Comparative pathogenicity of Malaysian variant and very virulent infectious bursal disease viruses in chickens.

Variant infectious bursal disease virus (vaIBDV) has been identified in various countries with significant economic losses. Recently, the first identification of a variant strain in Malaysia was reported. The pathogenicities of the Malaysian variant, UPM1432/2019, and very virulent infectious bursal disease virus (vvIBDV), UPM1056/2018 strains were comparatively evaluated in specific-pathogen-free (SPF) chickens based on gross and histopathological examinations and viral load. Four-week-old SPF chickens were randomly divided into three groups; group 1 served as the control, while groups 2 and 3 birds were challenged with the vaIBDV and vvIBDV, respectively. Three birds from each group were weighed, euthanized and necropsied at 2, 3, 4, 5, 7 and 21 days post-challenge (dpc). Unlike UPM1056/2018 group, birds from UPM1432/2019 group did not show clinical signs or death. UPM1056/2018 strain caused 11% mortality rate in the infected chickens. The bursal body index (BBIX) for UPM1432/2019- and UPM1056/2018-infected groups was <0.7 from 2 dpc and continued to decrease to 0.49 and 0.45, respectively, at 21 dpc. UPM1432/2019 strain was more persistent in the bursa than UPM1056/2018 strain. Both strains induced similar pathological lesions in SPF chicks. These results indicate that the Malaysian vaIBDV severely damaged the immune organs of chickens and was more persistent in bursal tissue than vvIBDV. The study provides insight into the pathogenicity of the variant strain as further study may be required to evaluate the efficacy of the currently available IBD vaccines in Malaysia against the strain. RESEARCH HIGHLIGHTSEmerging Malaysian variant IBDV caused severe bursal damage without mortality.Atypical vvIBDV induced bursal atrophy with inflammatory response and caused low mortality.Malaysian variant IBDV was more persistent in bursal tissue than vvIBDV.

Infectious bursal disease virus tissue tropism and pathogenesis of the infection in chickens by application of in situ PCR, immunoperoxase and HE staining.

Infectious bursal disease is one of an OIE list of notifiable diseases. Chicken is the only host that manifests clinical signs and its pathogenicity is correlated with the distribution of antigens in organs. This study was conducted to determine disease pathogenesis and virus tissue tropism by in situ PCR, immunoperoxidase staining (IPS), and HE staining. Twenty four chickens were infected with very virulent Infectious Bursal Disease Virus (vvIBDV). Fifteen chickens were kept as a control group. Infected chickens were sacrificed at hrs 2, 4, 6, 12, days 1, 2, 4, and 6 post-inoculation (pi). While, control chickens were euthanized on days 0, 1, 2, 4, and 6 pi. Different tissues were collected, fixed in 10% buffered formalin, and processed. At hr 2 pi, virus was detected in intestinal, junction of the proventriculus and gizzard, cecal tonsil, liver, kidney, and bursa of Fabricius. At hr 4 pi, virus reached spleen, and at hr 6 pi, it entered thymus. At hr 12 pi, virus concentration increased in positive tissues. The latest invaded tissue was muscle on day 1 pi. Secondary viraemia occurred during 12-24 h pi. In situ PCR was the most sensitive technique to highlight obscure points of infection in this study.

Velogenic newcastle disease virus tissue tropism and pathogenesis of infection in chickens by application of in situ PCR, immunoperoxase staining and HE staining.

Limited deep studies are available in the field of early stages of pathogenesis of Newcastle disease virus (NDV) infection and tissue tropism of NDV. In this study, 24 specific pathogen free (SPF) chickens of white leghorn breed were infected with Newcastle disease (ND) by intranasal administration of 105 50% EID50/0.1 mL of velogenic NDV (vNDV). A second group of 15 chickens were kept as a control group. Chickens were monitored every day to record clinical signs. Infected chickens were euthanized by cervical dislocation at successive times, namely at hours (hrs) 2, 4, 6, 12, days 1, 2, 4, and 6 post-inoculation (pi). Whereas, control group chickens were euthanized on days 0, 1, 2, 4, and 6 pi. Tissues of brain, trachea, lung, caecal tonsil, liver, kidney, spleen, heart, proventriculus, intestine, and thymus were collected, fixed in 10% buffered formalin, embedded in paraffin, and sectioned. HS staining, immunoperoxidase staining (IPS) and in situ PCR were applied. It was concluded that at hr 2 pi, virus seemed to be inclined to trachea and respiratory tract. Meanwhile, it attacked caecal tonsils, intestine and bursa of Fabricus. While primary viraemia was ongoing, virus created footing in kidney and thymus. At hr 4 pi, proventriculus, liver, and spleen were attacked. However, at hr 6 pi, brain and heart were involved. Secondary viraemia probably started as early as hr 12 pi since all collected tissues were positive. Tissue tropism was determined in trachea, caecal tonsil, liver, bursa of Fabricius, intestine, proventriculus, lung, spleen, thymus, kidney, heart, and brain.

In vitro characterization of chicken bone marrow-derived dendritic cells following infection with very virulent infectious bursal disease virus.

Infectious bursal disease is caused by infectious bursal disease virus (IBDV), an immunosuppressive virus that targets immune cells such as B cells and macrophages. However, the involvement of dendritic cells (DCs) during IBDV infection is not well understood. In this study the in vitro effects of live and inactivated very virulent IBDV (vvIBDV) UPM0081 on bone marrow-derived DCs (BM-DC) were characterized and compared with BM-DC treated with lipopolysaccharide (LPS). Morphologically, BM-DC treated with LPS and vvIBDV showed stellate shape when compared to immature BM-DC. In addition, LPS-treated and both live and inactivated vvIBDV-infected BM-DC expressed high levels of double positive CD86 and major histocompatibility complex class II antigens (>20%). vvIBDV-infected BM-DC showed significantly higher numbers of apoptotic cells compared to LPS. Replication of vvIBDV was detected in the infected BM-DC as evidenced by the increased expression of VP3 and VP4 IBDV antigens based on flow cytometry, real-time polymerase chain reaction and immunofluorescence tests. Levels of different immune-related genes such as interleukin-1ß (IL-1ß), CXCLi2 (IL-8), IL-18, interferon gamma (IFN-γ, IL-12α, CCR7 and Toll-like receptor-3 (TLR3) were measured after LPS and vvIBDV treatments. However, marked differences were noticed in the onset and intensity of the gene expression between these two treatment groups. LPS was far more potent than live and inactivated vvIBDV in inducing the expression of IL-1ß, IL-18 and CCR7 while expression of Th1-like cytokines, IFN-γ and IL-12α were significantly increased in the live vvIBDV treatment group. Meanwhile, the expression of TLR3 was increased in live vvIBDV-infected BM-DC as compared to control. Inactivated vvIBDV-treated BM-DC failed to stimulate IFN-γ, IL-12α and TLR3 expressions. This study suggested that BM-DC may serve as another target cells during IBDV infection which require further confirmation via in vivo studies.

Detection and molecular characterization of infectious spleen and kidney necrosis virus from major ornamental fish breeding states in Peninsular Malaysia.

'Gold standard' OIE reference PCR assay was utilized to detect the presence of infectious spleen and kidney necrosis virus (ISKNV) in freshwater ornamental fish from Malaysia. From total of 210 ornamental fish samples representing 14 species, ISKNV was detected in 36 samples representing 5 fish species. All positive cases did not show any clinical signs of ISKNV. Three restriction enzymes analyses showed that the fish were infected by identical strains of the same virus species within Megalocytivirus genus. Major capsid protein (MCP) genes of 10 ISKNV strains were sequenced and compared with 9 other reference nucleotide sequences acquired from GenBank. Sequence analysis of MCP gene showed that all strains detected in this study were closely related to the reference ISKNV with nucleotide sequence identity that was ranging from 99.8% to 100%. In addition, phylogenetic analysis of MCP gene revealed that viruses from genus Megalocytivirus can be divided into three genotypes: genotype 1 include reference ISKNV and all other strains that were detected in this study, genotype 2 include viruses closely related to red sea bream iridovirus (RSIV), and genotype 3 include viruses closely related turbot reddish body iridovirus (TRBIV).

The effects of polymorphisms in 7 candidate genes on resistance to Salmonella Enteritidis in native chickens.

Salmonella enterica serovar Enteritidis infection is a common concern in poultry production for its negative effects on growth as well as food safety for humans. Identification of molecular markers that are linked to resistance to Salmonella Enteritidis may lead to appropriate solutions to control Salmonella infection in chickens. This study investigated the association of candidate genes with resistance to Salmonella Enteritidis in young chickens. Two native breeds of Malaysian chickens, namely, Village Chickens and Red Junglefowl, were evaluated for bacterial colonization after Salmonella Enteritidis inoculation. Seven candidate genes were selected on the basis of their physiological role in immune response, as determined by prior studies in other genetic lines: natural resistance-associated protein 1 (NRAMP1), transforming growth factor ß3 (TGFß3), transforming growth factor ß4 (TGFß4), inhibitor of apoptosis protein 1 (IAP1), caspase 1 (CASP1), lipopolysaccharide-induced tumor necrosis factor (TNF) α factor (LITAF), and TNF-related apoptosis-inducing ligand (TRAIL). Polymerase chain reaction-RFLP was used to identify polymorphisms in the candidate genes; all genes exhibited polymorphisms in at least one breed. The NRAMP1-SacI polymorphism correlated with the differences in Salmonella Enteritidis load in the cecum (P = 0.002) and spleen (P = 0.01) of Village Chickens. Polymorphisms in the restriction sites of TGFß3-BsrI, TGFß4-MboII, and TRAIL-StyI were associated with Salmonella Enteritidis burden in the cecum, spleen, and liver of Village Chickens and Red Junglefowl (P < 0.05). These results indicate that the NRAMP1, TGFß3, TGFß4, and TRAIL genes are potential candidates for use in selection programs for increasing genetic resistance against Salmonella Enteritidis in native Malaysian chickens.

Changes in heat shock protein 70, blood parameters, and fear-related behavior in broiler chickens as affected by pleasant and unpleasant human contact.

An experiment was conducted to determine the effects of combining both pleasant and unpleasant contacts with human beings on physiology and behavior of broiler chickens. Birds were subjected to the following treatments: (i) received no physical or visual contact with humans (control); (ii) from d 1 to 28, chicks were individually stroked gently for 30 s once daily (PL); (iii) from d 1 to 28, chicks were picked up individually, suspended by both legs, exposed to recorded noise, and swung gently for 15 s once daily (UNPL); (iv) from d 1 to 14 and from d 15 to 28, chicks were subjected to PL and UNPL, respectively (PL-UNPL); and (v) from d 1 to 14 and from d 15 to 28, chicks were subjected to UNPL and PL, respectively (UNPL-PL). On d 42, birds from each treatment group were road-transported for 3 h. Heat shock protein (hsp) 70 expression, plasma levels of corticosterone, serum creatine kinase concentration, heterophil/lymphocyte ratios (HLR), and tonic immobility duration were determined pre- and posttransit. There were significant (P < 0.05) duration of transportation × human contact treatment interactions for HLR and hsp 70 density. Following transit, the PL chicks had significantly (P < 0.05) lower HLR and greater hsp 70 density than the other groups. The corticosterone of PL and UNPL chicks were lower than their control, PL-UNPL, and UNPL-PL counterparts. The PL and PL-UNPL treatments were effective in shortening tonic immobility duration significantly (P < 0.05). Except for UNPL-PL, the serum creatine kinase activity of PL was significantly lower than the other groups. In conclusion, subjecting birds to pleasant human contact reduced stress and fear reactions to transportation by enhancing the ability to express hsp 70 in the brain. Unpleasant human contact had adverse effect on the birds' response to transportation. Early age pleasant experience with humans failed to negate the adverse effects of subsequent unpleasant contact.

Epigenetic modification: possible approach to reduce Salmonella enterica serovar enteritidis susceptibility under stress conditions.

Stressors may influence chicken susceptibility to pathogens such as Salmonella enterica. Feed withdrawal stress can cause changes in normal intestinal epithelial structure and may lead to increased attachment and colonization of Salmonella. This study aimed to investigate modulatory effects of epigenetic modification by feed restriction on S. enterica serovar Enteritidis colonization in broiler chickens subjected to feed withdrawal stress. Chicks were divided into four groups: ad libitum feeding; ad libitum feeding with 24-h feed withdrawal on day 42; 60% feed restriction on days 4, 5, and 6; and 60% feed restriction on days 4, 5, and 6 with 24-h feed withdrawal on day 42. Attachment of S. Enteritidis to ileal tissue was determined using an ex vivo ileal loop assay, and heat shock protein 70 (Hsp70) expression was evaluated using sodium dodecyl sulphate-polyacrylamide gel electrophoresis and western blotting. Feed withdrawal stress increased S. Enteritidis attachment to ileal tissue. However, following feed withdrawal the epigenetically modified chickens had significantly lower attachment of S. Enteritidis than their control counterparts. A similar trend with a very positive correlation was observed for Hsp70 expression. It appears that epigenetic modification can enhance resistance to S. Enteritidis colonization later in life in chickens under stress conditions. The underlying mechanism could be associated with the lower Hsp70 expression in the epigenetically modified chickens.

The role of heat shock protein 70 in resistance to Salmonella enteritidis in broiler chickens subjected to neonatal feed restriction and thermal stress.

Environmental stressors may influence chicken performance and susceptibility to pathogens, such as Salmonella enteritidis. This study was conducted to determine the effects of heat shock protein (Hsp)70 expression on resistance to Salmonella enteritidis infection in broiler chickens subjected to heat exposure. Chicks were divided into 3 feeding regimens: ad libitum feeding (control); 60% feed restriction on d 4, 5, and 6 (FR60); and 60% feed restriction on d 4, 5, and 6 plus 1,500 mg/kg of quercetin (FR60Q). On d 35, all of the chickens were individually inoculated with 1 mL of Salmonella enteritidis (1.5 × 10(8) cfu/bird) and exposed to an ambient temperature of 37 ± 1°C and 70% RH for 3 h/d. The FR60 and FR60Q chickens had significantly lower Salmonella enteritidis colonization and lower Hsp70 expression than that of the control chickens following the heat exposure period. The least colonization was observed in the FR60Q group (1.38 log(10) cfu/g in the spleen and 1.96 log(10) cfu/g in the cecal content) and the highest was in the control group (2.1 log(10) cfu/g in the spleen and 4.42 log(10) cfu/g in the cecal content). It appears that neonatal feed restriction can enhance resistance to Salmonella enteritidis colonization in heat-stressed broiler chicks, and the underlying mechanism could be associated with the lower expression of Hsp70.

Dietary methionine and n-6/n-3 polyunsaturated fatty acid ratio reduce adverse effects of infectious bursal disease in broilers.

This study was carried out to investigate the modulatory effects of dietary methionine and n-6/n-3 polyunsaturated fatty acids (PUFA) ratio on immune response and performance of infectious bursal disease (IBD)-challenged broiler chickens. In total, 350 one-day-old male broiler chicks were assigned to 1 of the 6 dietary treatment groups in a 3 × 2 factorial arrangement. There were 3 n-6/n-3 PUFA ratios (45, 5.5, and 1.5) and 2 levels of methionine (NRC recommendation and twice NRC recommendation). The results showed that birds fed with dietary n-6/n-3 PUFA ratio of 5.5 had higher BW, lower feed intake, and superior FCR than other groups. However, the highest antibody response was observed in birds with dietary n-6/n-3 PUFA ratio of 1.5. Lowering n-6/n-3 PUFA ratio reduced bursa lesion score equally in birds fed with n-6/n-3 PUFA ratio of 5.5 and 1.5. Supplementation of methionine by twice the recommendation also improved FCR and reduced feed intake and bursa lesion score. However, in this study, the optimum performance (as measured by BW, feed intake, and FCR) did not coincide with the optimum immune response (as measured by antibody titer). It seems that dietary n-3 PUFA modulates the broiler chicken performance and immune response in a dose-dependent but nonlinear manner. Therefore, it can be suggested that a balance of moderate level of dietary n-6/n-3 PUFA ratio (5.5) and methionine level (twice recommendation) might enhance immune response together with performance in IBD-challenged broiler chickens.

Egg production, faecal pH and microbial population, small intestine morphology, and plasma and yolk cholesterol in laying hens given liquid metabolites produced by Lactobacillus plantarum strains.

1. Various dosages of metabolite combinations of the Lactobacillus plantarum RI11, RG14 and RG11 strains (COM456) were used to study the egg production, faecal microflora population, faecal pH, small intestine morphology, and plasma and egg yolk cholesterol in laying hens. 2. A total of 500 Lohmann Brown hens were raised from 19 weeks to 31 weeks of age. The birds were randomly divided into 5 groups and fed on various treatment diets: (i) basal diet without supplementation of metabolites (control); (ii) basal diet supplemented with 0·3% COM456 metabolites; (iii) basal diet supplemented with 0·6% COM456 metabolites; (iv) basal diet supplemented with 0·9% COM456 metabolites; and (v) basal diet supplemented with 1·2% COM456 metabolites. 3. The inclusion of 0·6% liquid metabolite combinations, produced from three L. plantarum strains, demonstrated the best effect in improving the hens' egg production, faecal lactic acid bacteria population, and small intestine villus height, and reducing faecal pH and Enterobacteriaceae population, and plasma and yolk cholesterol concentrations. 4. The metabolites from locally isolated L. plantarum are a possible alternative feed additive in poultry production.

Cloning and expression of highly pathogenic avian influenza virus full-length nonstructural gene in Pichia pastoris.

Avian influenza (AI) is a highly contagious and rapidly evolving pathogen of major concern to the poultry industry and human health. Rapid and accurate detection of avian influenza virus is a necessary tool for control of outbreaks and surveillance. The AI virus A/Chicken/Malaysia/5858/2004 (H5N1) was used as a template to produce DNA clones of the full-length NS1 genes via reverse transcriptase synthesis of cDNA by PCR amplification of the NS1 region. Products were cloned into pCR2.0 TOPO TA plasmid and subsequently subcloned into pPICZαA vector to construct a recombinant plasmid. Recombinant plasmid designated as pPICZαA-NS1 gene was confirmed by PCR colony screening, restriction enzyme digestion, and nucleotide sequence analysis. The recombinant plasmid was transformed into Pichia pastoris GS115 strain by electroporation, and expressed protein was identified by SDS-PAGE and western blotting. A recombinant protein of approximately ~28 kDa was produced. The expressed protein was able to bind a rabbit polyclonal antibody of nonstructural protein (NS1) avian influenza virus H5N1. The result of the western blotting and solid-phase ELISA assay using H5N1 antibody indicated that the recombinant protein produced retained its antigenicity. This further indicates that Pichia pastoris could be an efficient expression system for a avian influenza virus nonstructural (NS1).

Nutrition and immunity: the effects of the combination of arginine and tryptophan on growth performance, serum parameters and immune response in broiler chickens challenged with infectious bursal disease vaccine.

To explore the effects of the combination of tryptophan (Trp) and arginine (Arg) on growth performance, serum parameters and immune response of broiler chickens challenged with intermediate plus strain of infectious bursal disease virus vaccine, an in vivo experiment was conducted. A corn-soybean meal-based diet containing different levels of Arg and Trp was used. Cobb500 male broiler chickens from 0 to 49 days of age were subjected to a diet supplemented with the combination of Trp and Arg. Growth performance parameters and serum parameters were measured at 27 and 49 days of age. To evaluate the immunomodulatory effects of the combination of Trp and Arg on the challenged chickens, we measured the serum levels of interferon-α, interferon-γ and immunoglobulin G at 27, 35, 42, and 49 days of age. The results showed that the three evaluated immune system parameters including interferon-α, interferon-γ and immunoglobulin G were significantly enhanced after treatment. This enhancement resulted in the recovery of infectious bursal disease virus-infected chickens compared with controls as confirmed by histopathological examinations. Moreover, serum parameters such as albumin and total protein increased, whereas the treatment decreased (P<0.05) the feed:gain ratio, aspartate amino-transferase, alkaline phosphatase, lactic dehydrogenase, triglyceride and cholesterol. These findings suggest that the combination of Arg and Trp has a regulatory effect on growth performance. Moreover, it modulates the systemic immune response against infectious bursal disease.

Inactivation of Different Salmonella enteriditis Phage Types and Safety and Efficacy of Inactivated Products in Chicken.

This study was conducted to inactivate Salmonella enteriditis phage types (SE pt) and to determine the safety and efficacy of inactivated SE pt in chickens. SE pt 1, 3A, 6A, 7, and 35 were inactivated and inoculated (0.20 mL) in 124 chickens divided into 6 groups (CV1, CV3A, CV6A, CV7, CV35, and CV0 as a control). Sampling was conducted on day 14 after inoculation (pi). Eight chickens from each group were separated on day 14 pi for oral challenge with 0.20 mL/chicken (1010 cfu/mL) SE pt 6A and designated CV1C, CV3AC, CV6AC, CV7C, CV35C, and CV0C as control chickens. On days 7 and 14 postchallenge (pc), 4 chickens from every group were sacrificed for sampling. There was no significant difference in the body weight between different groups. In challenged groups, there was no significant association between different tissues and isolation of Salmonella on days 7 and 14 pc. There was significance (p < 0.05) in isolation of Salmonella when CV0C group was compared with other challenged groups. Significance was not observed between different tissues with respect to induction of microscopic changes. Significance was not observed between day 7 pc and day 14 pc with respect to scoring of lesions induced. Clinical signs and gross lesions were also recorded. ELISA was applied. Only in CV3AC group, the mean antibody titer was 1359 on day 14 pc. The conclusion was that inactivated SE pt 3A and 6A were safe and efficacious for protection against Salmonella enteriditis infection in chickens.

Efficacy of a bacteriophage isolated from chickens as a therapeutic agent for colibacillosis in broiler chickens.

The efficacy of bacteriophage EC1, a lytic bacteriophage, against Escherichia coli O78:K80, which causes colibacillosis in poultry, was determined in the present study. A total of 480 one-day-old birds were randomly assigned to 4 treatments groups, each with 4 pens of 30 birds. Birds from the control groups (groups I and II) received PBS (pH 7.4) or 10(10) pfu of bacteriophage EC1, respectively. Group III consisted of birds challenged with 10(8) cfu of E. coli O78:K80 and treated with 10(10) pfu of bacteriophage EC1 at 2 h postinfection, whereas birds from group IV were challenged with 10(8) cfu of E. coli O78:K80 only. All the materials were introduced into the birds by intratracheal inoculation. Based on the results of the present study, the infection was found to be less severe in the treated E. coli-challenged group. Mean total viable cell counts of E. coli identified on eosin methylene blue agar (designated EMB + E. coli) in the lungs were significantly lower in treated, E. coli-challenged birds than in untreated, E. coli-challenged birds on d 1 and 2 postinfection. The EMB + E. coli isolation frequency was also lower in treated birds; no E. coli was detectable in blood samples on any sampling day, and E. coli were isolated only in the liver, heart, and spleen of treated chickens at a ratio of 2/6, 1/6, and 3/6, respectively, at d 1 postinfection. The BW of birds from the E. coli-challenged group treated with bacteriophage EC1 were not significantly different from those of birds from both control groups but were 15.4% higher than those of the untreated, E. coli-challenged group on d 21 postinfection. The total mortality rate of birds during the 3-wk experimental period decreased from 83.3% in the untreated, E. coli-challenged birds (group IV) to 13.3% in birds treated with bacteriophage EC1 (group III). These results suggest that bacteriophage EC1 is effective in vivo and could be used to treat colibacillosis in chickens.

Selected microbial groups and short-chain fatty acids profile in a simulated chicken cecum supplemented with two strains of Lactobacillus.

Among the bacterial fermentation end products in the chicken cecum, butyrate is of particular importance because of its nutritional properties for the epithelial cell and pathogen inhibitory effects in the gut. An in vitro experiment, operated with batch bioreactor, was conducted to quantify butyric-producing bacteria in a simulated broiler cecum supplemented with Lactobacillus salivarius ssp. salicinius JCM 1230 and Lactobacillus agilis JCM 1048 during 24 h of incubation. Selected bacterial species were determined by real-time PCR and short-chain fatty acids and lactate concentrations were monitored. The results showed that after 24 h of incubation, Lactobacillus supplementation significantly increased the number of lactobacilli, bifidobacteria and Faecalibacterium prausnitzii in medium containing cecal content and lactobacilli supplementation (Cc + L) compared with the control (Cc). Addition of lactobacilli did not alter Escherichia coli and Clostridium butyricum, whereas it significantly (P < 0.05) reduced Salmonella in treatment Cc + L compared with the Cc treatment. Propionate and butyrate formation were significantly (P < 0.05) increased in treatment Cc + L as compared with the Cc treatment. Lactate was only detected in treatment containing 2 Lactobacillus strains. After 24 h of incubation, acetate concentration significantly (P < 0.05) decreased in all treatments. It was suggested that lactate produced by Lactobacillus in the cecal content improved the growth of butyric producers such as F. prausnitzii, which significantly increased butyrate accumulation. Additionally, the results showed that butyrate and propionate inhibited Salmonella without influencing the E. coli profile.

Gastrointestinal tract morphological alteration by unpleasant physical treatment and modulating role of Lactobacillus in broilers.

1. An experiment was conducted to determine the effects of supposedly unpleasant physical treatment on broiler performance, small intestinal development and ameliorating role of probiotics. 2. The following treatments were applied from day one: (1) chicks exposed to normal human contact fed basal diet (control); (2) chicks were exposed to unpleasant physical treatment and fed basal diet (UPT-BD); and (3) chicks were exposed to unpleasant physical treatment and fed basal diet supplemented with Lactobacillus (UPT-BDL). Chicks were exposed to UPT from days 1 to 21. Different segments of gastrointestinal tract were sampled at 14, 28, 35 and 42 d of age. 3. Broilers of UPT-BD had lower feed consumption compared with control group at 7 d of age. Overall, UPT-BDL birds showed higher body weight gain (BWG) and better feed conversion ratio (FCR) over the course of the experiment. 4. Birds of UPT-BD had lower concentrations of lactic, propionic and butyric acids in the caecum as compared with other groups at 14 d of age. Acetic acid concentration was profoundly decreased in both UPT groups compared to the control. 5. Duodenal villus height of UPT-BD broilers showed a slight reduction compared to the control and UPT-BDL birds at 14 d of age. Afterwards until day 42, UPT-BDL birds showed the highest villus height among treatments in different parts of the small intestine. 6. The results suggested that, even though UPT did not have significant inhibitory effects on the development of the small intestine and broiler performance, it negatively affected bacterial metabolic end products in the caecum, which could be ameliorated by the addition of Lactobacillus.

In vitro fermentation of broiler cecal content: the role of lactobacilli and pH value on the composition of microbiota and end products fermentation.

AIM: To assess the probiotic effects of Lactobacillus agilis JCM 1048 and L. salivarius ssp. salicinius JCM 1230 and the pH on the cecal microflora of chicken and metabolic end products. METHODS AND RESULTS: An in vitro system, operated with batch bioreactor, was used for this assessment. Selected bacterial species were monitored at two pH values, over 24 h of batch culture incubation. The concentration of short chain fatty acids (SCFA) and lactate in the fermented material was also determined. The addition of L. agilis JCM 1048 and L. salivarius ssp. salicinius JCM 1230 into vessel 2 (Cc + P) increased the total anaerobes, lactobacilli and bifidobacteria after 24 h incubation. Moreover, lactobacilli supplementation decreased the total aerobes and streptococci, but it did not have any effects on coliforms. The supplementation of lactobacilli in vessel 2 (Cc + P) was found to significantly increase the production of lactate, propionate and butyrate. Furthermore, pH did not alter the formation of butyrate, whereas the production of acetate and propionate was significantly decreased at pH = 5.8. CONCLUSIONS: L. agilis JCM 1048 and L. salivarius ssp. salicinius JCM 1230, as probiotic bacteria, have the ability to re-establish proper microbial balance by the formation of lactate as well as propionate, and stimulate butyrate-producing bacteria to produce butyrate in the chicken cecum. SIGNIFICANCE AND IMPACT OF THE STUDY: This study was the first to report this under in vitro conditions, highlighting the probiotic roles of the two Lactobacillus strains in broiler cecal fermentation at different initial pH. These useful data can be helpful in improving the fermentation process in chicken cecum.

Effects of feeding metabolite combinations produced by Lactobacillus plantarum on growth performance, faecal microbial population, small intestine villus height and faecal volatile fatty acids in broilers.

1. Four combinations of metabolites produced from strains of Lactobacillus plantarum were used to study the performance of broiler chickens. 2. A total of 432 male Ross broilers were raised from one-day-old to 42 d of age in deep litter pens (12 birds/pen). These birds were divided into 6 groups and fed on different diets: (i) standard maize-soybean-based diet (negative control); (ii) standard maize-soybean-based diet + Neomycin and Oxytetracycline (positive control); (iii) standard maize-soybean-based diet + 0.3% metabolite combination of Lactobacillus plantarum RS5, RI11, RG14 and RG11 strains (com3456); (iv) standard maize-soybean-based diet + 0.3% metabolite combination of L. plantarum TL1, RI11 and RG11 (Com246); (v) standard maize-soybean-based diet + 0.3% metabolite combination of L. plantarum TL1, RG14 and RG11 (Com256) and (vi) standard maize-soybean-based diet + 0.3% metabolite combination of L. plantarum TL1, RS5, RG14 and RG11 (Com2356). 3. Higher final body weight, weight gain, average daily gain and lower feed conversion ratio were found in all 4 treated groups. 4. The addition of a metabolite combination supplementation also increased faecal lactic acid bacteria population, small intestine villus height and faecal volatile fatty acids and faecal Enterobacteriaceae population.

Comparison of Sybr Green I, ELISA and conventional agarose gel-based PCR in the detection of infectious bursal disease virus.

The current available molecular method to detect infectious bursal disease virus (IBDV) is by reverse transcriptase-polymerase chain reaction (RT-PCR). However, the conventional PCR is time consuming, prone to error and less sensitive. In this study, the performances of Sybr Green I real-time PCR, enzyme-linked immunosorbent assay (ELISA) and conventional agarose detection methods in detecting specific IBDV PCR products were compared. We found the real-time PCR was at least 10 times more sensitive than ELISA detection method with a detection limit of 0.25pg. The latter was also at least 10 times more sensitive than agarose gel electrophoresis detection method. The developed assay detects both very virulent and vaccine strains of IBDV but not other RNA viruses such as Newcastle disease virus and infectious bronchitis virus. Hence, Sybr Green I-based real-time PCR is a highly sensitive assay for the detection of IBDV.