Pathogenicity of a Malaysian infectious bronchitis virus isolate in specific pathogen free chickens

Infectious bronchitis (IB) is one of very important diseases in chicken. It is caused by IB virus (IBV) from the Coronaviridae family. This disease is a worldwide, acute and highly contagious disease. The virus easily changes in nature and emerges as a new strain and causes problems. In Malaysia, the nephrogenic strain was detected in 1995 and the new variant QX strain was reported recently in 2009. These 2 strains caused high mortality and loss of production. Hence, the objectives of this study were to isolate and identify IBV from chickens during field outbreaks of the disease in 2009 and determine the clinical signs, gross and histological lesions of specific pathogen free (SPF) chicken infected with the IBV isolate. Samples of lungs, kidneys and trachea from suspected IB birds were tested by reverse transcriptase polymerase chain reaction (RT-PCR) assay and then inoculated to 10-day old SPF embryonated chicken eggs via allantoic route for virus isolation, propagation and preparation of the IBV inoculum. The allantoic fluid and chorioallantoic membranes (CAM) from the inoculated eggs were then tested for the presence of IBV before inoculated into SPF chicken. Seventy-two day old SPF chicken were divided into 3 groups known as Groups A, B and C. Groups A and B were inoculated with IBV inocula 0.1 mL via intranasal route while group C remained as uninoculated and served as control group. Four chickens were sacrificed prior to IBV inoculation. At days 1, 3, 5, 7, 14 and 21 post-inoculation (pi), four chickens from group A and C were sacrificed. The bodyweight was noted prior to sacrifice. On necropsy, gross lesions were recorded and samples of trachea and kidneys were collected and fixed in 10% buffered formalin for histological examination. Mortality and any abnormal clinical signs were recorded at least twice a day. Feed and drink were given ad libitum. The results showed that IBV was successfully detected from the trachea, lung and kidney samples of the chickens by RT-PCR. Infectious Bronchitis Virus was successfully isolated and propagated in SPF embryonated chicken eggs and detected in the allantoic fluids and CAM. The pathogenicity study showed that the IBV isolate caused severe respiratory signs and high mortality (60%) up to day 14 pi. Mild to severe gross and histological lesions were recorded in the trachea and kidneys up to day 14 pi. However, signs of recovery with mild respiratory signs, absence of mortality and mild kidney lesions were recorded at day 21 pi. It was concluded that the IBV isolate is highly pathogenic and might be a new or variant strain of IBV

Analysis of genetic variation of inducible nitric oxide synthase and natural resistance-associated macrophage protein 1 loci in Malaysian native chickens

The genetic diversity of 100 Malaysian native chickens was investigated using polymerase chain reaction-restriction fragment polymorphism (PCR-RFLP) for two candidate genes: inducible nitric oxide synthase (INOS) and natural resistance-associated macrophage protein 1 (NRAMP1). The two genes were selected because of their important role in chicken’s immune system. INOS and NRAMP1 PCR products were digested by AluI and SacI restriction enzymes, respectively. The restriction digests produced fragment sizes of 322 and 173 bp for INOS and 722 and 79 bp for NRAMP1 as one allele and an undigested PCR product as the other allele. Both loci were polymorph, however only INOS gene showed Hardy-Weinberg equilibrium. Average heterozygosity and the Shannon information index (I) was 0.43 and 0.62 for INOS and 0.48 and 0.68 for NRAMP1 genes, respectively. The observed polymorphism in this study shows the ability of these candidate genes in marker assisted selection and introgression programs to increase resistance to diseases in both Malaysian native and commercial chickens.Key words: Malaysian native chickens, polymorphism, inducible nitric oxide synthase (INOS), natural resistance-associated macrophage protein 1 (NRAMP1)

Sequence analysis and expression of VP3 gene of chicken anaemia virus

Chicken anaemia virus (CAV) isolate BL-5 VP3 gene was amplified, sequenced and expressed in E. coli as a fusion protein together with a six-histidine tag. The deduced amino acid sequence of VP3 was identical to other CAV isolates Cux-1, A2 and CIA-1 except for a single amino acid substitution at position 12. SDS-PAGE and Western blot analysis indicated that the expressed protein is insoluble and was found primarily from the cell lysate fraction. Expression of the protein was detected as early as 1 h, with maximum expression (~12% of the total protein) at 6 h post induction with IPTG. This study indicates that VP3 protein is highly expressed in insoluble form in E. coli. However, the biological function of the protein remains to be studie

Molecular characterization of fowl adenoviruses isolated from inclusion body hepatitis outbreaks in commercial broiler chickens in Malaysia

Fowl adenoviruses (FAdVs), belonging to the Aviadenovirus genus of the family Adenoviridae, have been classified into five species (A to E) and further divided into 12 serotypes. The objective of this study was to identify the serotype classification of five Malaysian FAdV isolates obtained from field outbreaks of IBH in commercial broiler chickens. Hexon-based polymerase chain reactions (PCR), combined with restriction enzyme analysis (REA), were applied. Viral DNA reacted positively with H1/H2 and H3/ H4 primer pairs which hybridised to highly conserved regions of the hexon genes. The restriction enzyme profiles of the H1/H2 fragment digested with HaeII and the H3/H4 fragment digested with HpaII revealed that all five isolates shared identical patterns and are characterised as being FAdV-8b, species E. Meanwhile, sequence analysis of the L1 loop region of the hexon gene revealed 98.1% identity with FAdV-8b strain 764. High bootstrap values in phylogenetic analysis supported the clustering of the Malaysian FAdV isolates into FAdV species E. The present study has provided a very useful reference for further studies of FAdVs in Malaysia. Vaccination strategies should be developed against FAdVs infection in commercial broiler chickens to prevent IBH outbreaks in the country

Effects of putrescine supplementation on growth performance, blood lipids and immune response in broiler chickens fed methionine deficient diet

The polyamines including putrescine (PUT), spemidine (SPD) and spermine (SPM) have been shown to play an important role in basic cellular processes. Methionine (Met) and arginine are required for polyamine biosynthesis. This experiment was carried out to study the effects of Met deficient diet with or without supplemental PUT on the performance, blood lipids and glucose and humoral immunity in broiler chickens. A total of 192 day-old chicks were allocated into factorial arrangement (2 × 2) of four dietary treatments including two Met levels (Normal and Low) and two PUT levels (0 and 0.03%). Body weight and feed intake were measured weekly. At the age of 28 d, 10 birds per treatment were challenged with infectious bursal disease vaccine orally (10 dosage/bird, 2 ml). Blood samples of the challenged birds were collected for antibody determination and white blood cell count. Blood cholesterol, triglyceride, glucose, hematocrit and protein of non-challenged birds were measured at the ages of 24, 33 and 40 d. Low Met decreased body weight gain, protein efficiency ratio and feed conversion ratio (P<0.05). In grower period, energy efficiency ratio was improved by PUT supplementation in the chicks fed with normal Met level. Feed conversion ratio was badly affected (P<0.05) by low Met during grower period. PUT decreased body weight gain of chickens fed low Met significantly (P<0.05), indicating the importance of adequate Met level when high dietary PUT is offered. Met deficient diet decreased plasma protein level significantly (P<0.05). Five days post challenged, PUT supplementation elevated antibody level significantly (P<0.05) in the chicks fed normal level of Met. However, Met deficient diet in challenged chicks, caused significantly (P<0.05) lower monocyte ratio. In conclusion, growth performance declined due to Met deficiency, particularly during starter period (0–21 d) and when dietary PUT was supplemented. PUT showed the potential to increase blood antibody level and better dietary energy efficiency ratio

In vitro fermentation of broiler cecal content : the role of oactobacilli 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. sali-varius 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. Further- more, pH did not alter the formation of butyrate, whereas the production of acetate and propionate was significantly decreased at pH = 5Æ 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

Characterization of chicken splenic-derived dendritic cells following vaccine and very virulent strains of infectious bursal disease virus infection

Studies have shown that infectious bursal disease virus (IBDV) infects lymphoid cells, mainly B cells and macrophages. This study was aimed to examine the involvement of chicken splenic-derived dendritic cells (ch-sDCs) in specific-pathogen-free chickens following inoculation with IBDV vaccine strain (D78) and a very virulent (vv) strain (UPM0081). Following IBDV infection, enriched activated ch-sDCs were collected by using the negative selection method and were examined based on morphology and immunophenotyping to confirm the isolation method for dendritic cells (DCs). The presence of IBDV on enriched activated ch-sDCs was analyzed based on the immunofluorescence antibody test (IFAT), flow cytometry, and quantitative real-time PCR (RT-qPCR) while the mRNAs of several cytokines were detected using RT-qPCR. The isolated ch-sDCs resembled typical DC morphologies found in mammals by having a veiled shape and they grew in clusters. Meanwhile, the expression of DC maturation markers, namely CD86 and MHCII, were increased at day 2 and day 3 following vvIBDV and vaccine strain inoculation, respectively, ranging from 10% to 40% compared to the control at 2.55% (P < 0.05). At day 3 postinfection, IBDV VP3 proteins colocalized with CD86 were readily detected via IFAT and flow cytometry in both vaccine and vvIBDV strains. In addition, enriched activated ch-sDCs were also detected as positive based on the VP4 gene by RT-qPCR; however, a higher viral load was detected on vvIBDV compared to the vaccine group. Infection with vaccine and vvIBDV strains induced the enriched activated ch-sDCs to produce proinflammatory cytokines and Th1-like cytokines from day 3 onward; however, the expressions were higher in the vvIBDV group (P < 0.05). These data collectively suggest that enriched activated ch-sDCs were permissive to IBDV infection and produced a strong inflammatory and Th1-like cytokine response following vvIBDV infection as compared to the vaccine strain