypanosome DNA. Infection rates of tsetse with Trypanosoma vivax universal, Trypanosoma congolense savannah, T. congolense forest and T. congolense kilifi were 4.2% (23/550), 4.7% (26/550), 1.1% (6/550) and 1.6% (9/550), respectively. To determine the mammalian hosts of T. congolense and T. vivax infections from the tsetse flies, mammalian mitochondrion DNA of blood meal in these flies were analyzed by PCR and subsequent gene sequence analysis of the amplicons. Sequence analysis showed the presence of cytochrome b gene (cyt b) of 7 different mammalian species such as human, elephant, buffalo, goat, warthog, greater kudu and cattle. Goats which were main livestock in these areas were further examined to know the extent of its contribution in spreading the infection. We examined the prevalence of trypanosome infections in the domestic goat population in 6 settlements in Chiawa alone. Of the 86 goats sampled, 4 (4.6%), 5 (5.8%), 4 (4.6%) and 4 (4.6%) were positive for T. vivax universal, T. congolense savannah, forest and kilifi, respectively. These findings showed that the host-source of trypanosome infections in vector fly give a vital information about spread of infection. The result of this study will certainly contribute in elucidating more the epidemiology of trypanosomosis.

Serological survey of Toxoplasma gondii in wild waterfowl in Chukotka, Kamchatka, Russia and Hokkaido, Japan.

Antibodies to Toxoplasma gondii were assayed by ELISA in 22 experimentally inoculated domestic ducks. In addition, a serological assay was carried out at Obihiro, Hokkaido, Japan, in 2004 and 2005, on 221 wild ducks of 5 species: Anas platyrhynchus (n = 111); A. poecilorhyncha (n = 27); A. acuta (n = 58); A. penelope (n = 16); and A. crecca (n = 9). Assays were also conducted using sera from 197 wild geese of 2 species, i.e., Anser albifrons (n = 162) and Ans. fabalis (n = 35). Birds were collected between 2003 and 2005 from 3 different areas: Lake Miyajima-numa, Hokkaido, Japan, regions around Anadyr city of Chukotka autonomous okrug, and Lake Makobetukoe, Kamchatka oblast, Russia. The ELISA cutoff value (OD) was > or =0.395 based on results from uninfected ducks; the final dilution ratio recognized as positive was represented by the end titer. The end titer in the experimentally infected ducks ranged from 1:400 to 1:3,200. Antibodies to T. gondii were found in 49 of the 221 wild duck samples from Japan: A. platyrhynchus (22/74); A. poecilorhyncha (2/15); A. penelope (3/16); A. acuta (4/58); and A. crecca (0/9), all in 2004. In 2005, T. gondii was found in A. platyrhynchus (13/37); and A. poecilorhyncha (5/12). Thirty-two of 197 wild goose samples were seropositive, i.e., Ans. albifrons (7/51) in 2004 and (11/72) in 2005 in Miyajima-numa, Japan and 9/39 in Chukotka, Russia as well as in Ans. fabalis (5/35) in Kamchatka, for which the end titer ranged from 1:100 to 1:3,200. In immunoblotting, the A. platyrhynchus samples showed specific IgG antibody binding to several antigens in the T. gondii lane, i.e., at 30 and 43 kDa, but not in the Neospora caninum lane. No specific bands were noted in samples for which antibody activity was not detected. These results suggest that wild waterfowl inhabiting Hokkaido, Chukotka, and Kamchatka may be exposed to T. gondii.

C57BL/6 mice infected with Neospora caninum during administration of progesterone show bias toward type 2 immune response.

To clarify the role of progesterone in the development of immune responses during pregnancy against Neospora caninum infection, C57BL/6 mice were given a progesterone pellet, and measured on Interferon-gamma and interleukin-4 production following the infection. IFN-gamma production in the prescribed group was significantly lower than that in the intact group on day 40 post administration. IL-4 producing cell population in the prescribed group was larger than that in the intact group. These results suggest that progesterone may alter the balance of cytokine production, and that the bias toward type 2 immune response may remain for a certain period after the infection.

Prevalence of Toxoplasma gondii and Neospora caninum in sika deer from eastern Hokkaido, Japan.

Brain and serum were collected from 120 and 12 free-ranging sika deer (Cervus nippon yesoensis), respectively, from six regions in eastern Hokkaido during controlled hunts in the autumn of 2003. Brains were tested for Neospora caninum and Toxoplasma gondii DNA by polymerase chain reaction (PCR) assays. Antibodies to Toxoplasma gondii were measured by means of a latex agglutination test. No brain tested positive for either type of DNA, and no antibody to Toxoplasma gondii was detected in serum, suggesting a low prevalence of infection with these organisms in free-ranging sika deer from eastern Hokkaido. Further examination of multiple tissues by PCR and serologic surveys will be necessary to confirm this.