Identification and characterization of Marek's disease virus serotype 1 (MDV1) ICP22 gene product: MDV1 ICP22 transactivates the MDV1 ICP27 promoter synergistically with MDV1 ICP4

A previous report [Virus Genes 6 (1992) 365-378] has shown that the US1 gene of Marek's disease virus serotype 1 (MDV1) encodes a homologue of herpes simplex virus type 1 infected cell protein No. 22 (ICP22). In the present study, we expressed and identified a product of the MDV1 US1 gene in chicken embryo fibroblasts (CEFs) with the aid of a recombinant baculovirus expressing a Flag epitope-tagged MDV1 US1 gene, under control of the SRalpha promoter (composed of the enhancer region of the simian virus 40 early promoter and the R region of the human T-cell leukaemia virus type 1 long terminal repeat). In CEF infected with the recombinant baculovirus, MDV1 ICP22 was specifically and efficiently expressed in the presence of n-butyric acid. The apparent M(r) of the expressed protein was 30,000. Reporter gene assays revealed that MDV1 ICP22 by itself transactivated an MDV1 ICP27 promoter/reporter construct weakly but specifically, and furthermore, worked synergistically with MDV1 ICP4 to efficiently up-regulate the MDV1 ICP27 promoter. MDV1 ICP22 may be a regulatory protein that stimulates viral promoters in co-operation with other viral regulatory proteins such as MDV1 ICP4.     

Characterization and epitope mapping of monoclonal antibodies to the nucleocapsid protein of severe acute respiratory syndrome coronavirus

The sudden emergence of severe acute respiratory syndrome (SARS) at the end of 2002 resulted in 774 reported deaths from more than 8000 cases worldwide. As no effective vaccines or antiviral agents are available, the most effective measure to prevent the expansion of a SARS epidemic is the rapid diagnosis and isolation of SARS patients. To establish specific diagnostic methods, we generated nine clones of monoclonal antibodies to nucleocapsid protein (NP) of SARS-coronavirus (SARS-CoV). On immunofluorescent antibody assay and Western blotting analysis, none of the monoclonal antibodies showed cross-reactivity to authentic and recombinant NPs of human coronavirus (HCoV) 229E strain. To determine the region on the NP molecule where the monoclonal antibodies bind, we generated four truncated recombinant NPs and analyzed the reactivity between monoclonal antibodies and truncated NPs. Two monoclonal antibodies reacted with a truncated NP covering from amino acid residues 111 to 230, and seven reacted with another truncated NP covering from amino acid residues 221 to 340. Epitope mapping analysis indicated that monoclonal antibody SN5-25 recognized the amino acid sequence Q(245)TVTKK(250) On SARS-NP. Within the epitope, Q245, T246, V247, K249, and K250 appeared to form an essential motif for monoclonal antibody SN5-25 to bind. The information about binding sites and epitopes of monoclonal antibodies may be useful for the development of new diagnostic methods for SARS and for analyzing the function of N protein of SARS-CoV.     

Fertility after artificial insemination using a soybean-based semen extender in sheep

The present study aimed to compare the fertility of ewes intrauterinally inseminated with frozen-thawed semen using a soybean-based semen extender (AndroMed) with those of ewes intrauterinally inseminated with frozen-thawed semen using a Tris-based extender containing either egg yolk or BSA. Suffolk ewes (n=104) were treated with an intravaginal sponge containing 40 mg fluoroprogesterone acetate (FGA) for 12 days and an intramuscular injection of 500 IU equine chorionic gonadotropin to induce estrus and ovulation during the non-breeding season (July, 2007). Intrauterine insemination was carried out 40-46 h after removal of the FGA sponge (n=90), regardless of the incidence of estrus. The pregnancy rates were not significantly different among the semen extenders containing egg yolk (64.5%) or BSA (58.6%) and AndroMed extender (56.7%). The lambing rates (64.5, 55.2 and 56.7% for the semen extenders containing egg yolk, BSA and AndroMed, respectively) and prolificacy (1.59 to 1.75) were also not significantly different. The present results indicate that an egg yolk-containing semen extender can be replaced with the non-animal derived extender AndroMed, which could be used for intrauterine insemination using frozen-thawed ram semen without reducing fertility.     

Effect of parental genotypes and paternal heterosis on litter traits in crossbred goats.

The effect of parental genotype and paternal heterosis on litter size (LS), total litter birth weight (TLW) and average litter birth weight (ALW) was analysed utilizing data from a crossbreeding programme involving the exotic German Fawn goats and local Katjang goats in Malaysia. In this study, these traits were regarded as traits of the litter to consider the effect of service sire genotype. The results revealed that LS was significantly influenced by the genotype of sire. The genotypes of sire and dam had significant effects on TLW and ALW. Estimates of crossbreeding parameter showed significant and negative influence of paternal heterosis on TLW and ALW while there was no significant effect of paternal heterosis on LS. The results of this study stress the need to reconsider the use of local males in the tropics.     

Effects of the butyric acid‐producing strain Clostridium butyricum MIYAIRI 588 on broiler and piglet zootechnical performance and prevention of necrotic enteritis

The objective of this study was to assess the effects of a probiotic strain Clostridium butyricumMIYAIRI 588 (CBM588) on broiler and weaned piglet health and zootechnical performance. Five field studies were carried out in broilers and five in weaned piglets under European feed additive guidelines. Each study followed a randomized blocked design with two treatments: Control (basal diet) and CBM588 supplemented groups. The zootechnical performance parameters selected were body weight, daily gain, feed intake and feed efficiency (feed:gain). Broilers fed diets with CBM588 gained significantly more weight (+2%, p < .001) and exhibited significantly better feed efficiency (?1.6%, p < .001) in comparison with Controls. Similarly, analysis of pooled data of weaned piglet trials showed that CBM588‐fed piglets were significantly heavier than Controls (+2.6%, p = .014), exhibited significantly higher mean daily gain (+4.7%; p = .004), and significantly improved feed efficiency (?4.2%, p = .001). In addition to the zootechnical efficacy studies, the preventive effect of CBM588 on necrotic enteritis (NE) was assessed in a natural challenge model in broilers where CBM588 reduced the incidence and severity of NE lesions. These data indicate the potential of CBM588 to improve broiler and weaned piglet zootechnical performance, and to make a positive contribution to animal health.     

Inclusion of Bacillus amyloliquefaciens strain TOA5001 in the diet of broilers suppresses the symptoms of coccidiosis by modulating intestinal microbiota

Coccidiosis is an intestinal parasitic infection and one of the most prevalent and economically damaging diseases of chickens. Furthermore, coccidia‐induced mucogenesis promotes secondary colonization by Clostridium perfringens, a major pathogen of chickens that causes necrotic enteritis. Our previous work found that supernatant of a culture of Bacillus amyloliquefaciens strain TOA5001 (BA) inhibited the growth of C. perfringens on Gifu anaerobic broth medium. Accordingly, we evaluated the effectiveness of dietary BA administration in inhibiting C. perfringens colonization of the intestine in broilers that were experimentally infected with coccidia. Ten healthy broilers from a BA‐supplemented (2 × 10⁵ colony‐forming units/g of feed) broiler group and 10 from a non‐treated group were challenged with Eimeria tenella and E. maxima (5000 oocysts of each species/chick) at 28 days old. At 36 days old, five chicks from each group were slaughtered, whereas the remaining five in each group were killed at 49 days old. Dietary BA administration into Eimeria‐challenged birds reduced coccidial symptoms such as intestinal lesions. It also modified the cecal microbiota through suppressing C. perfringens and E. coli colonization, and inducing domination of Faecalibacterium prausnitzii, the Lactobacillus group and unknown Lachnospiraceae genera by bacterial DNA‐based metagenome analyses. B. amyloliquefaciens TOA5001 supplementation suppressed the symptoms of coccidiosis by modulating cecal microbiota in Eimeria‐challenged broilers.     

Generation of anti‐porcine CD69 monoclonal antibodies and their usefulness to evaluate early activation of cellular immunity by flow cytometric analysis

T cell‐mediated cellular immunity and humoral immunity are equally important for the prevention of diseases. To assess activation of human and mouse cellular immunity, early activation markers of lymphocytes are often used in flow cytometry targeting expression of CD69 molecules. Response of humoral immunity against infection or vaccination has been well investigated in pigs, but that of cellular immunity has been largely neglected due to lack of direct evaluation tools. Thus, in pig research a proper assay of antibody reacted with porcine CD69 is still unavailable. In the present study, two anti‐porcine CD69 mAb‐producing mouse hybridomas, 01‐14‐22‐51 (IgG2b–κ) and 01‐22‐44‐102 (IgG2a–κ), both showing fine reactivity with phorbol 12‐myristate 13‐acetate (PMA) and ionomycin‐stimulated porcine peripheral blood lymphocytes in flow cytometry, were established. When porcine peripheral blood lymphocytes were activated with PMA and ionomycin and analyzed by flow cytometry, it was found that both mAbs generated in this study stained about 70% of lymphocytes. In contrast, after an identical procedure, only 5% and 13.5% of lymphocytes were stained with anti‐interferon‐γ mAb and anti‐tumor necrosis factor‐α mAb, respectively. These results indicate that evaluation of cellular immunity activation turns more sensitive after using our newly generated mAbs.     

Dietary administration of probiotics to sows and/or their neonates improves the reproductive performance,incidence of post‐weaning diarrhea and histopathological parameters in the intestine of weaned piglets

Probiotics have gained considerable attention with respect to their beneficial effects on livestock performance and health. The most significant effects of probiotics on the gut microbiota and the host animals take place when they are included in diets during particularly stressful periods such as weaning and/or at the beginning of the lactation period. The probiotics Bacillus mesentericus strain TO‐A at 1 × 10⁸ colony forming units (CFU)/g, Clostridium butyricum strain TO‐A at 1 × 10⁸ CFU/g and Enterococcus faecalis strain T‐110 at 1 × 10⁹ CFU/g were used. Litter weight at delivery and ratio of return to estrous improved significantly (17% and 24% improvement, respectively) by probiotic administration to sows (0.2% (w/w)). Furthermore, the feed intake of the probiotics‐administered sows was greater than that of the control sows during the late lactation period. Post‐weaning diarrheal incidence and growth performance was improved by probiotics administration to neonates (0.02% (w/w)), while the combined use of probiotics in sows and their neonates induced the enlargement of villous height and prevented muscle layer thinning in the small intestine of weaning piglets. The administration of probiotics of three species of live bacteria improved the porcine reproductive performance around stressful periods of sows (farrowing) and piglets (weaning). [Corrections added on 26 April 2016, after first online publication: ‘Enterococcus faecalis strain T‐100’ has been corrected to ‘Enterococcus faecalis strain T‐110’ in the above paragraph and in the ‘Probiotics’ section under the Materials and Methods heading.]     

Chemical characterization of milk oligosaccharides of the common wombat (Vombatus ursinus)

Previous structural characterizations of marsupial milk oligosaccharides have been performed in the tammar wallaby, red kangaroo, koala, common brushtail possum and the eastern quoll. To clarify the homology and heterogeneity of milk oligosaccharides among marsupial species, which could provide information on their evolution, the oligosaccharides of wombat milk carbohydrate were characterized in this study. Neutral and acidic oligosaccharides were isolated from the carbohydrate fractions of two samples of milk of the common wombat and characterized by ¹H‐nuclear magnetic resonance spectroscopy. The structures of six neutral saccharides were found to be Gal(β1‐4)Glc (lactose), Gal(β1‐3)Gal(β1‐4)Glc (3'‐galactosyllactose), Gal(β1‐3)Gal(β1‐3)Gal(β1‐4)Glc (3',3”‐digalactosyllactose), Gal(β1‐3)Gal(β1‐3)Gal(β1‐3)Gal(β1‐4)Glc, Gal(β1‐3)Gal(β1‐3)[Gal(β1‐4)GlcNAc(β1‐6)]Gal(β1‐4)Glc (galactosyl lacto‐N‐novopentaose I) and Gal(β1‐3)[Gal(β1‐4)GlcNAc(β1‐6)]Gal(β1‐3)[Gal(β1‐4)GlcNAc(β1‐6)]Gal(β1‐4)Glc (lacto‐N‐novooctaose), while those of six acidic saccharides were Neu5Ac(α2‐3)Gal(β1‐3)Gal(β1‐4)Glc. (sialyl 3'‐galactosyllactose), Neu5Ac(α2‐3)Gal(β1‐3)Gal(β1‐3)Gal(β1‐4)Glc (sialyl 3',3”‐digalactosyllactose), Neu5Ac(α2‐3)Gal(β1‐3)[Gal(β1‐4)GlcNAc(β1‐6)]Gal(β1‐4)Glc (sialyl lacto‐N‐novopentaose a), Gal(β1‐3)[Neu5Ac(α2‐3)Gal(β1‐4)GlcNAc(β1‐6)]Gal(β1‐4)Glc (sialyl lacto‐N‐novopentaose c), Neu5Ac(α2‐3)Gal(β1‐3)Gal(β1‐3)Gal(β1‐3)Gal(β1‐4)Glc,, Neu5Ac(α2‐3)Gal(β1‐3)Gal(β1‐3)[Gal(β1‐4)GlcNAc(β1‐6)]Gal(β1‐4)Glc and Gal(β1‐3)Gal(β1‐3)[Neu5Ac(α2‐3)Gal(β1‐4)GlcNAc(β1‐6)]Gal(β1‐4)Glc. In addition, small amounts of sulfated oligosaccharides but no oligosaccharides containing Neu5Gc or α(2–6) linked Neu5Ac were detected.     

Effects of land use on population presence and genetic structure of an amphibian in an agricultural landscape

Context

Species distributions are a function of an individual’s ability to disperse to and colonize habitat patches. These processes depend upon landscape configuration and composition.

Objectives

Using Blanchard’s cricket frogs (Acris blanchardi), we assessed which land cover types were predictive of (1) presence at three spatial scales (pond-shed, 500 and 2500 m) and (2) genetic structure. We predicted that forested, urban, and road land covers would negatively affect cricket frogs. We also predicted that agricultural, field, and aquatic land covers would positively affect cricket frogs.

Methods

We surveyed for cricket frogs at 28 sites in southwestern Ohio, USA to determine presence across different habitats and analyze genetic structure among populations. For our first objective, we examined if land use (crop, field, forest, and urban habitat) and landscape features (ponds, streams, and roads) explained presence; for our second objective, we assessed whether these land cover types explained genetic distance between populations.

Results

Land cover did not have a strong influence on cricket frog presence. However, multiple competing models suggested effects of roads, streams, and land use. We found genetic structuring: populations were grouped into five major clusters and nine finer-scale clusters. Highways were predictive of increased genetic distance.

Conclusions

By combining a focal-patch study with landscape genetics, our study suggests that major roads and waterways are key features affecting species distributions in agricultural landscapes. We demonstrate that cricket frogs may respond to landscape features at larger spatial scales, and that presence and movement may be affected by different environmental factors.