INVESTIGATION OF FIVE E. COLI SEROGROUPS (APEC) ISOLATED FROM CASES OF ARTHRITIS OF COMMERCIAL MEAT CHICKEN IN MIDDLE AND COASTAL REGIONS OF SYRIA

INVESTIGATION OF FIVE E. COLI SEROGROUPS (APEC) ISOLATED FROM CASES OF ARTHRITIS OF COMMERCIAL MEAT CHICKEN IN MIDDLE AND COASTAL REGIONS OF SYRIA

NAWAL ALKARAWANI and AZZAM KURDI

Master Degree of Poultry Diseases,

Faculty of Veterinary Medicine, AL Baath University, Syria

INTRODUCTION

Colibacillosis refers to any localized or systemic infection caused entirely or partly by avian pathogenic Echerichia coli (APEC), including colisepticemia, coligranuloma, yolk sac infection, air sacculitis, salpingitis, peritonitis, arthritis and others (Sharada et al., 2010). The most common forms of colibacillosis arthritis occurs among 2-10 week – old chickens (Aggad et al., 2006).

Escherichia coli is a part of the non-pathogenic normal inhabitants of intestinal and respiratory tracts in birds (Jordan and Pattison, 1996), so, it was considered as a potential pathogen. However, Escherichia coli infections in various farms are responsible for significant economic losses due to lower corporal development, insufficient feed conversion, increasing mortality, and higher cost of medicine (Rocha et al., 2008). Escherichia coli was the major cause of infections causing condemnation of processed chickens in Switzerland (Jacob et al., 1998).

However, arthritis caused by E. coli is a major cause of lameness in chicken. E. coli has been cultured from osteomyelitis, bacterial chondronecrosis and synovitis sites in previous studies (McNamee et al., 1998). Osteomyelitis may be seen in flock outbreaks of       E. coli colibacillosis (Reece, 1992; Thorp, 1996) in association with septicemia, arthritis and tenosynovitis.

(Maurer et al., 1998) described that many E. coli isolates commonly associated with colibacillosis in poultry belong to serogroups (O1, O6, O8, O15,O78), so these serogroups are more frequently recovered from Septicemic clinical cases (Heller and Drabkin, 1977; Cheville and Arp, 1998).

The rate of infection with E.coli bacteria (colibacillosis) is formed approximately 30-40% of the other diseases in poultry (Cheville and Arp, 1998). Serogroups (O1, O6, O8, O15, O78) are more pathogenic than other and more frequently associated with arthritis clinical cases (Barbour et al., 1970; Peighambari et al., 1995; Gomis et al., 2001). The previous studies have shown that hemolytic activities of APEC isolates correlated to the virulence of avian E. coli. In addition, approximately 90% of the isolates showed the hemolysis (alpha, beta, gamma) (Moon  et al., 2006). Also, several authors (McNamee et al., 1998) had reported that E. coli which was isolated from femur of lameness birds was hemolytic (86.4%).

The flagella, which are thin surface appendices, give motility to gram positive and negative bacteria in aqueous media. Their rotating movements allow microorganisms to approach adjacent epithelial cells, crossing the mucus barrier and causing adhesion, multiplication, colonization and infection (Stenutz    et al., 2006).

This study aims to investigate the five frequently strains from O serogroups (O1, O6, O8, O15, O78) and to test the motility and hemolysis characteristics of virulence factors.

MATERIALS and METHODS

Samples Collection:

During the period February 2012 to March 2013, a total of 188 joint samples of arthritis cases were collected from poultry farms in the middle and coastal regions of Syria (Hama and Tartos). Joints were subjected to cleaning and disinfection of the skin, then, exudates from swollen joints or foot pads were collected by sterilized swabs.

Samples Preparation:

Upon reaching the laboratory, samples were cultured in nutrient broth (HiMedia) overnight at 37°C.

Cultural and Biochemical characterization:

The samples were cultured, using asterilized loop, on specific solid media MacConkey agar - EMB agar), then, incubated at 37°C for 24 hours.

All bacterial colonies were selected from each sample. These colonies were isolated in pure culture for further identification.

A cultural suspension with 5 ml of physiological saline was prepared from each isolate and compared with McFarland standard to have a right turbidity. This material was used to inoculate HiMotility biochemical kit for E. coli (HiMedia) which include the following tests; Motility, Indole. Citrate, Glocoronidase, Nitrate, ONPG, Lysine, Lactose, Glucose, Sucrose and Sorbitol.

Kits were inoculated, incubated, handled, and analyzed according to the manufacturer^’s instructions.

Motility test:

The primary hanging Drop Method, as described by (Cowan, 1985), was carried out to detect the motility of all isolates. Isolates which didn^’t show motility had been retested by using SIM agar and incubated at 37°C for 24 hours (Quinn et al., 1994). In addition, results were supported with Biochemical kits which include a motility test.

Hemolytic Test:

The hemolysis of isolates was tested by culturing on blood agar and incubated at 37°C for 24 hours. The isolates which showed a complete hemolysis (Beta) on the blood agar had been recorded, as well as for the partial hemolysis (Alpha) and for the negative results (Gamma) (Quinn et al., 1994).

Serotyping:

We used five Serotypes (serum) of pathogenic E.coli (O1, O6, O8, O15, O78) [DENKA SEIKEN Co.Ltd, Tokyo, Japan]) (Veterinary Institute et al., 2009). Isolates were serotyped by agglutination test with specific antisera of somatic antigen to five serogroups (O1, O6, O8, O15, O78).

RESULTS

The articular region of affected hock, knee, hip and foot pad joints often appeared enlarged due to accumulated exudates distending into the joint cavity. Swelling was also observed as a result of periarticular inflammation of soft tissues and simultaneous tendovaginitis. The bacterial isolation from 188 cases of arthritis in chickens is shown in Table 1.

Table 1: Bacteriological results obtained from 188 investigated joints from chicken condemned due to arthritis.

Collected exudates from joints had been cultured on MacConkey agar and all colonies were primary classified as E. coli based on the colonies characteristics (Table 2).

Table 2: Culture characteristics on MacConkey agar

The examination of 188 joint samples collected from Hama and Tartos revealed, that 95% of Hama and 93% of Tartosjoint samples were classified as E. coli isolates according to the results of Biochemical tests (using Bio.Kits) (Table 3).

Table 3-a: E. coli isolates after Biochemical tests (HiMotility Kits)

E. coli was gram negative rod. Catalase test was positive and oxidase test was negative. Indole production and Methyl-Red test were positive. Voges-Proskauer test and Simon^,s citrate were negative.

Table 3-b: Results of primary biochemical tests of E. coli

Table 3-c: Results ofbiochemical tests (HiMotility Kits)

MOTI, Motility; IND, Indole; CIT, Citrate utilization; GLOR, Glocoronidase; NIT, Nitrate; ONPG,Ortho Nitrophenyl-βD-G alactopyranosidas; LDC, Lysine decarboxylase; LAC, Lactose; SUC, sucrose; GLU, Glucose; SOR, Sorbitol.                             negative reaction     -                                   +  positive reaction

The detection of motility showed that, 95.3% of Hama and 95.1%  of Tartosisolates were motile (Table 4).

Table 4: E. coli motility.

Hemolysis test showed that, 95.7% of Hama isolates were hemolytic and 71.3% of them were type beta, while 94.6% of Tartos isolates were hemolytic and 75.1% were type beta (Table 5).

Table 5: Hemolysis on blood agar.

88.4% of Hama and 80.4% of Tartos isolates were motile and hemolytic at the same time.

Only 5 isolates from Hama and 5 isolates from Tartos were motile and non-hemolytic.

Only 5 isolates from Hama and 5 isolates from Tartos were hemolytic and non motile (Table 6).

Table 6: Motility and hemolysis.

The five serogroups (O1, O6, O8, O15 and O78) formed a difference percentage in Hama and in Tartosisolates. O-serotype of pathogenic E. coli was O1(45 strains), O6(43), O8(5), O15(32), O78(12) (Table7).

Table 7: O-serotype of pathogenic E. coli

DISCUSSION

In this study, the isolation trials resulted in detaining of 86 and 51 E. coli isolates (of 188 samples) from Hama and Tartos arthritis cases, respectively.

The results of this study showed that the most affected joints were hock and knee joints (88.3%, 20.4%) respectively, this result is consistent with (Mutalib et al., 1996).  On the other hand, (McNamee et al., 1998) reported that (13.6%) of hip joints was affected, this result is similar with our^,s(14.6%).

A percentage of 95.3% of Hama and 95.1% of Tartos isolates were motile, these percentages show a high level comparing with the results of other studies. In a recent study (Rocha et al., 2008), motility was detected in 54.1% of the samples and was36.8% higher than that had been reported by (McPeake       et al., 2005).

By testing the hemolysis of all isolates we found that percentage of hemolysis for the two regions isolates were approximately close even the percentage of hemolysis type, for example; 95.7% of Hama isolates was hemolytic and that was 94.6% for Tartos isolates.

The Beta hemolysis for Hama isolates was 71.3% and for Tartos isolates was 75.1%. According to a recent study in Korea (Moon et al., 2006), it was found that 72% (48 isolates) of the APEC – isolated from birds with colibacillosis- revealed α or β hemolysis on blood agar plates. In addition, approximately 90% of the isolates, which showed the hemolysis, harbored one or more virulence genes (Moon et al., 2006). So, this result gives a reasonable idea about the relationship between hemolysis characteristic and pathogenicity of strains.

The relationship between the two characteristics, motility and hemolysis were observed as 88.4% of Hama isolates and 80.4% of Tartos isolates which were found motile and hemolytic.

On the other hand, only 5 isolates from Hama and 5 isolates from Tartos were found motile and non-hemolytic, while 5 isolates from Hama and 5 isolates from Tartos were found hemolytic and non-motile.

In conclusion, the majority of the motile isolates (76 isolates/86 for Hama and 41 isolates/51 for Tartos) were carrying the hemolytic characteristic and showed a Beta or Alpha hemolysis on blood agar.

The results showed that the five serogroups exist in the two regions with different percentages. The most predominant serotype was O1(45 strains) accounting for 32.8% and O6(43), O15(32), O8(5), O78(12). (Gomis et al., 2001) reported that E. coli of serogroups O1, O2, O78 isolated from avian colibacillosis predominantly in Canada. (Sojka and Carnaghan, 1961; Dho-Moulin and Fairbrother, 1999) described that many E. coli isolates commonly associated with colibacillosis in poultry belong to serogroups O1, O2, O78. The serotypes of E. coli in the present study were in similar with (Sojka and Carnaghan, 1961; Dho-Moulinand Fairbrother, 1999; Gomis et al., 2001). On the other hand, (Sharada      et al., 2010) reported that serotyping of isolates revealed redominantly O11, O79, O111 accounting for 26.15%, but O15, O78 were with very low percentage. So, we should more examine serotypes of isolated E. coli.

The serotype of E. coli O8 was isolated from 5 isolates and (Raji et al., 2003) reported that O8strain showed 100% mortality in 1 day-old chickens and was the most pathogenic serotype.

In this study, it was found that several serotypes were determined. This report is the first report of E. coli serotyping isolated from poultry with lameness and arthritis in Syria.

In conclusion, utilization of Biochemical Kits for detecting the characteristics of E. coli strains is more suitable and easier than using the standard techniques. It gives accurate results and save time, although it is more expensive.

It is remarkable that five serogroups (O1, O6, O8, O15, O78) were existed in Hama and Tartos regions of Syria with a high percentage and this isolates were holding pathogenic characteristics like hemolysis and motility. Our results were similar to the previous reports, so, these five serogroups are considered the most prevalent and pathogenic serogroups in Syria.

We need a more detailed study in order to determine the relationship between different strains which isolated in the middle, coast and other regions of Syria. So, we need to establish the pathogenic characteristics. Ultimately, identification of potential virulence traits may allow for it as specific markers for the diagnosis of pathogenic strains.

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