Infectious coryza outbreak in a table egg layer flock in Alberta

Abstract

Infectious coryza (IC) is an economically important, acute, and highly contagious respiratory disease of chickens caused by Avibacterium paragallinarum. A case of 27-week-old table egg layers was submitted to the Diagnostic Services Unit, Faculty of Veterinary Medicine, University of Calgary (Alberta). The chickens had a history of swollen faces, eyelids, combs, and wattles; lacrimation; and nasal discharge with increased mortality and an acute drop in egg production of up to 14%. Avibacterium paragallinarum was cultured from infraorbital sinuses and wattle samples. Whole-genome sequencing based multilocus sequence typing identified A. paragallinarum sequence type-8, and genomic analysis of HMTp210 gene predicted Page serovar C. In the same time frame, IC outbreaks were also recorded in 3 other flocks in Alberta and some flocks in Saskatchewan and Manitoba. In addition to IC, infectious bronchitis virus was identified. The disease was linked to the introduction of subclinically infected pullets from another province into this flock of naïve birds. This is the first reported case of IC in recent times in Alberta. Screening birds for infectious disease status should be a critical practice if a carrier state exists in long-lived birds.

Key clinical message:

This outbreak underscores the need for strict biosecurity. Avoid introducing new birds to closed flocks. If necessary, rule out infections with carrier states through appropriate screening.

Infectious coryza (IC) is an economically important disease of intensively raised commercial chickens worldwide. It is an acute, sometimes chronic, contagious, upper respiratory tract disease that results in airsacculitis and condemnation in broiler chickens and reduced egg production (up to 10 to 40%) in breeders and layers (1). Infectious coryza is caused by Avibacterium paragallinarum (AP). Both pathogenic and apathogenic strains exist (2). The bacterium is typed into 3 serovars (Page scheme) (3), A, B, and C; and several subgroups, A1 to A4, B1, and C1 to C4, by modified Kume’s schemes, respectively (4). Authors of recent publications have tried to correlate the bacterial genotypes to serovars based on sequence analysis of HMTp210 gene (1,5) or the multilocus sequence typing (MLST) scheme (6). The disease is prevalent worldwide (6). In the United States, recent outbreaks have been reported in several states (1,2,7). Serovar C has been predominant (1,8), whereas serovars A and B have been documented in the past (3,5,9,10). The disease has been listed as an annually notifiable disease by the Canadian Food Inspection Agency (11). Occasional reports have been recorded in small flocks; however, no published reports have identified IC in Canada. We report a case of IC introduced into an egg layer flock in Alberta that had important health implications in the affected flock.

CASE DESCRIPTION

A 27-week-old flock of 15 000 table egg layer chickens experienced a 14% drop in egg production and increased mortality following the introduction of 920 pullets from another province in February 2024. Clinical signs included facial swelling, lacrimation, and nasal discharge. Three similar outbreaks were reported in Alberta, 2 in Saskatchewan, and 6 in Manitoba (internal field communication). These flocks also received pullets from the same source. Two culled birds were submitted for diagnostic workup at the Diagnostic Services Unit, Faculty of Veterinary Medicine at the University of Calgary (Alberta) with a suspicion of IC.

Gross lesions included swollen faces, eyelids, and wattles with marked subcutaneous edema (Figure 1 A); yellow mucoid exudate in the infraorbital sinuses (Figure 1 B); and marked necrosis and hemorrhage in the wattles (Figure 1 C). Other gross pathological findings were cloudy air sacs and multifocal hepatic necrosis. Histological lesions included lymphoplasmacytic and mucoid sinusitis (Figure 1 D, E) and necrotizing and hemorrhagic wattle dermatitis (Figure 1 F). Other lesions included necrotizing cranial osteomyelitis, cellulitis, multifocal hepatitis, and airsacculitis. Based on clinical history and gross and histological lesions, AP was at the top of the differential diagnosis list. Other differentials included Pasteurella multocida, Ornithobacterium rhinotracheale, Gallibacterium anatis, Mycoplasma gallisepticum, Mycoplasma synoviae, infectious bronchitis virus (IBV), infectious laryngotracheitis virus (ILTV), avian metapneumovirus (AMPV), Newcastle disease virus, avian influenza, and Escherichia coli infection concurrent with respiratory diseases.

FIGURE 1.

Gross and histopathological lesions in a laying hen confirmed to be infected by infectious coryza. A — Swollen face, eyelid, comb, and wattles. B — Mucoid exudate in the infraorbital sinus. C — Necrosis and hemorrhage in a section of wattle. D — Mucoid exudate and cellular debris in the infraorbital sinus and inflamed epithelium. Stain: hematoxylin and eosin (H&E); magnification: 20×. E — Higher-magnification view of epithelium, showing edema and infiltration of lymphocytes, plasma cells, and a few heterophils. Stain: H&E; magnification: 400×. F — Hemorrhage in the center and necrosis of the tissue around the periphery in the wattle section. Stain: H&E; magnification: 100×.

Samples of necrotic wattles and swabs from the infraorbital sinuses were submitted to the bacteriology section of the Diagnostic Services Unit, Faculty of Veterinary Medicine, University of Calgary. Samples were also sent to the Animal Health Laboratory (Guelph, Ontario) for PCR testing for IBV, Mycoplasma spp., ILTV, and AMPV. Briefly, samples were inoculated onto 5% sheep blood agar plates (Oxoid, Nepean, Ontario) and chocolate agar plates (Oxoid) incubated at 35°C for 48 h in an environment containing 5% CO₂, and onto MacConkey agar plates (Oxoid) incubated as described under atmospheric conditions. Bacterial identification was done using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS; Bruker Daltonics, Bremen, Germany). Additional isolate characterization was done through MLST and HMTp210 gene analysis based on whole-genome sequencing. Whole-genome sequencing of AP isolate was done on the MiSeq System (Illumina, San Diego, California, USA) using the Nextera XT DNA library preparation and MiSeq reagent v3 (2 × 300-bp paired-end) kits. The pubMLST was used for MLST, whereas BioNumerics software v7.6.3 (Applied Maths, Austin, Texas, USA) was used for HMTp210 gene analyses, including creation of concatenated sequences of HMTp210 region 1 and the HMTp210 hypervariable region (5). The HMTp210 gene sequence of this isolate was submitted to GenBank under accession number PV550778. A multiple alignment-based single-linkage tree of concatenated HMTp210 gene from a representative of each genotype (GT) group (accession numbers provided in Table 1) was used to determine the GT of this isolate and to predict its Page serovar.

TABLE 1.

List of strains and genotypes used to prepare the phylogenetic tree.

Strain identification	Genotype	Accession numbers
Strain_221	GT-I	ON937733a, ON959452b
Strain_2403	GT-II	ON937735a, ON959454b
Strain_E-3C	GT-III	ON937738a, ON959457b
Strain_HP-14	GT-IV	ON937740a, ON959459b
Strain_Modesto	GT-V	ON937742a, ON959461b
Strain_HP-60	GT-VI	ON937741a, ON959460b
TW09	GT-VII	MT050503c
Strain_SA-3	GT-VIII	ON937743a, ON959462b
Strain_Spross	GT-IX	ON937744a, ON959453b
Strain_ESV-135	GT-X	ON937739a, ON959458b
Strain_2671	GT-XI	ON937736a, ON959455b
TW11	GT-XII	MT050504c
TW08	GT-XIII	MT050502c
AVP_CH_NL_GD_14787_2015	GT-XIV	ON937692a, ON959411b

^aHMTp210 region 1 accession number.

^bHMTp210 hypervariable region accession number.

^cAccession number.

Avibacterium paragallinarum was isolated in mixed culture in moderate numbers (2+) from the infraorbital sinus and in small numbers (1+) from the wattle tissue. All bacterial species isolated from the infraorbital sinus and wattle tissue are summarized in Table S1 (available online from: Supplementary Materials) with their MALDI-TOF MS scores and quantities. Multilocus sequence typing analysis identified sequence type-8 (ST-8). Analysis of concatenated HMTp210 gene further revealed that this isolate belonged to genotype X (GT-X), sharing 98.35% homology with the concatenated HMTp210 gene of strain ESV-135, the representative of the GT-X group (Figure 2). Members of the GT-X group correlate with Page serovar C isolates (5).

FIGURE 2.

A multiple alignment-based single-linkage tree was generated using concatenated HMTp210 gene sequences each representing a single Avibacterium paragallinarum genotype (GT). To determine its genotype, concatenated HMTp210 gene sequence of isolate 24-094350 (this case) was included into the alignment. Genotype and strain designation for each isolate are shown next to the circle representing individual concatenated HMTp210 sequence. Square root scaling was used to scale the branch lengths.

The PCR tests for Mycoplasma spp., ILTV, and AMPV subtypes A, B, and C were all negative, ruling out these pathogens as causative agents in this case. Tracheal samples were weakly positive for IBV via real-time PCR (ct = 31), indicating a low nucleic acid amount; hence, sequencing could not be completed.

DISCUSSION

Several diseases, alone or concurrently, can mimic the clinical presentation observed in the case. In past, serovar was determined using the serotyping technique (3,4) Due to the cumbersome technique and lack of commercially available antisera for typing of AP isolates (5), this method is restricted to very few testing facilities. Due to a lack of sera in the present case, we used molecular methods to determine the serovar. First, we determined the geographic origin of the isolate. The AP isolate was typed as ST-8 by MLST, confirming the previous report that AP isolates are related geographically, as all isolates from North America, including Mexico, were reported to belong to this sequence type (6). Although this technique provided insights into epidemiological and genetic relatedness of AP strains, serovar identification was not possible.

Therefore, a few attempts using various genetic targets were made to correlate GTs with serovars. The most frequent genetic target used for this correlation was either the full length or various regions of the HMTp210 gene (1,5). Although most reports indicated good correlation among Page serovars and HMTp210 regardless of the region analyzed, there were still some conflicting results regarding the correlation with Kume serovars. Recently, a new genotyping method was proposed, based on concatenated sequence of region 1 and hypervariable region 2 of HMTp210 gene and dividing AP isolates into GTs I through XIV (5). Using this method, we established that our isolate belonged to GT-X. However, since we used only a single representative of each GT group for our analyses, we submitted our HMTp210 sequence for independent analysis to Buter and Dijkman, who confirmed our findings using a maximum parsimony tree built from > 120 AP isolates (personal communication). Although there was a good correlation between Page serovars and GTs, in the absence of serological data to confirm our results, we predicted that our isolate was likely Page serovar C. However, more work is required to predict the Kume serovar because some isolates belonging to GT-X were reported as Kume serovar C-2 based on genetic analysis of the hypervariable region of HMTp210 (1), whereas most other isolates were classified as Kume serovar C-1 (5).

In this case scenario, a shortage of birds due to a change in quota (bird allocation to producers) in the province and ongoing outbreaks of highly pathogenic avian influenza increased the need for bird movement between sites, including delivery of pullets from mixed sources to producers. This was ultimately suspected to be the cause of the disease outbreak. Although birds were SE-negative, they were carriers of AP, which was spread to affected flocks in western Canada.

Antimicrobial interventions were not instituted in Alberta as they do not eliminate the disease and may promote establishment of a carrier state, increasing the risk of future relapse (12). Further, use of antimicrobials in laying flocks is restricted due to egg withdrawal times, which prohibit their use in flocks laying eggs for human consumption. Consequently, affected flocks were either culled or placed under strict quarantine and managed with all-in-all-out protocols. Recommended additional control measures included heightened biosecurity, no addition or removal of birds, and screening of new placements. Currently, there are no national surveillance data available on IC in Canada and no vaccination program is established in the industry. Commercial vaccines are available, but their use is only recommended if the disease becomes endemic.

To our knowledge, this is the first reported case of IC in Alberta. Using MLST, it was confirmed that this isolate belonged to ST-8. Further, it belonged to GT-X and was likely Page serovar C. This outbreak highlighted the importance of biosecurity and that caution must be exercised when adding new birds from another source. Screening of birds for IC and other respiratory diseases (ILT and fowl cholera) for which a carrier state exists should be implemented.