Effects of high‑grain diet feeding on fatty acid profiles in milk,blood,muscle,and adipose tissue,and transcriptional expression of lipid‑related genes in muscle and adipose tissue of dairy cows

Background High-grain(HG)diets affect lipid metabolism in the liver and mammary tissue of dairy cows,but its effects on muscle and adipose tissue have not been wide evaluated.Thus,the aim of this study is to clarify this issue.Methods Twelve Holstein cows were randomly divided into two groups:conventional diet group(CON,n=6)and the HG diet group(n=6).On day 7 of week 4,rumen fluid was sampled to measure pH,milk was sampled to meas-ure components,and blood was sampled to measure biochemical parameters and fatty acid composition.After the experiment,cows were slaughtered to collect muscle and adipose tissue for fatty acid composition and transcriptome analysis.Results HG feeding decreased the ruminal pH,milk’s fat content and long-chain fatty acid proportion(P<0.05)and increased the proportion of short-and medium-chain fatty acids in the milk(P<0.05)as compared with CON diets.The concentrations of blood cholesterol,low-density lipoprotein,and polyunsaturated fatty acids in the HG cows were lower than those in CON cows(P<0.05).In muscle tissue,HG feeding tended to increase the triacylglycerol(TG)concentration(P<0.10).Transcriptome analysis revealed changes in the biosynthesis of the unsaturated fatty acids pathway,the regulation of lipolysis in the adipocytes pathway,and the PPAR signalling pathway.In adipose tissue,HG feeding increased the concentration of TG and decreased the concentration of C18:1 cis9(P<0.05).At the transcrip-tome level,the fatty acid biosynthesis pathway,linoleic acid metabolism pathway,and PPAR signalling pathway were activated.Conclusion HG feeding leads to subacute rumen acidosis and a decreased milk fat content.The fatty acid profiles in the milk and plasma of dairy cows were changed by HG feeding.In muscle and adipose tissue,HG feeding increased TG concentration and up-regulated the expression of genes related to adipogenesis,while down-regulated the expression of genes related to lipid transport.These results complement our knowledge of the fatty acid composi-tion of muscle and adipose tissue in dairy cows and expand our understanding of the mechanisms by which HG diets affect lipid metabolism in muscle and adipose tissue.

Isolation of bacteria from fermented food and grass carp intestine and their efficiencies in improving nutrient value of soybean meal in solid state fermentation

Background: Soybean meal is an excellent and cost-effective protein source; however, its usage is limited in the piglet due to the presence of anti-nutritional factors and the antigens glycinin and β-conglycinin. The objective of the current study was to screen and select for bacteria that can be efficiently adopted to ferment soybean meal in order to solve this problem.Results: Bacteria were isolated from fermented soy foods and the grass carp intestine, and strains selected for high protease, cellulase and amylase activities. The isolated bacteria were characterized as Bacillus cereus, Bacillus subtilis and Bacilus amyloliquefacien, respectively. Fermentation with food-derived Isolate-2 and fish-derived F-9 increased crude protein content by 5.32% and 8.27%, respectively; improved the amino acid profile by increasing certain essential amino acids, broke down larger soy protein to 35 k Da and under, eliminated antigenicity against glycinin and β-conglycinin, and removed raffinose and stachyose in the soybean meal following a 24-h fermentation.Conclusions: Our results suggest these two B. amyloliquefaciens bacteria can efficiently solid state ferment soybean meal and ultimately produce a more utilizable food source for growing healthy piglets.

Innovative approaches to genome editing in avian species

The tools available for genome engineering have significantly improved over the last 5 years, allowing scientist to make precise edits to the genome. Along with the development of these new genome editing tools has come advancements in technologies used to deliver them. In mammals genome engineering tools are typically delivered into in vitro fertilized single cell embryos which are subsequently cultured and then implanted into a recipient animal.In avian species this is not possible, so other methods have been developed for genome engineering in birds. The most common involves in vitro culturing of primordial germ cells(PGCs), which are cells that migrate through the embryonic circulatory system to the developing gonad and colonize the gonad, eventually differentiating into the gonadocytes which produce either sperm or ova. While in culture the PGCs can be modified to carry novel transgenes or gene edits, the population can be screened and enriched, and then transferred into a recipient embryo. The largest drawback of PGC culture is that culture methods do not transfer well across avian species, thus there are reliable culture methods for only a few species including the chicken. Two newer technologies that appear to be more easily adapted in a wider range of avian species are direct injection and sperm transfection assisted gene editing(STAGE).The direct injection method involves injecting genome engineering tools into the circulatory system of the developing embryo just prior to the developmental time point when the PGCs are migrating to the gonads. The genome engineering tools are complexed with transfection reagents, allowing for in vivo transfection of the PGCs. STAGE utilizes sperm transfection to deliver genome engineering tools directly to the newly fertilized embryo. Preliminary evidence indicates that both methodologies have the potential to be adapted for use in birds species other than the chicken, however further work is needed in this area.

Effects of galacto-oligosaccharides on growth and gut function of newborn suckling piglets

Background: Most research on galacto-oligosaccharides(GOS) has mainly focused on their prebiotic effects on the hindgut,but their beneficial effects on the small intestine(SI) have received little attention.Since jejunum is the important place to digest and absorb nutrients efficiently,optimal maturation of the jejunum is necessary for maintaining the high growth rate in the neonate.Therefore,this study investigates the effect of the early intervention with GOS on the intestinal development of the jejunum.Methods: A total of 6 litters of neonatal piglets(10 piglets per litter; Duroc × Landrace × Large White) with an average birth weight of 1.55 ± 0.05 kg received 1 of 2 treatments based on their assignment to either the control(CON) group or the GOS(GOS) group in each litter.Piglets in the GOS group were orally administrated 10 mL of a GOS solution(reaching 1 g GOS/kg body weight) per day from the age of 1 to 7 d; the piglets in the CON group were treated with the same dose of physiological saline.All piglets were weaned on d 21.On d 8 and 21 of the experimental trial,1 pig per group from each of the 6 litters was euthanized.Results: The early intervention with GOS increased the average daily gains in the third week(P < 0.05).Decreased crypt depth was also observed in the jejunum of the piglets on d 21(P < 0.05).The early intervention with GOS increased the jejunal lactase activity on d 8,maltase activity and sucrase activity on d 21(P < 0.05).In addition,the early intervention with GOS also facilitated the mRNA expression of Sodium glucose co-transporter 1(SGLT1) on d 8 and the m RNA expression of Glucose transporter type 2(GLUT2) on d 21(P < 0.05).It was further determined that GOS up-regulated the m RNA expression of preproglucagon(GCG),insulin-like growth factor 1(IGF-1),insulin-like growth factor 1 receptor(IGF-1 R) and epidermal growth factor(EGF).GOS also up-regulated the protein expression of glucagon-like peptide-2(GLP-2) and EGF in the jejunum of the piglets.Furthermore,it was also found that GOS enhanced the protein expression of ZO-1 and occludin on d 8(P < 0.05),as well as increased the mRNA expression of TGF-β and decrease the mRNA expression of IL-12(P < 0.05).Conclusions: These results indicate that GOS have a positive effect on piglet growth performance in addition to decreasing the crypt depth and enhancing functional development in jejunum of suckling piglets.

Long-term effects of early antibiotic intervention on blood parameters,apparent nutrient digestibility, and fecal microbial fermentation profile in pigs with different dietary protein levels

Backgroud： This study aimed to determine the effects of early antibiotic intervention（EAI） on subsequent blood parameters, apparent nutrient digestibility, and fecal fermentation profile in pigs with different dietary crude protein（CP） levels. Eighteen litters of piglets（total 212） were randomly allocated to 2 groups and were fed a creep feed diet with or without in-feed antibiotics（olaquindox, oxytetracycline calcium and kitasamycin） from postnatal d 7 to d 42. On d 42, the piglets within the control or antibiotic group were mixed, respectively, and then further randomly assigned to a normal-（20%, 18%, and 14% CP from d 42 to d 77, d 77 to d 120, and d 120 to d 185,respectively） or a low-CP diet（16%, 14%, and 10% CP from d 42 to d 77, d 77 to d 120, and d 120 to d 185,respectively）, generating 4 groups. On d 77（short-term） and d 185（long-term）, serum and fecal samples were obtained for blood parameters, microbial composition and microbial metabolism analysis.Results： EAI increased（P 〈 0.05） albumin and glucose concentrations in low-CP diet on d 77, and increased（P 〈 0.05） urea concentration in normal-CP diet. On d 185, EAI increased（P 〈 0.05） globulin concentration in normal-CP diets, but decreased glucose concentration. For nutrient digestibility, EAI increased（P 〈 0.05）digestibility of CP on d 77. For fecal microbiota, the EAI as well as low-CP diet decreased（P 〈 0.05） E. coli count on d 77. For fecal metabolites, on d 77, EAI decreased（P 〈 0.05） total amines concentration but increased skatole concentration in low-CP diet. On d 185, the EAI increased（P 〈 0.05） putrescine and total amines concentrations in low-CP diets but reduced（P 〈 0.05） in the normal-CP diets. The low-CP diet decreased the concentrations of these compounds.Conclusions： Collectively, these results indicate that EAI has short-term effects on the blood parameters and fecal microbial fermentation profile. The effects of EAI varied between CP levels, which was characterized by the significant alteration of glucose and putrescine concentration.

Morphological adaptation of sheep's rumen epithelium to high-grain diet entails alteration in the expression of genes involved in cell cycle regulation, cell proliferation and apoptosis

Background: The objectives of this study were to characterize changes in the relative m RNA expression of candidate genes and proteins involved in cell cycle regulation, cell proliferation and apoptosis in the ruminal epithelium(RE) of sheep during high-grain(HG) diet adaptation.Results: Twenty sheep were assigned to four groups with five animals each. These animals were assigned to different periods of HG diet(containing 40% forage and 60% concentrate mix) feeding. The HG groups received an HG diet for7(G7, n = 5), 14(G14, n = 5) and 28 d(G28, n = 5), respectively. In contrast, the control group(CON, n = 5) was fed the forage-based diet for 28 d. The results showed that HG feeding linearly decreased(P < 0.001) the ruminal p H, and increased the concentrations of ruminal total volatile fatty acid(linear, P = 0.001), butyrate(linear, P < 0.001), valerate(quadratic P = 0.029) and the level of IGF-1(quadratic, P = 0.043) in plasma. The length(quadratic, P = 0.004), width(cubic, P = 0.015) and surface of the ruminal papillae(linear, P = 0.003) were all enlarged after 14 d of HG diet feeding.HG feeding cubically increased the number of cell layers forming the stratum corneum(SC, P < 0.001) and the thickness of the SC(P < 0.001) and stratum basale(P < 0.001). The proportion of basal layer cells in the RE decreased(linear, P < 0.001) in the G0/G1-phase, but it increased linearly(P = 0.006) in the S-phase and cubically(P = 0.004) in the G2/M-phases. The proportion of apoptosis cells in G7, G14 and G28 was reduced compared to the CON(quadratic, P <0.001). HG diet feeding linearly decreased the m RNA expression of Cyclin E1(P = 0.021) and CDK-2(P = 0.001) and(P = 0.027) the protein expression of Cyclin E1. Feeding an HG diet linearly increased the m RNA expression of genes IGFBP-2(P = 0.034) and IGFBP 5(P < 0.009), while linearly decreasing(P < 0.001) the IGFBP 3 expression. The expression of cell apoptosis gene Caspase 8 decreased(quadratic, P = 0.012), while Bad m RNA expression tended to decrease(cubic, P = 0.053) after HG feeding.Conclusions: These results demonstrated sequential changes in rumen papillae size, cell cycle regulation and the genes involved in proliferation and apoptosis as time elapsed in feeding a high-grain diet to sheep.

Low-protein diets supplemented with casein hydrolysate favor the microbiota and enhance the mucosal humoral immunity in the colon of pigs

Background:High-protein diets can increase the colonic health risks.A moderate reduction of dietary crude-protein(CP)level can improve the colonic bacterial community and mucosal immunity of pigs.However,greatly reducing the dietary CP level,even supplemented with all amino acids(AAs),detrimentally affects the colonic health,which may be due to the lack of protein-derived peptides.Therefore,this study evaluated the effects of supplementation of casein hydrolysate(peptide source)in low-protein(LP)diets,in comparison with AAs supplementation,on the colonic microbiota,microbial metabolites and mucosal immunity in pigs,aiming to determine whether a supplementation of casein hydrolysate can improve colonic health under very LP level.Twenty-one pigs(initial BW 19.90±1.00 kg,63±1 days of age)were assigned to three groups and fed with control diet(16%CP),LP diets(13%CP)supplemented with free AAs(LPA)or casein hydrolysate(LPC)for 4 weeks.Results:Compared with control diet,LPA and LPC diet decreased the relative abundance of Streptococcus and Escherichia coli,and LPC diet further decreased the relative abundance of Proteobacteria.LPC diet also increased the relative abundance of Lactobacillus reuteri.Both LP diets decreased concentrations of ammonia and cadaverine,and LPC diet also reduced concentrations of putrescine,phenol and indole.Moreover,LPC diet increased total short-chain fatty acid concentration.In comparison with control diet,both LP diets decreased protein expressions of Toll-like receptor-4,nuclear factor-κB,interleukin-1βand tumor necrosis factor-α,and LPC diet further decreased protein expressions of nucleotide-binding oligomerization domain protein-1 and interferon-γ.LPC diet also increased protein expressions of G-protein coupled receptor-43,interleukin-4,transforming growth factor-β,immunoglobulin A and mucin-4,which are indicators for mucosal defense activity.Conclusions:The results showed that supplementing casein hydrolysate showed beneficial effects on the colonic microbiota and mucosal immunity and barrier function in comparison with supplementing free AAs in LP diets.These findings may provide new framework for future nutritional interventions for colon health in pigs.

Bats and Viruses:a Brief Review

Bats,probably the most abundant,diverse and geographically dispersed vertebrates on earth,have recently been shown to be the reservoir hosts of a number of emerging viruses responsible for severe human and livestock disease outbreaks. Flying foxes have been demonstrated to be the natural reservoir for Hendra and Nipah viruses. Evidence supporting the possibility of bats as potential reservoirs for SARS coronavirus(SARS-CoV) and Ebola virus has also been reported. The recent discovery of these viruses and other viruses occurring naturally in the bat population provides a unique insight into a diverse pool of potentially emergent and pathogenic viruses. The factors which influence the ability of zoonotic viruses to effectively cross the species barrier from bats to other animal populations are poorly understood. A brief review is provided here on the recently emerged bat viruses and on current and future strategies for research in this area.

The community structure of Methanomassiliicoccales in the rumen of Chinese goats and its response to a highgrain diet

Background: The newly proposed methanogenic order ‘Methanomassiliicoccales' is the second largest archaeal population in the rumen, second only to the Methanobrevibacter population. However, information is limited regarding the community of this new order in the rumen.Methods: This study used real-time PCR and 454 pyrosequencing to explore the abundance and community composition of Methanomassiliicoccales in the rumen of Chinese goats fed a hay(0% grain, n = 5) or a high grain(65% grain, n = 5) diet.Results: Real-time PCR analysis showed that the relative abundance of Methanomassili coccales(% of total archaea) in the goat rumen was significantly lower in the high-grain-diet group(0.5% ± 0.2%) than that in the hay-diet group(8.2% ± 1.1%, P < 0.05). The pyrosequencing results showed that a total of 208 operational taxonomic units(OTUs) were formed from ten samples at 99% sequence identity. All the sequences were identified as Methanomassiliicoccaceae at the family level, and most of the sequences(96.82% ± 1.64%) were further classified as Group 8, 9, and 10 at the Methanomassiliicoccales genus level in each sample based on the RIM-DB database. No significant differences were observed in the number of OTUs or Chao1's, Shannon's or Pielou's evenness indexes between the hay-and high-grain-diet groups(P ≥ 0.05). PCoA analysis showed that diet altered the community of Methanomassiliicoccales.At the genus level, the relative abundances of Group 10(67.25 ± 12.76 vs. 38.13 ± 15.66, P = 0.012) and Group 4(2.07 ± 1.30 vs. 0.27 ± 0.30, P = 0.035) were significantly higher in the high-grain-diet group, while the relative abundance of Group9 was significantly higher in the hay-diet group(18.82 ± 6.20 vs. 47.14 ± 17.72, P = 0.020). At the species level, the relative abundance of Group 10 sp.(67.25 ± 12.76 vs. 38.13 ± 15.66, P = 0.012) and Group 4 sp. MpT 1(2.07 ± 1.30 vs. 0.27 ± 0.30,P = 0.035) were significantly higher in the high-grain-diet group, while the relative abundance of Group 9 sp. ISO4-G1 was significantly higher in the hay-diet group(12.83 ± 3.87 vs. 42.44 ± 18.47, P = 0.022).Conclusions: Only a few highly abundant phylogenetic groups dominated within the Methanomassiliicoccales community in the rumens of Chinese goats, and these were easily depressed by high-grain-diet feeding. The relatively low abundance suggests a small contribution on the part of Methanomassiliicoccales to the rumen methanogenesis of Chinese goats.

Ileum terminal antibiotic infusion affects jejunal and colonic specific microbial population and immune status in growing pigs

Background: Compared with oral antibiotics(primarily disrupt foregut microbiota), the present study used antibiotics with ileum terminal infusion to disrupt the hindgut microbiota, and investigated the changes in specific bacterial composition and immune indexes in the jejunum and colon, and serum of growing pigs. Twelve barrows(45 d of age, 12.08 ± 0.28 kg) fitted with a T-cannula at the terminal ileum, were randomly assigned to two groups and infused either saline without antibiotics(Control) or with antibiotics(Antibiotic) at the terminal ileum. After 25 d experiment, all pigs were euthanized for analyzing bacterial composition and immune status.Results: Ileum terminal antibiotic infusion(ITAI) altered dominant bacteria counts, with a decrease in Bifidobacterium, Clostridium cluster IV and Clostridium cluster IV in the colon(P < 0.05), and an increase in Escherichia coli in the jejunum(P < 0.05). ITAI decreased(P < 0.05) short-chain fatty acids concentrations in the colon. ITAI decreased interleukin-8(IL-8), IL-10 and secretory immunoglobulin A(sIgA) concentrations, and down-regulated IL-10, Mucin-1(MUC1), Mucin-2(MUC2) and zonula occludens-1(ZO-1) mRNA expression in the colonic mucosa(P < 0.05). In the jejunal mucosa, ITAI decreased interferon-γ(IFN-γ), tumor necrosis factor-α(TNF-α), s IgA and IgG levels together with down-regulation of IFN-γ, TNF-α, MUC2 and ZO-1 mRNA expression(P < 0.05). Furthermore, ITAI decreased IL-10, INF-γ, TNF-α, IgA and IgG concentrations in serum(P < 0.05). Correlation analysis revealed that the change in intestinal microbiota was correlated with alterations of Ig and cytokines.Conclusions: ITAI affected jejunal and colonic specific bacteria counts, and altered some immune markers levels in the jejunal and colonic mucosa and serum. These findings implicate the potential contribution of hindgut bacteria to immune response in the intestinal mucosa and serum of growing pigs.

Transcriptomic analysis reveals the molecular mechanisms of rumen wall morphological and functional development induced by different solid diet introduction in a lamb model

Background:This study aimed to elucidate the molecular mechanisms of solid diet introduction initiating the cellular growth and maturation of rumen tissues and characterize the shared and unique biological processes upon different solid diet regimes.Methods:Twenty-four Hu lambs were randomly allocated to three groups fed following diets:goat milk powder only(M,n=8),goat milk powder+alfalfa hay(MH,n=8),and goat milk powder+concentrate starter(MC,n=8).At 42 days of age,the lambs were slaughtered.Ruminal fluid sample was collected for analysis of concentration of volatile fatty acid(VFA)and microbial crude protein(MCP).The sample of the rumen wall from the ventral sac was collected for analysis of rumen papilla morphology and transcriptomics.Results:Compared with the M group,MH and MC group had a higher concentration of VFA,MCP,rumen weight,and rumen papilla area.The transcriptomic results of rumen wall showed that there were 312 shared differentially expressed genes(DEGs)between in“MH vs.M”and“MC vs.M”,and 232 or 796 unique DEGs observed in“MH vs.M”or“MC vs.M”,respectively.The shared DEGs were most enriched in VFA absorption and metabolism,such as peroxisome proliferator-activated receptor(PPAR)signaling pathway,butanoate metabolism,and synthesis and degradation of ketone bodies.Additionally,a weighted gene co-expression network analysis identified M16(2,052 genes)and M18(579 genes)modules were positively correlated with VFA and rumen wall morphology.The M16 module was mainly related to metabolism pathway,while the M18 module was mainly associated with signaling transport.Moreover,hay specifically depressed expression of genes involved in cytokine production,immune response,and immunocyte activation,and concentrate starter mainly altered nutrient transport and metabolism,especially ion transport,amino acid,and fatty acid metabolism.Conclusions:The energy production during VFA metabolism may drive the rumen wall development directly.The hay introduction facilitated establishment of immune function,while the concentrate starter enhanced nutrient transport and metabolism,which are important biological processes required for rumen development.

Characterization and application of monoclonal antibodies against Shewanella marisflavi, a novel pathogen of Apostichopus japonicus

Shewanella marisflavi strain AP629 was certified as a novel pathogen of the sea cucumber Apostichopusjaponicus. In this study, four monoclonal antibodies （MAbs） （3C1, 3D9, 2F2, 2A8） against strain AP629 were developed by immunizing Balb/C mice. 3C1 and 3D9 recognized S. marisflavi only, showing no cross reactivity to other gram-negative bacteria, However, 2F2 and 2A8 showed cross reactivity to all tested bacteria. Indirect immunofluorescence, and immunogold electron microscopy, showed the binding antigens of 3C1 and 3D9 were located at the secretion on the surface of strain AP629. The binding antigens of 2F2 and 2A8 were noted on the membrane of the cells. MAbs 3C1 and 3D9 recognized the lipopolysaccharide fraction of strain AP629, and 2F2 and 2A8 recognized in western-blotting protein antigens with molecular weights of 113 and 128 kDa respectively. MAbs 3C1 and 3D9 have the potential for use in pathogen diagnosis, epidemiology and studies on the mechanism of how S. marisflavi infects A. japonicus. Imrnunohistochemistry with 3C1 or 3D9 identified strain AP629 in the body wall of infected A. japonicus. In the adult sea cucumbers that were infected via body wall injection, positive signals were observed at the site of skin ulceration, and at the connective tissue of the non-ulcerated body wall. In addition, some large blue-stained cells aggregated at the connective tissue colonized by large numbers of bacteria. In juveniles infected via immersion infection, positive signals were observed at the cuticle of the body wall only. Our results suggest that 3C1 and 3D9 could be used in various immunological assays to study the invasion mechanism of strain AP629 in A. japonicus, the law of bacterial colonization, proliferation in different tissues of A. japonicus, and correlation between secretion on the surface of strain AP629 and its pathogenesis to A. japonicus.

Identification of Immunogenic Determinants of the Spike Protein of SARS-like Coronavirus

Bat SARS-Iike coronavirus （SL-CoV） has a genome organization almost identical to that of SARS-CoV, but the N-terminus of the Spike （S） proteins, which interacts with host receptor and is a major target of neutralizing antibodies against CoVs, of the two viruses has only 63-64% sequence identity. Although there have been reports studying the overall immunogenicity of SsL, knowledge on the precise location of immunodominant determinants for SSL is still lacking. In this study, using a series of truncated expressed SsL fragments and SsL specific mouse sera, we identified two immunogenic determinants for SSL. Importantly, one of the two regions seems to be located in a region not shared by known immunogenic determinants of the SSARS. This finding will be of potential use in future monitoring of SL-CoV infection in bats and spillover animals and in development of more effective vaccine to cover broad protection against this new group of coronaviruses.

The effect of increased atmospheric temperature and CO_2 concentration during crop growth on the chemical composition and in vitro rumen fermentation characteristics of wheat straw

This experiment was conducted to investigate the effects of increased atmospheric temperature and CO2 concentration during crop growth on the chemical composition and in vitro rumen fermentation characteristics of wheat straw. The field experiment was carried out from November 2012 to June 2013 at Changshu （31°32′93″N, 120°41′88″E） agro-ecological experimental station. A total of three treatments were set. The concentration of CO2 was increased to 500 pmol/mol in the first treatment （CO2 group）. The temperature was increased by 2℃ in the second treatment （TEM group） and the concentration of CO2 and temperature were both increased in the third treatment （CO2 ＋ TEM group）. The mean temperature and concentration of CO2 in control group were 10.5 ℃ and 413μmol/mol. At harvesting, the wheat straws were collected and analyzed for chemical composition and in vitro digestibility. Results showed that dry matter was significantly increased in all three treatments. Ether extracts and neutral detergent fiber were significantly increased in TEM and CO2 ＋ TEM groups. Crude protein was significantly decreased in CO2＋TEM group. In vitro digestibility analysis of wheat straw revealed that gas production was significantly decreased in CO2 and CO2 ＋ TEM groups. Methane production was significantly decreased in TEM and CO2 ＋ TEM groups. Ammonia nitrogen and microbial crude protein were significantly decreased in all three treatments. Total volatile fatty acids were significantly decreased in CO2 and CO2 ＋ TEM groups. In conclusion, the chemical composition of the wheat straw was affected by temperature and CO2 and the in vitro digestibility of wheat straw was reduced, especially in the combined treatment of temperature and CO2.

Reverse genetics: Unlocking the secrets of negative sense RNA viral pathogens

Negative-sense RNA viruses comprise several zoonotic pathogens that mutate rapidly and frequently emerge in people including Influenza, Ebola, Rabies, Hendra and Nipah viruses. Acute respiratory distress syndrome, encephalitis and vasculitis are common disease outcomes in people as a result of pathogenic viral infection, and are also associated with high case fatality rates. Viral spread from exposure sites to systemic tissues and organs is mediated by virulence factors, including viral attachment glycoproteins and accessory proteins, and their contribution to infection and disease have been delineated by reverse genetics; a molecular approach that enables researchers to experimentally produce recombinant and reassortant viruses from cloned cD NA. Through reverse genetics we have developed a deeper understanding of virulence factors key to disease causation thereby enabling development of targeted antiviral therapies and well-defined live attenuated vaccines. Despite the value of reverse genetics for virulence factor discovery, classical reverse genetic approaches may not provide sufficient resolution for characterization of heterogeneous viral populations, because current techniques recover clonal virus, representing a consensus sequence. In this review the contribution of reverse genetics to virulence factor characterization is outlined, while the limitation of the technique is discussed withreference to new technologies that may be utilized to improve reverse genetic approaches.

Dose and time response of ruminally infused algae on rumen fermentation characteristics,biohydrogenation and Butyrivibrio group bacteria in goats

Background： Micro-algae could inhibit the complete rumen BH of dietary 18-carbon unsaturated fatty acid （UFAs）. This study aimed to examine dose and time responses of algae supplementation on rumen fermentation, biohydrogenation and Butyrivibrio group bacteria in goats. Methods： Six goats were used in a repeated 3 x 3 Latin square design, and offered a fixed diet. Algae were infused through rumen cannule with 0 （Control）, 6.1 （L-AIg）, or 18.3 g （H-AIg） per day. Rumen contents were sampled on d 0, 3, 7, 14 and 20. Results： H-AIg reduced total volatile fatty acid concentration and acetate molar proportion （P 〈 0.05）, and increased propionate molar proportion （P 〈 0.05）, whereas L-AIg had no effect on rumen fermentation. Changes in proportions of acetate and propionate in H-AIg were obvious from d 7 onwards and reached the largest differences with the control on d 14. Algae induced a dose-dependent decrease in 18：0 and increased trons-18：1 in the ruminal content （P 〈 0.05）. H-AIg increased the concentrations of t9, t] 1-18：2 and tl 1, cl 5-18：2 （P 〈 0.05）. L-AIg only seemed to induce a transient change in 18-carbon isomers, while H-AIg induced a rapid elevation, already obvious on d 3, concentrations of these fatty acid rose in some cases again on d 20. Algae had no effect on the abundances of Butyrivibfio spp. and Butyrivibrio proteoclosdcus （P 〉 0.10）, while H-AIg reduced the total bacteria abundance （P 〈 0.05）. However, this was induced by a significant difference between control and H-AIg on d 14 （-4.43 %）. Afterwards, both treatments did not differ as increased variation in the H-AIg repetitions, with in some cases a return of the bacterial abundance to the basal level （d 0）. Conclusions： Changes in rumen fermentation and 18-carbon UFAs metabolism in response to algae were related to the supplementation level, but there was no evidence of shift in ruminal biohydrogenation pathways towards t1 0-18：1 L-AIg mainly induced a transient effect on rumen biohydrogenation of 18-carbon UFAs, while H-AIg showed an acute inhibition and these effects were not associated with the known hydrogenating bacteria.

First report on molecular characterization of Leishmania species from cutaneous leishmaniasis patients in southern Khyber Pakhtunkhwa province of Pakistan

Objective: To report presence of Leishmania major in Khyber Pakhtunkhwa of Pakistan, where cutaneous leishmaniasis(CL) is endemic and was thought to be caused by Leishmania tropica only. Methods: Biopsy samples from 432 CL suspected patients were collected from 3 southern districts of Khyber Pakhtunkhwa during years 2011–2016. Microscopy on Giemsa stained slides were done followed by amplification of the ribosomal internal transcribed spacer 1 gene. Results: Leishmania amastigotes were detected by microscopy in 308 of 432 samples(71.3%) while 374 out of 432 samples(86.6%) were positive by ribosomal internal transcribed spacer 1 PCR. Subsequent restriction fragment length polymorphism confirmed Leishmania tropica in 351 and Leishmania major in 6 biopsy samples. Conclusions: This study is the first molecular characterization of Leishmania species in southern Khyber Pakhtunkhwa. It confirmed the previous assumptions that anthroponotic CL is the major CL form present in Khyber Pakhtunkhwa province. Furthermore, this is the first report of Leishmania major from a classical anthroponotic CL endemic focus identified in rural areas of Kohat district in southern Khyber Pakhtunkhwa.

Application of High-Throughput Sequencing: Discovery of Informative SNPs to Subtype <i>Bacillus anthracis</i>

Single Nucleotide Polymorphisms (SNPs) are the most common and abundant genetic variation found in the genome of any living species, from bacteria to humans. In bacterial genotyping, these evolutionarily stable point mutations represent important DNA markers that can be used to elucidate deep phylogenetic relationships among worldwide strains, but also to discriminate closely related strains. With the advent of next generation sequencing (NGS) technologies, affordable solutions are now available to get access to the complete genome sequence of an organism. Sequencing efforts of an increasing number of strains provide an unprecedented opportunity to create comprehensive species phylogenies. In this study, a comparative analysis of 161 genomes of Bacillus anthracis has being conducted to discover new informative SNPs that further resolves B. anthracis SNP-based phylogenetic tree. Nine previously unpublished SNPs that define major groups or sub-groups within the B. anthracis species were selected and developed into SNP discriminative assays. We report here a cost-effective high-resolution melting-based genotyping method for the screening of 27 canonical SNPs that includes these new diagnostic markers. The present assays are useful to rapidly assign an isolate to one sub-lineages or sub-groups and determine its phylogenetic placement on the B. anthracis substructure population.

Peste des Petits Ruminants Vaccine (Nigerian Strain 75/1) Confers Protection for at Least 3 Years in Sheep and Goats

The present study reports the duration of immunity and protective efficacy of Peste des Petits Ruminants (PPR) vaccine (Nigerian strain 75/1) in sheep and goats. A total of 105 sheep and goats were divided into three groups A, B and C. Group A received normal recommended dose (1.0 ml) of PPR vaccine, group B received half dose (0.5 ml) of PPR vaccine and group C was kept as unvaccinated control group in contact with vaccinated animals. The post vaccination dynamics of antibodies against PPR virus was studied. It was found that significant antibody titres persisted for 3 years post vaccination in sheep and goats vaccinated with either full dose or half dose of PPR vaccine. The challenge protection studies were carried out in experimental animals at 24 and 36 month post vaccination. The vaccinates withstood challenge and were found completely resistant clinically and virologically to virulent PPR virus for 24 and 36 months post vaccination. The unvaccinated control animals developed typical clinical signs of PPR and the challenged virus was detected in ocular, nasal and oral secretions of these animals. This study demonstrated that a single immunization with PPR vaccine conferred solid protection in sheep and goats for 3 years.

Epidemiological Analysis of Peste des Petits Ruminants (PPR) Outbreaks in Pakistan

The current study reports the outbreaks of Peste des Petits Ruminants (PPR) in the small ruminant population of Pakistan. The objectives were to understand the clinical picture of disease under field conditions, estimate the basic epidemiological parameters for the local population of small ruminants and to determine the spatial and temporal distribution of PPR during 2005 to 2007 in Pakistan. A total of 62 outbreaks were investigated among sheep and goat flocks in the five provinces of Pakistan and Azad Jammu & Kashmir (AJK). The PPR virus activity in these outbreaks was demonstrated by clinical picture and presence of PPR virus specific antibodies by employing cELISA. The combined estimates of mean cumulative morbidity and mortality for sheep and goats were estimated 65.37% and 26.51% respectively with a case fatality of 40.40%. The species specific mean cumulative morbidity, mortality and case fatality for goats were 68.80%, 29.45% and 42.75% respectively, while these estimates for sheep were 48.77%, 14.98% and of 26.16% respectively. These estimates for goats were significantly higher (P < 0.001 to P = 0.001) than those for sheep. It was concluded that PPR is wide spread throughout the country and epidemiological picture suggest that disease has established as an endemic infection in the country.