Gallibacterium anatis infection in poultry: a comprehensive review

Gallibacterium anatis (G. anatis), a member of the Pasteurellaceae family, normally inhabits the upper respiratory and lower genital tracts of poultry. However, under certain circumstances of immunosuppression, co-infection (especially with Escherichia coli or Mycoplasma), or various stressors, G. anatis caused respiratory, reproductive, and systemic diseases. Infection with G. anatis has emerged in different countries worldwide. The bacterium affects mainly chickens; however, other species of domestic and wild birds may get infected. Horizontal, vertical, and venereal routes of G. anatis infection have been reported. The pathogenicity of G. anatis is principally related to the presence of some essential virulence factors such as Gallibacterium toxin A, fimbriae, haemagglutinin, outer membrane vesicles, capsule, biofilms, and protease. The clinical picture of G. anatis infection is mainly represented as tracheitis, oophoritis, salpingitis, and peritonitis, while other lesions may be noted in cases of concomitant infection. Control of such infection depends mainly on applying biosecurity measures and vaccination. The antimicrobial sensitivity test is necessary for the correct treatment of G. anatis. However, the development of multiple drug resistance is common. This review article sheds light on G. anatis regarding history, susceptibility, dissemination, virulence factors, pathogenesis, clinical picture, diagnosis, and control measures.

According to several studies, Gallibacterium caused deaths in domestic birds and occasionally people, suggesting it was more dangerous than an opportunistic infection (Driessche et al. 2020).The importance of G. anatis was underestimated for a long time due to an incomplete understanding of the pathogenesis, virulence, and growth kinetics (Kristensen et al. 2011).However, the overall rate of G. anatis disease increased in layer, breeder, and boiler chicken flocks in previous years (Elbestawy et al. 2018;Krishnegowda et al. 2020;Algammal et al. 2022a).These days, this newly developing illness poses a grave threat to the world's chicken meat and egg industry (Antenucci et al. 2020;Algammal et al. 2021a;Elewa, 2021;Sorescu et al. 2021).The disease is widely distributed in American, European, Australian, African, and Asian countries.Although G. anatis may be regarded as a
Infection was reported even in non-avian species, namely, cattle, horses, pigs, sheep, and rabbits (Kristensen et al. 2011;Lawal et al. 2017).Furthermore, some G. anatis infections in humans have been detected (Aubin et al. 2013;Driessche et al. 2020).Regarding age susceptibility, older research studies have reported that young birds are less susceptible to G. anatis than adults (Bisgaard 1977;Mushin et al. 1980)

Infection and dissemination
The primary route of G. anatis infection is the horizontal one through the respiratory tract (Bisgaard 1977).Therefore, this bacterium is primarily prevalent in flocks with low biosecurity measures (Bojesen et al. 2003a).Isolation of G. anatis from the different body organs indicates the systemic circulation and spread of the bacterium from its natural habitat (Zepeda et al. 2010).Affection of the reproductive organs may be associated with ascending infections from the cloaca (Bojesen et al. 2003a;Neubauer et al. 2009).There is experimental evidence of vertical transmission in embryonated eggs through the transovarian/oviduct route or the trans-eggshell route (Matthes and Hanschke 1977;Persson and Bojesen, 2015;Wang et al. 2018).G. anatis was isolated from the egg yolk and ovaries of hens 10 days after the experimental infection (Shapiro et al. 2013;Paudel et al. 2014a).In embryonated eggs, G. anatis may penetrate the eggshell pores and infect the growing embryo causing mortality (Wang et al. 2018).Besides, venereal transmission has been suggested since G. anatis damages semen quality and causes epididymitis (Paudel et al. 2014b).Semen of G. anatis infected cockerels may function in transmission among adult birds and probably to their offspring.
Intranasal inoculation of cockerels with G. anatis resulted in the presence of the pathogen in the testis and epididymis within a week post-infection (pi).In addition, the sperm density was lower, total and progressive motility were lower, and membrane integrity was worse in the semen, all indicating a detrimental effect on fertility.The study of Neubauer et al. (2009) revealed that G. anatis affected 13-layer flocks, and the birds suffered from cannibalism (extensive wound pecking around the cloaca).

Zoonotic importance
It is presumed that G. anatis is a food contaminant that transmits to humans.In 2013, in France, the first case of bacteremia initiated by G. anatis was reported in an immunocompromised woman infected through consuming contaminated food.The predominant symptoms include fever, severe abdominal pain, anemia, diarrhea, and neutropenia (Gautier et al. 2005;Aubin et al. 2013).Driessche et al. (2020) reported that poultry and cattle have a likely risk for zoonotic transmission of G. anatis, and further research should be conducted to establish their zoonotic potential.Recently, Wang et al. (2023) reported a case of acute watery diarrhea (7-8 times daily) brought on by G. anatis in a 62-year-old man with type 2 diabetes and hypertension.There was no urgency, no bloating or pain in the abdomen, no dread of a cold or fever, and only nausea and vomiting were the symptoms of the illness.The bacteria were identified using 16S rRNA sequencing and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS).

Virulence factors
The different virulence factors of G. anatis are illustrated in Fig. 1.

Gallibacterium toxin A
Gallibacterium toxin A (GtxA) is a vital virulence protein that is responsible for the hemolytic property of G. anatis biovar hemolytica (Kristensen et al. 2011).It has a leukotoxic impact on chicken macrophage cell line HD11 and lyses red blood cells (RBCs) (Kristensen et al. 2010;Persson and Bojesen 2015), and stimulates the immune response (Bager et al. 2014).This toxin has two domains, the C-and N-; both are needed for hemolytic activity.The C-terminus is similar to repeat in toxins (RTX) of the Pasteurellaceae family, while the N-terminal has RBCs lytic and leukocidal actions (Yang et al. 2020).The GtxA knockout mutant bacterium was exposed to a reduction of pathogenicity.It is comparable to the cytoskeletal protein talin, which binds integrins to the actin and vacuolar cytoskeleton and is crucial for interactions between the host and the bacteria (Kristensen et al. 2010).Actin can support cell motility, recognition, adhesion, phagocytosis, signal modulation of immune cells, and free radical generation.When GtxA binds to actin in host immune cells, the cell shape can be altered, and cellular signal transmission is impeded, and as a result, the bacteria can elude the host immune system (Aktories et al. 2011).Nassik et al. (2019) demonstrated that all the molecularly identified strains of G. anatis expressed the virulent GtxA.Tang et al. (2020) implied that GtxA plays a crucial function in an acute cytokine-mediated Th2-like response versus G. anatis infection in the ovarian tissue, helping in the pathogenesis of G. anatis infections in laying hens.Additionally, Tang and Bojesen (2020) attempted to explain the immunosuppressive effect of G. anatis GtxA during the interaction with chicken macrophage-like HD11 cells by encouraging cell adhesion and invasion, reducing the host inflammatory response based on an initial over-expression of interleukin (IL)-10 and a corresponding low-level expression of tumor necrotizing factor (TNF)-α, and concluded that GtxA induces cell death (apoptosis) without revealing clear causes.Fimbriae.

Haemagglutinin
Some strains of Gallibacterium can agglutinate RBCS of avian and mammalian species (Zepeda et al. 2009;Ramirez-Apolinar et al. 2012).Though most strains of G. anatis can agglutinate rabbits' RBCs, few strains may agglutinate the RBCs of chickens and quails due to the expression of haemagglutinins or adhesins binding receptors on the cells' surface (Bager et al. 2013a;Johnson et al. 2013).Moreover, hemagglutinin protein was detected in biofilms and outer membrane vesicles (OMVs) liberated from G. anatis (Montes-Garcia et al. 2016).

Outer membrane vesicles
Proteins, lipopolysaccharides, and DNA are found in the budding regions of the outer cell membrane, OMVs, of several Gram-negative bacteria (Mashburn-Warren and Whiteley 2006; Kulp and Kuehn 2010;MacDonald and Kuehn 2012).The enormous collection of core proteins found in the OMVs of G. anatis strains is mainly unaffected by the various in vitro growth conditions, but certain environmental stimuli greatly influence their expression.They facilitate bacterial adhesion and colonization, the formation of biofilms, and the removal of several antibiotic compounds (Bager et al. 2013a).Additionally, G. anatis releases hemagglutinin in the OMVs to agglutinate avian RBCs (Zepeda et al. 2009;Bager et al. 2013aBager et al. , 2014;;Johnson et al. 2013) and trigger a robust immunological response (Pors et al. 2016b).Bojesen et al. (2007a) identified and characterized the genetic elements responsible for capsule biosynthesis in Gallibacterium for the first time.The capsule biosynthetic locus of Gallibacterium resembles the Escherichia coli (E.coli) group 2 capsule in structure and is considered a critical virulence factor for the pathogenesis of G. anatis infection.Some Gallibacterium strains have a polysaccharide capsule that adds virulence (Persson and Bojesen 2015).Capsule in G. anatis has an essential role in the adhesion and interaction of the bacterium with the surface of the host and immune evasion (Bojesen et al. 2011b;Singh et al. 2011;Harper et al. 2012).Although this capsule can be seen using electron microscopy (Bojesen et al. 2011c), it disappears following sub-cultures (Kjos-Hansen 1950).A capsule-knockout mutant (ΔgexD) became more virulent than its wild-type equivalent (Bojesen et al. 2011c).

Biofilm formation
Biofilms mainly comprise proteins, polysaccharides, nucleic acids, and amyloid proteins (Costerton et al. 1999;Larsen et al. 2007;Lopez-Ochoa et al. 2017).Biofilm formation begins through the bacterium's capacity to bind the cell's inert surface.The adhesive capacity of G. anatis as a tool for colonization of tissue surfaces and for allowing infection to persist inside the host was studied by scanning electron microscopy, and the findings implied that all the used isolates had formed robust biofilms on polystyrene and glass within the first 3 h of exposure (Vaca et al. 2011).Strains of G. anatis are classified into 3 categories: weak, moderate, and strong groups according to the biofilm formation capability (Johnson et al. 2013).This may indicate that the development of biofilms is significant for particular bacterial clades.Additionally, the biofilm is crucial for the chronicity and duration of infections and for reducing their sensitivity to antibiotics (Costerton et al. 1999;Donlan and Costerton 2002;Persson and Bojesen 2015).It has been found that proteins in biofilms are capable of interacting with several host proteins, including fibronectin, fibrinogen, laminin, and plasminogen, and consequently alter the host's homeostasis (Epstein and Chapman 2008;Lopez-Ochoa et al. 2017).

Metalloproteases
Metalloprotease enzymes are essential in G. anatis bacterium for proteolysis, increasing virulence, colonization, nutrient acquisition, and degradation of immunoglobulin (Ig) (G), along with bacterial invasion into the systemic circulation (Garcia-Gomez et al. 2005;Chavez et al. 2017b).It was found that these enzymes can down-regulate the immune response by acting on antibodies and complement system (Miyoshi and Shinoda 2000) that helps in colony establishment and bacterial transmission to blood circulation.In addition, proteases may be accountable for the hostspecific pathogenicity of G. anatis strains (Zepeda et al. 2010).Metalloproteases such as metal-dependent endonuclease domain, zinc, and ATP-dependent metalloprotease are encoded in the G. anatis genome (Johnson et al. 2013).

Elongation factor Tu
Elongation factor Tu is a protein released through vesicle formation, possesses amyloid characters, and is consequently included in the pathogenesis of G. anatis (Lopez-Ochoa et al. 2017).

Clustered regularly interspaced short palindromic repeats (CRISPR)
According to Johnson et al. (2013), the bacterial innate defense system known as clustered regularly interspaced short palindromic repeats (CRISPR) breaks down invasive foreign and phage and plasmid nucleic acids.It can obstruct transformation, indicating that different strains of G. anatis have different levels of innate ability (Kristensen et al. 2012).

Integrative conjugative elements
Integrative conjugative elements have specific distinguishing characteristics, such as a site-specific integrase, transfer genes, and genes that control excision and transfer.Genes encoded inside these elements have been used to identify them in the G. anatis genome (Wozniak et al. 2009;Johnson et al. 2013).They have genes for antibiotic resistance, which spreads resistance to other bacteria (Bojesen et al. 2011b).

Small colony variants
Small colony variants are detected in the cultures of G. anatis as they have hemolytic activity (Greenham and Page 7 of 20 383 Hill 1962).Moreover, they might aid in bacterial survival, recurrent infections, and the development of antibiotic resistance (Proctor et al. 2006).

Other virulence genes
G. anatis pathogenicity has been linked to the genes cps16A, 16B, and 16F that encode the enzymes glycosyltransferase, hyaluronidase, and UDP-glucose 6-dehydrogenase, respectively (Bosse et al. 2017), through the cloning and characterization of the gene encoding G. anatis fnrG (a homolog of the global regulator gene conferring a hemolytic phenotype in E. coli) and its function in the production of the E. coli silent hemolysin.Bojesen (2003) investigated the relationship between G. anatis and E. coli.sheA was studied by creating an E. coli-sheA, a null mutant of the silent hemolysin, from a 4.2 kilobase pair hind segment harboring fnrG.The fact that fnrG activates sheA and produces a non-hemolytic transformant led him to conclude that fnrG is probably a member of the fnr global regulatory protein family.

Pathogenicity
The bacterium was isolated from apparent healthy chickens' nasal and tracheal passages and cloaca as a part of their normal microbiome (Bojesen et al. 2003a).Several studies verified the existence of G. anatis as a key bacterial pathogen in both natural and experimental infections.The association of G. anatis with septicemia, oophoritis, salpingitis, egg abnormalities, pericarditis, hepatitis, tracheitis, and elevated mortality in pullets signifies that at least some strains of Gallibacterium possess pathogenic possibility in chickens (Bojesen et al. 2004;Neubauer et al. 2009;Jones et al. 2013;Paudel et al. 2013Paudel et al. , 2014aPaudel et al. , b, 2015;;Elbestawy 2014;Persson and Bojesen 2015).The differentiation between pathogenic and non-pathogenic isolates of G. anatis using embryo lethality assay failed to obtain any significant results because both G. anatis isolates from healthy and sick chickens caused hemorrhagic lesions and death of embryos in 70 to 100% of inoculated eggs (Trampel and Nalon, 2008).
Despite the previous pathological findings of G. anatis, it was found that this pathogen may colonize the upper respiratory and reproductive tracts without initiating substantial signs or lesions (Paudel et al. 2013(Paudel et al. , 2014a, b), b).Many virulence factors enable G. anatis to invade, adhere, and colonize the host's surface epithelium.After oropharyngeal and oviduct epithelial cells infection with G. anatis, the pathogen adheres firmly via the reaction between the adhesin and the host's cell surface receptor, followed by rampant multiplication, colonization, and syntheses of virulence factors (Vaca et al. 2011;Lucio et al. 2012;Bager et al. 2013a,b).G. anatis F149T can express fimbriae responsible for mucosal attachment and colonization to the epithelium of the oropharynx (Lucio et al. 2012).The in vitro study of Zhang et al. (2017) showed that higher adhesion to primary chicken oviduct epithelial cells and increased generation of inflammatory cytokines have been observed in the highly pathogenic strains of G. anatis [IL-6, TNF-α, and interferon (IFN-c)], resulting in inflammation and tissue injury.Many other specific virulence factors can influence the pathogenicity of G. anatis, such as IgG-degrading proteases, biofilm, hemagglutinin, fimbriae, capsule, GtxA, OMVs, metalloproteases, elongation factor Tu, and clustered regulatory short palindromic repeats (Bojesen et al. 2004;Garcia-Gomez et al. 2005;Christensen et al. 2007b;Zepeda et al. 2009;Kristensen et al. 2010;Lopez-Ochoa et al. 2017).
Three G. anatis isolates from Mexico, China, and Austria were evaluated for their differences in pathogenicity in a specific pathogen-free Lohmann layer chicken population collected from various geographic locations.The results indicated that the Mexican isolate had a slightly higher pathogenicity than the two other strains, suggesting a geographical pathogenicity difference (Paudel et al. 2013).Regarding the genetic variety of isolates of G. anatis, Bojesen et al. (2003b) obtained 114 G. anatis isolates from tracheal and cloacal swab samples of organic flocks, egg-producing flocks, and layer parent chicken flocks.The findings showed higher genetic similarity (more than 94%) in the organic flock isolates.On the contrary, the layer parent flock isolates were divided into two subclusters containing each tracheal or cloacal isolated, with similarity above 90%.This genetic diversity, indicating clonal lineages, may have altered to several spots inside the same bird, granting them additional capability to initiate a disease (Bojesen and Shivaprasad 2007;Johnson et al. 2008).

Clinical manifestations
Infection with G. anatis is associated with multiple and variable clinical pictures but is often non-specific.In broilers, the pathogen causes respiratory manifestations in the form of nasal discharge, swelling and shaking of the head, cough, rales, dyspnea, diarrhea, and emaciation (Bojesen et al. 2008;El-Adawy et al. 2018;Elbestawy et al. 2018).In addition, G. anatis infection may induce a 3-18% reduction in egg production in layers (Jones et al. 2013), while in breeders, it causes an increasing mortality rate of 0.06-4.9%(Elbestawy et al. 2018).Mixed respiratory and reproductive disorders could be noticed in layers and breeder chicken flocks (Ataei et al. 2017;Chavez et al. 2017a;Abd El-Hamid et al. 2018).Elewa (2021) diagnosed G. anatis in layer chickens suffering from sneezing, rales, gasping, coughing, head swelling, and a 5-10% drop in egg production.Moreover, Bojesen et al. (2004) investigated the pathology of G. anatis virulent strain 12656-12 by intravenous or intraperitoneal inoculation in normal or immunosuppressed commercial brown laying chickens aged 15 weeks old.The author found that intravenously infected birds had severe septicemic lesions in both the normal and immunosuppressed birds.Mortality (73%) was recorded with severe depression and reluctance to move chickens.While Hui-min et al. (2009) reported the relationship between G. anatis and layer oviduct cysts, they did not indicate whether these lesions were co-associated with other viral diseases such as infectious bronchitis and low pathogenic avian influenza (LPAI-H9N2) or not.The clinical signs and gross lesions of 42 naturally infected layers with G. anatis were observed and recorded.Furthermore, a combined infection among G. anatis and other pathogens was also investigated.Hyperemia, swelling, and epithelium cell degeneration or necrosis were found in the mucosa of the respiratory tract and mucosa of the oviduct that presented many neutrophilic granulocytes and ovarian cysts.Most importantly, the severity of the clinical manifestations induced by G. anatis is exaggerated by co-infection with other pathogens, such as A. paragallinarum in chickens.Paudel et al. (2017a) observed that chickens infected with both G. anatis and A. paragallinarum had more severe nasal secretions and swelling of infraorbital sinuses than those with a single infection.In addition, in the presence of E. coli infection, G. anatis caused higher egg embryonic mortality than eggs with a single infection (Wang et al. 2018).
Under experimental infection conditions, G. anatis induced typical respiratory signs and weight losses in broiler chickens (Abd El-Hamid et al. 2018).However, layer chickens exhibited whitish diarrhea with a 66 to 47% drop in egg production (Paudel et al. 2014a).Specific pathogen-free cockerels showed no signs following experimental infection with G. anatis, but there was an alteration in the semen quality (Paudel et al. 2014b).Intranasal inoculation of G anatis showed an extensive and regular bacterial distribution in the respiratory and reproductive tract till 28 days pi (dpi) vs. one dpi in intravenously inoculated birds (Jones et al. 2013;Paudel et al. 2013).
Following experimental induction with G. anatis, layer chickens exhibited variable gross lesions such as hemorrhagic or ruptured ovarian follicles, pericarditis, multifocal hepatic necrosis, egg deformities in the oviduct, and fibrinous peritonitis (Paudel et al. 2014a).It has been noted that G. anatis biovar 3 caused purulent oophoritis, swollen blood vessels in the ovary, oviduct, and peritoneum, as well as purulent or fibrinous exudates in the peritoneum.Further experimental studies of G. anatis in chickens represented regression and deformity of ovaries, focal or diffuse salpingitis, catarrhal to purulent tracheitis, congestion of lungs, air sacculitis, pericarditis, liver congestion, and ascites (Paudel et al. 2013;Pors et al. 2016a;Abd El-Hamid et al. 2018).
G. anatis biovar haemolytica had been isolated from the nasal sinuses of infected cases with a nephropathogenic strain of infectious bronchitis and had respiratory signs (Franca et al. 2011).As they cause more severe peritonitis, salpingitis, and oophoritis, concurrent E. coli and G. anatis infections might worsen lesions compared to a single infection (Pors and Bojesen 2011).Besides, another study confirmed the localization of Gallibacterium mainly in the trachea and ovaries (Huangfu et al. 2012).G. anatis was isolated at a meager rate (2.7%) from pododermatitis cases in numerous layer flocks in Denmark in 2015 (Olsena et al. 2018).

Diagnosis
The different diagnostic tools of G. anatis are shown in Fig. 3.

Isolation and phenotypic identification
The absence of pathognomonic respiratory and reproductive signs and lesions caused by G. anatis necessitates a specific diagnosis to confirm the infection.Isolation and phenotypic identification of G. anatis are the basis for diagnosing G. anatis infections in almost all cases (Christensen et al. 2003).However, overgrowth by other bacteria, mainly E. coli, significantly hinders the isolation process (Wang et al. 2016).Furthermore, phenotypic characterization techniques can produce heterogeneous, time-and labor-intensive findings.Consequently, molecular diagnostic methods that depend on identifying 16S-23S rRNA sequences are growing (Bojesen et al. 2007b;Alispahic et al. 2012;Huangfu et al. 2012).
Species of Gallibacterium are usually grown at 37°C for 24-48 h on blood agar medium (with 5-10% citrated bovine blood) under facultative anaerobic/microaerophilic conditions (Christensen et al. 2003).Most G. anatis isolates produce a wide β-hemolytic zone with butyrous, circular, smooth, greyish, non-pigmented, and shiny colonies (El-Adawy et al. 2018).After 24-48 h of incubation, G. anatis develops with a modest granular deposit and weak turbidity in a nutrient broth supplemented with 5% horse serum and 3% glucose.Better bacterial growth is observed in brain heart infusion broth when 5% horse serum is added.
Biochemically, several tests are used to identify G. anatis, as detailed in Table 2 (Christensen et al. 2003;Bisgaard et al. 2009).

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS)
MALDI-TOF-MS is an effective technique for identifying G. anatis biomarkers (El-Adawy et al. 2018).This test has an impressive prospective for a routine laboratory diagnosis of the bacteria as it is rapid, can proceed with many samples concurrently, and needs small sample sizes.This test identified one clonal lineage of G. anatis in various flocks (Alispahic et al. 2011(Alispahic et al. , 2012;;Allahghadry et al. 2021).The genetic diversity of Gallibacterium isolated from 13 farms with various biosecurity measures and management techniques was examined using phylogenetic analysis of incomplete rpoB sequences and biotyping using MALDI-TOF-MS.The findings showed significant variability among isolates from farms with poor biosecurity standards and those with greater biosecurity standards (more closely linked and grouped).Low biosecurity standards allow viruses to spread horizontally, and gene transfer creates genetic diversity (Lozica et al. 2020).

Molecular detection
Molecular diagnosis of G. anatis has been recently developed, being fast, easy, highly specific, sensitive, and reliable (Ataei et al. 2017).Molecular assays such as polymerase chain reaction (PCR) are confirmatory to both phenotypically detected G. anatis strains and the negative samples following initial isolation (Bisgaard et al. 2009;Elewa 2021).Primers, including 16S to 23S rRNA internal transcribed Table 2 Phenotypic and biochemical reactions for the identification of G. anatis biotypes ONPG β-galactosidase, ONPG β-Glucosidase Characteristics are scored as: + , all strains positive within 1-2 days; ( +), all strains positive within 14 days; − , all strains negative after 14 days; W, weakly positive; D, some strains positive, while others are negative (number of strains positive/number of strains tested) (Christensen et al. 2003;Bisgaard et al. 2009)
When phenotypic identification of Gallibacterium is challenging, classification based on the gene sequence of the DNA-dependent RNA-polymerase (rpoB) subunit might be utilized (Korczak et al. 2004;Christensen et al. 2007b).The gtxA-encoding gene, the fimbrial gene, and the gyrase subunit B gene may all be found using the PCR approach (Sorour et al. 2015;Krishnegowda et al. 2020).
Real-time quantitative PCR (qPCR) is used as a speciesspecific identification and quantifying method for G. anatis (Huangfu et al. 2012;Wang et al. 2016Wang et al. , 2018)).The protein gyrase subunit B gene is essential for the function of the DNA replication enzyme because it encodes the ATPase domain of this enzyme.The qPCR is more rapid, highly specific, sensitive, reproducible, and cost-effective, and it needs a lower DNA concentration than conventional PCR or phenotypic characterization methods (Wang et al. 2016;Huangfu et al. 2012).
Real-time loop-mediated isothermal amplification PCR assay has recently been used to detect G. anatis.This assay targets the sodA gene.It has been described as sensitive, rapid, and specific for G. anatis identification.Moreover, it is quicker and cheaper than quantitative PCR (Stępień-Pyśniak et al. 2018).

Other diagnostic techniques
Agar gel precipitation test Galaz-Luna et al. (2009) used an agar gel precipitation test for serotyping and cross-reactivity and found no relationship between biovar and serovar among the species of Gallibacterium.

Enzyme-linked immunosorbent assay
Serological testing is helpful for flock monitoring or diagnosing Gallibacterium infection.Wang (2012) developed an indirect enzyme-linked immunosorbent assay (ELISA) to recognize all antibodies against G. anatis in chickens.The response curve was established when the optical density 450 nm values varied along with the time.The antibody level peaked in 2-month pi, but for a short period, and then gradually dropped.Nowadays, ovotransferrin can be detected in chicken serum using ELISA as an acute phase protein marker in experimental G. anatis infections (Roy et al. 2014).Acute-phase proteins are good markers for diagnosis and prognosis.

Hemolysis and cytotoxicity assay
The hemolysis assay of G. anatis can be adopted using washed bovine blood RBCs and detected using ELISA, while the cytotoxicity assay can be applied on HD11 cells in 96-well tissue culture plates (Kristensen et al. 2010).The GtxA is the cause of cytotoxicity and hemolysis (Kristensen et al. 2011).

Immunofluorescence microscopy
The culture of G. anatis is fixed with paraformaldehyde on glass slides and labeled with anti-fimbriae immune serum.The conjugated goat anti-rabbit secondary antibodies are added, the slides are mounted, and the images are captured using laser scanning microscopy (Bager et al. 2013b).

In situ hybridization
The 16S rRNA of Gallibacterium is the target of an in situ hybridization probe dyed cyanine.The pathogenic changes in the spleen and liver tissues of experimentally infected hens have been studied using this hybridization approach Page 13 of 20 383 (Bojesen et al. 2003a(Bojesen et al. , 2004)).This technique is vital for the detection of G. anatis pathogenies and dissemination.

Immuno-gold electron microscopy
This method is processed as an immunofluorescence assay, but the secondary antibodies are gold particles on nickel grids coated with Formvar carbon (Bager et al. 2013b).Electron microscopy is used for the detection of G. anatis.

Histopathology and immunohistochemistry
Several studies reported the histopathological examinations.Following the intravenous or intraperitoneal inoculation of virulent G. anatis strains in normal or immunosuppressed 15-weeks-old brown laying chickens, the liver lesions in the intravenously infected birds included basophilic aggregates (Gallibacterium microcolonies) bordered by necrotic hepatocytes, non-identifiable necrotic cells, proteinaceous fluid, and eosinophilic and basophilic aggregates in the ellipsoids in the spleens.After 12 and 24 h pi, the histopathological changes included forming multinucleated giant cells around some of the ellipsoid lesions in the spleen and liver.At the same time, the intraperitoneally infected chickens with a normal immune status showed diffuse purulent peritonitis and fibrinous perihepatitis 12 h pi (Bojesen et al. 2004;Zepeda et al. 2010).A combined infection of G. anatis and other pathogens was studied, and the results indicated changes in the lung, trachea, oviduct, and ovary regarding hyperemia, swelling, epithelium cell degeneration, and necrosis in the mucosa of the respiratory tract.The mucosa of the oviduct presented several neutrophilic granulocytes and ovarian cysts (Hui-min et al. 2009).The different histopathological severity degrees were due to the differences in virulence among the used G. anatis biovar hemolytica in chickens (Zepeda et al. 2010;Abd El-Hamid et al. 2016;Mataried 2016).
Immunochemistry was used to detect the ability of G. anatis isolates to adhere to or invade the chicken oviduct epithelial cells using polyclonal antibodies.The results of this assay revealed that G. anatis could attach epithelial cells without invasion (Zhang et al. 2017).

Prevention and control strategies
Adopting effective biosecurity measures, "all in-all out," in poultry farms prevents the horizontal transmission of G. anatis, the gene transfer, and consequently, the heterogeneity or the diversity of this pathogen (Lozica et al. 2020).Treatment of a mixed infection with other bacteria using a specific drug, controlling of other immunosuppressive agents, and amelioration of stress conditions are the must to prevent or control G. anatis infection (Mataried 2016;Paudel et al. 2017a, b;Abd El-Hamid et al. 2018).Eggs or hatchery cleanliness is crucial to prevent trans-eggshell transmission caused by fecal contamination with G. anatis (Wang et al. 2018).

Antibiotic treatment
The administration of effective antimicrobials is needed to treat G. anatis infections.As of now, the antigenic diversity among G. anatis strains and MDR causes treatment failure and hinders vaccination-based prophylaxis (Bojesen et al. 2003b(Bojesen et al. , 2007c;;Christensen et al. 2003;Bojesen et al. 2011a, b;Johnson et al. 2013;Jones et al. 2013;Chavez et al. 2017b;Hess et al. 2019).In this regard, Elbestawy (2014) detected absolute resistance of 20 isolates of G. anatis to oxytetracycline and lincospectin and owed this resistance to the extensive exposure of layer chicken flocks in Egypt to long courses of these antibiotics in feed as a rational prophylactic program.A similar resistant pattern was obtained in Morocco by Nassik et al. (2019), who found that all G. anatis strains were resistant to ampicillin, erythromycin, oxytetracycline, and sulfamethoxazole-trimethoprim.
Besides, Bojesen et al. (2011b) reported that field strains of G. anatis were resistant to sulfamethoxazole and tetracycline at rates of 97% and 92%, respectively, while reference strains were resistant to the previous antimicrobials at rates of 42% and 67%, respectively.Alarmingly, MDR has markedly increased worldwide in the last decade, which is deemed a public health threat.Numerous recent epidemiological studies established the occurrence of extensive drug resistance and MDR bacterial pathogens from distinct origins, including humans, fish, food products, and poultry (Abd El-Ghany, 2021;Algammal et al. 2021b, c;Hetta et al. 2021;Algammal et al. 2022b).
The antimicrobial resistance (AMR) of G. anatis strains isolated from chickens in different localities were studied using PCR and cloning and sequencing of plasmid-mediated resistant gene of streptomycin, sulfamethoxazole, and fluoroquinolones (Bojesen et al. 2011a, b;Guo, 2011;Gao, 2012;Monita et al. 2011).The antimicrobial susceptibility for 84 chicken isolates of G. anatis from broiler and breeder flocks was studied in the USA compared to P. multocida isolates.Amoxicillin, neomycin, and sulfonamide-trimethoprim resistance levels were superior in G. anatis than in P. multocida (Jones et al. 2013).Resistance to tetracycline could be due to the genes tetB, tetH, and tetL, which have been detected in strains of G. anatis (Abd El Tawab et al. 2018).
Owing to the emergence of widespread MDR strains of G. anatis, traditional antimicrobials are not suggested for treating G. anatis infection.In the study by Prasai et al. (2017), feed supplementation with zeolites (aluminosilicate minerals) showed an effective reduction of G. anatis in chickens.In addition, Zhang et al. (2019) reported that specific chicken egg yolk IgY produced against recombinant N-terminal of GtxA revealed significant protection against G. anatis infection and decreased the severity of the lesion in the liver and intestine.Moreover, a recent study by Zhang et al. (2021) showed that a strain of Leuconostoc mesenteroides (QZ1178) exerts potential in vitro antibacterial effects against G. anatis through lactic acid formation.

Vaccines
Prior to now, the effectiveness of conventional vaccinations in protection against G. anatis infection was limited due to the antigenic diversity reported by Krishnegowda et al. (2020).A bacterin including 3 different biovars accounting for 95% of the outbreaks in Mexico has been developed by Boehringer Ingelheim, Mexico.The serological response was monitored from week 9 to week 40 using a custommade ELISA test for G. anatis.The results indicated that the birds vaccinated with G. anatis bacterin were high in antibody titers and egg production (Gonzalez et al. 2003;Vazquez et al. 2003).The prepared autogenous bacterin was very effective in protecting against clinical disease and decreasing egg production and performance in layers (Castellanos et al. 2007) and in broiler breeder flocks (Calderon and Thomas 2009).
The vaccination potential of many immunogenic antigens such as GtxA, OMVs, fimbriae, capsules, elongation factor Tu, CRISPR, and recombinant proteins (GtxA and fimbriae) has been studied (Bager et al. 2013a(Bager et al. , 2014;;Pedersen et al. 2015;Pors et al. 2016a, b).Moreover, the efficacy of various immunogenic proteins such as GtxA, fimbriae, Gab_1309, and Gab_2348 for epitope recognition and prediction was investigated.The results revealed that these immunogenic proteins could stimulate the immune response (Ataei et al. 2019).Vaccination of layer chickens with OMVs of G. anatis decreased the severity of lesions and increased the serum titers of specific immunoglobulin (Y) (Pors et al. 2016b).Immunization with OMVs and the fimbrial protein of G. anatis could protect against the disease (Persson et al. 2018).A killed bivalent commercial vaccine (bacterin), including A. paragallinarum and G. anatis antigens, can induce complete protection in immunized chickens (Paudel et al. 2017a,b).Finally, Allahghadry et al. (2021) detected three major immunogen genes, gtxA, Gab_1309, and Gab_2312, in the examined G. anatis isolates, which possibly exemplify efficient vaccine targets, and the authors suggested the use of the gtxA gene (the most conserved sequence in all G. anatis strains) for the future vaccine development purposes (recombinant vaccines).
In conclusion, periodic studies regarding Gallibacterium infections should be applied to control their economic losses and improve poultry production.These studies should investigate the current epidemiological situation of the disease in the locality, the sources of infection in terms of public health, the antibiogram profile of the isolated strains, and the development of future vaccines.In addition, it is crucial to discuss the precise biology and pathophysiology of Gallibacterium infection and how this bacterium switches from being a normal inhabitant flora to a pathogen producing a disease condition in the host.

Fig. 2
Fig. 2 Characteristic signs and lesions of G. anatis infection

Table 1
The first detection, incidence rate, and diagnostic methods of G. anatis infection worldwide (after establishment of new taxonomy,Gallibacterium, in 2003)