PROBLEM OF ESCHERICHIA COLI INFECTION IN BROILERS IN QENA PROVINCE

Assiut University web-site: www.aun.edu.eg

PROBLEM OF ESCHERICHIA COLI INFECTION IN BROILERS IN QENA PROVINCE

SHIMAA A.M. AHMED²; AHMED I.A.¹; NABILA OSMAN¹ and

DINA M.W. SHIBAT EL-HAMD²

¹ Department of Poultry Diseases, Faculty of Veterinary Medicine, South Valley University, Qena, Egypt.

² Animal Health Research Institute, Qena, Egypt.

Received: 22 March 2017;       Accepted:  30 April 2017

INTRODUCTION

Colibacillosis is an economically important disease, which is prevalent in Egypt and throughout the world and may constitute a great hazard to poultry industry causing high poultry mortality, loss of weight and reduction of egg production (Bandyopadhay and Dhawedkar, 1984; Kaul et al., 1992; Barnes and Gross, 1997; Margie and Lawrence, 1999; Radwan et al., 2014 and Ammar et al., 2015). More than 1000 E. coli serotypes have been reported but only small percentages have been implicated in poultry diseases. The pathogenic and non-pathogenic strains in poultry are differentiated based on the virulence. Hence, detection of these strains becomes important for effective colibacillosis treatment and control (Cloud et al., 1985 and Suthantha et al., 2001). E. coli species are divided serologically intoserogroups   and   serotypes   on   basis   of    their

Corresponding author: Dr. AHMED I.A.

E-mail address: ibrahim.ahmed@lycos.com

Present address: Department of Poultry Diseases, Faculty of Veterinary Medicine, South Valley University, Qena, Egypt.

antigenic   composition   (somatic   or   O antigens for serogroups and flagella or H antigens for serotypes). Many strains express a third class of antigens (capsular or K antigens) (Compos et al., 2004). Pathogenic E. coli strains have been divided into intestinal pathogenic E. coli and extra intestinal pathogenic E. coli (ExPEC) depending on the location of the infection. Avian pathogenic E. coli (APEC) strains belong to the ExPEC group is a major pathogen responsible for morbidity and mortality in chickens. It induces different syndromes in poultry, including systemic and localized infections, such as respiratory colibacillosis, acute colisepticemia, yolk sac infection, enteritis, arthritis, omphalitis, swollen-head syndrome, coli granuloma, salpingitis and oophritis accounting for about 5-50% mortality in poultry flocks. The most common form of colibacillosis is characterized by an initial respiratory disease in 3-6 week-old broiler chickens. It is usually followed by a systemic infection with characteristic fibrinous lesions (airsacculitis, perihepatitis, and pericarditis) and fatal septicemia (Ewers et al., 2003; Roy et al., 2006; Sharada et al., 2010 and Abd El Tawab et al., 2014).

The pathogenicity of E. coli is generally initiated by several predisposing factors, such as mycoplasma infections, viral infections, environmental factors and immune-suppressive diseases (Ewers et al., 2003; Bopp et al., 2005 and Gomes et al., 2005).

In addition, the zoonotic potential of APEC raises food safety concerns (Mora et al., 2009; Tivendale et al., 2010 and Chanteloup et al., 2011). ExPECs that can cause neonatal meningitis, urinary tract infections and septicaemia in humans share high levels of genomic similarity and some key virulence factors with APECs (Moulin-Schouleur et al., 2006, 2007; Johnson et al., 2008; Mora et al., 2012 and Olsen et al., 2012).

Several virulence factors have been associated with the virulence of APEC, including those encoding for adhesions, toxins, autotransporters, iron acquisition systems, sugar metabolism, serum resistance proteins, and capsule as well as lipopolysaccharide complexes (Ewers et al., 2003; Li et al., 2005 and Schouler et al., 2012). However, numerous studies have demonstrated that these virulence factors are rarely all present in the same isolate and that they can occur either individually or polygenically with varying frequencies in clinical isolates, indicating that APEC strains constitute a heterogeneous group (Delicato et al., 2003; Vandekerchove et al., 2005 and Mbanga and Nyararai, 2015). The accurate identification of virulent strains of E. coli and the virulence genes they possess is essential if genes that can serve as vaccine targets are to be identified. Virulence gene studies are therefore important as they not only aid in the characterization of pathogenic strains of E. coli but may eventually lead to the development of effective vaccines (Mbanga and Nyararai, 2015).

Epidemiological tracing of E. coli strains is of considerable importance in veterinary microbiology. The data can be used to monitor trends in the occurrence of pathogenic strains or to identify possible source of infection. Autologous bacterins provide limited serotype- specific protection, because multiple serogroups are associated with disease, especially O1, O2 and O78 among many others (Dziva and Stevens, 2008 and Roshdy et al., 2012).

The precise set of virulence factors possessed by Avian Pathogenic E. coli (APEC) isolated from infected birds is not clearly established in the isolates of Qena Governorate. Detection of Avian Pathogenic E. coli (APEC) and their virulence factors that causes infections in poultry becomes important for effective treatment with antimicrobial therapy and control schemes resulting in reducing both the incidence and mortality. So, this study was carried out in order to record the prevalence of E. coli infection in Qena province by bacteriological characterization of E. coli isolates in chickens, identification of the prevalent serotypes of E. coli isolates and detection of some virulence genes of the isolated strains by using PCR.

MATERIALS AND METHODS

Collection of samples:

A total of 600 freshly dead and diseased chicken samples categorized as 332 from baby chicks, from 268 broilers were collected from 81 farms at different Governmental and private poultry farms in five different centers at Qena province (Qena, Qift, Deshna, Qus and Nag-Hammadi). Bacteriological Samples were taken from diseased and freshly dead chicken (liver; spleen; nasal cavity; trachea; lung; air sacs; kidneys; yolk sacs; pericardium; and intestine from cases exhibiting perihepatitis, pneumonia, airsacculitis, yolk sac infection, pericarditis, and enteritis) after clinical and postmortem examination. Each examined organ was taken alone in sterile plastic bag, kept in icebox and be transferred with minimum delay to the laboratory.

Bacteriological examination:

Under complete aseptic conditions, samples (after sterilization of their surfaces using heated spatula), were inoculated into nutrient broth and incubated at 37 ^°C for 24 hours. This step was followed by subculturing on E. coli selective media (MacConkeyʼs agar and Eosin methylene blue (EMB) agar), with incubation at 37 ^°C for another 24 hours. Suspected purified colonies to be E. coli were picked up and streaked on nutrient agar slopes and kept for morphological and biochemical identification (Konemann et al., 1997; Quinn et al., 2002 and Lee and Nolan, 2008).

Serological typing of E.coli:

Fifty E.coli suspected isolates “biochemically identified” were subjected to serotyping according to (Edwards and Ewing, 1972 and Lee et al., 2009) by Slide agglutination test, using Polyvalent and monovalent diagnostic E.coli antisera.

Detection of E. coli virulence genes by PCR:

Multiplex PCR was applied by using five sets of primers for detection of five virulence genes playing a role in the virulence of APEC. These genes were eaeA (intimin or E.coli attaching and effacing gene); ompA (outer membrane protein); stx2 (shiga-toxin2 gene); papC (pyelonephritis associated Pili gene); tsh (temperature sensitive heamagglutinin gene). Multiplex PCR was applied on isolated E. coli Following QIA amp DNA mini kit instructions (Catalogue no.51304); Dream Taq Green PCR Master Mix (2X) (Thermo Scientific) Cat No. K1081 and agarose gel electrophoreses (Sambrook et al., 1989).

RESULTS

Prevalence of E. coli isolates among chickens:

The number of the positive samples with E. coli isolation was 50 out of 600 isolates (8.3%); 30 isolates (9.0%) from baby chicks (up to one week), 20 isolates (7.4%) from broilers (from one week to 6 weeks), as revealed from table (1). The bacteriological examination of studied organs revealed that E. coli were isolated fromlung samples (17.5%); kidney and ureter samples (16.7%); air sac samples (14%); yolk sac samples (12.5%); intestinal samples (11.8%); spleen samples (10%); heart samples (7.9%); liver samples (3.4%) and nasal cavity &  tracheal samples (2.5%) as shown in table (2).

Serotyping of E.coli isolates:

Serological examination was carried out on biochemically identified E. coli isolates (50 isolates) for species identification. Among the fifty investigated isolates, 37 typable E. coli isolates (74%), and 13 untypable isolates (26%) were identified. Among the typable isolates, serogroup O27 was the most frequently observed (8%) followed by O44 (6%), O125 (6%), O152 (6%) and O159 (6%) followed by serogroup O1 (4%), O6 (4%), O20 (4%), O26 (4%) and O166 (4%).Then, O8 (2%), O86a (2%), O115 (2%), O128 (2%), O136 (2%), O146 (2%), O158 (2%), O164 (2%) and O167(2%), as described in table (3).

Detection of some virulence genes of E.coli isolates by PCR:

Multiplex PCR results shown in table 4 and figure 1 to 4, revealed that ompA virulence gene was detected in all serogroups except O8 giving a PCR product of 919 bp. Moreover, papC virulence gene was detected in all serogroups except O8, O86a, O136, O146, O167 and O169 giving a PCR product of 501 bp. While Stx2 virulence gene was detected only in serotypes O27 & O125 giving a PCR product of 779 bp. The eaeA gene was amplified in serogroups O1, O20, O26, O115, O152, O166, O168 and O169 giving a PCR product of 248 bp. Finally, tsh virulence gene was detected in all serogroups except O20, O26, O86a, O115, O128, O167 and O169 giving a PCR product of 620 bp. Although we notice some exceptions from these results, that some samples having the same strain but different in carrying the same virulence genes. For example, the strain O27, there are 4 samples represent it, samples No. 24, 15, 11 and 47. Sample No. 24 has 4 virulence genes,  ompA, papC, tsh and Stx2,  although sample No. 15 has only 3 virulence genes ompA, papC and tsh, on the other hand, sample No. 11 has only 2 virulence genes, ompA and papC. Interestingly, sample No.47 lacks any of them. This is the same with the strain O125 which represented with 3 samples No. 23, 22 and 48.Sample 23 has 4 virulence genes, ompA, papC, tsh and Stx2,  although sample No. 22 has only 3 virulence genes ompA, papC and tsh, but the sample No.48 also lack all of them. This result makes the samples No. 47 and 48 a good chance for possibility of preparation of an effective vaccine from these samples after testing them for pathogenicity test.

Table 1: Incidence of E. coli recovered from examined chickens samples.

Table 2: Incidence of E. coli in different organs of examined chicken samples (Calculated according to the total number of each samples).

Table 3: Results of isolates serotyping.

Table 4: Results of virulence genes of E. coli isolates by PCR.

Fig. 1: Amplification of eaeA gene of E. coli serogroups

Lane L: 100-600bp DNA Ladder. Neg.: Negative control. Pos.: Positive control. Lane 5: E.coli O1 (Positive). Lane 16: E.coli O152 (Positive). Lane 18: E.coli O115 (Positive).

Fig. 2: Amplification of tsh gene of E. coli serogroups

Lane L: 100-1000 bp DNA Ladder. Neg.: Negative control. Pos.: Positive control. Lane 5: E.coli O1 (Positive). Lane 12: E.coli O44 (Positive). Lane 15: E.coli O27 (Positive).

Fig. 3: Amplification of papC, stx2 and ompA genes of E. coli serogroups

- Lane L: 100-1500bp DNA Ladder. Neg.: Negative control. Pos.: Positive control.

- ompA gene (919 bp): Lane 5: E.coli O1 (Positive). Lane 12: E. coli O44 (Positive). Lane 15: E. coli O27 (Positive).

- papC gene (501 bp): Lane 5: E. coli O1 (Positive). Lane 12: E. coli O44 (Positive). Lane 15: E. coli O27 (Positive).

Fig. 4: Amplification of stx2 gene of E. coli serogroups

stx2 gene (779 bp): Lane 23: E. coliO125 (Positive). Lane 24: E. coliO27 (Positive).

DISCUSSION

The number of positive E.coli isolates was50 out of 600 isolates (8.3%) were collected from 81 farms at different Governmental and private poultry farms in five different centers at Qena province (Qena, Qift, Deshna, Qus and Nag-Hammadi). These finding were almost similar to those obtained by (Abd EL-Samie, 2015) who said bacteriological isolation showed that 34 out of 420 specimens  was E. coli positive (8 %).

On the other hand, higher incidence (26.9%) was published by (Ramadan et al., 2016). Interestingly, more recent investigations revealed much higher rates of E. coli for chicken (60%) obtained by (Eid et al., 2016).

In the present study, the incidence of E. coli varied according to age that, 30 isolates (9.0%) were from baby chicks (up to one week) and 20 isolates (7.4%) were from broilers (from one week to 5 weeks). These results agreed with (Shahat, 2015) who found that the incidence of E.coli in one week old chicks was (14.3%), while in one-three weeks it was (9.8%) and in age over three weeks (8.9%). Our results also agreed with (Naliaka, 2011), who found that the prevalence of disease decreased with increase in bird age in isolates from broiler chicken, Also, This is in agreement with earlier studies (Smith et al., 2006 and Moussa et al., 2007) that confirmed this relation.

The bacteriological examination revealed that higher rates of isolation of E. coli was from  lung (17.5%); followed by kidneys and ureters (16.7); air sac (14%); yolk sac (12.5%); intestinal tract (11.8%); spleen (10%); heart (7.9%); liver (3.4%); and finally nasal cavity & trachea (2.5%). These results agreed with (Abd EL-Samie, 2015) who said that the incidence of E. coli in lung (60%) is higher than heart (20%) and spleen (20%). Moreover, (Yousef et al., 2015) disagreed this result that he found the incidence of E. coli in the lung was the lowest in compare with heart and liver.

In case of comparing the results of incidence of E. coli in liver and heart, (Abd El Tawab et al., 2015) agreed with our result that the incidence of E. coli in heart is higher than that in liver. But, (Eid and Erfan, 2013) disagreed with this result.

In case of comparing the results of incidence of E. coli in intestine and liver (Abd El Tawab et al., 2014) agreed with our result. But, (Sharada et al., 2010) disagreed with this result.

In the present study, among the fifty investigated isolates, 37 typable E. coli isolates (74%) could be serotyped in 21 serogroups. The predominant serogroup was O27 (8%). These results go hand to hand with the previous study of (Shahat, 2015) who found that E. coli serogroup O27 was the most predominate (15%). El-jakee et al. (2012), identified E. coli serogroup O27 that recovered from diarrheic chicken from Egypt.

In the present study, other serogroups followed the O27, were O44 (6%), O125 (6%), O152 (6%) and O159 (6%). These results were agreed with that obtained with (Abd El Tawab et al., 2015) who mentioned that themost commonly detected E. coli serogroups were O44 (11.3%), O125 (7.5%). These results also matched with those by (Suwanichkul and Panigrahy, 1988; Gross, 1991 and Bosch et al., 1993), who reported that serogroup O44 and other strains were traditionally associated with colibacillosis in poultry. Also, (Roshdy et al., 2012) mentioned that O44 was the predominate strain (9.3%) followed by O125 (5.5%). Moreover, (Eid and Erfan, 2013) found that O125 was the second predominate with percentage (14.29%) but O44 was found with lower percentage (3.57%). This disagreed with (Eid et al., 2016) who found O125 and O44 in the lowest percentage.

E. coli serogroups O1 (4%), O6 (4%), O20 (4%), O26 (4%) and O166 (4%) was also identified in this study. This agreed with (Saif, 2003) whoreported that only 15% of the strains belonged to the serogroups O1, O2, O35, O36, and O78 that have been associated previously with avian colibacillosis were isolated from diseased birds, suggesting that this might signal the emergence of new pathogenic serotypes. Also, this is agreed with (Yousef et al., 2015; Eid et al., 2016 and Ramadan et al., 2016) who found O1 strain in low percentages. Our result disagreed with (Hassan, 2016) who found O1 in a high percentage (23.5%).

Our results nearly matched with (Eid et al., 2016) who reported that O26 found in a percentage (6.67%) and this is the same with (Ramadan et al., 2016) who found O26 (6.89%). Yousef et al., 2015, disagreed our result that they found O26 was the most predominate strain.

Da Silveira et al. 2002, identified 2.3% of APEC serogroup O6 among isolates from chicks with omphalitis, (Vandemaele et al., 2003) investigated 100 APEC strains from 83 Belgian poultry farms, detecting only three serogroup O6 strains, these results agreed our results. (Knöbl et al., 2012) disagreed these result that they found 31 strains (62%) belonged to serogroup O6.

In the present study, other serogroups were detected:  O8 (2%), O86a (2%), O115 (2%), O128 (2%), O136 (2%), O146 (2%), O158 (2%), O164 (2%) and O167 (2%). These results agree with (Yousef et al., 2015 and Eid et al., 2016) that O128 present in a very low percentage.

In the present study, the percentage of Untypable E. coli strains was low (26%). This result close to the results of (Shahat, 2015), who found that 2 strains out of 20 strains (10%) and also (Khan et al., 2005), who found that three strains of total 19 strains (15.8) isolated from poultry litter were not serogrouped to any known serogroup.

On the other hand, higher percentage (60%) was mentioned by (Zhao et al., 2005) were non-typable with standard O antisera, this similar to (Cloud et al., 1985 and Allan et al., 1993), who found that the large percentage of Untypable E. coli strains was common characteristics of all groups of E. coli recovered from avian colibacillosis regardless of geographical location.

Enteric E. coli are both natural flora of humans and important pathogens causing significant morbidity and mortality worldwide. Traditionally enteric E. coli have been divided into 6 pathotypes, (Enteropathogenic E. coli (EPEC), verotoxin (Shiga-like toxin)-producing or enterohaemorrhagic E. coli (VETC or EHEC), Enterotoxigenic E. coli (ETEC), Enteroaggregative E. coli (EAEC), Enteroinvasive E. coli (EIEC) and Diffusely adherent E. coli (DAEC) (Clements et al., 2012). Three of these pathotypes can invade the intestine, they are EIEC, DAEC and VTEC. In EIEC, there is the invasive plasmid with the capability to invade host tissues. DAEC is also responsible for recurring urinary tract infections. VTEC travels to kidney through the bloodstream damaging renal endothelial cells resulting in renal inflammation (Hussain, 2015). So, their isolation from any organ other than intestine is very logic because they have the ability to invade the intestine and travel to any organ. But the other three pathotypes “ETEC, EPEC and EAEC” have unknown mechanisms for invasion of intestine, because their action occur only in the intestine. So, it is very interesting to isolate these strains from organs other than the intestine. This occurred with O27, O128, O146, O168 and O169 which are ETEC as shown in table (3). Also, this occurred with O44, O125, O166, O128 and O158 which are EPEC and in the same time are EAEC except O166 and O158 which only are EPEC. All of them isolated from organs other than intestine like liver, heart, lung, air sac, kidney and spleen. This is explained by (Elsas et al., 2011) who mentioned that horizontal gene transfers have been involved in the spread of the virulence-related genomic islands across different E. coli strains.

It is difficult to compare between the incidences of pathogenic E. coli along with the distribution of its serotypes among different countries owing to the contribution of many conditions such as geographic area, seasonal variation, sampling techniques, and the conventional methods used for its isolation. However, it might be helpful in the prediction of certain outbreaks caused by these serotypes, especially in countries of the same geographical and climatic conditions, with a subsequent application of a suitable control regime (Ramadan et al., 2016). These variations reflect that E. coli serogroups are country specific and also it may differ within different localities in the same country and this beneficial in bacterines preparation as it must be specific to the predominant serotypes, also it is clear that avian E. coli represented by known and few serotypes (Abd El Tawab et al., 2015).

In the present study, Multiplex PCR results recovered that ompA virulence gene was detected in all serogroups except O8 giving a PCR product of 919 bp. This result almost similar to that of (Johnson et al., 2008; Zhao et al., 2009 and Abd El Tawab et al., 2014) who said that ompA gene was found in all APEC isolates.

Moreover, papC virulence gene was detected in all serogroups except O8, O86a, O136, O146, O167 and O169 giving a PCR product of 501 bp. These results come in accordance with (Jan Ben et al., 2001; Zhao et al., 2009; Qabajah and Yaqoub, 2010 and Abd El Tawab et al., 2014). Also our results agree with those by (Roussan et al., 2014) who mentioned that the gene papC, indicating the existence of the papC, was present in 50% of all investigated strains.

While Stx2 virulence gene was detected only in serotypes O27 & O125 giving a PCR product of 779 bp. Our result agreed with those obtained by (Abd El Tawab et al., 2014) that he found the stx2 gene was amplified in both serotypes O55 and O125 giving a PCR product of 779bp. This also nearly agree with the results obtained by (Jan Ben et al., 2001; Fujioka et al., 2009; Hideki et al., 2009; Zhao et al., 2009; Dutta et al., 2011 and Al- Ajmi, 2011). On the contrary, these results disagreed with (Kobayashi et al., 2005 and Shimaa, 2013) who could not detect shiga toxin genes in chicken samples.

The eaeA gene was amplified in serogroups O1, O20, O26, O115, O152, O166, O168 and O169 giving a PCR product of 248 bp. These results came in accordance with those recorded (Frank et al., 1998; Osek, 2003; Ahmed et al., 2007; Fujioka et al., 2009; Hideki et al., 2009; Dutta et al., 2011; Al-Ajmi, 2011 and Abd El Tawab et al., 2014). Meanwhile these results disagreed with others who found no eaeA gene detected in all APEC isolates (Olsen and Christensen, 2011 and Shimaa, 2013).

 Finally, tsh virulence gene was detected in all serogroups except O20, O26, O86a, O115, O128, O167 and O169 giving a PCR product of 620 bp. This result goes in parallel with those obtained by (Gomis et al., 2001; Jan Ben et al., 2001; Fujioka et al., 2009; Qabajah and Yaquoub, 2010 and Abd El Tawab et al., 2014).

Although we notice some exceptions from these results, that some samples having the same strain but different in carrying the same virulence genes. For example, “the strain O27”, there are 4 samples represent it, samples No. 24, 15, 11 and 47. Sample No. 24 has 4 virulence genes, ompA, papC, tsh and Stx2,  although sample No. 15 has only 3 virulence genes ompA, papC and tsh, on the other hand, sample No. 11 has only 2 virulence genes, ompA and papC. Interestingly, sample No.47 lacks any of them. This is the same with the strain O125 which represented with 3 samples No. 23, 22 and 48.Sample 23 has 4 virulence genes, ompA, papC, tsh and Stx2, although sample No. 22 has only 3 virulence genes ompA, papC and tsh, but the sample No.48 also lack all of them. This result makes the samples No. 47 and 48 a good chance for possibility of preparation of an effective vaccine from these samples after testing them for pathogenicity test. These results typically agree with (Delicato et al., 2003; Vandekerchove et al., 2005 and Mbanga and Nyararai, 2015) who said that numerous studies have demonstrated that these virulence factors are rarely all present in the same isolate and that they can occur either individually or polygenically with varying frequencies in clinical isolates. This all indicates that APEC strains constitute a heterogeneous group. The accurate identification of virulent strains of E. coli and the virulence genes they possess is essential if genes that can serve as vaccine targets are to be identified. Virulence gene studies are therefore important as they not only aid in the characterization of pathogenic strains of E. coli but may eventually lead to the development of effective vaccines.

CONCLUSION

Prevalence studying of E. coli is very helpful for disease controlling. The combination of serotypic analysis and detection of the virulence factors of E. coli is more valuable as an epidemiological tool for identification of isolates. The multiplex PCR is a rapid and specific diagnostic tool used for genetic characterization of avian pathogenic E. coli. 

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