Avibacterium paragallinarum, the Causative Agent of Infectious Coryza: A Comprehensive Review

The bacterial species name Avibacterium paragallinarum (AP) is used today for the bacterium previously known as Haemophilus paragallinarum. The name change happened after phenotypic and genotypic analyses revealed the bacterium belonged to a different taxonomy phylum (1). This review will be referring to the bacterium as AP, even though many of the referenced literature may refer to the bacterium as H. paragallinarum.

De Blieck in 1931 was the first to isolate the bacterium from chickens showing IC clinical signs. He then named this organism as Bacillus Haemoglobinophilus coryzae gallinarum (2). Shortly after this discovery, similar organisms were discovered by McGaughey (3) and Nelson (4). McGaughey reported the bacterium to belong to the genus Haemophilus (5) and Eliot and Lewis in 1934 (6) proposed naming the bacterium as Haemophilus gallinarum; however, this name was quickly changed to H. paragallinarum. When the bacterium was first identified, it was shown to require both X (hemin) and V (NAD) factors to be cultured (7,8). The V factor is a co-enzyme used in metabolism known as nicotinamide adenine dinucleotide (NAD+) (9), and the X factor was found to be hemin, a common bacterial iron source acquired from erythrocytes (10). AP is a Gram-negative, bipolar staining nonmotile bacteria (10,11). It is pleomorphic and appears as rod-shaped and/or cocci-shaped cells when viewed under the microscope. Individual bacterial cells can range from 1 to 3 μm in length and 0.4 to 0.8 μm in width. Cultures that are older than 48–60 hr will have cells that start to degrade, leaving only fragments and undefined forms behind (11,12). Since its discovery, the identification and characterization of avian Haemophilus-like organisms, has been surrounded by a cloud of mystery and confusion (5). This confusion continues to date after more than 90 yr since their initial observation. Eliot and Lewis (6) classified the newly discovered X- and V-dependent bacteria as Haemophilus gallinarum. However, it was later discovered to be only V-factor (NAD) -dependent, which led to the renaming of the bacterium as H. paragallinarum (13,14). This name remained until the entire family Pasteurellaceae was analyzed phenotypically and genotypically with the use of the 16S rRNA. The results proposed a new genus ending with the renaming of the bacterium to Avibacterium paragallinarum. AP differs from the rest of the Avibacterium species by its ability to ferment D-xylose and trehalose while also its lacks catalase (1). However, other bacterial species in the genus Avibacterium are closely related to each other, contributing to the diagnostic and taxonomic confusion of AP. This is discussed in more detail in Part II of this review.

IC in chickens is a disease characterized by an upper respiratory tract infection (11,15,16). Although AP has been isolated from other avian species (11,17,18,19,20), chickens are the main natural host in which clinical disease occurs after natural infection (16). Chickens of all ages are susceptible to the infection (1,7,14,); however, this disease affects mature egg-laying hens more frequently and more severely with drops in egg production of up to 40% (1,14). IC can also lead to significant economic losses in broilers as it results in growth retardation and most importantly increased condemnations in the processing plant due to airsacculitis (7). At the flock level, the first noticeable signs of IC are usually a severe drop in water and feed consumption (21). Additionally, there is a noticeable decrease in flock vocalization, and birds appear to be quiet and depressed. Consequently, within 24–48 hr, a sharp drop in egg production occurs, which could continue for 7–21 days (7,22,23,24,25) in uncomplicated and up to 9 wk in complicated cases of the disease (21). Morbidity in a flock is usually high, with the majority of individuals in the flock showing the clinical disease (16). Mortality can increase but usually remains low if uncomplicated (26). However, complicated IC cases either by environmental stressors, primary, or secondary pathogens can result in more severe clinical signs, higher mortality, and prolonged course of the disease (26,27). At an individual-bird level, clinical signs of IC usually start with nasal discharge, ocular discharge, sneezing, and facial swelling. Mild to moderate or severe swelling can occur in either one or both infraorbital sinuses because of mucoid or fibrinous exudate with rhinitis, as well as subcutaneous edema in the tissues surrounding the eyes with conjunctivitis. The exudate can travel with gravity through the subcutaneous space, causing inflammation and swelling of the intermandibular space and wattles. The inflammatory exudate can also go through the choanal cleft, into the oral cavity and to the trachea causing tracheitis. If it travels further down the respiratory system, AP can cause lower respiratory lesions such as airsacculitis and pneumonia, and eventually lead to systemic infection (Fig. 1) (1,21). The incidence of AP becoming a systemic infection is low; the disease is usually limited to the upper respiratory system; however, bacterial isolation from nonrespiratory organs has confirmed this observation (21,27). The severity of IC and course of the clinical signs can vary based on multiple factors: AP strain, age, AP vaccination status, environmental stressors, and infections with other respiratory pathogens (27,28,29). The term “mixed infection,” which refers to the involvement of more than one pathogen in an infected flock, can complicate the detection of any one individual pathogen (28,30). In mixed infections, other pathogens can compromise the respiratory or alter the immune system of the birds, exacerbating the clinical signs of IC and extending the duration of the disease (7,26,27,31). Clinical signs of IC are also dependent on the specific AP isolate involved in the outbreak; different serovars have shown significantly different levels of virulence (7,32,33). Young chickens can show severe clinical signs; however, severe disease is more frequently reported in mature laying hens (1). Traditionally the incidence of the disease is much higher in warm weather geographies, leading to severe economic losses in developing countries (15,16,17); however, this has recently changed, as discussed later in this review.

Similar to other poultry bacterial pathogens, AP has an acute phase in which clinical signs can be observed, followed by a chronic or carrier phase in which the host does not display clinical signs, but still carries and sheds the bacteria. Chronic carrier chickens are the reservoir of the infection (16). AP is transmitted horizontally, and there is no published evidence to support that vertical transmission occurs. Within a flock, the infection is transmitted to susceptible chickens through direct or indirect contact from chronic carriers or acutely infected chickens. Between flocks on the same site or in close proximity, water or airborne infections are proposed to play a major role in IC transmission (34). Between sites over a long distance, the main source of infection is usually infected carriers (34,35). This was evident in the recent outbreak in the Midwestern states in the United States. Transmission of IC from one state to another was tracked to the movement of chronic carrier replacement flocks. This understanding of AP transmission dynamics informs the approach to disease prevention in commercial flocks. Prevention is generally attempted through biosecurity, avoiding any direct (birds) or indirect (vectors such as water, feed, equipment, etc.) contact between susceptible flocks and chronic carriers or acutely infected chickens. However, this can be challenging, especially in face of high infectious pressure because of expanding outbreaks in densely populated areas (21,36). In environments with high challenge levels, proper vaccination becomes an essential tool to protect infected flocks from clinical disease. Although vaccination can reduce bacterial shedding in infected flocks, by itself it is incapable of eliminating the challenge in highly contaminated environments. This is why biosecurity, cleaning and disinfection, and proper vaccination (number of doses and serotype match) should be used to reduce high infectious challenge. This pressure reduction allows better control of the disease.

Clinical signs and gross lesions are characteristic and highly suggestive of IC. However, IC diagnosis confirmation requires bacterial isolation and/or direct detection of AP nucleic acid via PCR or metagenomic sequencing. Although a positive PCR result means pathogen detection, it does not always mean disease. PCR results can be more informative if they are paired with clinical signs suggestive of IC. AP is a fastidious microorganism and is difficult to isolate (37). The selection of birds is critical for successful isolation of AP. Birds early in the course of infection have a higher bacterial load, which increases isolation success rate. AP grows on blood tryptose agar plates, that are incubated in microaerophilic conditions if NAD is added (38,39,40). Staphylococcus epidermidis isolates are commonly used as the nursing colony to supply the NAD (V-factor) required for the growth of AP. In recent outbreaks in the United States, isolation has been more successful on Chocolate agar (21). NAD-dependent AP are tiny, dewdrop, almost transparent, colonies that grow adjacent to the nursing colony in what is known as the “satellite” colony morphology of AP (Fig. 2) (41,42). Bacterial isolate identification can be achieved by biochemical tests (22,23,24), PCR, or sequencing. More recently, matrix-assisted laser desorption ionization time-of-flight mass spectrometry, commonly known as MALDI TOF, has been increasingly available in veterinary diagnostic laboratories and can be used for bacterial colonies identification. However, MALDI TOF database needs to include enough species and isolates to differentiate between Avibacterium species.

NAD-independent isolates were first discovered in Kwa-Zulu Natal, South Africa in 1989 in poultry that showed clinical signs of IC (43,44,45). These isolates were cultured without the presence of a nursing colony and therefore do not show the typical satellite growth (35). At the time, the isolates were not considered to be AP (7,46,47). However, Mouahid and collaborators (44) were able to confirm that these isolates were AP with the use of phenotypic and molecular studies. Additionally, NAD-independent isolates reacted with AP monoclonal antibodies, leaving no doubt that they were correctly classified as AP (45,48). Later studies indicated that the NAD-independence was caused by a transmissible plasmid (45). When the plasmid was transformed into NAD-dependent isolates, they were rendered independent (45). However, not long after the transformation, the plasmid was lost which resulted in the bacterium losing its NAD-independence properties (45). When serotyped, 58% of the NAD-independent strains belonged to Page Serovar A (49). The NAD-independent strains were much more contagious and quickly became the predominant isolates in South Africa, between 1989 and 1993 (47,50). However, the use of commercial vaccines has shown to protect against NAD-independent strains from the A and C serogroup (51). NAD-independent AP isolates have been isolated in Mexico, Peru, and South Korea (43,44,45,52,53,54). Challenge studies used to investigate the virulence of NAD-independent variants showed that these isolates were less virulent when compared with the NAD-dependent strains from the same serogroup (33).

Serotyping is one of the most important pieces of information necessary for understanding the epidemiology of any specific IC outbreak and necessary for downstream decisions regarding outbreak control and vaccine selection. The serological properties of AP were first established in the United States by Page (55). Using a plate agglutination test, Page identified three distinct serovars, namely A, B, and C, which were later identified worldwide (56,57,58). There are three different antigens detected in the agglutination test; the first is Labile “L” which is heat unstable at 65°C for 30 min, the second is Heat Labile, trypsin resistant “HL,” and the third antigen is heat stable, trypsin resistant “HS”. The L antigen has three serologically different forms; L1, L2 and L3 which are responsible for serogroup specificity. Page Serovar A possess L1 and L3 antigen, whilst Page Serovar C possess L2 and L3. In addition to L3 antigen, HL and HS are common antigens between the Serovars A and C (59). Cross-reactions between the serovars in other related studies using the agglutination tests for serotyping can be explained by the presence of these shared or common antigens between the serovars (14,57). Additionally, it was found that 15%–38% of the isolates could not be serotyped using the Page scheme, as they were either non-agglutinating or auto-agglutinating (35) resulting in the need for an update to the Page serotyping scheme.

Kume and co-workers (1983) proposed a serotyping scheme in which AP isolates are treated with thiocyanate and sonicated allowing the exposure of the hemagglutinins (5,60). The antigen detected in the hemagglutination-inhibition (HI) assay is the “HA-L,” which is heat labile, trypsin sensitive, hyaluronidase resistant, and active against glutaraldehyde-fixed chicken erythrocytes. HA-L has several distinct serologic forms, which could be classified into three serogroups (I, II, and III) and seven serovars (HA-1 to HA-7) (60). The seven different serovars were grouped as follows, HA-1 to HA-3 fall under Serogroup I, HA-4 to HA-6 fall under Serogroup II, and HA-7 falls under Serogroup III. An additional form was later discovered and named HA-8, and was placed into Serogroup I (61). In 1990, a seminal study published by Blackall et al. (62) reported the discovery of another HAL form (HA-9) and placed it under Serogroup II. Most importantly that study found that Kume Serogroup I resembled Page Serovar A, Kume Serogroup II resembled Page Serovar C, and Kume Serogroup III resembled Page Serovar B (62). Therefore, the Kume Serogroups I, II, and III were renamed Serogroups A, C, and B, respectively. Additionally, Serovars HA-1 to HA-9 under each of the serogroups were renamed accordingly (Table 1) and remain as the nomenclature used for AP serogroups and serovars to date. When comparing the HI to the plate agglutination method, HI was able to identify 21/23 previously unidentified isolates by plate agglutination (62). Therefore, Blackall and co-workers proposed the use of HI assay instead of plate agglutination for AP serotyping. The main utility of these typing systems is vaccine selection, because there is lack of cross protection between serogroups. One additional utility for serotyping assays is the ability to track the infection, determine the scale of and better understand risk events for specific outbreak transmission. However, AP serotyping has its challenges. Standardized reference antisera and/or AP strains are not widely accessible. Even when HI is properly conducted, field isolates occasionally are not typed in any of the currently known reference serovars and reported as untypable (63,64). AP isolates genetically or antigenically distinct from standard isolates can lead to IC outbreaks because of failure of protection by commercial vaccines. Multiple examples in different parts of the world have been published documenting such vaccination failures (36,63,65,66,67). Recent advances in sequencing technologies and molecular characterization of AP bacterial isolates (68,69) can address some of these issues, better identify and characterize novel isolates, and potentially predict if they are divergent enough to escape vaccine protection. We will expand on recent advances in AP sequence typing in the Genotyping of AP for serotype prediction and epidemiologic investigation section of this review.

A number of serological tests have been developed to detect antibodies against AP in chickens for both diagnosis and for vaccine evaluation. These tests include two different HI assays (70,71), a plate or tube agglutination test (72,73,74,75), an agar gel precipitation test (AGP) (70,76) and a latex agglutination test (77).

Agglutination titers can be detected in chickens as early as 7–14 days after vaccination or infection and usually titers last for 3 mo to a year, respectively (70,73,74,76) . One of the main drawbacks of the agglutination tests is not being serovar-specific (59). Therefore, agglutination titers do not correlate with protection against a specific AP isolate (72,75,78). Similarly, AGP test can detect antibodies as early as 2 wk following infection or vaccination. Antibodies can be detected for at least 3 mo (70,72). AGP test can be a sensitive assay; however, results obtained from AGP tests, similar to agglutination tests, are not serotype specific (72).

For a better predictive correlation between detected antibodies and their protective ability type-specific assays, i.e., HI should be used. The first HI assay developed uses freshly cultured and washed AP bacterial cells to detect Type I (Serogroup A) antibodies in infected and vaccinated birds (70,79). This assay, while more specific, compared to the agglutination and AGP tests, can only detect antibodies against Serovar A (80). The second HI assay detects antibodies produced against HA-L antigen, the same antigen used for the detection of Kume’s serovars (71). This HI assay can be used to detect antibodies in chickens that were vaccinated with inactivated vaccines for both Serogroups A and C (71). Surprisingly, this assay is able to detect only antibodies from naturally infected chickens with Serogroup A, but not Serogroup C (81). In spite of its limitation, this assay could be a good option for vaccine antibody response evaluation.

The latex agglutination test is not commonly used; it does, however, offer serovar-specific agglutination results in vaccinated chickens. Antibody titers detected by latex agglutination assay peak between 1 and 2 wk and decrease over time (77). The relationship between latex agglutination antibody titer and immunity of vaccinated chickens are yet to be evaluated; furthermore the accuracy of the test in naturally infected chickens is also unknown (5) .

In general, serological tests developed for IC are not commonly used, partially because they are inconsistent, unscalable, and lack specificity. In that regard, more standardized and scalable tests, such as ELISA would improve the accessibility and the utility of IC serological testing. To date, no commercial ELISA kits are available to use for IC diagnostic or surveillance purposes.

Chickens exposed to natural or experimental infection become resistant to reinfection as early as 2 wk postexposure (72) with either homologous or heterologous serovars (82). However, immunity induced by the use of inactivated whole bacterins is serogroup specific (83). A proposed explanation to broad protection conveyed by live replicating bacteria could be that there are cross-protective antigens produced during in vivo replication of the bacterium that are not activated during in vitro replication. Given its importance in HA activity and in serotyping of AP, the HMTp210 surface protein has long been considered an immune dominant antigen. Correlation between HI titers and serogroup-specific protection has been established (71,81,84,85). However, protection against AP could be achieved even without high HI titers (86,87) suggesting that other antigens could also play a role in immune protection. For example, the capsular polysaccharides are immunologically active (88) and can provide serotype specific immunity (80).

In a series of studies, immune-related gene expression was followed after infection and development of clinical signs (89,90,91). It was demonstrated that the innate immune response plays a significant role in the outcome of the disease. The initial recognition of the pathogen was mediated by Toll-like receptors (TLR) 2 and 4. During the early stages of the disease in presence of mild clinical signs, the immune response was driven by TLR4 in the nasal mucosa, followed by a T-helper 2 (Th2) response and activation of the MyD88-dependent pathway, resulting in inflammatory cytokine production and more severe clinical signs (90). Boucher and co-workers in 2015 (90) demonstrated upregulation of immune genes as clinical signs progressed. This finding might explain, partially, some differences in virulence presented by some serovars for example, C-3. The reviewed data also suggest that oxidative stress might be involved in the initial response to the challenge (89).

These are molecules important for the microbial pathogenesis and its ability to cause disease (92). Pathogenicity of AP has been attributed to a number of virulence factors (93,94,95,96). In this section we will review the main factors including: the hemagglutinin antigen (97), the capsule (98,99), the outer membrane (80,96), and putative toxins.

The hemagglutinin (HA) antigen is very important in the immunogenicity and pathogenicity of AP (65,100). Two genes, hagA and HMTp210 were first believed to encode an outer-membrane protein that functions as the HA antigen (101,102). However, several studies have reported that the HMTp210 gene encodes the protein that behaves as HA (84,85,103,104). This was confirmed as AP isolates that lacked HMTp210 gene also lost their HA activity not eliciting HI antibodies when inoculated into chickens (103). The HA antigen is a trimeric adhesin that binds to the surfaces of infected cells and contributes to biofilm formation (100,101,105). Initially, it was reported that the Serogroup B strains Spross and 0222 were nonpathogenic, potentially due to the lack of HA (74,97). However, it was later discovered by Kume and co-workers that these strains possess the HA antigen (106) adding complexity to the pathogenic determination. The HMTp210 gene sequence and its relation to serotyping is explained in more detail in the Genotyping of AP for Serotype Prediction and Epidemiological Investigation section of this review.

The polysaccharide capsule plays an important role in virulence, as it is involved in colonization as well as lesion formation when IC infection occurs. In an early study investigating the ability of AP to adhere to chicken embryo fibroblasts, it was discovered that a “fuzzy material” present on pathogenic cells increased the bacterial adherence to the host cells (107). A few years later, Sawata et al. (99) investigated two phenotypic forms of one strain of AP, the mucoid (M) encapsulated and rough (R) unencapsulated variants. His characterization showed that while the M variant was pathogenic the R variant was not. Pathogenicity could be explained by the presence of capsule allowing the bacterium to adhere to the surface of the nasal mucosa and effectively infect the upper respiratory tract (32).

Gram-negative bacteria release outer membrane vesicles (OMVs) during infection. These contain proteins involved in cell interaction, activation (108), cytokine secretion (109), and apoptosis (110,111). It has also been reported that OMVs can damage host tissues and innate defense systems, causing inflammation, allowing pathogens better colonization of the host (111,112,113). These vesicles can also contain virulence factors (95,114). Separate from OMVs and similar to other Gram-negative bacteria, at least five different secretion systems have been described for AP. Their goal is to release molecules used in colonization, communication, and to obtain nutrients from the environment (115,116,117).

Repeats-in-toxins (RTX) are members of a rapidly expanding family of proteins (118) commonly produced by Gram-negative bacteria (119). The presence of 110–120 kDa putative RTX proteins (120,121,122) along with metalloproteases (123) has been reported in AP. On mucosa, these proteases can degrade host tissues and structures leading to edema and inflammation (124). The metalloprotease produced by AP did not degrade IgG, but still contributed as a virulence factor to AP pathogenicity (95,125). The avx operon is one of the most conserved RTX operons and has been found in some AP strains (122). Pan and co-workers (121) discovered a novel toxin named Avxl. This toxin was produced by Serovar C strains isolated from Taiwan. This toxin did not show cytotoxicity against peripheral blood mononuclear cells. However, a variant of Avxl, the bivalent serine-protease-RTX toxin named Avibacterium RTX toxin (AvxA) was reported to have a strong cytotoxic activity against avian macrophages (122).

When investigating whole-genome sequences (WGS) of two AP reference strains (221 and H18) along with two AP field isolates, the cytolethal distending toxin (CDT) gene was detected (126). This toxin contributes to the persistence of infection by impairing the host’s ability to eliminate the pathogen (127).

Similar to any other pathogen, certain AP isolates have higher or lower virulence levels. The pathogenicity of specific AP isolates has been studied (7,32). In general, Serogroup C strains are more pathogenic when compared to other serovars (33,94,128,129,130). On the other hand, from the 1960s to the 1980s, Serogroup B is regarded as the least pathogenic (74,97). However, subsequently, many Serogroup B isolates have been proven to be highly virulent and variant B strains have been reported to escape the immunity of vaccinated birds (66,67,106,131). Additionally, NAD-independent isolates have lower virulence compared to NAD-dependent isolates (33) .

Whole-cell inactivated bacterins are the only commercially available vaccine against IC (67,105). These are usually bivalent, trivalent, or tetravalent, representing two or three serogroups, that is, A, B, and C (67,87,105). The rule of thumb is that each serogroup represents a separate immunovar, meaning that immunity induced by inactivated vaccines provides protection to the serovars within the same serogroup (serogroup specific) (83). However, multiple reports indicate that protection provided by inactivated vaccines within each of the immunovars is not always complete (18,64,65,132). This represents a challenge to producing universally protective vaccines against all different serovars, in different geographical regions across the world (64,132,133). This lack of cross-protection can render vaccine programs ineffective with the emergence of variant AP strains. However, this is usually corrected by using field-isolated variants in autogenous inactivated vaccines. In South Africa an increase on the detection of Serovar C-3 occurred because of vaccines lacking this serovar (64). The situation was controlled when the C-3 isolates were incorporated in vaccination programs (36). Identifying the specific serovars causing disease in a certain region can ensure success of a vaccination program. This emphasizes the need for a system that can accurately identify AP serovars (7,105). In this regard, molecular characterization of AP isolates can potentially offer a solution for accurate serovar prediction.

IC is considered an important disease in commercial poultry in countries with a tropical or subtropical climate (18,27,134,135,136). However, in the past two decades IC breaks in commercial poultry have extended further north in Europe. In addition, since 2018 in North America, the disease has moved north into midwestern states. This geographical expansion of the disease has created challenges to a commercial poultry industry that was not prepared to facing and controlling IC. Therefore, —there was a lack of epidemiological understanding, an absence of vaccine stocks, and a lack of laboratories capable of diagnosing and typing the disease. This created a need for applied research to detect, characterize, and understand the bacterium behavior and additionally to make sure that the vaccine choice was adequate in lieu of this unprecedented outbreak.

Until 2008, reports of IC in the commercial poultry industry in Europe were scarce. However, during the nearly two decades since, there has been an increase in the incidence of AP causing clinical IC outbreaks in commercial poultry in the continent. Reports indicate that the earliest cases of IC in commercial poultry in Europe appear to be in the Netherlands in 2008 (137) with more outbreaks in commercial poultry in the following years. In 2010 Welchman and co-workers (138) reported for the first time a clinical outbreak of IC in hobby and small mixed layer flocks in the United Kingdom. They also speculated that the outbreak was related with the introduction of carrier birds through the trading of hobby and backyard birds. Additionally, an increase in cases was noticed in poultry in Sweden (139) and a more recent report described the involvement of a Serovar A strain in broilers in Poland (140). To understand this IC outbreak, 18 isolates from outbreaks in layer flocks over different geographical areas in the Netherlands were studied in 2019 (141). Serovar A-1 was dominant in these outbreaks; however, Serovars A-2 and B-1 were also detected. In the same study, the pathogenicity of the A-1 serovar was tested in chickens, under experimental conditions, and the clinical signs were reproduced with moderate clinical score. To understand the epidemiology of the outbreak, the study compared AP sequences focusing on a 0.5-kb fragment of the HPG2 region (142) in addition to the enterobacterial repetitive intergenic consensus-based polymerase chain reaction (ERIC-PCR) typing method, which was traditionally used for AP epidemiological studies (62,128,143,144,145,146). Both HPG2 and ERIC-PCR typing methods agreed, showing the involvement of five different genotypes in these outbreaks. However, one genotype was dominant. Furthermore, AP isolates in these outbreaks showed a nucleotide change from G to A at nucleotide position 1516. This nucleotide change led to a mismatch that altered the detection of the AP when using the nonmodified PCR described by Corney et al. (147). After 2019, Serovar C-4 was frequently involved in IC outbreaks in layers in the Netherlands. An autogenous vaccine based on this isolate as well as a commercial registered trivalent vaccine was shown to be effective in reducing clinical signs and was used in the field to control these outbreaks (148).

IC has always been considered an endemic disease of chickens in Florida, Texas, and California, but was rare in mid-western states (149). However, in December 2018 the disease emerged in Pennsylvania and rapidly spread in the commercial poultry population (21). Between December 2018 and December 2019, 68 farms were affected by IC, involving approximately 14 million birds (21). Most affected farms housed layer chickens (37/68), and a smaller number of broiler flocks (19/68), pullet (10/68), and layer breeder farms (2/68) were affected. Layer flocks ranged in age from 18 to 110 wk, with 61% being over 50 wk of age (average: 54.8 wk). Pullets ranged in age from 8 to 16.5 wk (average: 11.9 wk) and broilers ranged in age from 33 to 50 days (average: 39.9 days). Since the introduction and use of vaccines in 2019, the incidence of IC cases has been greatly reduced. However, given the complex and diverse poultry industry in Pennsylvania, IC became endemic and new cases continued to be reported to date.

In most flocks, the first clinical sign is a significant drop in feed and water consumption followed by drops in egg production with subsequent respiratory disease and increased mortality. Affected birds appeared depressed and had variable levels of facial swelling centered around the infraorbital sinus area, swollen wattles, mucoid nasal discharge, and respiratory rales. Other lesions, which are often present in conjunction with secondary bacterial pathogens, included caseous pericarditis, caseous perihepatitis, mucoid or hemorrhagic tracheitis, caseous material in the lungs, caseous airsacculitis, egg yolk peritonitis, and occasionally subcutaneous caseous material in the neck. The course of the disease, egg production parameters, and mortality varied between farms. In layer flocks, clinical disease lasted up to 8–9 wk. Egg production drops in layer flocks ranged between 8% and 43%, with mortality approximately 3–4 times higher than average. The range of mortality in broilers was 1.9%–25.6% with condemnation due to airsacculitis ranging from 0.25% to 3.40%. Weight and uniformity issues were apparent at processing because of the decrease in feed and water consumption.

Presumptive diagnosis of IC was based on clinical signs, lesions, and confirmed via bacterial culture and/or quantitative real-time PCR (qPCR) (150). During 2018–2019, AP was isolated not only from the upper respiratory tract (infraorbital sinuses, choanal cleft, and trachea), but also from heart, liver, and air sac lesions. In addition to AP, other bacteria were also isolated. The main bacterial co-infections included Escherichia coli, Ornithobacterium rhinotracheale (ORT), and Gallibacterium anatis. Additionally, Avibacterium endocarditidis was isolated from 12% of the cases.

Delmarva is a peninsula on the east coast of the United States, and is divided between three states,—Delaware, Maryland. and Virginia. It is one of the largest poultry production and most densely populated areas in the United States. Given the connections between the poultry industry in Delmarva and Pennsylvania, monitoring efforts started after seeing an increase of cases in the neighboring state. The first cases reported in Delmarva were in winter of 2019. In May 2019, qPCR was adopted as a confirmatory testing for IC (147). The first backyard flock detection was made in July 2019 in Delaware. In a retrospective analysis, AP was detected in samples obtained from nine backyard flocks prior to September 2019, from cases with and without respiratory disease. In September 2019, the first Maryland table egg layer farm with AP was reported, followed 2 wk later by another farm nearby in Delaware. The movement of Pennsylvania raised pullets was thought to be the route of introduction to the Delmarva region. An epidemiologic investigation listed vendors (trash, propane, deliveries, and manure haulers) in addition to employees of both egg farms as possible mechanisms of transmission between the two layer sites. In January 2020, AP was detected in a commercial broiler flock in Delaware. The broiler index case presented with no facial swelling. At necropsy, caudal abdominal airsacculitis was observed. Concurrent detections of infectious bronchitis virus and ORT, were common during the winter months. Subsequent cases of AP in broiler flocks seemed to be linked to the movement of workers between farms. In the initial broiler cases, mortality went from 1.5/1000/day to 20/1000/day. Investigation of the relatedness of IC cases on Delmarva using multilocus sequence typing (MLST) (151) showed that backyard cases were unrelated to commercial cases, and supported the epidemiologic evidence that the Delmarva cases were related to the Pennsylvania outbreak.

For several months, IC seemed to be limited to Pennsylvania and Delmarva. However, in December of 2020, one house in a large multiage layer complex in Ohio showed typical clinical signs of IC. To stamp out the infection, the flock was depopulated. Unfortunately, a few days later, clinical signs were observed in other houses, spreading throughout the complex. Similar to the Delmarva outbreak, sequence typing information indicated its relatedness to the Pennsylvania outbreak; however, an epidemiological link was not established. Significant biocontainment efforts as well as vaccination of all the incoming pullets into this multiage layer complex with a double dose of inactivated vaccines was carried, resulting in the containment of the outbreak in this complex in Ohio. In November 2022, another layer site in Ohio was positive for IC. This second Ohio outbreak did have a direct epidemiological link to the Pennsylvania outbreak, through bird movement. In May 2023, again because of bird movement, IC spread to a poultry densely populated area in western Ohio and eastern Indiana that is populated with approximately 25 million layers. Once in the area, the outbreak spread rapidly (within 6 wk) to approximately 70% of the layer population. In September 2023, IC spread to one multiage layer complex in eastern Iowa. This latest spread to Iowa was also connected to layer pullet movement from Ohio. Sequence typing confirms that all recent IC outbreaks in the midwest are related to each other and to the original outbreak in Pennsylvania. Based on the concatenated sequences of region 1 and the hypervariable region (HVR) of HMTp210 gene (69), the predicted serotype of all the cases in the midwest and Delmarva is the C-1 serovar. Along with epidemiologic evidence, this outbreak is spread from commercial-industry-to-commercial-industry primarily through infected birds’ movements. Although there are backyard cases detected in proximity to this spreading outbreak, the sequencing as well as epidemiologic investigation data do not suggest there is IC cross transmission between backyard and commercial poultry.

In late 2020, after the spread of IC from Pennsylvania to Ohio, an outbreak investigation was conducted to define AP dissemination in the region. Several multiage layer complexes were screened for AP using pooled choanal swabs (five birds per pool/six pools per flock/site) with the qPCR assay available at the time (147). Samples were collected from apparently healthy layer flocks with no history of IC. Despite that, multiple flocks yielded positive qPCR results, with moderately high C_T values (25.6–34.1). These results were surprising, because of the absence of IC clinical signs in naïve susceptible chicken population. These qPCR tests were repeated using a recently developed assay targeting a genetic segment unique to AP in the recN gene (150). The new assay confirmed the previous results with comparable C_T values. To investigate further, 30, 10-wk-old naïve susceptible sentinel birds, with no previous exposure or vaccination against AP were placed into these caged layer flocks. Sentinel birds were qPCR negative before placement. Fifteen days after placement, the sentinel birds were submitted for necropsy and choanal swabs pools were collected (five birds per pool) and tested by qPCR for the presence of AP (147,150). No clinical respiratory disease was observed during gross pathology. However, qPCR yielded positive results, with C_T values similar to the layer flocks. Pure NAD-dependent AP was isolated from both the sentinel birds and caged layer flocks and they were dubbed non-pathogenic Avibacterium paragallinarum (npAP). WGS was performed on the acquired AP isolates. Distinct sequence differences were detected in these strains compared with commonly isolated AP. Unique HMTp210 insertions, the lack of capsular polysaccharide loci (including hctA) were some of the findings. These strains are still considered within the AP species, with a genomic average nucleotide identity (ANI) score of 96% (152,153,154,155,156); however, because of the large insertions in the HMTP210 gene, serotype prediction by sequencing was not possible. The presence of these strains in any chicken flock can lead to diagnostic confusion, with severe consequences in the face of a fast-spreading IC. A prevalence study was conducted to estimate the prevalence of these strains in the commercial layer population across the United States (157). A total of 710 oropharyngeal (OP) swab pools (five swabs/pool) were collected and tested from 80 naïve-healthy laying chicken sites across 13 states. In addition, positive qPCR samples were re-tested by a newly developed qPCR assay able to differentiate these strains from conventional strains (158). Results showed that 231 out of 710 total pools were positive for npAP (32.5%) representing 28 out of the 80 total sites (35%). Multiage layer complexes showed the highest percent positivity (23/40 or 57.5%) compared to all-in-all-out production systems (5/40 or 12.5%). This pilot study suggests a prevalence of over 30% for npAP in naïve-healthy layer flocks in the United States. Currently, a combination of isolation and WGS is the only diagnostic approach capable of completely differentiating between these two AP populations, which indicates the immediate need for improvements in the current diagnostic assays.

Commercially available licensed vaccines against AP in the United States are inactivated whole cell bacterins containing the three Serogroups A, B, and C. On the other hand, autogenous vaccines in the United States are formulated using isolates representing local or regional AP strains. Before the 2018–2019 outbreak in Pennsylvania, the use of commercial inactivated IC vaccines in the United States was limited to states where the disease is endemic. However, in the face of such large outbreak vaccine supply was short. To fill the supply gap, producers resorted to the use of autogenous inactivated vaccines. To formulate autogenous vaccines, local isolates should be characterized, that is, serotyped and pathotyped before being included in these products (159). Historically, the inclusion of more than one strain per serotype in the vaccine had provided successful results (51,67,133); this was especially true for B and C serotypes, which show increased diversity (66,132). Two doses with a 4-wk interval is needed for a proper and sustained protection (160); the two doses are usually applied between 10 and 20 wk of age (160) and even earlier when the challenge is higher. One of the challenges facing autogenous vaccine formulation, is the inefficient growth of wild type AP in vitro, compared with laboratory adapted strains such as Spross, H-18, or Modesto. This might be responsible in part for the lower antigenic load sometimes observed in autogenous vaccines. De Bliek reported changes in the shape of AP after subsequent passages in artificial media (161). Similar morphological changes were correlated to variations in virulence by Sawata and Kume (32), which can be associated with reduced immunogenicity. Bacterial destruction has also been suggested during the inactivation, homogenization process, or storage conditions (162). Future research should focus on these associations and bring more guidance to autogenous vaccine producers in terms of growth, virulence, antigenicity, proper inactivation, adjuvants, and storage of these products.

Two trivalent and one tetravalent commercially available vaccine were evaluated in their efficacy to prevent disease when vaccinated birds are challenged with U.S. C and A serotypes. In addition to conventional A, B, and C strains the tetravalent vaccine included a B variant serovar. The protection of SPF pullets against a contemporary C serovar challenge was stronger when birds were vaccinated twice at 7 and 11 wk of age (163,164). Similar results were observed when one of the trivalent vaccines containing Serovars A (083), B (Spross), and C (H-18) was tested against the challenge with an A contemporary strain obtained in the United States. A significant reduction in shedding was detected in pullets vaccinated twice (164). Studies on vaccine administration have shown that intramuscular and subcutaneous routes give similar immunization results (165); other authors have shown better antibody responses on birds where vaccines were applied IM, followed by SC in the inguinal fold, and finally SC in the base of the neck (166).

Subunit vaccines have shown a close correlation between the HI antibody levels and their protective activity (74). However, the challenge with using subunit vaccines is generally the low antigen load per dose (84,85,102). The HVR of the HMTp210, from A and C serogroups, was targeted to produce subunit proteins and used to immunize chickens. Conflicting results were reported on the efficacy of these HVR subunit proteins to produce HI protective antibodies (103,104). A potential explanation for these differences could be antigenic load and adjuvant differences in different experiments. Unlike other poultry bacterial pathogens such as Mycoplasma gallisepticum, Mycoplasma synoviae, Pasteurella multocida, E. coli, and Salmonella, no commercially available modified live vaccines (MLVs) against AP are available to the poultry industry. There is evidence that AP MLVs can provide a quicker onset of a broader spectrum protection (72,82). Additionally, MLVs could have the advantage of being administered via mass application methods. Research in the field of vaccine development and application is needed for a better prevention and control of this pathogen.

Latin America (LA) in this review is used to refer to Mexico and the entirety of Central and South America. Demand for poultry products throughout LA is rapidly growing (167). However, IC is endemic in all LA countries, and clinical disease is reported in layers and broilers (160,168,169). The highest incidence of IC outbreaks in LA is mostly in warm geographies; however, it shows seasonality with higher incidence in fall and winter (160). Rains and high humidity may increase the chances of survivability of AP in the environment. The serovar distribution in LA can be described as follows: A-1, A-2, B-1, C-1, and C-2 have been identified in Mexico (170) and Peru (171); A-3 identified in Brazil (60); A-3, B-1 and C-1 in Ecuador (172); B-1 in Panama (135) and Costa Rica (69). Of note, un-typable isolates have been reported in Argentina (63); Brazil (64); Ecuador (172), and Mexico (170). Given the limited accessibility to conventional serotyping assays, different techniques have been used for molecular typing. The concatenated sequences of region 1 and HVR of HMTp210 gene resulted the clustering of all Mexico isolates into two clusters: Cluster I including all Serovar A-1 isolates and Cluster X including all C-1 isolates from Mexico. Additionally, two isolates from Costa Rica identified as B-1 corresponded to a different cluster than an isolate from Guatemala identified as Serovar B-1 (69). Isolates from Costa Rica have been characterized by the sequencing and analysis of the HVR of HMTp210 gene and they appear to be related to the reference strain H18 and 0083 (173).

Commercially available vaccines in LA are frequently formulated with international reference strains (160). Nevertheless, commercial vaccines may be produced using local strains. Certain AP serovars may become more prevalent than others (63,169,174,175). In these situations, commercial vaccine efficacy is questioned; for example, only one among all the evaluated vaccines provided good protection level in vaccinated chicken against a specific C-1 isolate from Mexico (175,176) . Expectedly, this isolate was reported to have high virulence (177). Similarly, multiple variant B strains with differences in virulence and antigenicity have been reported (67,174,178,179). NAD-independent strains have been reported in Mexico and Peru (53,180,181). Therefore, and to improve protection against antigenic variants, some vaccines may contain more than one strain from the same serogroup; the most common examples are quadrivalent vaccines including a B variant strain (64,66,67,174,178).

Antibiotic use is a common practice to combat IC outbreaks; continuously monitoring AP antimicrobial susceptibility patterns and optimizing their use during an outbreaks is required (182,183). Differences in the antimicrobial sensitivity patterns for AP isolates obtained in Ecuador, Mexico, Panama, and Peru have been evaluated, finding isolates showing multidrug resistance (MDR) phenotypes in 57.5% of the samples (183). However, in Mexico, AP isolates from Sonora State were susceptible to the majority of antibiotics tested (184). Differences in Minimum Inhibitory Concentration (MICs) have been detected between isolates from different continents, with isolates from the United States and Europe showing higher MICs than isolates from Africa. Additionally, isolates obtained in America showed elevated MICs for aminoglycosides, quinolones, tetracyclines, and/or trimethoprim/sulfamethoxazole (185).

Asia. IC was initially reported in India in the 1950s, making it one of the earliest Asian countries to document the disease (186) causing significant economic losses due to decreased egg production, growth retardation, and treatment (187). In Malaysia, the first detection of AP was in 1960 by Chong (188). In Japan, IC was first reported and isolated in 1962 (189). Early research on IC was extensive in Japan. Several recognized reference strains were isolated there, that is, strains 221 (serovar A) (189) and H-18 (serovar C) (190). From the 1960s to the 1980s, the strain H-18 (C-1) was the most prevalent serotype in Japan (60). At the beginning of the 21st century, most reports were mainly about the progress of bacterial molecular typing and other research aspects, whereas case reports became rare. Additionally, in 2013, there was a report in quails showing typical clinical signs of IC. A total of eight AP isolates were isolated from quails, displaying 100% resistance to drugs such as ampicillin, neomycin, furazolidone, streptomycin, and cephalexin (20).

Since 1980, cases of IC have been reported in China; however, Serovar A was first reported in 1987, Serovar C in 1993, and Serovar B in 2003 (192). Prior to 2000, the majority of isolated strains belonged to Serovar A. Since then, Serovar C strains have been increasing in frequency. Between 2012 and 2017, Serovar B was most commonly isolated (131). In recent years, Serovars A and C have become predominant (191,192). Lately, poor vaccine protection has been reported, indicating that variants might be causing problems (105,193). Among the isolated strains, the majority are NAD-dependent, with a few reports of NAD-independent strains (194).

In Korea, only NAD-dependent AP strains belonging to Page Serovar A have been identified since it was first reported in the 1980s (28,195). The only available vaccines were bacterins of the strain 221. These remain effective in controlling the infection (personal communication). With the prohibition of antibiotic use in feed in 2011 (Korean Ministry of Agriculture, Food and Rural Affairs), AP can be more frequently isolated from chicken flocks with a suspicion of IC. The field-isolated strains showed resistance to enrofloxacin, ampicillin, gentamicin, cloxacillin, and sulfamethoxazole-trimethoprim (28). In 2017, Korea reported the first case in Asia of nontypeable variants of AP with NAD-independent variant strains, and they were more resistant to antibiotics compared to NAD-dependent strains (54) .

The disease has been reported in south Asia since 1950s (186) and to date is present in India (37,196), in Pakistan (197,198), and Bangladesh (10,199). Additionally, AP has been reported to circulate in all of southeast Asia. In Indonesia, reports of IC are in all ages, including village chickens (kampung) (18,200). By 2000, all three Page serovars (A, B, and C) had been confirmed in Indonesia. Given that the majority of IC vaccines used in Indonesia contain only Serovars A and C, the detection of Serovar B in chickens suggests that currently available commercial vaccines may not effectively address the incidence of IC (18). Furthermore, reports of quail infections with Serovar B have also emerged (19). Other southeast Asian countries that reported the disease include Thailand (87,201,202), the Philippines (203), Vietnam (204), and Malaysia (205). In West Asia, IC has been reported in Iran (206,207,208) in Iraq (29) and in Turkey (209).

Africa. IC was first reported in 1968 in the Republic of South Africa (SA) (210) and quickly became the most impactful poultry bacterial disease in the country. Since the early 1970s, vaccines have been used in SA (210). Before any IC vaccines were used in SA, there was an almost equal distribution of Serovar A-1, C-2, and C-3 strains. In the early 1980s, vaccination against IC started with a bacterin containing Serovar A-1 (210). Later, additional vaccines were introduced into SA containing Serovars A-1, B-1, and C-2. In the 1980s vaccines were no longer effective in preventing the disease in commercial poultry (33). Bragg and co-workers in 1996 (33) attributed the vaccine failure to a shift in the dominant AP population to serovar C-3. The inclusion of the SA C-3 strain successfully controlled the disease (211,212). NAD-independent isolates were first isolated in Kwa-Zulu Natal, in SA in 1989 (38,39,40). Other African countries such as Zimbabwe (129), Morocco (44), Uganda (213), and Egypt (214) have reported the occurrence of cases.

A general disadvantage of all serotyping assays is being labor and time consuming, with low repeatability within and between laboratories (63,64,172). Additionally, serotyping assays often fail to capture the diversity of the entire bacterial species, resulting in a number of nontypeable strains (63,64,172). A specific challenge for AP serotyping is the limited availability of antigens and antisera available in only a handful of laboratories around the world. Sequence typing can be a potential solution to the lack of availability and reproducibility of the conventional serological typing of AP.

The HMTp210 protein is a 210-kDa Trimeric autotransporter adhesin (215), which plays a key role in hemagglutination, and contributes significantly to the immunogenic and pathogenic characteristics of AP (100). The length of the HMTp210 gene is nearly 6 kb, and the majority of full-gene sequences deposited on GenBank are 6117 bp long, ranging from 6066 to 6261 bp (14,68,84,85,216,217). The initial 20%–25% of the HMTp210 gene, region 1 (nucleotides 1 to ∼1,300–1,500), bears most of the genetic variability that can be used to differentiate between Page Serogroups A, B, and C (218). A HVR between nucleotides 3,319 and 4,939 was determined as a potential target for the genotyping of AP (68). There is a broad correlation between the HVR gene phylogenetic clustering and the Kume serovars (69,219,222,224), but it was inaccurate in predicting all of the nine serovars, particularly those belonging to Serogroup C (179,220). Nevertheless, Buter et al. (69) showed that when HVR sequence was concatenated with the initial 1200 bp of HMTp210 gene (region 1) resulted in a much more accurate prediction of the nine currently known Kume serovars. This concatenated sequence typing assay uncovered and corrected some of the conventional serotyping discrepancies previously reported for specific B-1 and C-1 isolates (179,220). Furthermore, this assay shows the potential ability to resolve and classify some of the previously untypeable isolates. In this study, AP was classified into 14 genotypes (GTs) (GTI–GTXIV) (Table 2) within which each of the reference strains was separated in a different GT, indicating a high correlation between GTs and Page and Kume serovars. Therefore, this sequence typing scheme can accommodate the current and the future additional diversity of serovars beyond the known Page and Kume serovars. Additional efforts to use the entire sequence of HMTp210 gene (217), proposed to classify strains into four genogroups: I, II, III, and IV, that somewhat correspond to Page Serogroups B, C2, C1, and A. The C serogroup is divided into two clusters—Genogroup II containing mostly North American C-2 strains, and Genogroup III containing various C types detected worldwide (217).

Although much of the focus on the molecular typing of AP has been driven by the need for vaccine selection, there has been limited progress in molecular typing to understand the relatedness between AP strains and the epidemiology of outbreaks. In the past, DNA fingerprinting schemes such as ERIC-PCR and single locus-based schemes such as HPG2 and HagA have been utilized (141,163,184) with challenges. Therefore, the recent re-emergence of AP (141,152,184,221) led to the simultaneous and independent development of three different multilocus sequence typing (MLST) schemes, each using a different number of loci and targets. The first scheme (151) was developed using six loci and could classify 75 samples from 13 different countries into 31 unique sequence types and identified eight clonal clusters. It demonstrated greater discriminatory power than existing HPG2-based methods and showed high congruency with ad hoc core genome MLST results for WGS samples aligning with the epidemiological data such as geographical origin. The obtained data suggest that outbreaks in commercial poultry in the United States are caused by closely related events. Moreover, backyard outbreaks in Delmarva, were different from each other and different from the commercial outbreak strains. Additionally, this scheme was able to differentiate between prevalent outbreak strains and nontypical strains reported in some flocks (152). The second MLST scheme (222) was developed to utilize five loci and resulted in 29 unique alleles and 11 sequence types. Limited information is available about this scheme. The third MLST was developed by Guo et al. (223) and is based on sequencing fragments of seven housekeeping genes, the scheme was used to type 59 isolates and WGS available on GenBank representing serovars (A, B, and C) from five different countries. Strains were classified into eight sequence types (STs) and grouped into three clonal complexes. The data of this MLST suggest that the chosen loci were not sufficiently variable (224). The limited sample diversity likely contributed to the low discriminatory power and regional bias, potentially restricting the scheme's applicability in other contexts.

Currently, there is no well-established method for typing AP based on WGS, such as a core genome multilocus sequence typing (cgMLST) scheme. However, Roh et al. (225) generated WGS data from 46 AP isolates obtained during a Pennsylvania IC outbreak in 2019 and conducted a pan-genome analysis. They identified a total of 4,284 genes, with 1,396 core genes present in all 46 isolates. Although this analysis provided insights into the relatedness of these isolates, the limited diversity of the sequenced isolates poses a significant limitation for using these core genes to analyze other AP outbreaks. Therefore, developing a standardized and reproducible method for typing AP based on diverse genomes set, including the reference isolates, is still necessary. Sequence typing assays are increasing in popularity due to their power, affordability and flexibility. Despite this, to adequately work they depend on databases containing good quality sequences to perform comparisons.

Bacterial WGS is poised to surpass traditional bacterial typing methods, refining epidemiological investigations and clarifying bacterial classification. The close genetic relationship among Avibacterium species (226) has caused a significant confusion about the accurate classification and characterization of multiple AP isolates. Since the initial identification of AP there has been a continuous emergence of variants and new Avibacterium species (152,227).

Aside from AP, the number of sequenced genomes for other species within the genus Avibacterium remains very limited (228). Even for AP, the most important species for the poultry industry, the generation of sufficient number of genomes has lagged behind that of many other avian pathogenic bacteria (Table 3). The currently available genomes for different Avibacterium species are limited in number and originate from only 10 countries. Additionally, the quality of some genomes is often suboptimal, as revealed by quality assessments (Supplemental Table S1). For example, multiple genomes (n=10) have lower depth coverage than the recommended standard (≥50×) (229) for high-quality assembly. Furthermore, eight AP whole genome sequences (strains: Z1S-2-1, Z2S-2-1, AV37, AV36, AV20, AV12, AV11, and IVL_IC.A) were mislabeled as AP, whereas ANI score calculations suggested their reclassification as another species (Supplemental Table S1).

Overall, among the 88 genomes confirmed as AP (ANI > 95 to AP type strain), the whole genome size ranged from 2,337,807 to 2,869,246 nucleotides, with an average size of 2,570,033.4 nucleotides. The G+C content varied from 40.8% to 41.3%, with an average of 40.9%. While draft genomes offer valuable preliminary data on AP genomes, complete genomes present a more accurate representation of the bacterial genome. This includes all coding and non-coding regions, ensuring that no genetic information is missing or misassembled. All of the complete genomes for AP (n = 15) possessed only a single circular chromosome with only one strain (AG21-0333; GenBank accession CP104914.1) has 2 plasmids (GenBank accessions CP104915.1 and CP104916.1). There were some previous reports for other AP strains harboring one or two plasmids (230,231) but genome sequences for these strains are not available. Within the 15 AP complete genomes, two genomes from npAP (152) exhibited multiple genomic differences compared to ESV-135 reference strain. However, two of these differences are key findings that could explain the lack of pathogenicity of the npAP (158).

Based on the genome sequence, a tetracycline-resistance-associated transposon (Tn10) was found in AP isolated from a broiler outbreak (232). Luis Tataje-Lavanda et al also reported the full-genome sequence of a NAD-hemin-independent AP serovar C-2 strain, FARPER-174, isolated from layer hens in Peru, and identified that the genome contained 12 potential genomic islands that include ribosomal protein-coding genes, a nadR gene, hemocin-coding genes, sequences of fagos, an rtx operon, and drug resistance genes (233). Cao et al. (234) performed the genomic-based antimicrobial resistance analysis of AP isolates in Guangdong Province, China. They analyzed the antibiotic resistance genes of isolates using the genomic method and found that the isolates possessed a collective total of 14 genes associated with antibiotic resistance such as tet (B), catP, and floR. In 2007, Hsu et al. (230) ollected 18 AP isolates in Taiwan from 1990 to 2003 and tested the resistance to antimicrobial agents. Interestingly, they found that 72% of isolates contained plasmids (pYMH5 and/or pA14). Plasmid pYMH5 encoded functional streptomycin, sulfonamide, kanamycin, and neomycin resistance genes, which is the first MDR plasmid reported in AP and it may facilitate the spread of antibiotic-resistance genes between bacteria. According to the sequence analysis, pYHM5 contains four open reading frames, including StrA gene encoding aminoglycoside phosphotransferase, StrB gene encoding aminoglycoside phosphotransferase, sulII gene encoding dihydropteric acid synthase, apA1 gene encoding aminoglycoside phosphotransferase, mbeCy gene encoding mobilization protein, and a partial ORF (235). During the comparative genomic analysis of AP isolate P4chr1, Xu et al. (236) demonstrated that the genomic size of AP P4chr1 was approximately 2.77 Mb with a 25-kb tolerance island that covered 6 types of antibiotics and 11 antibiotic resistance genes (ARGs).

Once a bacteriophage genome is integrated into the host-cell genome it is referred to as a prophage (91). Bacterial genomes frequently contain regions with prophage-associated genes, which include satellite phage-like elements known as phage-inducible chromosomal islands (PICIs). These elements play a significant role in horizontal gene transfer, host adaptation, and virulence in numerous pathogens (237). Within the family Pasteurellaceae various bacteriophages have been reported in Haemophilus influenzae, Actinobacillus actinomycetemcomi-tans, Pasteurella multocida, and Mannheimia haemolytica (91). Roodt et al. (91) described prophage and prophage remnants detected in AP. Genome annotation revealed the presence of prophage and prophage remnant sequences in the Modesto strain. Complete sequences of Mu-like bacteriophage and HP2-like bacteriophage were identified. Genomic studies of AP remain scarce and several key areas that still require investigation, including, but not limited to, mobile genetic elements, genomic evolution, genome plasticity, phylogenomic analysis and recombination detection within AP genomes.

The recent expansion of AP geographical range into northern latitudes with colder climates in Europe and North America rejuvenated interest in studying AP and contributed to the generation of new knowledge about the poultry bacterial pathogen. Some of the most significant advances is the development of sequence typing assays with the potential to provide a more reliable alternative to the use of conventional serotyping assays. Additionally, the availability of WGS platforms has the potential to provide deeper insights into the determinants of antigenicity, virulence factors and antimicrobial resistance.

Despite the recent advances, some knowledge gaps persist. Since its first reports and isolation, confusion about AP classification and variants emergence have been one of the biggest challenges to characterize this poultry pathogen fully. Immune variants, nontypeable, and NAD-independent strains are some of the examples. Most recently, npAP were discovered in North America representing a diagnostic challenge in areas where IC was not an endemic pathogen. The genus Avibacterium contains closely related bacterial species, some of which are poorly defined. Perhaps, this is part of the reason of the misclassification and confusion. Other Avibacterium species can be normal flora and do not appear to be primary poultry pathogens; however, more research is needed on these species, to shed light on the less understood parts of this genus and to classify ambiguous AP isolates better.

Research on new vaccine platforms, including the possibility of MLV, vaccine delivery, vaccine take assessments, is needed. Diagnostic confirmation of IC can be achieved more sensitively by a number of AP qPCR assays; however, bacterial isolation remains necessary and remains a challenge. Selective media that can allow AP to compete against the other fast-growing commensals and contaminants could significantly improve isolation rates. Having bacterial isolates is necessary to produce autogenous vaccines, for generating high-quality WGS, studying the antigenicity, discovery of new variants, and development of typing strategies. Additionally, there are no commercially available, scalable serological assays to assess antibody levels in chicken serum. ELISAs have been developed and standardized for the great majority of infectious poultry pathogens, but not for AP so far. The availability of AP commercial ELISA assays can insignificantly improve surveillance and vaccine evaluations. In summary, AP still is a challenging poultry pathogen that requires the attention of the scientific community to improve its prevention and control.

Supplemental data associated with this article can be found at https://doi.org/10.1637/aviandiseases-D-24-00105.s1.

Abbreviations:

AGPT =

agar gel precipitation test;

AP =

Avibacterium paragallinarum;

ARG =

antibiotic resistance genes;

CDT =

cytolethal distending toxin;

ERIC-PCR =

enterobacterial repetitive intergenic consensus-based polymerase chain reaction;

GT =

genotypes;

HA =

hemagglutinin;

HI =

hemagglutination inhibition;

HVR =

hypervariable region;

IC =

infectious coryza;

MALDI TOF =

matrix-assisted laser desorption ionization time-of-flight mass spectrometry;

MDR =

multidrug resistance;

MLST =

multilocus sequence typing;

MLV =

modified live vaccines;

NAD =

nicotinamide adenine dinucleotide;

npAP =

nonpathogenic Avibacterium paragallinarum;

OMVs =

outer membrane vesicles;

OP =

oropharyngeal;

pAP =

pathogenic Avibacterium paragallinarum

PICIs =

phage-inducible chromosomal islands;

qPCR =

quantitative real-time PCR;

RTX =

repeats-in-toxin;

SPF =

specific pathogen free;

TLR =

toll-like receptors;

WGS =

whole genome sequences