The use of Microarray Technology to screen for immune related genes regulated during infection with Avibacterium paragallinarum serovar C-3
ABSTRACT: Avibacterium paragallinarum is the causative agent of Infectious Coryza, an acute upper respiratory tract disease in chickens. The occurrence of outbreaks has emphasized that the disease can be significant in broiler as well as layer chickens. In developing countries, infectious coryza is commonly complicated by a range of other infections, resulting in severe disease and significant economic losses. There are vaccines on the market but with limited efficiency, due to the serological differences amongst the different serogroups of A. paragallinarum. Recent advances in genomics have led to whole genome sequencing of the chicken, creating an opportunity for the use of high-throughput technology such as microarrays. The objectives of this study was to screen for gene expression patterns across clinical scores associated with the immune response in chickens infected with A. paragallinarum serovar C3, as well as to establish which biological pathways are stumulated when infected with A. paragallinarum. A total of 15 birds (male, leghorn) were obtained at 10 weeks of age. The birds were kept in separate layer cages up until 21 weeks, this is the susceptible age for infectious coryza. Twelve (12) birds were challenged via the infra-orbital eye area with 0.1ml challenge bacterium (A.paragallinarum serovar C3), Three were left unchallenged, which served as the control. As symptoms developed birds were scored, with score1 representing mild symptoms, score2, moderate and score3 representing severe symptoms. Each score is represented by two biological replicates. Thus a total of 8 samples were hybridized, two representing control.
Project description:Infectious bursal disease virus (IBDV) is a highly contagious dsRNA virus (Birnaviridae) which causes immuno-suppression in chickens. Although largely controlled by vaccination, new, virulent strains of the virus mean that infectious bursal disease (IBD or ëGumboroí disease) still remains a threat to the poultry industry. The virus infects dividing IgM+ B-lymphocytes and the main site of viral replication is the bursa of Fabricius where B cells are produced. Infection is spread orally via contaminated feed and water. IBDV affects young birds, with the disease usually being diagnosed in 3-6 week old birds. Younger birds do not show clinical signs but are immuno-suppressed. Symptoms include anorexia, depression, diahorrea, ruffled feathers, immuno-suppression and bursal lesions. The disease peaks between 2-5 days post infection and is practically cleared by day 7. Mortality is variable but can be up to around 70% with very virulent strains of the disease. Even if birds survive, the resulting immuno-suppression and effect on egg production in layer birds is significant. Being able to breed commercial lines of birds for enhanced genetic resistance to IBDV is an obvious goal in the fight against the disease. Three-week-old chicks were inoculated with virus via an intra-nasal route and tissue samples were collected at 2, 3 and 4 days post-inoculation. Bursa and spleen tissues were examined from control and infected birds at 2, 3 and 4 days post-infection in birds known to be either susceptible or resistant to the virus. As well as understanding the host immune response to IBDV, we are interested in identifying genes involved in disease resistance and so we have analysed the gene expression profiles at these times, when the innate immune response is active. We assume that genes underlying resistance will be involved at this early stage of the host immune response.
Project description:In March 2017, H7N9 highly pathogenic avian influenza (HPAI) virus was detected in 2 broiler breeder farms in the state of Tennessee, USA. Subsequent surveillance detected the low pathogenicity avian influenza (LPAI) virus precursor in multiple broiler breeder farms and backyard poultry in Tennessee and neighboring states. The pathogenesis of the H7N9 LPAI virus was investigated in commercial broiler breeders, the bird type mostly affected in this outbreak. Infectivity, transmissibility, and pathogenesis of the H7N9 HPAI and LPAI viruses were also studied in 4-week-old specific pathogen free (SPF) leghorn chickens. The mean bird infectious doses (BID50) for the LPAI isolate was 5.6 log10 mean egg infectious dose (EID50) for broiler breeders and 4.3 log10 EID50 for SPF layer chickens, and no transmission to contact-exposed birds was observed. In both bird types, virus shedding was almost exclusively from the oropharyngeal route. These findings suggest sub-optimal adaptation for sustained transmission with the H7N9 LPAI isolate, indicating that factors other than the birds genetic background may explain the epidemiology of the outbreak. The BID50 for the HPAI isolate in SPF layer chickens was more than 2 logs lower (<2 log10 EID50) than the LPAI isolate. Also, the HPAI virus was shed by both the oropharyngeal and cloacal routes and transmitted to contacts. Greater susceptibility and easier transmission of the H7N9 HPAI virus are features of the HP phenotype that could favor the spread of HPAI over LPAI viruses during outbreaks.
Project description:BACKGROUND:Poultry farming is widely practiced by rural households in Vietnam and the vast majority of domestic birds are kept on small household farms. However, smallholder poultry production is constrained by several issues such as infectious diseases, including avian influenza viruses whose circulation remains a threat to public health. This observational study describes the demographic structure and dynamics of small-scale poultry farms of the Mekong river delta region. METHOD:Fifty three farms were monitored over a 20-month period, with farm sizes, species, age, arrival/departure of poultry, and farm management practices recorded monthly. RESULTS:Median flock population sizes were 16 for chickens (IQR: 10-40), 32 for ducks (IQR: 18-101) and 11 for Muscovy ducks (IQR: 7-18); farm size distributions for the three species were heavily right-skewed. Muscovy ducks were kept for long periods and outdoors, while chickens and ducks were farmed indoors or in pens. Ducks had a markedly higher removal rate (broilers: 0.14/week; layer/breeders: 0.05/week) than chickens and Muscovy ducks (broilers: 0.07/week; layer/breeders: 0.01-0.02/week) and a higher degree of specialization resulting in a substantially shorter life span. The rate of mortality due to disease did not differ much among species, with birds being less likely to die from disease at older ages, but frequency of disease symptoms differed by species. Time series of disease-associated mortality were correlated with population size for Muscovy ducks (Kendall's coefficient τ = 0.49, p-value < 0.01) and with frequency of outdoor grazing for ducks (τ = 0.33, p-value = 0.05). CONCLUSION:The study highlights some challenges to disease control in small-scale multispecies poultry farms. The rate of interspecific contact and overlap between flocks of different ages is high, making small-scale farms a suitable environment for pathogens circulation. Muscovy ducks are farmed outdoors with little investment in biosecurity and few inter-farm movements. Ducks and chickens are more at-risk of introduction of pathogens through movements of birds from one farm to another. Ducks are farmed in large flocks with high turnover and, as a result, are more vulnerable to disease spread and require a higher vaccination coverage to maintain herd immunity.
Project description:Egyptian chickens, representing 2 breeds and 7 strains, were genotyped using the PCR-RFLP and sequencing techniques for detection of a non-synonymous dimorphism (G/A) in exon 14 of chicken Myxovirus resistance (Mx) gene. This dimorphic position is responsible for altering Mx protein's antiviral activity. Polymerase Chain reactions were performed using Egyptian chickens DNA and specific primer set to amplify Mx DNA fragments of 299 or 301 bp, containing the dimorphic position. Amplicons were cut with restriction enzyme Hpy81. Genotype and allele frequencies for the resistant allele A and sensitive allele G were calculated in all the tested chickens. Results of PCR-RFLP were confirmed by sequencing. The three genotypes AA, AG, GG at the target nucleotide position in Mx gene were represented in all the studied Egyptian chicken breeds and strains except Baladi strain which showed only one genotype AA. The average allele frequency of the resistant A allele in the tested birds (0.67) was higher than the sensitive G allele average frequency in the same birds (0.33). Appling PCR-RFLP technique in the breeding program can be used to select chickens carrying the A allele with high frequencies. This will help in improving poultry breeding in Egypt by producing infectious disease-resistant chickens.
Project description:Poultry meat and eggs contaminated with Salmonella enterica serovar Enteritidis or Salmonella enterica serovar Typhimurium are common sources of acute gastroenteritis in humans. However, the exact nature of the immune mechanisms protective against Salmonella infection in chickens has not been characterized at the molecular level. In the present study, bacterial colonization, development of pathological lesions, and proinflammatory cytokine and chemokine gene expression were investigated in the liver, spleen, jejunum, ileum, and cecal tonsils in newly hatched chickens 6, 12, 24, and 48 h after oral infection with Salmonella serovar Typhimurium. Very high bacterial counts were found in the ileum and cecal contents throughout the experiment, whereas Salmonella started to appear in the liver only from 24 h postinfection. Large numbers of heterophils, equivalent to neutrophils in mammals, and inflammatory edema could be seen in the lamina propria of the intestinal villi and in the liver. Interleukin 8 (IL-8), K60 (a CXC chemokine), macrophage inflammatory protein 1 beta, and IL-1 beta levels were significantly upregulated in the intestinal tissues and in the livers of the infected birds. However, the spleens of the infected birds show little or no change in the expression levels of these cytokines and chemokines. Increased expression of the proinflammatory cytokines and chemokines (up to several hundred-fold) correlated with the presence of inflammatory signs in those tissues. This is the first description of in vivo expression of chemokines and proinflammatory cytokines in response to oral infection with Salmonella in newly hatched chickens.
Project description:Vaccination of chickens has become routine practice in Asian countries in which H5N1 highly pathogenic avian influenza (HPAI) is endemically present. This mainly applies to layer and breeder flocks, but broilers are usually left unvaccinated. Here we investigate whether vaccination is able to reduce HPAI H5N1 virus transmission among broiler chickens. Four sets of experiments were carried out, each consisting of 22 replicate trials containing a pair of birds. Experiments 1-3 were carried out with four-week-old birds that were unvaccinated, and vaccinated at day 1 or at day 10 of age. Experiment 4 was carried out with unvaccinated day-old broiler chicks. One chicken in each trial was inoculated with H5N1 HPAI virus. One chicken in each trial was inoculated with virus. The course of the infection chain was monitored by serological analysis, and by virus isolation performed on tracheal and cloacal swabs. The analyses were based on a stochastic SEIR model using a Bayesian inferential framework. When inoculation was carried out at the 28th day of life, transmission was efficient in unvaccinated birds, and in birds vaccinated at first or tenth day of life. In these experiments estimates of the latent period (~1.0 day), infectious period (~3.3 days), and transmission rate parameter (~1.4 per day) were similar, as were estimates of the reproduction number (~4) and generation interval (~1.4 day). Transmission was significantly less efficient in unvaccinated chickens when inoculation was carried out on the first day of life. These results show that vaccination of broiler chickens does not reduce transmission, and suggest that this may be due to the interference of maternal immunity.
Project description:Many livestock and human vaccines are leaky because they block symptoms but do not prevent infection or onward transmission. This leakiness is concerning because it increases vaccination coverage required to prevent disease spread and can promote evolution of increased pathogen virulence. Despite leakiness, vaccination may reduce pathogen load, affecting disease transmission dynamics. However, the impacts on post-transmission disease development and infectiousness in contact individuals are unknown. Here, we use transmission experiments involving Marek disease virus (MDV) in chickens to show that vaccination with a leaky vaccine substantially reduces viral load in both vaccinated individuals and unvaccinated contact individuals they infect. Consequently, contact birds are less likely to develop disease symptoms or die, show less severe symptoms, and shed less infectious virus themselves, when infected by vaccinated birds. These results highlight that even partial vaccination with a leaky vaccine can have unforeseen positive consequences in controlling the spread and symptoms of disease.
Project description:Infectious bronchitis virus (IBV) is one of the foremost causes of a persistent economic burden to poultry industries worldwide. IBV belongs to the genus Gammacoronavirus within the family Coronaviridae. The IBV infection leads to respiratory and nephrogenic symptoms in broiler chickens. In addition, its infection leads to reduced fertility and hatchability in layer birds. We determined the first complete genome sequence of a variant IBV from an outbreak in Haryana state of the Northern part of India using next generation sequencing. On phylogenetic analysis of the IBV isolate, it clustered with genotype I lineage 1 (GI-1). The deduced amino acid sequence of S gene of IBV isolates showed a high level of identity with strains isolated from Tamil Nadu and the reference vaccine strains. Our result suggests that the IBV virus isolated from unvaccinated chicken flocks in North India might be a revertant strain originally evolved from the live attenuated vaccine strains used in the region. Determination of the complete genome sequence of additional IBV isolates from India is necessary to understand the epidemiology of IBV in India.
Project description:Salmonella enterica serovar Enteritidis is a major foodborne pathogen in the United States, causing gastroenteritis in humans, primarily through consumption of contaminated eggs. Chickens are the reservoir host of S. Enteritidis. In layer hens, S. Enteritidis colonizes the intestine and migrates to various organs, including the oviduct, leading to egg contamination. This study investigated the efficacy of in-feed supplementation with trans-cinnamaldehyde (TC), a generally recognized as safe (GRAS) plant compound obtained from cinnamon, in reducing S. Enteritidis cecal colonization and systemic spread in layers. Additionally, the effect of TC on S. Enteritidis virulence factors critical for macrophage survival and oviduct colonization was investigated in vitro. The consumer acceptability of eggs was also determined by a triangle test. Supplementation of TC in feed for 66 days at 1 or 1.5% (vol/wt) for 40- or 25-week-old layer chickens decreased the amounts of S. Enteritidis on eggshell and in yolk (P<0.001). Additionally, S. Enteritidis persistence in the cecum, liver, and oviduct in TC-supplemented birds was decreased compared to that in controls (P<0.001). No significant differences in feed intake, body weight, or egg production in birds or in consumer acceptability of eggs were observed (P>0.05). In vitro cell culture assays revealed that TC reduced S. Enteritidis adhesion to and invasion of primary chicken oviduct epithelial cells and reduced S. Enteritidis survival in chicken macrophages (P<0.001). Follow-up gene expression analysis using real-time quantitative PCR (qPCR) showed that TC downregulated the expression of S. Enteritidis virulence genes critical for chicken oviduct colonization (P<0.001). The results suggest that TC may potentially be used as a feed additive to reduce egg-borne transmission of S. Enteritidis.
Project description:Stocking densities for domestic chickens (Gallus gallus domesticus) are regulated by the Council Directives of the European Union for both laying hens and broiler chickens. For layer pullets no regulation of stocking density has been established yet. Based on the existing Council Directives for laying hens (1999/74/EC), broiler chickens (2007/43/EC) and calculations of the floor space that is required for the respective chicken's body, we exemplarily calculated maximum stocking densities for layer pullets. Based on the calculations we obtained absolute additional spaces for birds of different live body mass classes, i.e., useable floor space that the birds have additionally available to the space covered by their body. This allowed us to calculate the relative additional space per individual. We suggest the relative additional space to be a key parameter to derive requirements for a maximum stocking density in layer pullets. We analysed several scenarios for pullets under consideration of the Council Directives for laying hens and for broiler chickens, coming to the conclusion that layer pullets at the end of their rearing period should be provided ideally with a relative additional space of about 40-60%.