Isolation, conventional and molecular characterization of Salmonella spp. from newly hatched broiler chicks

Salmonella is an important pathogen for poultry production as well as for human due to zoonotic importance. It has more than 2600 identified serovars despite of this identification and classification of Salmonella isolates into different serovars is critical for study of incidence and surveillance. This study investigates the epidemiology and molecular characterization of Salmonella isolates in broiler chicks during 1st week of life. A total of (n = 1000) samples including liver, intestine, yolk sac, spleen and heart blood were collected from El-Gharbia, El-Behera, Kafr-Elshikh, Alexandria, Marsamatroh Provinces in Egypt and tested through bacteriological, biochemical, serological and molecular examinations. Incidence of Salmonella was demonstrated on 75 positive samples from 1000 samples and the predominance of Salmonella that isolated from internal organs of newly hatched chicks was highest from yolk sacs (10%), liver and intestines (9%) followed by the spleen (7.5%) then heart blood (2%). Serotyping of the isolated strains using slide agglutination test revealed that 24 isolates belonging to S. enteritidis (1,9,12 g.m 1,7), while, 14 isolates belonging to S. virchow (6,7 r 1,2), in addition to, 12 isolates belonging to S. typhimurium (1,4,5,12.i.1,2) and 8 isolates belonging to S. kentucky (6,8.I,z). Enterobacterial Repetitive Intergenic Consensus (ERIC) PCR revealed that two S. enteriditis isolates were identical and one isolate differ by 40%, while two S. typhimurium isolates were identical by 80% and one isolate was similar by 20% to the other two isolates, in addition, two S. virchow isolates were identical by 80% and the two S. kentucky isolates were different.


Introduction
Salmonella isolates are considered as the most circulating and frequent bacterial agents causing disease poultry and other avian species. It is associated with high economic losses because of high mortality, morbidity and impaired productions. It is considered as a major food-borne pathogen in most countries of the world especially in developing countries ( Soultose et al. 2003;Carraminana et al. 2004). Salmonella contamination of poultry and poultry products are frequently occurred and can be transmitted to humans through transportation and consumption of undercooked poultry meat (Bailey and Cosby 2003;Kimura et al. 2004). Wide variations of Salmonella serovars commonly infect poultry and one serovar may be common in a country for number of years before it is substituted by another isolate. The serovars may vary geographically, but the most common serovars reported globally are S. typhimurium and S. enteritidis as reported by World Health Organization (2006). Salmonellosis has been associated with infection of broiler flocks that has ability of vertical transmission to progeny (Irshad et al. 2013). The predominant serotypes have been identified in Egyptian poultry farms are Salmonella enterica serovar Typhimurium and S. enterica serovar Enteritidis (Abd El-Ghany et al. 2012).
Serotyping is a basic biomarker to investigate the epidemiological situation of Salmonella infections and it is Open Access *Correspondence: shessin@ksu.edu.sa 3 Department of Animal Production, College of Food and Agriculture Sciences, King Saud University, P.O. Box 2460, Riyadh 11451, Saudi Arabia Full list of author information is available at the end of the article commonly used to trace back the contamination sources during outbreaks. White and Kauffmann developed the serotyping scheme on 1920 that was based on the flagella H, somatic O antigens and the observed phase-shift in flagella antigen (Molbak et al. 2006). This method is worldwide and it is considered as the standard method for Salmonella serotypes identification. The advantages of identifying Salmonella serotypes include providing information about the disease severity, contamination source and the resistance pattern (Molbak et al. 2006). Moreover, molecular techniques have been used to differentiate the strains of Salmonella isolates including pulsed field gel electrophoresis (PFGE), enterobacterial repetitive intergenic consensus (ERIC) PCR, Random Amplification of Polymorphic DNA (RAPD), Single Strand Conformation Polymorphism (SSCP), hybridization and ribotyping-PCR (Anjay et al. 2015). Due scarce knowledge available on conventional and molecular identification of Salmonella species, this investigation was designed to follow the epidemiology of Salmonella isolates through biochemical, serological and molecular methods.

Sample collection
A total of one thousand samples including liver, intestine, yolk sac, spleen and heart blood of newly hatched chicks during first week of life were collected aseptically from 25 poultry farms located in five different governorates in Egypt (El-Gharbia, El-Kafr-Elshikh, El-Behera, Alexandria and Matroh) with 10 chicks for each farm as shown in Table 1. The samples were collected in separate sterile plastic bags and immediately transported to the laboratory in ice box (4 °C).

Bacterial isolation
The collected samples were cultured on 1% peptone broth then 1 ml selenite F. broth and incubated aerobically at 37 °C for 18 h then were subcultured to MacConkey, Salmonella shigella agar and/or XLD media. The cultured plates were incubated at 37 °C for 24 h. Suspected colonies were picked up, preserved into semi solid agar as stock medium and into slant agar for further biochemical and serological identification.

Biochemical identification
Dry heat fixed smears of suspected colonies were stained using Gram's stain then were examined, revealing the presence of Gram negative bacilli. The suspected isolates were identified biochemically (Hossain et al. 2006) by applying catalase test, oxidase test and IMViC group of biochemical tests. The identified isolates as Salmonella species were cultivated on triple sugar iron agar (TSI).

Serological identification
Serogrouping of identified bacterial isolates was performed according to Kauffmann-White method (Aribam et al. 2015).

Molecular identification
Biochemically, identified Salmonella isolates were then serotyped and further characterization was done by using ERIC PCR for intra-serotyping of Salmonella isolates. DNA was extracted from studied isolates according to QIAamp DNA mini kit instructions and PCR Master Mix was prepared according to Emerald Amp GT PCR master mix (Tarkara) Code.No.RR310Akit using the following primer set ERIC-DG111-F with primers sequences ATG TAA GCT CCT GGG GAT TCA C and ERIC-DG112-R with primers sequences AAG TAA GTG ACT GGG GTG AGC G. Amplification of primers was done by using thermal cycling (Fendri et al. 2013). Briefly, denaturation at 94 °C for 2 min, annealing at 49 for and extention at 72 for 2 min followed by 35 cycles including 94 °C for 1 min, 56 °C for 1 min and 72 °C for 2 min and final extension at 72 °C for 5 min. After that the amplified product was loaded on 1.5% agrose gel using 100 bp gene ruler for 1 h at 5 V and the gel was visualized by chemical documentation (Bio Red).

Morphological identification of the isolated organisms
Morphology revealed the 75 samples out of one thousands appeared on MacConkey agar, colorless and translucent, though they sometimes have dark centers. Gram's stain smears from suspected colonies showed Gram-negative rod-shaped motile bacteria, or bacillus. On XLD, they were pink with or without black centers while, colonies on S.S agar media appeared as white colonies with black center.

Biochemical identification of the isolated organisms
The isolated micro-organisms were positive for methyl red, catalase, TSI, citrate utilization test, lysine iron agar, oxidase and christensen citrate while negative to indole, Phenol red, sucrose, and Voges-Proskauer. They ferment variety of sugar types but remain negative on KCN medium and ONPG reaction as illustrated in Table 2.

Incidence of Salmonella in different organs
Revealing to traditional identification on media and biochemically identification the proportion of isolates result as Salmonella isolates from various organs of newly hatched chicks represented by 7.5% total distribution in various organ as shown in Table 3.

Serological identification of the isolated organisms
The serotyping investigated the S. typhimurium, S. enteritidis, S. virchow and S. kentucky with O antigen are 4, 3, 2 and 2 while presence of H factor only in S. enteritidis. S. typhimurium and S. virchow as shown in Table 4.

Strain wise distribution of Salmonella species
Serotyping revealed that the distribution of S. entritidis was comparatively higher than S. Virchow, S. typhimurium and S. Kentucky as 2.4, 1.4, 1.2 and 0.8% while 1.7% strains were untypable as illustrated in Table 5. ERIC-PCR revealed that two Salmonella enteritidis were found identical while one was different i-e; lane S.  Table 6 and Fig. 1.

Discussion
Salmonella is considered as one of the major pathogenic agents which infect the variety of avian species specially poultry birds including layer as well as broiler reared in the modern intensive system with higher biosafety, biosecurity and standard management. Any contributions for elimination of Salmonella incidences and infection in birds could have a major influence in reducing the populations of the organism under natural conditions. One thousand samples were collected from different farms including liver, intestine, yolk sac, spleen and heart blood of newly hatched chicks at El-Gharbia, El-Behera, Kafr-Elshikh, Alexandria, Marsamatroh Provinces. The samples were examined bactriologically to isolate the Salmonella isolates. In this study, 75 samples out of 1000 samples (7.5%) were found positive. The higher percentage of isolation from the internal organs from yolk sacs (10%) then from livers (9%) and from 20 intestines (9%) were the same, spleen (7.5%), and finally heart blood (2%) ( Table 3). These results are in contrary to (Ahmed et al. 2008;Islam et al. 2016) who showed that the prevalence of avian Salmonellosis was highest in adult layer (53.25%) followed by brooding (14.55%) then growing (16.10%) and pullet (16.10%). The prevalence rate of Salmonella spp. in different poultry farm were different i-e; the 80 samples were tested from the clinically healthy birds showed 44 (55%) positive (Ahmed 14 ). Moreover the samples from birds having diarrhea infection rate (66.67%) (Hossain et al. 2006).
Ten Salmonella isolates belonging to 4 serotypes produced ERIC PCR fingerprints that were distinct for each serotype (Table 6). ERIC PCR found that three S. enteriditis isolates (isolates 2 and 3 identical in 1200, 1070, 700, 445, 166 bands but isolate 1 different from it in 708,450 bands) so two S. enteriditis isolates were identical and one isolate was different from it by 40%. Three S. typhimurium isolates (isolate 1 belonging to 1909, 1200, 700, 391 and 232 bands it was different from isolates 2 and 3 while isolates 2 and 3 identical in 1214, 700, 395, 235 bands and the difference in 1968 and 1938 bands) so two S. typhimurium isolates were identical by 80% and one isolate was similar by 20% to the other two isolates two S. virchow isolates were identical in 1034, 760, 445, 266 bands and the difference in 1372 and 1357 bands) so two S. virchow isolates were identical by 80%. Two S. kentucky isolates (isolate 1 belonging to 958, 576, 378, 277, 149 bands and it was different from isolates 2 which belonging to 948,581,378,280,151 bands) so two S. kentucky isolates were not identical. ERIC-PCR is a useful and recent method for DNA typing for analysis and evaluation of fingerprinting. It is used in epidemiology of Salmonella enteritidis (Suh and Song 2006). Using specific ERIC primers, a total of 30 strains of Salmonella enteritidis of four main clusters had found 60% similarity.
This study found that the Serotyping of the isolated strains revealed that 24 isolates belonging to S. enteritidis (1,9,12 g.m 1,7), while, 14 isolates belonging to S. virchow (6,7 r 1,2), in addition to, 12 isolates belonging to S. typhimurium (1,4,5,12.i.1,2) and 8 isolates belonging to S. kentucky (6,8.I,z). ERIC-PCR revealed that two S. enteriditis isolates were identical and one isolate was different from it by 40%, while two S. typhimurium isolates were identical by 80%and one isolate was similar by 20% to the other two isolates, in addition to, two S. virchow isolates were identical by 80% and the two S. kentucky isolates were not identical. This study will help future researchers to uncover new and critical methods that should be used to improve diagnosis and control measures for prevention zoonotic infections of Salmonella species.