Virulence Characteristic of Avian Pathogenic Escherichia coli (APEC) Isolates Karakteristik Virulensi Isolat Avian Pathogenic Escherichia coli (APEC)

Avian pathogenic Escherichia coli (APEC) is a cause of colibacillosis in poultry, one of the respiratory disease that causes serious problems in the poultry industry. The APEC can cause high mortality and culling, decreased production, and high costs of treatment. Manifestations of colibacillosis are airsacculitis, perihepatitis, and pericarditis. The APEC serotypes that are widely identified in the field are O1K1, O2K1, and O78K80. Embryo lethality assay (ELA) is a method for to determine the virulence of APEC serotypes. The aim of this study is to determine the virulence characteristic of APEC isolates. Five APEC serotypes O1K1, O2K1, O78K80, O157H7, and unknown serotype were used for ELA method by inoculated E. coli into chorioallantoic of specific Sruti Listra Adrenalin, et.al.


Introduction
Respiratory infection is one of the main problems in the poultry industry. The infection causes economic loss due to the increase of mortality and culling, decrease of carcass quality, production, and high costs of treatment. Colibacillosis is one of the respiratory infections caused by APEC. The APEC is the extraintestinal group of E. coli which causes various internal organ lesions/ septicemia such as airsacculitis, perihepatitis, pericarditis, cellulitis, egg peritonitis, salpingitis, coligranuloma, omphalitis, and osteomyelitis/ arthritis (Chansiripornchai et al., 2011;Ghunaim et al., 2014;Gross, 1991;Kemmett et al., 2013;Roseliza et al., 2016). This systemic infection occurs when a large number of pathogenic E. coli into blood vessels through the respiratory or digestive tract (Chansiripornchai, 2009).
Prevention of APEC infections has been carried out by vaccination of other respiratory agents and E. coli itself, reducing stress in chickens, predisposition factors, and treatment with antibiotics (Chansiripornchai, 2009;Cortes et al., 2010;Ghunaim et al., 2014;Wibowo and Amanu, 2009). The increase of antibiotics resistance to E. coli and residues in meat should be the concern (Gregersen et al., 2010). Whereas E. coli vaccination provides a better result if the vaccine serotypes are homolog with field strain (Ahmed et al., 2015;Dho-Moulin and Fairbrother, 1999;La Ragione et al., 2013). In addition, there is no diagnostic tool to differentiate the E. coli isolates from primary (highly virulent), secondary (moderately secondary), or non-pathogenic (avirulent) isolates (Wooley et al., 2000;Oh et al., 2012).
Effective and reliable methods for bacterial virulence testing require a research tool (Andersson et al., 2015). Embryo lethality assay (ELA) can be used to differentiate between virulent and avirulent E. coli isolates (Nolan et al., 1992). The ELA method is a sensitive method to determine E. coli virulence in diagnostic laboratories, by the percentage of embryo mortality after inoculation (Oh et al., 2012).

Inoculum
The inoculum was an isolate that confirmed as E. coli on EMB media, Gram staining, and biochemical tests. The E. coli then grown on brain heart infusion (BHI) media at 37 0 C for 24 hours, then washed with PBS solution, and compared with standard McFarland 0.5. The number of bacteria inoculated is 100-500 CFU/ 0.1 ml (Nolan et al., 1992;Powell & Finkelstein, 1966;Wooley et al., 2000). The determination of the bacteria number was by the total plate count (TPC) method on nutrient agar (NA), with sample dilutions had been standardized with McFarland 0.5.

Embryo Lethality Assay (ELA)
Fifty-five of specific pathogen free 12-days old embryos were divided into 5 groups based on E. coli serotypes (O1K1, O2K1, O78K80, O157H7, and unknown serotype). Each group of 10 embryos treatment and an embryo control was inoculated pathogen free 12-days old embryos. Each group of 10 embryos, inoculated E. coli dose of 100-500 CFU/ 0.1 ml. Candling was carried out for 6 days (18-days old embryo) to determined the mortality and pathological lesions. The percentage of embryo mortality post-inoculated with APEC O1K1, O2K1, unknown serotypes were 100% (10/10), O78K80 serotype was 90% (9/10), and O157H7 serotype was 70% (70%). Lesions of all embryos were cranial and extremity hemorrhage. In this study, E. coli isolates had high virulence.
Key words: avian pathogenic E. coli (APEC); E. coli serotypes; virulence; embryo lethality assay with PBS. Each treatment group was inoculated with each E. coli serotype in chorioallantoic. Candling is carried out for 6 days (18-days old embryo) to determined the mortality. Mortality was recorded and observed for pathological lesions (Gibbs et al., 2003;Nolan et al., 1992;Oh et al., 2012;Powell & Finkelstein, 1966;Wooley et al., 2000). There were virulent characteristic as described by previous study (Wooley et al., 2000), where the mortality of embryos < 10% is avirulent, 10-29% is moderate virulence, and > 29% is high virulence. Reisolation of chorioallantoic was on EMB media, as confirmation of the cause of death was E. coli.

Escherichia coli Isolates
The E. coli serotypes O1K1, O2K1, O78K80, O157H7, and unknown serotype on EMB showed typically colony of metallic sheen (Figure 1a). The Gram staining showed a small rod Gram-negative ( Figure 1b). This result is according to Swayne et al. (2013), Gordon and Jordan's (1982) where the growth of E. coli in EMB is metallic sheen, compared with the other Escherichia species. The cell morphology of bacterium is a small rod, Gram-negative, and does not have spores.
The results of biochemical tests, serotypes O1K1, O2K1, O78K80, O157H7, and unknown serotype on TSIA media could ferment all carbohydrates (glucose, lactose, sucrose) and have CO 2 gas. Urea, VP, and citrate were negative results. The bacterial positive on semisolid media, because E. coli have flagella. All isolates could fermented carbohydrates (sorbitol, glucose, lactose, sucrose, and mannitol), changed the color of the media to yellow, except unknown serotype which cannot ferment sucrose. All biochemical results according to Abott et al. (2003) and Nataro et al. (2011). The biochemical results are presented in Table 1 Embryo Lethality Assay (ELA) The number of APEC serotypes O1K1, O2K1, O78K80, O157H7, and unknown  serotype as much as 100-500 CFU/ 0,1 ml were determined by the TPC method, then inoculated on chorioallantoic of 12-days old embryos, each 10 embryos/ serotype. After 6 days (18-days old), the result of E. coli serotypes O1K1, O2K1, and unknown serotype were the highest percentage of embryo mortality (100%) than the other APEC serotypes. The O78K80 serotype has 90% of mortality, and the O157H7 serotype has 70% ( Table 2). The chickens in the control group did not have mortality. Virulence characteristics based on the percentage of embryo mortality according to Wooley et al. (2000), all the E. coli serotypes O1K1, O2K1, O78K80, O157H7, and unknown serotype were highly virulent. The high virulence of serotypes O1K1, O2K1, and O78K80 were in line with Wahyuwardani et al. (2014) with a case of broiler colibacillosis in Yogyakarta and Bogor, which were O1K1, O2K1, and O78K80 all of the 14 samples of E. coli isolates.
The 10 embryos used in each isolate to determine the E. coli virulence by previous research (Nabbut and Khatib, 1978). The percentage of mortality post-inoculated was significantly high, medium, or avirulent of APEC isolates (Wooley et al., 2000). The different mortality shows the ability of each isolate to be able to kill the embryo (Gibbs et al., 2003).
Although O1, O2, and O78 are the main E. coli serotypes that cause colibacillosis, there were many studied for other serotypes that cause colibacillosis, including O25, O125, O153 serotypes. This is proved that E. coli serotype is not an important factor for the pathogenesis of colibacillosis in chickens (Ozaki and Murase, 2009). The E. coli O157 serotype is one of the pathogenic serotypes in chickens experimentally. The E. coli O157 serotype is not only pathogens that infect poultry, but also in humans. Several studied have shown that poultry already has and persistently infected with E. coli O157 serotype (El-Sawah et al., 2018). The O157 serotype has also been reported isolated from pigeons, have lesions and histopathology that characterizes APEC infections (Dutta et al., 2013).

Embryonic Lesions and Reisolation of Bacteria
Lesions in dead embryos were cranial, skin, and extremity hemorrhages (Figure 2). These results according to Wooley et al. (2000), skin and cranial hemorrhages to encephalomalacia in embryos inoculated with virulent isolates. The presence of hemorrhage in the embryo is due to the massive growth and multiplication of bacteria in the tissues, the production and release of hemolytic factors, and other toxin factors (Nabbut and Khatib, 1978).
The iss gene is a virulence factor and strongly correlates with virulent E. coli isolates (Nolan et al., 1992). The iss gene of isolates can predict the percentage of embryo mortality. The iss genes, resistant complement, and CoIV production are strongly associated with virulence potential by chicken embryo lethality method. The chicken embryo can be used to distinguish APEC from commensal E. coli (Gibbs et al., 2003).
According to Wooley et al. (2000) control of colibacillosis is difficult because the lack of a diagnostic tool to determine the virulence of isolates. The results of this study are easy to do to determine virulence among isolates, because of the significant number of mortality and percentages (high ability to kill embryos). Most embryos would died 3-4 days post-inoculation, the most mortality usually 2 days post-inoculation (Gibbs et al., 2003;Nabbut and Khatib, 1978;Nolan et al., 1992;Powell and Finkelstein, 1966).