Infectious Coryza Research Articles

Transcriptional profiling of chicken immunity-related genes during infection with Avibacterium paragallinarum

Avibacterium paragallinarum is the causative agent of Infectious Coryza (IC), which is an upper respiratory tract disease in chickens. The occurrence of outbreaks has emphasized the significance of the disease globally in the chicken industry. Studies have demonstrated that early immune responses are critical in defining the severity and physiological outcome of an infection. This prompted the need to investigate the regulation of immune functions by the number of genes that are expressed during the chickens’ response to A. paragallinarum serovar C3 insult.This study consisted of 15 male leghorn birds that were scored into groups (score 1, 2, 3) according to severity of symptoms after they were challenged.Expression patterns of immunity-related genes were followed as symptoms progressed from a disease score of 1 to 3. The data proposed that initial pathogen recognition was either through Toll-like receptors 2 or 4. Unique expression patterns were observed such as the up-regulation of TLR7 which recognizes viral-like particles. This substantiated the presence of prophages reported in the genome of A. paragallinarum. Significant down-regulation of metabolic pathways was observed, which led us to hypothesize that the host may rely on an oxidative stress response as initial immune response.The data sheds light onto the mechanisms that govern the immune system towards infection and/or towards the initial response to infections with highly virulent A. paragallinarum.

Isolation and molecular identification of Avibacterium paragallinarum in suspected cases of infectious coryza

Isolation and identification of Avibacterium paragallinarum, the causative agent of infectious coryza, is considered a challenging task in laboratories with limited specialties. In the present study, 14 commercial layer fowls showing the typical symptoms of infectious coryza were subjected to primary isolation followed by polymerase chain reaction confirmation of suspect colonies (culture-PCR). Direct PCR assays on infraorbital sinus swab samples were also carried out. Thirty-five suspected cases of infectious coryza in commercial broiler chickens were also screened using direct PCR on infraorbital sinus swabs. In culture-PCR, only 1 of the 4 suspected isolates was confirmed as Av. paragallinarum. In comparison, in direct PCR, 5 layer samples were shown to be positive for Av. paragallinarum. All of the broiler samples were negative in the direct PCR assay. Our findings indicate that primary isolation in combination with PCR can be a simple method for diagnosis of infectious coryza, although with a lower sensitivity than direct PCR. While direct PCR is comparably the more rapid and sensitive test, there will be instances in which the bacterial isolate is needed for further use. Hence, the culture-PCR method can be a practical and simple approach, especially in laboratories with limited specialty in identification of this fastidious organism.

Identification and functional analysis of the cytolethal distending toxin gene from Avibacterium paragallinarum

Avibacterium paragallinarum is the causative agent of infectious coryza, an important respiratory disease of chickens. Cytolethal distending toxins (CDTs) are a family of protein cytotoxins that cause cell cycle arrest and apoptosis in eukaryotic cells. Whole-genome sequencing analysis showed that Av. paragallinarum contains cdtABC genes. Filter-sterilized lysates prepared from Av. paragallinarum or from recombinant Escherichia coli expressing cdtABC genes exhibited CDT activity on HeLa cells and chicken embryo fibroblast (DF-1) cells. In vitro DNase assays showed that purified recombinant CdtB has DNase activity. Polymerase chain reaction and sequencing analysis revealed that the cdtABC genes are present in all strains of Av. paragallinarum examined in this study. This is the first report of the identification and functional analysis of cdtABC genes from Av. paragallinarum. The gene products of cdtABC genes may be involved in the pathogenesis of the disease caused by Av. paragallinarum.

Draft Genome Sequence of the Virulent Avibacterium paragallinarum Serotype A Strain JF4211 and Identification of Two Toxins

Avibacterium paragallinarum is an important pathogen of chicken livestock causing infectious coryza. Here, we report the draft genome sequence of the virulent A. paragallinarum serotype A strain JF4211 (2.8 Mbp and G+C content of 41%) and the two toxin operons discovered from the annotation of the genome.

Investigation on infectious coryza of layer chicken in Bangladesh with isolation, identification and antibiogram study.

Among infectious diseases, infectious coryza is one of the major problems affecting commercial poultry industry in the developing country like Bangladesh. The present study was conducted for isolation, identification and characterization of Haemophilus paragallinarum from layer chicken in Bangladesh. A total of 122 samples (Nasal / tracheal swab, visceral organs like liver, lung, heart)) were collected from from Rangpur (Paragon Poultry Farm ), Takhurgaon (North Egro Poultry Farm) and Dinajpur (Nizam Poultry Farm) districts of Bangladesh during the period from March 2011 to February, 2012 . The samples were collected from suspected birds based on age, sex, breed, temporal and spatial differences for the isolation and identification of Haemophilus paragallinarum by morphology, staining, cultural and biochemical properties. A total of 122 samples were screened by epidemiological study, of which the overall prevalence of Haemophilus paragallinarum was detected as 47.54 %. The prevalence was very high in laying hen (52.8%) and growing birds (42.8) in compare with the prelaying stage (16.6%). The prevalence of Haemophilus paragallinarum in Dinajpur, Rangpur and Thakurgaon were found 86.67%, 25%, and 34.21% respectively. The isolates were resistant to norfloxacin and tylosin. It was evident that amoxycillin and gentamicin can be of better value in the treatment of infectious coryza in layer chickens in Bangladesh.

Pathogenesis of Infectious Coryza in Chickens (Gallus gallus) by Avibacterium paragallinarumn Isolate of Bangladesh

An investigation was conducted to isolate and identify the causal agent of infectious coryza (IC) with pathogenesis study by local isolate of Avibacterium paragallinarum in chicks in Bangladesh. One isolate of A. paragallinarum was used to study the experimental pathogenesis. For this, 14 days old 24 chicks were grouped into two (A and B) and each group contained 12 birds. Chicks of group A were inoculated with 1ml of 2 days old nutrient broth and were kept as control group while group B were inoculated with 1 ml of 2 days old culture broth of A. paragallinarum. To study the pathology, 4 birds from each group were sacrificed on day 3, 5 and 7 of post inoculation. Sacrificed birds of group A did not reveal any clinical sign and lesion. Chicks of group B showed mild nasal discharge, conjunctivitis, depression and inability to move. The gross lesions of the chicks of group B included mucous in nasal passage, conjunctivitis, swelling of sinuses and face and congested lungs. The microscopic lesions of the chicks of this group were acanthosis and congested blood vessels of nasal passage, pneumonic lesion of lung, focal hepatitis of liver and fatty change and lipid nodules in macrophages of heart which were progressively prominent on day 7 of bacterial inoculation. A. paragallinarum was reisolated from day 7 of post inoculation (PI) from nasal passage of chicks in which lesions were prominent. The proposed experimental pathogenesis was after intranasal inoculation with A. paragallinarum, rhinitis developed, bacteria entered into blood, reached different organs producing lesions. The lesions which are discussed here (rhinitis in association with focal hepatitis, fatty change in heart with lipid granuloma, progressive pneumonic lesions) are not usually present in adult and young birds. DOI: http://dx.doi.org/10.3329/agric.v11i1.15240 The Agriculturists 2013; 11(1) 39-46

Genome sequence and comparative analysis of Avibacterium paragallinarum

Background: Avibacterium paragallinarum, the causative agent of infectious coryza, is a highly contagious respiratory acute disease of poultry, which affects commercial chickens, laying hens and broilers worldwide. Methodology: In this study, we performed the whole genome sequencing, assembly and annotation of a Peruvian isolate of A. paragallinarum. Genome was sequenced in a 454 GS FLX Titanium system. De novo assembly was performed and annotation was completed with GS De Novo Assembler 2.6 using the H. influenzae str. F3031 gene model. Manual curation of the genome was performed with Artemis. Putative function of genes was predicted with Blast2GO. Virulence factors were identified by comparison with the Virulence Factor Database. Results: The genome obtained has a length of 2.47 Mb with 40.66% of GC content. Seventy five large contigs (>500 nt) were obtained, which comprised 1,204 predicted genes. All the contigs are available in Genbank [GenBank: PRJNA64665]. A total of 103 virulence factors, reported in the Virulence Factor Database, were found in A. paragallinarum. Forty four of them are present in 7 species of Haemophilus, which are related with pathogenesis, virulence and host immune system evasion. A tetracycline-resistance associated transposon (Tn10), was found in A. paragallinarum, possibly acting as a defense mechanism. Discussion and conclusion: The availability of A. paragallinarum genome represents an important source of information for the development of diagnostic tests, genotyping, and novel antigens for potential vaccines against infectious coryza. Identification of virulence factors contributes to better understanding the pathogenesis, and planning efforts for prevention and control of the disease.

Draft Genome Sequence of Avibacterium paragallinarum Strain 221

Avibacterium paragallinarum is the causative agent of infectious coryza. Here we report the draft genome sequence of reference strain 221 of A. paragallinarum serovar A. The genome is composed of 135 contigs for 2,685,568 bp with a 41% G+C content.

Listeria monocytogenes Infection in Poultry and its Public Health Importance with Special Reference to Food Borne Zoonoses

Listeriosis is a disease that causes septicemia or encephalitis in humans, animals and birds. Although, the disease is rare and sporadic in poultry but if occurs then causes septicemia or sometimes localized encephalitis. Occasionally, the disease is seen in young chicks and the causative agent, like in humans and animals, is Listeria monocytogenes. The organism is capable to infect almost all animals and poultry; however, outbreaks of listeriosis are infrequent in birds. It is widely distributed among avian species and chickens, turkeys, waterfowl (geese, ducks), game birds, pigeons, parrots, wood grouse, snowy owl, eagle, canaries, which appear to be the most commonly affected. Chickens are thought to be the carriers of Listeria and also the prime reservoirs for the infection and thus contaminate the litter and environment of the poultry production units. Listeriosis is often noticed along with other poultry diseases such as coccidiosis, infectious coryza, salmonellosis, campylobacteriosis and parasitic infections, signifying the opportunistic nature of the organism. Intestinal colonization of poultry and the presence of L. monocytogenes in feces represent a potential source of the organism for listeriosis in ruminants. Man gets infection from raw broiler meat due to Listeria contamination and unhygienic conditions of the processing area, rather than acquiring direct infection from birds. With the changing food habits of the people, the health consciousness is also increasing and since listeriosis has now been recognized as an emerging food borne zoonoses. Therefore, this review has been compiled to make aware the poultry producers and the consumers of poultry meat/products regarding the importance of the disease and its public health significance.

Safety and efficacy studies on trivalent inactivated vaccines against infectious coryza

The safety and efficacy of an inactivated oil-emulsion infectious coryza vaccine containing three Avibacterium paragallinarum isolates (one each of Page serovars A, B, and C) was evaluated. The safety of six batches of the vaccine was confirmed by testing with chickens vaccinated with a single large dose or vaccinated repeatedly with a normal dose. Efficacy tests were carried out on three batches of vaccine using both specific pathogen free (SPF) chickens and conventional chickens. In SPF chickens given a single vaccination at 42 days of age, the protection rate against all three serovars of Av. paragallinarum was at least 80% at 30 days post vaccination. The conventional chickens, which were immunized at 42 and 110 days of age, were challenged at 9 months post the second vaccination and the protection rate was at least 80% for all three serovars. The effect of storage on the vaccine was evaluated in SPF chickens using three batches of vaccine stored at 4–8°C for 1 year. The protection rate against challenge from all three serovars (single vaccination at 42 days of age and challenge at 30 days post-vaccination) was at least 80%.

AvxA, a composite serine-protease-RTX toxin of Avibacterium paragallinarum

Avibacterium paragallinarum, the etiological agent of infectious coryza in chicken, was found to encode a bivalent serine-protease - RTX-porin toxin named AvxA. This toxin is encoded on a classical RTX operon structure with the activator gene avxC, the structural serin-protease-RTX toxin gene avxA, and the genes for a proper type I secretion system avxBD. AvxA is activated by the product of the avxC gene, secreted by the avxBD specified type I secretion system and proteolytically processed leaving a 95 kDa RTX moiety that is found in culture supernatants of A. paragallinarum serovars A, B and C. The RTX moiety of AvxA (AvxA-RTX) is cytotoxic against the avian macrophage like cell line HD11 but not against bovine macrophage cell line BoMac. Purified IgG from hyper-immune rabbit anti-AvxA-RTX serum made by immunization with recombinant AvxA-RTX from a serotype A strain fully neutralizes the cytotoxic activity of recombinant active AvxA-RTX and of A. paragallinarum serotypes A, B and C. This indicates that AvxA is a common major virulence attribute of all A. paragallinarum serotypes.

Identification of Prophages and Prophage Remnants within the Genome of Avibacterium paragallinarum Bacterium

Bacterial whole genome sequencing has delivered an abundance of prophage sequences as a by-product and the analysis of these sequences revealed ways in which phages have affected the genome of their host bacteria in various bacterial species. The aim of this study was to identify the phage-related sequences in the draft assembly of the Avibacterium paragallinarum genome, the causative agent of infectious coryza in poultry. Whole genome assembly was not possible due to the presence of gaps and/or repeats existent on the ends of contigs. However, genome annotation revealed prophage and prophage remnant sequences present in this genome. From the results obtained, a complete Mu-like bacteriophage could be identified that was termed AvpmuC-2M. A complete sequence of HP2-like bacteriophage, named AvpC-2M-HP2, was also identified.

Development of a recombinant vaccine against infectious coryza in chickens

Infectious coryza is an acute respiratory disease of chickens caused by Avibacterium paragallinarum, and this infection is associated with growth retardation and reduced egg production. Previous studies have shown that HMTp210, a 210-kDa outer-membrane protein, is the major protective antigen of Av. paragallinarum both serovars A and C. Region 2 is a serovar-specific domain in the HMTp210 protein. Although the serovar C region 2 has been reported to be an effective vaccine antigen for infectious coryza, there have been no reports on the efficacy of region 2 from serovar A. In the current study, region 2 from serovars A and C was expressed as a fusion peptide. Chickens inoculated with vaccine consisting of 0.6μg of the fusion peptide showed no clinical signs of disease after challenge with either serovar A or C, and there were no side effects such as swelling at the injection site. These results demonstrate that the recombinant fusion peptide derived from HMTp210 could be useful for producing effective and safe vaccines against infectious coryza in chickens.

Identification and Characterization of an RTX Toxin–Like Gene and Its Operon from Avibacterium paragallinarum

Avibacterium paragallinarum is the causative agent of infectious coryza, an important respiratory disease of chickens. Whole-genome sequencing analysis showed that A. paragallinarum strain H18 contains an RTX toxin-like operon with strong similarity to the RTX operons of other members of the Pasteurellaceae. Four genes, designated avxIC, avxIA, avxIB, and avxID, were found in this operon. The avxIA gene encodes the structural RTX toxin-like protein, which has a predicted molecular mass of about 250 kDa. The AvxIA protein contains a peptidase S8 domain and a proprotein convertase P-domain, neither of which has been found in other RTX toxins. Recombinant AvxIA proteins expressed in Escherichia coli showed neither hemolytic nor cytotoxic activity. Polymerase chain reaction and sequencing analysis revealed that the avxIA gene was present in all strains and field isolates of A. paragallinarum examined in this study. Sera collected from chickens exposed to A. paragallinarum exhibited strong reactivity to the AvxIA protein, which suggests that AvxIA is immunogenic. This is the first report of the identification of an RTX toxin-like operon from A. paragallinarum. The gene products of this operon may be related to disease pathogenesis and potentially represent a useful vaccine target of A. paragallinarum.

Serovar Identification, Antimicrobial Sensitivity, and Virulence of Avibacterium paragallinarum Isolated from Chickens in Thailand

Avibacterium paragallinarum causes infectious coryza in chickens, an acute respiratory disease that has worldwide economic significance. The objectives of this study were to determine the serovars, antimicrobial resistance, and pathogenicity of A. paragallinarum isolated from chickens in Thailand. Eighteen field isolates of A. paragallinarum were confirmed by PCR. When examined by serotyping in a hemagglutination inhibition test, 10 isolates were serovar A, five isolates were serovar B, and three isolates were serovar C. The susceptibility of the isolates to 16 antimicrobial agents was tested by a disk diffusion method. All isolates were susceptible to amoxicillin-clavulanic acid. There was a high level of resistance to lincomycin and erythromycin. All isolates were resistant to cloxacillin and neomycin. A study of bacterial entry into, and survival within, chicken macrophages showed variation between isolates but no clear connection to serovar. A virulence test was performed by challenging 4-wk-old layers via the nasal route with 400 dl of bacteria (10(8) colony-forming units/ml). Clinical signs were observed daily for 7 days, and the birds were subjected to a postmortem necropsy at 7 days postchallenge. All 18 field isolates caused the typical clinical signs of infectious coryza and could be re-isolated at 7 days after challenge. There was no significant difference in the clinical scores of the isolates except that two isolates (112179 and 102984, serovars A and B, respectively) gave a significantly higher score than did isolate CMU1009 (a serovar A isolate). No correlation between serovar and severity of clinical signs was found.

Development of an Enzyme-Linked Immunosorbent Assay for the Measurement of Antibodies Against Infectious Coryza Vaccine

Infectious coryza is an acute respiratory disease caused by infection with Avibacterium (Haemophilus) paragallinarum. It is characterized by nasal discharge and facial swelling and is associated with growth retardation and a reduction in egg production. Hemagglutination inhibition (HI) tests are used to estimate vaccine-induced immunity against infectious coryza in vitro; however, these procedures are complicated and their sensitivity is insufficient. To address these problems, an enzyme-linked immunosorbent assay (ELISA) technique using serovar-specific regions of HMTp210 (210 kDa), an outer-membrane protein of A. paragallinarum, was developed to measure the antibodies against infectious coryza. Chickens with an ELISA titer of 0.3 or more did not exhibit clinical signs of infectious coryza against challenge with A. paragallinarum, although their HI antibody titers were negative. On the other hand, chickens with an ELISA titer below 0.3 exhibited clinical signs of the disease with one exception. Antibody prevalence rates on ELISA were 80% and 60% against infection with serovars A and C, respectively, and ELISA also detected antibodies in chickens infected with A. paragallinarum with a sensitivity higher than that of HI tests. Taken together, the ELISA technique developed in this study is a valuable tool for the measurement of antibodies produced against the infectious coryza vaccine or in response to an infection with A. paragallinarum.

Development of an Enzyme-Linked Immunosorbent Assay for the Measurement of Antibodies Against Infectious Coryza Vaccine

Development of an Enzyme-Linked Immunosorbent Assay for the Measurement of Antibodies Against Infectious Coryza Vaccine

Development of a Multiplex PCR and PCR-RFLP Method for Serotyping of Avibacterium paragallinarum

Avibacterium (Haemophilus) paragallinarum (A. paragallinarum) is a causative agent of infectious coryza in chickens and is classified into three serovars by agglutination tests. In an effort to identify the serovars easily, PCR and PCR-RFLP were employed. As the target gene for PCR, the hypervariable region of HMTp210, which encodes the HA antigen, was used. PCR using primer sets around the hypervariable region amplified 0.8, 1.1 and 1.6 kbp fragments for serovars A, B and C, respectively. Alternatively, the 1.6 kbp fragments were amplified with another primer pair encompassing the hypervariable region and was subjected to digestion with Bgl II, which resulted in the detection of serovar-specific digestion patterns. These results indicate that PCR and PCR-RFLP using the hypervariable region of HMTp210 are alternative methods to identify the serovar of A. paragallinarum.

ERIC-PCR Genotyping of Emergent Serovar C-1 Isolates of Avibacterium paragallinarum from Mexico

Between 2008 and 2010, 14 isolates of Avibacterium paragallinarum were identified as serovar C-1 in Mexico. All isolates were obtained from commercial laying hens suffering infectious coryza despite a history of vaccination. The enterobacterial repetitive intergenic consensus-based PCR genotyping showed that all isolates had a common pattern. Until recently, serovars A-1, A-2, B-1, and C-2 were the serovars prevalent in Mexico. Serovar C-1 has been identified in Japan and recently in the Americas in Ecuador. Our current study suggests that Av. paragallinarum serovar C-1 is an emerging serovar in Mexico. Our results also indicate that the Mexican isolates of Av. paragallinarum serovar C-1 may have a clonal relationship. Knowledge of the genetic diversity of Av. paragallinarum may be of value in understanding vaccine performance and identifying the best combination to achieve broader protection.

Demonstration of antibiotic resistance genes strA, blaTEM, tetA, tetC and sul2 in Avibacterium paragallinarum

Avibacterium paragallinarum causes a highly contagious disease in chickens called infectious coryza. We have previously isolated A. paragallinarum from diseased chickens and demonstrated their virulence in experimental infection in different poultry species. In the present study, the isolates were screened for selected resistance genes for clinically relevant antibiotics. The study demonstrated resistance genes for sulphamethoxazole, tetracycline, streptomycin and ampicillin resistance in the isolates. Multiple resistance and resistance genes to streptomycin (strA), ampicillin (blaTEM), tetracycline (tetC and tetA) and sulphamethoxazole (sul2) in isolates of A. paragallinarum are reported. The demonstration of these genes in A. paragallinarum, similar to what has been demonstrated in other respiratory pathogens, is a concern for potential horizontal spread of multiple drug resistance leading to treatment failures in different respiratory diseases. Key words: Antibiotic resistance genes, Avibacterium paragallinarum.