Development and Validation of PCR Assays for Improved Diagnosis of Infectious Coryza by Differentiating Pathogenic and Nonpathogenic Avibacterium paragallinarum.

Infectious coryza (IC) caused by Avibacterium paragallinarum (AP) is an emerging infectious respiratory disease in the commercial chicken layer industry in the Midwestern US states. Outbreak investigations around positive index cases led to the discovery of nonpathogenic AP (npAP), which caused quantitative real-time PCR (qPCR) positive results in naïve-healthy layer (NHL) flocks. Therefore, the reliability of positive qPCR as confirmed diagnosis of IC became questionable and the poultry industry was deprived from an essential diagnostic tool in the face of an actively spreading outbreak. However, the prevalence of npAP in NHL flocks and the magnitude of this diagnostic challenge remained unclear. This pilot surveillance study aims to provide an initial estimate of npAP prevalence in the US commercial layer industry. Two differential qPCR assays were recently developed to differentiate pathogenic AP (pAP) and npAP. A total of 710 oropharyngeal (OP) swab pools (5 swabs/pool) were collected from 80 NHL sites across 13 US states and tested using qPCR assay targeting the recN gene as a screening test. Two hundred thirty-one out of 710 total pools were positive for npAP (32.5%) representing 28 positive sites out of the 80 total sites (35%). All positive qPCR samples from NHL flocks were confirmed to be due npAP. The differential qPCR was capable of confirming 85.71% of the npAP cases, while the remaining cases required further isolation and whole-genome sequencing (WGS). In conclusion, this pilot study indicates that the prevalence of npAP in NHL flocks in the United States is above 30%. Therefore, in flocks with no clinical signs, qPCR assays cannot be relied upon for IC diagnostic confirmation. Currently, isolation combined with WGS is the only diagnostic tool capable of completely differentiating between these two AP populations, which indicates the immediate need for improvements in the available diagnostic assays.

Infectious coryza (IC) caused by Avibacterium paragallinarum (AvP) is an upper respiratory disease in chickens and incurs a significant economic impact on laying hens. Control of IC requires reliable bacterial isolation, but AvP is a fastidious bacterium, and the currently used methods yield poor isolation. To address this need, we developed selective media for the efficient growth and isolation of AvP. Several basal media supplemented with various growth factors were explored, and Mueller Hinton agar (MHA) supplemented with fetal bovine serum (FBS) plus NAD yielded optimal AvP growth, eliminating the need for nurse bacteria. This medium (named MSN) was further supplemented with vancomycin and crystal violet to produce two selective media, named MSNV and MSCV, respectively, to inhibit commensal Gram-positive bacteria that reside in the upper respiratory tract of chickens. MSNV and MSCV were compared with the standard isolation methods used in veterinary diagnostic laboratories for AvP isolation using samples from IC-positive and IC-negative flocks verified by clinical observation and AvP-specific RT-PCR. The two selective media significantly increased the isolation of AvP from clinical cases (59.4% for MSNV and 46.9% for MSCV) compared to the conventional method (37.7%). In addition, MSNV and MSCV successfully isolated the recently discovered non-pathogenic AvP variants, which were difficult to obtain using the standard methods. Notably, both media drastically reduced background bacteria and improved the purity of AvP isolates. These results demonstrate the efficacy and usefulness of the selective media for AvP isolation, which will enhance IC diagnosis, antimicrobial susceptibility testing, and vaccine development.IMPORTANCEInfectious Coryza is an economically important disease in poultry, resulting in poor growth and a significant reduction in egg production. The causative agent (AvP) of the disease is difficult to isolate from clinical samples and often requires a nurse bacteria to provide essential nutrients for optimal growth. In addition, the currently used methods for isolation are inefficient and often result in contamination by residential bacterial species. The selective media (MSNV and MSCV) developed in this study solve these problems by eliminating the need for nurse bacteria and by inhibiting Gram-positive bacteria normally present in the respiratory tract of chickens. The media not only increase isolation efficiency but also improve the purity of the isolates. These advancements will facilitate IC diagnosis and the development of vaccines for effective control of this major poultry pathogen.

Infectious coryza is an upper respiratory disease of chickens caused by Avibacterium paragallinarum. Outbreaks of infectious coryza caused by Av. paragallinarum serovar C-1 isolates in coryza-vaccinated flocks in Ecuador and Mexico have been reported. In the current study, the protection conferred by four commercially available, trivalent infectious coryza vaccines in chickens challenged with a serovar C-1 isolate from an apparent coryza vaccine failure in a layer flock in Mexico was evaluated. Only one infectious coryza vaccine provided a good protection level (83%) in vaccinated chickens. These results might explain the infectious coryza outbreaks in vaccinated flocks that have been observed in the field.

This is the first extensive report on the identification and characterization of Avibacterium paragallinarum (AVP) isolates obtained from outbreaks of infectious coryza (IC) in IC-vaccinated layer flocks from Sonora State in Mexico. Isolates obtained from IC outbreaks during the years 2007, 2014, 2015, 2017, and 2019 were identified by conventional PCR test and 16S rRNA gene analysis, serotyped by Page serotyping and genotyped by the recently described partial sequence analysis of the HPG2 region. Furthermore, antimicrobial susceptibility profiles were determined by a recently improved minimal inhibitory concentration (MIC) test. The conventional PCR test and the 16S rRNA analyses confirmed the isolates as AVP. Serotyping results showed the involvement of isolates belonging to serotypes A, B, and C in the IC outbreaks. Genotyping of the HPG2 region revealed the presence of sequence type (ST)1, ST4, and ST11, of which the latter has also been identified in Europe. The MIC susceptibility test showed that all tested isolates were susceptible for the majority of tested antimicrobials, including erythromycin and tetracycline, which are important antibiotics for the treatment of IC. The IC situation in Sonora State, Mexico, is complex because of the presence of serotypes A, B, and C. This finding emphasizes the importance of biosecurity in combination with the application of the most optimal vaccination programs in the control of IC in Sonora State, Mexico.

Worldwide, Avibacterium paragallinarum is the aetiological agent of infectious coryza in poultry. Vaccines are the best means of control, helping reduce clinical signs and colonization of this bacterium. Most vaccines are based on international reference strains, or, lately, regional strains, but, generally, without any information regarding their virulence. The characterization of the pathogenicity of 24 Av. paragallinarum strains of the three Page serogroups, including four variant strains of serogroup B, all isolated from infectious coryza outbreaks in Peru, was performed. After experimental inoculation into the infraorbital sinuses, information regarding their capacity to induce infectious coryza typical clinical signs, spreading, and colonization was recorded. Furthermore, after intraperitoneal inoculation, septicaemia and death were registered. Differences among strains in these parameters were observed, even within strains from the same serogroup. Finally, the four most pathogenic strains, one from each serogroup, were chosen to formulate an experimental vaccine that was tested successfully against homologous challenges in reducing clinical signs and colonization in vaccinated birds compared to unvaccinated ones. This is the first time that Av. paragallinarum strains from Peru were studied thoroughly for their virulence in a search for improving vaccine formulation.

Simple SummaryThe incidence of infectious coryza has increased in China, even occurring in vaccinated chickens, causing serious economic losses. Several researchers believe that mutation, the increased virulence of Avibacterium paragallinarum, and improper vaccination procedures lead to poor protection from an inactivated vaccine. Based on this, the purpose of the current study was to evaluate the protective efficacy of a commercial infectious coryza trivalent inactivated vaccine against field infection of Avibacterium paragallinarum in China. Our results suggested that double vaccination showed better protection efficacy than single vaccination. The clinical symptoms, pathological changes, and bacterial shedding of chickens with double vaccination were significantly lower than those of control groups after challenge with field Avibacterium paragallinarum isolates of three serovars. No significant differences were found in body weight and egg production between the boost vaccination groups and the negative control group. The current commercial vaccine shows comparable efficacy in shortening the course of infection if administered twice. Our results provide a reference for infectious coryza clinical vaccine application in China. In addition, better ventilation strategies and biosecurity are important components of a strategic plan to prevent infectious coryza.Infectious coryza (IC) is an acute respiratory disease caused by Avibacterium paragallinarum (Av. paragallinarum). In recent years, there have been frequent outbreaks of IC in chickens vaccinated with an inactivated vaccine, causing huge losses to the poultry industry. In this study, the protective efficacy of the trivalent inactivated IC vaccine (PT Medion Farma Jaya) against the field isolates of three serovars of Av. paragallinarum was verified. After vaccination, the hemagglutination inhibition antibody titers in double-vaccinated groups (A2, B2, and C2) were higher than those in single-vaccinated groups (A1, B1, and C1). The highest antibody titer was 213.1 at 3 weeks after the booster vaccination in group A2. Consistent with the trend in hemagglutination inhibition antibody titers, the protective efficacy of double vaccination was better than that of single vaccination. The clinical symptoms and pathological changes were alleviated, or the bacterial shedding was significantly reduced with double vaccination after challenge with field isolates of three serovars (p < 0.05). In particular, the chickens with double vaccination showed no clinical symptoms, pathological changes, or bacterial shedding after challenge by the serovar C strain. There was no significant difference in body weight and egg production between the double-vaccinated groups and the negative control group (p > 0.05). Therefore, we recommend that the commercial IC vaccine should be double-vaccinated in clinical applications.

Avibacterium paragallinarum is a Gram-negative bacterium that causes infectious coryza (IC), a respiratory disease that is highly contagious among chickens. Through PCR testing and sequence analysis of the FlfA gene, a total of 10 Av. paragallinarum field isolates were identified, accounting for 5.7% of the samples. In this study, we assessed the virulence of field isolates by utilizing a rapid artificial intrasinus injection route model. Upon observation, we have found that all 10 field isolates from Egypt that were identified as type B strains possess virulent properties and have the ability to cause Infectious Coryza (IC) disease in chickens. The current study aimed to sequence the FlfA gene and conduct phylogenetic analyses to better understand the Avibacterium paragallinarum field isolates that have been circulating in Egypt in recent years. This research will provide valuable insights into the genetic makeup and evolution of this pathogen, which can inform future efforts to control and prevent its spread. The amino acid sequences of Avibacterium paragallinarum from Egypt and the reference sequence exhibit a remarkable degree of homology, ranging from 97.96% to 100%. This finding underscores the close relationship between the two sequences and highlights the potential for further research into the genetic makeup of this pathogen. Furthermore, the discovery of a fimbrial cluster that is identical to the Galli bacterium FlfA in the Av. paragallinarum genome indicates that these two species may have recently exchanged this fimbriae. This exchange could have occurred during the natural co-colonization of the chicken's upper respiratory tract. It is possible that this exchange could have implications for the pathogenicity and virulence of these bacteria. The FlfA protein has unique characteristics and was subjected to bioinformatics analysis to predict its properties and protein interactions. The FlfA protein structure is stable with many coiled regions. ..

Avibacterium paragallinarum (historically called Hemophilus paragallinarum) causes infectious coryza (IC), which is an acute respiratory disease of chickens. Recently, outbreaks of IC have been reported in Pennsylvania (PA) in broilers, layer pullets, and laying hens, causing significant respiratory disease and production losses. A tentative diagnosis of IC can be made based on history, clinical signs, and characteristic gross lesions. However, isolation and identification of the organism are required for a definitive diagnosis. Major challenges with the bacteriological diagnosis of A. paragallinarum include that the organism is difficult to isolate, slow-growing, and can only be successfully isolated during the acute stage of infection and secondary bacterial infections are also common. As there were very limited whole genomes of A. paragallinarum in the public databases, we carried out whole-genome sequencing (WGS) of PA isolates and based on the WGS data analysis; we designed a novel probe-based PCR assay targeting a highly conserved sequence in the recN, the DNA repair protein gene of A. paragallinarum. The assay includes an internal control, with a limit of detection (LOD) of 3.93 genomic copies. The PCR efficiency ranged between 90 and 97%, and diagnostic sensitivity of 98.5% compared with conventional gel-based PCR. The test was highly specific, and no cross-reactivity was observed with other species of Avibacterium and a range of other common poultry respiratory viral and bacterial pathogens. Real-time PCR testing on 419 clinical samples from suspected flocks yielded 94 positives and 365 negatives in agreement with diagnostic bacterial culture-based detection. We also compared the recN PCR assay with a previous HPG-2 based real-time PCR assay which showed a PCR efficiency of 79%.

In 2017, the Turlock branch of the California Animal Health & Food Safety laboratory system received a significant increase in infectious coryza (IC) necropsy cases, with a total of 54 submissions originating from commercial broilers (n = 40), commercial layers (n = 11), and backyard chickens (n = 3). Layer flocks positive for IC were distributed within the adjacent counties of Merced and Stanislaus, while broiler flocks were concentrated within Merced County. The backyard flocks were located in Alameda and Sacramento counties. The clinical and pathologic presentation was consistent with IC, although septicemic lesions were also noticed. Avibacterium paragallinarum was isolated and identified by PCR from the respiratory tract as well as from extrarespiratory sites. Polymicrobial infections involving other viral (infectious bronchitis virus, infectious bursal disease virus) and bacterial (Mycoplasma spp., Escherichia coli, Ornithobacterium rhinotracheale, Gallibacterium anatis biovar haemolytica) agents were commonly reported. Thirteen selected Av. paragallinarum isolates were successfully characterized as serovar C (Page scheme) and serovar C2 (Kume scheme). They shared a unique enterobacterial repetitive intergenic consensus (ERIC) PCR, differing from the four reference strains, and showed consistent high minimum inhibitory concentration values for tetracycline, suggesting a common origin from a single clone. Based on these results, high biosecurity standards and proper immunization of susceptible, multi-age flocks should always be implemented and adjusted as needed. The importance of backyard flocks should not be underestimated due to their unique epidemiologic role.

Infectious coryza (IC) caused by Avibacterium paragallinarum (AP) has risen in importance as a poultry disease over the past several years because of its increased incidence in commercial poultry in both Europe and the United States. Because of this rise in importance, more attention has been focused on diagnosis, isolation, and surveillance of this bacterial pathogen. As a result, new knowledge has been produced and published. This review was compiled with the main purpose of summarizing and presenting the updated knowledge available about AP. However, the new knowledge can only be understood in the context of previously known facts about the disease. Therefore, this review has been organized in two major parts. The first part is a review of the established knowledge about AP, followed by recent updates. In the first part, we summarize the established well-known as well as some of the less-known facts and literature about AP. The second section focuses on specifics of the latest IC outbreaks in commercial poultry in northern latitudes, particularly in Europe and in North America. Additionally, we reviewed the current geographical distribution of the disease in Asia, South America, and Africa. The crises created by emerging or re-emerging disease outbreaks ignite interest in understanding the disease and pathogen in order to combat it properly. This results in new knowledge that improves the understanding of the disease features, leading to improved disease prevention, control, and eradication. Although knowledge about AP has advanced, knowledge gaps about the disease still persist. Therefore, this review concludes with summarizing the current knowledge gaps as well as potential areas for future research.

PCR is the most effective method for detecting difficult-to-cultivate pathogens and pathogens that are part of mixed infections in animals, such as Ornithobacterium rhinotracheale, which causes bird ornithobacteriosis, or Avibacterium paragallinarum, which causes infectious coryza. In this work, we developed and validated two efficient and sensitive diagnostic assays for the rapid and accurate detection of A. paragallinarum and O. rhinotracheale DNA in bacterial isolates and clinical samples using real-time PCR with TaqMan-like probes. When designing the PCR assays, we performed in silico analysis, optimized DNA isolation methods and PCR conditions, and assessed the analytical and diagnostic performance of PCR. We designed primers and probes that have no mismatches with published whole-genome sequences of bacteria. The optimization of conditions showed that the PCR assays are sufficiently robust to changes in temperature and oligonucleotide concentration. The validation showed that the developed assays have high analytical and diagnostic sensitivity and specificity. These assays are expected to improve the differential diagnosis of respiratory diseases in chickens and turkeys.

The reemergence of infectious coryza (IC) caused by Avibacterium paragallinarum (AP) as an acute and occasionally chronic respiratory disease in domestic poultry has caused severe losses in several U.S. states. The disease is also associated with decreased egg production in layers and increased condemnations from air sac infections in broilers. A series of applied experiments were performed to elucidate the persistence of AP in infected broiler flocks, to genotype AP strains isolated from field cases, and to evaluate commercial and autogenous vaccine protection in commercial and specific-pathogen-free (SPF) chickens. Experimental evaluation of environmental persistence suggests that AP did not persist more than 12 hr in a hypothetically contaminated environment. Additionally, other detected potential pathogens such as Gallibacterium anatis and infectious bronchitis virus caused mild respiratory signs in the exposed birds. The HMTp210 and HagA genes of four IC field strains were sequenced and compared with published sequences of HMTp210 and HagA. The HMTp210 phylogeny showed a marginally imperfect clustering of the sequences in genogroups A, B, and C. Although not definitive, this phylogeny provided evidence that the four field strains aligned with previously characterized serovar C strains. Moreover, the base pair homology of the four strains was 100% identical to serovar C reference strains (H-18 and Modesto). HagA phylogeny was unclear, but interestingly, the IC field strains were 100% homologous to C-1 strains reported from Mexico and Ecuador. Finally, vaccine protection studies in commercial hens indicate that clinical signs are induced by a combination of IC and other concomitant pathogens infecting commercial birds. Additionally, vaccine protection experiments performed in SPF hens indicated that protection provided by the two commercial vaccines tested provided a reduction in clinical signs and bacterial shedding after two applications.

The total poultry population in Ethiopia is estimated to be 51.35 million. The Poultry sector is a fastest growing among the animal production activities offers an opportunity to feed the fastest growing human population and provide income resources for poor farmers. There are different diseases which can affect chicken. Among the bacterial diseases, Infectious Coryza (IC) is an acute and contagious respiratory disease of chickens caused by the bacteria, Avibacterium paragallinarum. IC is distributed worldwide and typically transmitted by direct contact, airborne droplets and contamination of drinking water. But Egg transmission does not occur. The initial step in the pathogenesis of infectious coryza is adherence to and colonization of the nasal mucosa. The diagnosis can be based on a history of rapid disease spread, clinical symptoms, and pathological changes caused by IC. Diagnostic methods for IC include direct isolation, the HI test for serovar A and Polymerase Chain Reaction (PCR). Diagnosis of Avibacterium paragallinarum have been proposed as alternatives to conventional serotyping by the Page scheme. The HI test, which is one of the most widely used serological test, is often utilized to detect changes in antibody titers in cases of field infection or vaccination, and is useful for evaluating the prevalence of IC in certain areas. Rapid and accurate detection of respiratory IC has become a challenge because of the involvement of more than one agent with similar clinical signs and lesions, which complicates diagnostic decisions, as well as treatment and control strategies.

Infectious Coryza (IC), caused by Avibacterium paragallinarum, is an infectious and highly contagious bacterial respiratory disease of poultry. The present communique deals with the pathological investigation of complicated cases of Infectious Coryza. The dead birds with nasal/lacrimal discharge and swollen sinuses were screened for the presence of Avibacterium paragallinarum and other concurrent bacterial infection by PCR and microbial culture, respectively. The birds (n=08) found positive for Avibacterium paragallinarum were also found to be infected with other bacteria like E. coli, Klebsiella, Salmonella and Pseudomonas. Pathologically, fibrinous deposits were seen in airsac, liver and heart of all the birds but more severe changes were seen in birds with concurrent E. coli infection. The histopathological lesions like tracheitis and deciliaton were consistently observed in the trachea, and lungs from all birds with complicated coryza and also showed interstitial pneumonic changes. All these cases were diagnosed as complicated cases of infectious coryza.

Infectious coryza, caused by Avibacterium paragallinarum, is an acute respiratory disease of poultry that can result in substantial morbidity, mortality, and economic losses. In March 2017, the Turlock branch of the California Animal Health and Food Safety laboratory system encountered an unusual clinical and pathologic presentation of infectious coryza in 6 live, 29-d-old, commercial broiler chickens that were submitted for diagnostic investigation. Antemortem evaluation revealed severe neurologic signs, including disorientation, torticollis, and opisthotonos. Swollen head-like syndrome and sinusitis were also present. Histologically, severe sinusitis, cranial osteomyelitis, otitis media and interna, and meningoencephalitis were noted, explaining the clinical signs described. A. paragallinarum was readily isolated from the upper and lower respiratory tract, brain, and cranial bones. Infectious bronchitis virus (IBV) was also detected by PCR, and IBV was isolated in embryonated chicken eggs. Based on sequencing analysis, the IBV appeared 99% homologous to strain CA1737. A synergistic effect between A. paragallinarum and IBV, resulting in exacerbation of clinical signs and increased mortality, may have occurred in this case. A. paragallinarum should be considered among the possible causes of neurologic signs in chickens. Appropriate media should be used for bacterial isolation, and the role of additional contributing factors and/or complicating agents should be investigated in cases of infectious coryza.