Diagnosis of Gallibacterium Anatis in Layers: First Report in Turkey

ABSTRACT

Gallibacterium anatis, a member of the Pasteurellaceae family, leads to decrease in egg-production, animal welfare and increase in mortality. This study aimed to diagnose G. Anatis, which caused economic losses in laying hens by using conventional and molecular techniques. In this study, G. anatis was examined from a total of 200 dead chicken tissues (heart, liver, lung, spleen and trachea) in laying hen farms that observed a decrease in egg production with respiratory system infection. Conventional methods based on colony morphology, sugar fermentation tests and hemolytic properties and molecular conformation using 16S rRNA-23S rRNA specific primers were performed to identify G. anatis. G. anatis was isolated in 20 (10%) of the examined samples and isolates were confirmed by conventional PCR. A total of 11 (2.2%) positivity was obtained as isolates were the result of PCR performed on tissues and organs directly. As a result, the presence of G. anatis was detected for the first time in Turkey by this study. It was thought that G. anatis may have a role in egg production losses due to respiratory tract infection in poultry and this situation may be a guide for poultry clinicians and microbiologists.

Keywords:
Gallibacterium anatis; isolation and identification; layers; PCR

INTRODUCTION

Major health problems of the poultry industry have certain effects on egg production. Especially, infectious diseases which may drop in egg production and egg quality by affecting the reproductive system directly and the health status of poultry indirectly (Clauer, 2009).

Gallibacterium anatis (G. anatis) has been known to be a part of the normal microflora of the lower genital and upper respiratory tract (Bojesen et al., 2004; Jones et al., 2013; Lawal et al., 2018; Persson & Bojesen 2015; Rzewuska et al., 2007). Paudel et al., 2013. In recent years, decreased egg production associated with oophoritis, follicule degeneration, salpingitis, respiratory system disorders and increased mortality in commercial layers has accelatered interest in G. anatis infections (Alispahic et al., 2011; Bisgaard et al., 2009; Bager et al., 2013; Bojesen et al., 2003; Bojesen, 2003; Sing, 2016; Chaveza et al., 2017; Johnson et al., 2013; Paudel et al., 2014). The epidemiology and bacteria-host interactions of Gallibacterium spp. are little understood due to a lack of published literature and previous uncertainty with regard to the identification of bacteria representing this genus (Bisgaard, 1993).

The aim of this study was to investigate the Gallibacterium anatis from commercial layers that suffered respiratory tract disease and decrease in egg production as well as determine a convenient microbiological and molecular diagnostic technique.

MATERIALS AND METHODS

G. anatis strains

G. anatis F149T (non-hemolytic strain, ATCC 43329) and 12656-12 strain (hemolytic strain) were obtained from Prof. Anders Miki Bojesen (Department of Biology, Department of Veterinary Diseases, Copenhagen University) and used in this study.

Sampling

G. anatis was examined from in a total of 200 dead hens tissue samples (heart, liver, lung, spleen and trachea) were collected from 31 commercial layer houses from three different cities (Afyonkarahisar, Kütahya, Gaziantep) during the period from August 2017 to January 2018 in Turkey. The number of samples collected are summarized in Table 1.

Table 1
Layer houses where samples were collected

Flocks	Breed	Age(week)	Number of sampled animal
1	Lohmann white	24	5
2	Lohmann white	76	3
3	Lohmann white	64-66	2
4	Lohmann white	64-66	3
5	Lohmann white	16	3
6	Supernick	18	19
7	Lohmann white	60	17
8	Lohmann white	65	4
9	Lohmann white	55	4
10	Lohmann white	85	12
11	Lohmann white	12	16
12	Lohmann white	52	2
13	Lohmann white	60	6
14	Lohmann white	85	11
15	Lohmann white	60	6
16	Lohmann white	48-50	6
17	Lohmann white	36	4
18	Lohmann white	70	5
19	Atak-S	32	2
20	Lohmann white	68	4
21	Supernick	75	14
22	Supernick	19	5
23	Lohmann white	65	9
24	Lohmann white	75	7
25	Lohmann white	35	5
26	Lohmann white	66	4
27	Lohmann white	75	3
28	Lohmann white	57	4
29	Lohmann white	30	6
30	Nick chick white	16	5
31	Lohmann white	36	4
Total			200

A total of 10 to 45.000 flock sized, 12-85 week-old laying hens were housed in 60 x 60 cm cages (n:5-8 birds in each cage) in a 40x10m farm building. The lit-ter of the poultry houses was of good quality, although ventilation by mechanical fans or windows was poor in some of these. Water and feed were provided ad libi-tum. Average body weight of birds was 1500-1600 g.

All of the examined dead birds included in the study had recent histories of respiratory disease and reproductive problems with a cumulative mortality rate during the week of sampling which ranged from0.4-0.7%.

Isolation and identification: Tissue samples were inoculated to 5% sheep blood (Oxoid, USA) and MacConkey agar (Oxoid, USA). The plates were incubated at 37°C for 18-24 hours aerobically. Beta haemolytic, circular, smooth, shiny and greyish suspect colonies were stained by Gram staining and biochemical tests were performed to identify the Gram negative rods (Bager et al., 2013; Bojesen & Shivaprasad, 2006). Gallibacterium isolates were suspensed in seven hundred microlitres were mixed with 300 μl sterile glycerol 50% and stored at -80 oC until further use (Bojesen et al., 2003).

Molecular identification: DNA extraction from G. anatis isolates and tissues (heart, lung, trachea, spleen) was performed according to the instructions of the GeneJET Genomic DNA Purification Kit (Thermo Scientific, USA) and the QIAamp DNA Stool Kit (Qiagen, Hilden, Germany). DNAs were stored for use as template DNA at -20°C until amplification.

A primer pair specific for 16S-23S rRNA genes [1133F(5’-TATTCTTTGTTACCARCGG-3’) and 114R (5’-GGTTTCCCCATTCGG-3’)] of G. anatis were selected. PCR was performed with the default settings of the thermocycler (Nyx Technik, A6-00150, USA) and the PCR assay was carried out in a 25 µl reaction solution containing 3 µl MgCl (25 mM), 0.5 µl dNTP (10 mM), 10 pmols of primers and 0.2 µl Taq polymerase (5U/µl). The following cycling conditions were used: 3 min at 94°C, followed by 30 cycles of 1 min at 94°C (denaturation) and 1 min at 54°C (primer annealing), 1 min at 72°C (extension), and 7 min at 72°C (final extension).

The amplification products (790 bp and 1080 bp for the G. anatis) were examined by the separation of PCR products during electrophoresis on 1.5 % agarose gel stained with safe dye (Jena Bioscience, Germany).

RESULTS

Isolation and Identification

In the present study, 20(10%) Gallibacterium spp. were isolated from tissue and organ specimens (trachea, heart, liver, lungs and spleen) from 8 out of 31 flocks. Gallibacterium spp. was isolated from lung (5%), heart (0.5%), liver (1%), and from trachea (3.5%) as shown in Table 2.

Table 2
Isolation and identification results from the tissue samples.

Positive flocks	Lung	Spleen	Heart	Liver	Trachea
6	6(%3)	-	1(%0.5)	1(%0.5)	1(%0.5)
7	1(%0.33)	-	-	-	1(%0.5)
10	-	-	-	-	2(%1)
11	-	-	-	-	1(%0.5)
14	-	-	-	1(%0.5)	1(%0.5)
23	2(%1)	-	-	-	-
24	1(%0.5)	-	-	-	-
31	-	-	-	-	1(%0.5)
Total	10(%5)	-	1(%0.5)	2(%1)	7(%3.5)

According to the tissue samples collected from the different provinces, 25.8% was isolated from Afyonkarahisar, while no Gallibacterium spp. was isolated from Gaziantep or Kütahya.

Beta haemolytic Gallibacterium spp. isolates (Figure 1), all tested catalase-positive and 9 (45%) tested positive in an oxidase test. Based on the results of biochemical tests (Table 3), a total of 20 isolates were identified as G. anatis, and 10 (5%) E. coli isolates were found during G. anatis isolation from 200 layers. Of these E. coli isolates, 6 (60%) were isolated from the lungs, 3 (30%) were isolated from the trachea and 1 (10%) was isolated from the heart.

Figure 1
G. anatis β-hemolytic colonies on sheep blood agar.

Table 3
G. anatis biochemical test results.

Isolates	Catalase	Oxidase	MacConkey	Sucrose	Arabinose	Lactose	Glucose	Sorbitol	Trehalose	Maltose	Fructose	Mannitol	Urea	Indole	ONPG
1	+	-	-	+	-	-	-	-	-	+	-	-	-	-	+
2	+	-	-	+	-	-	-	-	-	+	-	-	-	-	+
3	+	-	-	+	-	-	+	-	-	+	-	-	-	-	+
4	+	+	-	+	-	-	-	+	-	+	-	-	-	-	+
5	+	-	-	+	+	-	-	+	-	+	+	-	-	-	+
6	+	-	-	+	+	-	-	+	-	+	+	+	+	+	+
7	+	-	-	+	+	-	-	+	-	+	+	+	+	+	+
8	+	+	-	+	+	-	-	+	+	+	+	+	+	+	+
9	+	-	-	+	+	-	+	+	-	+	+	+	+	+	-
10	+	+	-	+	+	-	+	-	+	+	+	+	-	+	-
11	+	+	-	+	+	-	+	-	+	+	-	+	-	-	-
12	+	+	-	+	-	+	+	-	+	+	-	+	+	+	-
13	+	+	-	+	-	-	+	+	-	-	-	-	-	+	-
14	+	-	-	+	+	-	-	-	-	-	-	-	+	+	-
15	+	-	-	+	-	-	+	-	-	-	-	-	+	+	+
16	+	+	-	+	-	+	+	-	-	+	-	+	-	-	-
17	+	+	-	+	-	-	-	-	-	+	+	-	-	-	+
18	+	-	-	+	+	-	-	-	-	+	+	-	+	-	+
19	+	+	-	+	+	+	-	+	-	+	+	-	+	+	+
20	+	-	-	+	+	-	+	+	-	+	+	+	+	-	-

Molecular Diagnosis

Molecular diagnosis of biochemically confirmed G. anatis isolates (n=20) exhibit the desirable PCR product of 790 bp and 1080 bp size of 16S-23S rRNA primers (Figure 2). The conventional PCR directed to detect 11 (2.2%) G. anatis from lung (2.5%), trachea (2%), and liver (1%) of 200 layers revealed, the results of which are presented in Table 4.

Figure 2
PCR results of G.anatis (M=100bp marker; 1-5=positive isolates; 6= positive control; 7=negative control; 8-19=positive isolates; 20=positive control; 21= negative control).

Table 4
PCR results obtained from tissues.

Positive flocks	Lung	Spleen	Heart	Liver	Trachea
6	2(%1)	-	-	1(%0.5)	1(%0.5)
7	1(%0.5)	-	-	-	1(%0.5)
10	-	-	-	-	-
11	-	-	-	-	1(%0.5)
14	-	-	-	1(%0.5)	1(%0.5)
23	2(%1)	-	-	-	-
24	1(%0.5)	-	-	-	-
31	-	-	-	-	-
Total	5(%2.5)	-	-	2(%1)	4(%2)

DISCUSSION

G. anatis is an infectious agent that has been isolated from broiler and egg-laying chickens with salpingitis and peritonitis in various countries around the world in recent years, and is associated with economic losses due to the resulting decline in egg yield (Bojesen et al., 2003; Elbestawy et al., 2018).

G. anatis can be found in European, African and Asian countries, but has also been reported in China, India, Japan, and North and South America (Singh et al., 2016). No G. anatis infection has been reported in Turkey to date, and the present study is the first to report a prevalence rate of 10% in egg-laying chickens. It is thought that the reason why G. anatis has not been detected to date is due to the similarity of the symptoms of this infection to that of various respiratory tract infections, and particularly to the symptoms of fowl cholera, and the fact that the precise taxonomic classification of the bacteria was not established until 2003.

It has been reported that phenotypical characteri-zation for Gallibacterium species (G. genomospecies 1 and 2) is difficult and time-consuming due to their heterogeneity (Alispahic et al., 2011; El-Adawy et al., 2018; Sing, 2016). The present study investigated the presence of G. anatis in tissues and organs collected from chickens showing symptoms of respiratory tract infection along with a decrease in egg yield. Conventional methods based on hemolysis and carbohydrate fermentation (Christensen et al., 2003), and molecular methods based on the detection of 16S-23S rRNA sequences (Bojesen et al., 2007) were preferred as the diagnostic tools. G. anatis was isolated and identified from 10% of the lung, spleen, heart, liver and trachea specimens obtained from 200 chickens. The rate of bacterial isolation on a material basis was 5% for lungs and 3.5% for trachea, which isolates particularly being identified in the respiratory tract organs, which is consistent with the findings reported in other studies (Bisgaard, 1977; Bojesen et al., 2003; Mushin et al., 1979). In their study, Bojesen et al. (2003) collected tracheal and cloacal swabs from infected flocks, and identified a high isolation rate for G. anatis in the tracheal swabs. Elbestawy et al., (2018) identified six isolates of G. anatis (19.6%) in tracheal, ovarian and oviduct swabs obtained from egg-laying chickens with oophoritis, tracheitis, salpingitis and peritonitis. In a study conducted in China, Huangfu et al. (2012) collected tracheal, ovarian and oviduct samples and identified 33 (18.2%) isolates of G. anatis. In another study reported in Mexico, G. anatis isolates were identified from tracheal samples in 30%, and in egg follicules in 30% of 600 samples obtained from layer poultry houses (Chaveza et al., 2017). G. anatis was detected in egg-laying chickens with symptoms of salpingitis in Iran (Ataei et al., 2017). As was the case for Mexico and Iran, G. anatis was recently reported for the first time in Turkey (Ataei et al., 2017; Chaveza et al., 2017). Among the 31 investigated poultry houses located in the provinces of Afyonkarahisar, Gaziantep & Kütahya, only 8(25.80%) poultry houses were positive for the bacteria, all of which were located in Afyonkarahisar. It was considered that the high density of the egg-laying chicken population in this province compared to other provinces, and the fact that much of the sampling was particularly performed in this province, may explain the high isolation rate in Afyonkarahisar (Yumbir, 2018).

Molecular diagnostic methods have been widely used in the recent years for diagnosis and phenotyping, being fast, easy and with high specificity, sensitivity and reliability (Ataei et al., 2017; Bojesen et al., 2007). Similar to the studies of other researchers, the present study adopted the PCR method to confirm the identified G. anatis isolates and to further examine the tissue and organ specimens that tested negative in the initial isolation tests (Bojesen et al., 2010; Bisgaard et al., 2009; Christensen et al., 2003). Molecular confirmation of G. anatis was performed by using 16S rRNA-23S rRNA primers, which have previously been used in the literature and are known to be specific to G. anatis (Bojesen et al., 2007). These primers are especially preferred for differentiating from other species in the Pasteurellaceae family that may cause diagnostic confusion (Christensen et al., 2003). A conventional PCR confirmed 20 (10%) G. anatis isolates with bands at 790bp and 1080bp. A direct PCR analysis of organ and tissue samples revealed 11 (2.2%) G. anatis-specific bands. The PCR detection of G. anatis from 5 (2.5%) lungs, 2 (1%) hearts and 4(2%) trachea specimens, with simultaneous isolation of bacteria from the relevant specimens, is in parallel with the results of researchers who have conducted similar studies (Ataei et al., 2017; Chavez et al., 2017; Sorour et al., 2015).

According to the information gathered from the poultry house owners, ventilation problems in the poultry houses where G. anatis was isolated and identified represented an important stress factor for the animals. It has previously been suggested that while G. anatis is found in the normal respiratory microflora of animals, it becomes the cause of an opportunistic respiratory tract infection when the immune system of the animal is compromised and/or due to stress and unfavorable changes in the care and nutritional intake of the animals (Bojesen et al., 2003). The high rate of isolation from the lungs (5%) and trachea (7.5%) in the present study supports this hypothesis.

It was suggested that G. anatis could be the cause of both primary and secondary infections in animals, that G. anatis infections are often accompanied by E. coli infection, and that it is difficult to differentiate between these two microorganisms in animals with salpingitis and peritonitis (Bisgaard, 1977; Mirle et al., 1991). In the present study, E. coli isolates 10 (5%) were recovered during G. anatis isolation from 200 chickens. In support of previous studies, E. coli was detected in respiratory tract organs, with 6 (60%) isolates recovered from the lungs and 3 (30%) isolates recovered from the trachea (Carlson & Whenham, 1968; Gross, 1961; Neubauer et al., 2009).

It was concluded that to reduce the losses and to enhance productivity in poultry industry; other Gallibacterium species should be identified, the infection should be investigated in different age and breeding, the characteristics of the bacteria should be determined for future vaccines and additional studies to determine the sources of infection in terms of public health.

ACKNOWLEDGMENTS

This study was derived from the MSc. thesis of the first author.

We would like to thank Prof. Anders Miki Bojesen from from Department of Veterinary Disease Biology, University of Copenhagen, for his valuable contributions (positive control strains maintenance and professional advices) to the study.

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• Ethical approval and consent to participate

  This study was approved by Animal Research Ethics Committee of Burdur Mehmet Akif Ersoy University (Protocol No. MAKU-HADYEK/ 2017-314) and the institutional ethics committee gave permission to collect samples from laying houses.

• Consent for publication

  Not applicable.

• Availability of data and materials

  We allow the use of data and materials upon request to the corresponding author.

• Funding

  This research was supported by Burdur Mehmet Akif Ersoy University Scientific Research Projects Coordination Unit with 0468-YL-17 project number.

Publication Dates

• Publication in this collection
  11 Nov 2019
• Date of issue
  2019

History

• Received
  12 Feb 2019
• Accepted
  25 Mar 2019