Gallibacterium anatis Emerging in Indonesia: Isolation and Molecular Characterization From Chickens in a West Java Poultry Farm

TL;DR

This study identifies Gallibacterium anatis in Indonesian chickens, showing it causes respiratory and reproductive issues and has key virulence genes.

Contribution

First molecular evidence of Gallibacterium anatis in Indonesian poultry, including virulence gene detection and phylogenetic analysis.

Findings

14 out of 17 suspected isolates confirmed as G. anatis via molecular methods.

All isolates carried the gyrB virulence gene, with high prevalence of gtxA and flfA.

Sequence similarity of >98.65% with reference G. anatis strains in GenBank confirmed species identity.

Abstract

Gallibacterium anatis (G. anatis) is associated with decreased egg production and respiratory disorders in chickens. The presence of G. anatis in Indonesia has not been reported. This study aimed to identify G. anatis both phenotypically and genotypically in layer, broiler, and breeder chicken from farms in West Java. A total of 23 suspected gallibacteriosis cases were collected from chickens exhibiting respiratory and reproductive disorders between May 2020 and October 2023. Phenotypic characterization was conducted using blood agar culture, Gram staining, and biochemical tests (catalase, oxidase, and TSIA). Genotypic identification of G. anatis was performed using PCR targeting the 16S‐23S rRNA gene, followed by detection of virulence genes (gtxA, flfA, and gyrB). Sequencing and phylogenetic analyses were carried out using BLAST program and MEGA 7. Among the 23 collected samples, 17 samples showed phenotypic characteristics of G. anatis, and 14 of these isolates (73.9%) were confirmed molecularly. Virulence genes profiling revealed the presence of gyrB (100%), gtxA (93.3%), and flfA (71.4%). Sequence analysis demonstrated more than 98.65% similarity between all isolates and reference G. anatis strains in GenBank, supporting accurate species identification. This study provides the molecular evidence of G. anatis in layer, breeder, and broiler chickens from farms in West Java, Indonesia. The detection of key virulence genes further indicates the pathogen’s potential role in reduced production and respiratory disease in Indonesian poultry. These findings highlight the importance of routine surveillance and molecular diagnostics to support early detection and control strategies for gallibacteriosis in commercial poultry populations.

Figures

Distribution patterns of Gallibacterium anatis virulence genes. Each circle is representatives of the virulence gene, such as gtxA, flfA, and gyrB.

Phylogenetic tree of G. anatis isolates from Indonesia. This phylogenetic tree was constructed using neighbor‐joining with the MEGA 7 application from the 16S‐23S rRNA gene. The branches of the dendrogram show bootstrap values (%) with 1000 replications, and the scale represents one per 1000 nucleotide sequence substitutions of the 16S rRNA gene. () denotes the isolates tested. () denotes outgroup reference isolates.

Tables

TABLE 1. Specific primers used for polymerase chain reaction (PCR) to detect virulence genes of Gallibacterium anatis.

Target genes	Sequences (5′–3′)	Size (bp)	References
gtxA	F: CAA​ACC​TAA​TTC​AAT​CGG​ATG	1257	[9]
	R: TGC​TTC​AAT​AAT​TTT​CCA​TTT​TC
flfA	F: CAC​CAT​GGG​TGC​ATT​TGC​GGA​TGA​TCC	538	[9]
	R: ATT​CGT​ATG​CGA​TAG​TAT​AGT​TC
gyrB	F: TGT​GCG​TTT​CTG​GCC​AAG​TC	561	[10]
	R: CGC​TCA​CCA​ACT​GCA​GAT​TC

TABLE 2. Number of Gallibacterium anatis isolates from West Java during the period 2020–2023.

Origin of isolates	Number of samples	Number of phenotypic test	Number of molecular test
			Positive	Negative
Farm A (Breeder‐Layer)	5	5 (100%)	5 (100%)	0 (0%)
Farm 1 (Layer‐Ciampea)	3	0 (0%)	—	—
Farm 2 (Broiler‐Ciseeng)	3	0 (0%)	—	—
Farm 3 (Broiler‐Cibinong)	4	4 (100%)	3 (75%)	1 (25%)
Farm 4 (Layer‐Cibinong)	4	4 (100%)	3 (75%)	1 (25%)
Farm 5 (Layer‐Cibinong)	4	4 (100%)	3 (75%)	1 (25%)
Total	23	17 (73.9%)	14 (82.35%)	6 (17.64%)

TABLE 3. Number of virulence genes tested in Gallibacterium anatis.

Isolates name	Virulence genes
	gtxA	gyrB	flfA
001/BEBR/SBM/2020	+	+	−
002/BELY/CS–BG/2022	−	+	+
003/BELY/CS–BG/2022	+	+	+
004/BEBR/PR–BG/2022	+	+	+
005/LY/SBM/2022	+	+	−
012/BR/CB–BG/2023	+	+	+
014/BR/CB–BG/2023	+	+	+
015/BR/CB–BG/2023	+	+	+
016/LY/CB–BG/2023	+	+	+
017/LY/CB–BG/2023	+	+	+
018/LY/CB–BG/2023	+	+	+
020/LY/CB–BG/2023	+	+	+
021/LY/CB–BG/2023	+	+	−
022LY/CB–BG/2023	+	+	+

TABLE 4. Results of similarity of Gallibacterium anatis 16S‐23S rRNA.

Isolates name	Bacterial references	Access code	Similarity	Query cover
001/BEBR/SBM/2020	G. anatis strain ESV200	CP114281.1	99.38%	100%
002/BELY/CS–BG/2022	G. anatis strain ESV 200	CP114281.1	99.64%	100%
	G. anatis strain YU‐ZMD	GU475462.1	99.64%	100%
003/BELY/CS–BG/2022	G. anatis strain TH22	CP103874.1	99.53%	100%
	G. anatis strain YJ922	CP126978.1	99.53%	100%
	G. anatis strain YU‐ZMD	GU475462.1	99.53%	100%
004/BEBR/PR–BG/2022	G. anatis strain ESV200	CP114281.1	99.27%	100%
005/LY/SBM//2022	G. anatis strain TH22	CP103874.1	99.73%	100%
	G. anatis strain DFSO2	CP126977.1	99.73%	100%
	G. anatis strain YJ922	CP126978.1	99.73%	100%
	G. anatis strain BJF12	CP126975.1	99.73%	100%
	G. anatis strain ESV200	CP114281.11	99.73%	100%
	G. anatis strain UMN179	CP002667.1	99.73%	100%
013/BR/CB–BG/2023	G. anatis strain TH22	CP103874.1	99.62%	100%
	G. anatis strain DFSO2	CP126977.1	99.62%	100%
	G. anatis strain YJ922	CP126978.1	99.62%	100%
	G. anatis strain YU‐ZMD	GU475462.1	99.62%	100%
016/LY/CB–BG/2023	G. anatis strain TH22	CP103874.1	99.52%	100%
	G. anatis strain BJF12	CP126975.1	99.52%	100%
	G. anatis strain YU‐ZMD	GU475462.1	99.52%	100%
017/LY/CB–BG/2023	G. anatis strain IMT49310	CP110225.1	99.75%	100%

Full text

1. Introduction

Gallibacterium anatis (G. anatis) is a bacterium that causes gallibacteriosis [1], which plays a role in reducing egg production in laying hens by up to 3%–18%. G. anatis is increasingly recognized as an important pathogen associated with reduced egg production and reproductive tract lesions in laying hens [2]. Infections caused by this bacterium can be primary or secondary [3]. Besides targeting the reproductive organs, this bacterium is also present in various other organs such as the heart, spleen, upper respiratory tract, lower genital tract, and multiple systemic tissues of affected chickens, reflecting its broad tissue tropism [2–5].

Historically, G. anatis was classified as Pasteurella haemolytica; however, in 2003, it was reclassified into a new genus, Gallibacterium [4]. In recent years, reports of G. anatis infections have increased worldwide, indicating its rising clinical importance [2]. A new taxonomy of G. anatis has been identified in several countries [6]. However, there have been no documented reports on the occurrence of G. anatis in Indonesia.

Detection of G. anatis can be performed phenotypically and genotypically. Molecular identification remains essential due to its phenotypic similarity to other respiratory pathogens [2]. Genotypic detection typically uses PCR targeting the 16S and 23S rRNA genes, and identification can be confirmed with MALDI‐TOF; indeed, isolates from reproductive, respiratory, and serosal lesions have been characterized using these methods [4]. Classification using 16S rRNA gene targets is currently widely used in bacterial diagnostics [6]. Genomic analyses have shown considerable diversity among G. anatis isolates, including variation in virulence‐associated genes. Some studies report highly conserved RTX toxin systems as core virulence mechanisms [2, 7], while others identified multiple antibiotic resistance genes varying between organs and flocks [8, 9]. In Morocco, G. anatis was isolated from ovaries, trachea, and cloaca in layer chickens with decreased egg production, indicating its direct impact on productivity [5].

Recent epidemiological studies further underscore the significance of G. anatis. Surveys of poultry flocks in Poland (2022–2023) found a prevalence of 22.5%, with 20% of isolates exhibiting resistance to at least eight antibiotics [10]. All G. anatis biovar haemolytica strains carried the toxin gene gtxA and showed multidrug resistance [9], while additional studies revealed widespread resistance to 20 antimicrobials across respiratory, reproductive, and gastrointestinal isolates [11]. Moreover, G. anatis has been detected in pet birds (∼10.5% prevalence), with isolates showing both high antibiotic resistance and moderate biofilm formation, indicating persistence and treatment challenges [12].

West Java is a province that serves as a center for chicken farming in Indonesia [13], thus serving as a starting point for identifying potentially harmful infectious agents in the poultry industry. Information regarding the presence of G. anatis has not yet been reported in West Java or Indonesia. Therefore, the aims of this study were to isolate and identify the bacterium G. anatis phenotypically and genotypically on chicken farms in West Java, to molecularly characterize the virulence genes of G. anatis, and to analyze the genetic relationship among G. anatis bacteria.

2. Materials and Methods

2.1. Ethical Approval

Ethical approval on this study was obtained from Animal Ethics School of Veterinary Medicine and Biomedical Science, IPB University with number: 121/SKE/X/2023.

2.2. Sample Collection

The research samples consisted of a total of 23 suspected gallibacteriosis cases in West Java from May 2020 to October 2023. The field samples were obtained from several chicken flocks across five different farms that experienced respiratory disorders, accompanied by a 7%–12% reduction in egg production and abnormalities in eggshell formation. All isolates were obtained from tracheal swab, trachea, lung, ovary, and oviduct organs of broilers, layers, and breeder chickens that were suspected of having clinical symptoms of G. anatis infection. Organ samples were placed in sterile plastic, while swab samples were placed in Amies transport medium (Labware), which was then transported using a cold box not exceeding 48 h from sample collection to prevent bacterial growth.

2.3. Isolation and Identification of G. anatis

Samples were obtained from tracheal swabs and reproductive and respiratory organs of chickens exhibiting respiratory disorders or decreased production. Cultures were conducted on blood agar media from archived isolates and field samples, followed by incubation at 37°C for 24–48 h. Colonies were observed macroscopically and bacterial morphology was observed microscopically by Gram staining. Biochemical tests were performed to confirm G. anatis by the catalase test, the oxidase test, and triple sugar iron agar (TSIA) [14].

Isolates exhibiting the phenotypic characteristics of G. anatis were further molecularly confirmed. Confirmation of G. anatis was conducted using the 16S rRNA gene target 1133fgal (5′‐TATTCTTTGTTACCARCGG‐3′) as the forward primer and 23S rRNA 114r (5′‐GGT​TTC​CCC​ATT​CGG‐3′) as the reverse primer, which yielded a positive result for G. anatis at a band size of 1030 bp [14].

2.4. Detection of Virulence Genes of G. anatis

All samples confirmed as G. anatis by PCR testing were further subjected to the detection of virulence genes of G. anatis bacteria encoding gtxA (cytolytic–hemolytic gene), flfA (flagellar gene), and gyrB (gyrase subunit B). The primer sequences used are listed in Table 1.

2.5. Sequence and Phylogenetic Analyses

Eight isolates of G. anatis were utilized for sequencing analysis using Sanger sequencing with BigDye Terminator V3.1 (Thermo Fisher Scientific GmBH, Dreieich 63303, Germany) [15]. The nucleotide sequence of the G. anatis 16S rRNA gene was analyzed using Basic Local Alignment Search Tool (BLAST) to determine its identity and homology to other isolates in the GenBank database of the National Center for Biotechnology Information (NCBI). Phylogenetic analysis was conducted using the Molecular Evolutionary Genetics Analysis (MEGA) 7 application with the neighbor‐joining method [16]. Phylogenetic trees were constructed by selecting reference isolates with the highest BLAST analysis scores.

3. Results

3.1. Phenotypic and Genotypic Identification of G anatis

A total of 17 (73.9%) of 23 samples showed suspected growth of G. anatis. All of these demonstrated growth as small‐sized, nonmucoid, semitransparent colonies with a distinct hemolytic zone on blood agar (Figure 1). Morphological test results are Gram‐negative and pleomorphic coccobacilli. Biochemical test results were catalase‐positive, oxidase‐positive, and indole‐negative.

FIGURE 1Cultural and microscopic characteristics of Gallibacterium anatis. (a) Growth of G. anatis on blood agar. (b) Gram‐stained G. anatis demonstrating Gram‐negative coccobacilli under 1000× magnification.(a)(b)

Identification of 17 positive G. anatis samples based on phenotypic observation consisted of five sample isolates from a breeder–layer farm, four isolates from broiler farms in Cibinong, and eight isolates from layer farms in Cibinong (Table 2). Molecular confirmation of suspected G. anatis isolates showed that, out of the three layer chicken farms, two farms were confirmed to have G. anatis (Table 2). The farms confirmed to have G. anatis experienced a decrease in egg production ranging from 5% to 20%. G. anatis bacteria were identified in chicken farms in the same area, specifically in the Cibinong region. On the broiler chicken farm, out of the two farms sampled, one farm was confirmed to have G. anatis, with a positivity rate of 75% in chickens experiencing respiratory disturbances ranging from moderate to severe.

3.2. Presence Virulence Genes of G. anatis

Virulence gene analysis was conducted targeting three genes: gtxA, gyrB, and flfA. Among the 14 confirmed G. anatis isolates, a positive result for gtxA was detected in 93.3% (13/14), for gyrB in 100% (14/14), and for flfA in 71.4% (10/14) of the isolates (Table 3). Three isolates were negative for the flfA gene, originating from Sukabumi (broiler, breeder, and layer chicken) and Bogor (layer chicken). Only one isolate (002/BELY/CS–BG/2022) tested negative for gtxA. Analysis of the G. anatis virulence gene combinations showed that each isolate carried at least two virulence genes (28.57%), while the highest prevalence (71.42%) was observed in isolates harboring all three virulence genes (Figure 2).

Distribution patterns of Gallibacterium anatis virulence genes. Each circle is representatives of the virulence gene, such as gtxA, flfA, and gyrB.

3.3. Similarity and Phylogenetic Tree of G. anatis

Similarity analysis using BLAST revealed that the 16S rRNA gene sequence of the isolates were highly similar to G. anatis. Similarity values greater than 98.65% indicate that a bacterial isolate belongs to the same species, while values above 95% can identify the genus [17]. In this study, all nucleotide sequences showed a similarity exceeding 98.65% with G. anatis (Table 4).

The bacterial isolates analyzed here shared similarities with strains from various countries. Specifically, strains YU‐ZMD, TH22, YJ922, DFS02, and BJF12 originated from chickens in China; ESV200 from Mexico; UMN179 from the United States; and IMT49310 from Germany.

Confirmation of the taxonomic proximity of a bacterial isolate by using similarity values can be reinforced by constructing a phylogenetic tree. The phylogenetic tree showed that all isolates clustered within the G. anatis in‐group, forming two main supergroups, while three reference isolates from China and the United States formed a different supergroup. Outgroup species, G. salpingitidis and G. trehalosifermentans formed distinct clusters but remained within the overall Gallibacterium clade.

The phylogenetic tree also indicated that isolates 003/Breeder Layer/Cisarua–Bogor/2022 and 005/Layer/Sukabumi//2022 were more closely related, supported by a bootstrap value of 100% (Figure 3). However, other isolates showed variable relationships with both the tested and reference strains, with bootstrap values below 70%. Bootstrap values reflect the reliability of the phylogenetic tree, where values greater than 70% correspond to a confidence level exceeding 95% [18]. The highest bootstrap values in some relationship suggest that the formed tree could vary if alternative phylogenetic reconstruction methods are applied.

Phylogenetic tree of G. anatis isolates from Indonesia. This phylogenetic tree was constructed using neighbor‐joining with the MEGA 7 application from the 16S‐23S rRNA gene. The branches of the dendrogram show bootstrap values (%) with 1000 replications, and the scale represents one per 1000 nucleotide sequence substitutions of the 16S rRNA gene. () denotes the isolates tested. () denotes outgroup reference isolates.

4. Discussion

In the present study, G. anatis was successfully isolated from poultry farms in West Java, Indonesia. Notably, the presence of G. anatis has also been identified in Europe, Africa, China, India, Japan, North America, and South America [6], underscoring its global distribution. Recent reports further confirm its widespread emergence in commercial poultry systems, particularly in Asia and Europe, where multidrug‐resistant and highly virulent strains have been documented [9]. G. anatis can cause a decrease in egg production in layer and breeder chickens, as well as respiratory disorders in broilers. In this study, G. anatis was identified in layer farms in the Cibinong area. This is consistent with the findings of Yaman and Yapicier [6], who reported G. anatis in Turkish layer farms experiencing decreased egg production. A decrease in production was also observed on Farm 1 in the Ciampea region, but G. anatis was not identified. Such production drops may be linked to other bacterial or viral pathogens that were not identified in this study. Multifactorial drops in egg production have been frequently reported in the field, as coinfections with E. coli, Mycoplasma spp., Avibacterium paragallinarum, or viral pathogens often complicate accurate attribution of production losses [19, 20].

The molecular results for G. anatis in this study indicated that not all isolates showing phenotypic suspicion of G. anatis were successfully molecularly confirmed using the 16S‐23S rRNA gene. One sample from Farms 3, 4, and 5 showed negative results based on molecular analysis. This could be due to the fact that phenotypic testing relies on biochemical pathways and carbon source utilization, which may have similarities with other bacteria, especially within the same family. Bacteria with phenotypic characteristics similar to those of G. anatis include Pseudomonas aeruginosa and Mannheimia haemolytica [21]. These results highlight the importance of combining phenotypic and molecular methods for accurate identification. This challenge aligns with recent findings showing that G. anatis displays substantial phenotypic variability, which can lead to misidentification when relying solely on biochemical assays [22].

The virulence genes of G. anatis have previously been identified. The flfA gene encodes the fimbriae in G. anatis and plays a role in the adhesion process, especially in the oropharyngeal epithelial cells of chickens, which have three fimbrial gene clusters [23, 24]. The presence of flfA can be associated with tissue tropism [25], but the data in this study are not sufficient to establish this correlation. The upper respiratory tract, particularly the trachea, is the common site of G. anatis colonization [2]. However, G. anatis has been detected in internal organs such as the liver, heart, and lungs [14, 15]. These findings suggest that certain strains have the potential for extra‐respiratory dissemination, although the mechanisms underlying this spread remain to be fully elucidated.

The gtxA gene encodes a toxin that can cause inflammation and is responsible for hemolytic activity [26]. G. anatis bacteria are grouped into two biotypes: hemolytic and nonhemolytic. The presence of the gtxA gene can be used to classify isolates as belonging to the hemolytic biotype. This classification can also be observed through phenotypic testing, which results in hemolytic zones on blood agar media. In this study, Isolate 002/BELY/CS‐BG/2022 exhibited a hemolytic zone but tested negative for the gtxA gene. This is likely due to the low concentration of the gtxA gene in the medium during the late log phase of the bacterial growth cycle [27], making it undetectable in molecular testing. Additionally, other isolates that displayed hemolytic zones on blood agar correlated with the presence of the gtxA gene in this test. Similar discrepancies have been reported in recent studies, where gtxA expression was shown to be variable based on the growth phase and environmental conditions, leading to inconsistent molecular detection despite clear phenotypic hemolysis [11].

All isolated samples tested positive for encoding the gyrB, which is associated with virulence. The presence of gyrB in these isolates is significant because the gene encodes the ATPase domain of gyrase, which is essential for bacterial DNA replication [28]. The gyrB gene can be used as a discriminatory marker for identifying G. anatis and differentiating closely related Pasteurellaceae species [22, 29]. The presence of these virulence genes confirms the pathogenic potential of the isolates collected in this study.

Similarity analysis showed that Isolates 016/LY/CB–BG/2023 and 017/LY/CB–BG/2023 originated from the same type of chicken and area; however, they exhibited different reference bacterial strains based on similarity values. Isolate 014/BR/CB–BG/2023, the only broiler chicken tested in this study, showed similar testing results that can also be found in isolates originating from layer chickens or breeders. Interestingly, Isolate 017/LY/CB‐BG/2023, from layer chickens, showed high similarity to isolates from different host species, including cattle. This suggests that the 16S–23S rRNA gene is highly conserved and may not reflect host‐specific adaptation, indicating that sequence similarity does not necessarily correspond to the original host of the isolate. This is consistent with recent genomic studies showing that G. anatis forms host‐independent lineages and that cross‐species similarities are common, especially between poultry and ruminant isolates [9].

Phylogenetic analysis of G. anatis has been conducted extensively to classify this bacterium. Partial use of the 16S‐23S rRNA gene was unable to differentiate isolates based on their origin or epidemiology. Phylogenetic analysis using the 16S‐23S rRNA gene can only group isolates within the same species but not necessarily within the same group with good bootstrap values. In addition to using this gene, the flfA gene has also been utilized for bacterial analysis; however, it also fails to differentiate this gene based on sample origin. Current evidence strongly supports the use of multilocus sequence typing (MLST), core‐genome SNP analysis, or whole‐genome sequencing (WGS) to obtain clearer cluster separation and better infer transmission routes and virulence‐associated lineages [7].

These findings demonstrate that G. anatis is present in West Java poultry farms, with isolates carrying virulence genes that may contribute to decreased egg production and respiratory disorders. Future studies should include larger sample sizes, additional virulence or housekeeping genes, and WGS to better understand the epidemiology, host adaptation mechanisms, and pathogenicity of G. anatis in Indonesian poultry systems.

5. Conclusion

This is the first report to publish findings regarding the presence of G. anatis isolates in Indonesia. A total of 17 isolates were phenotypically identified as G. anatis. Molecular analysis confirmed that 14 of these isolates were G. anatis, and the virulence genes gtxA, gyrB, and flfA were detected, showing diverse patterns. Nucleotide analysis results indicated that the isolates had similarities to G. anatis with various strains. However, the G. anatis strain in this study could not be classified into a specific group in the phylogenetic tree constructed using the 16S‐23S rRNA gene sequence. The presence of G. anatis in chickens experiencing respiratory disorders or decreased production is similar to that of G. anatis in several countries, confirming the existence of this bacterium.

Funding

The publication of this journal was granted by the United States Agency for International Development (USAID) throughout the SEAOHUN One Health Scholarship Program. The contents are the responsibility of the authors and do not necessarily reflect the views of USAID or the United States Government.

Conflicts of Interest

The authors declare no conflicts of interest.