Isolation and Identification of Salmonella from Chickens Prepared for Slaughter in the State of Kuwait

Salmonella enterica serovar Enteritidis throughout the world, is frequently linked to food-borne illness. Products made from poultry are important sources of transmission. Therefore, this study aimed to identify the incidence of Salmonella species in Kuwait's broiler flocks and determine which antibiotics are the most effective against the various Salmonella serotypes. A total of 2064 chicken samples (liver, intestine, and caecum) were collected from dead carcasses raised ten broiler flocks, between January and December 2017. The results revealed that S. enterica were found in the chicken older than 7 days old even though they did not have any signs of a clinical illness. It means that S. enterica can enter the human food supply through slaughterhouses and the contaminate carcasses. There was significant difference between the rate of Salmonella isolation and seasons, where the winter season had higher rate of Salmonella isolation compared to the other seasons. Salmonella Enteritidis and S. typhimurium were the most frequently isolated serotypes. Antimicrobials susceptibility testing showed that 88% to 60% of the isolates were sensitive to Amoxicillin Clavulanic acid. However, most of Salmonella isolates revealed high resistance to Cefotaxime, Ampicillin, Amoxicillin, Sulfamethoxazole trimethoprim followed by Gentamycin, Spiramycin, Doxycycline and Chloramphenicol. The study highlights the implementation of unique biosecurity and biocontrol strategies for Salmonella management that can prevent the negative effects of antibiotics and can make the environment and foods derived from animals safe. ـــــــــــــــــــــــــــــــــــــــــ


INTRODUCTION
Salmonella species are significant contributor to foodborne illnesses globally (de Jong and Ekdahl, 2006). Their yearly economic loss estimate is $3.7 billion (USDA, 2016). The main serovars linked to human salmonellosis are Salmonella enterica serovars Enteritidis and Typhimurium, which cause the majority of illnesses (Dunkley et al., 2009). In the United States, S. enterica represents the major cause of microbial foodborne diseases (Scallan et al., 2011). According to the Public Health Agency of Canada (2006) and Collard et al., (2008), S. enterica serovar Enteritidis is mostly responsible for human salmonellosis outbreaks in the United States and Europe, and is associated with the ingestion of contaminated chicken products (Braden, 2006;Much et al., 2009). Salmonella species can cause self-limited, mild to moderate gastroenteritis, and in a rare number of the cases, severe disease may lead to death (Voetsch et al., 2004). Salmonella serotype prevalence varies between 6% and 30% in live birds (Bjerrum et al., 2005), while the incidence of Salmonella enterica in poultry and poultry products varies between 1% and 65.5% (Agunos, 2007).
For all ethnic groups, poultry and poultry products provide an affordable source of animal protein (Nidaullah et al., 2017). One of the most significant animal industries in Kuwait is the poultry sector, which provides around 50% of the chicken meat consumed in the State of Kuwait (Ministry of planning. Statistical and Census Sector, 2020). The most common poultry species worldwide is chicken. Additional, chicken meat is considered as Kuwait's main source of protein (Ministry of planning. Statistical and Census Sector, 2020). In Egypt, the prevalence of Salmonella enteritidis was (1.73%) and Salmonella typhimurium (0.43%) in imported ducklings (Sorour et al., 2023). Salmonella can spread horizontally and/or vertically in poultry. Examples of horizontal transmission include contamination from hatcheries, equipment, people, insects, rodents, feed, water, and litter (Gast et al., 2020). According to Agunos (Agunos, 2007) and Crump and Mintz (Crump and Mintz, 2010), vertical transmission occurs when the bacteria is passed on directly from colonized breeding flocks (via ovaries) to the offspring.
Salmonella is thought to be mostly found in live chickens since it may live in their skin, feathers, and gastrointestinal tracts. Salmonella most frequently contaminates poultry meat through the processing of intestinal contents feces, infected processing tools, contaminated water, and processing employees' hands. In poultry slaughter plants, defeathering, evisceration, chilling, and packaging processes can spread Salmonella throughout the entire plant (Thames and Sukumaran, 2020). Inadequate hygiene practices during chicken carcass cutting and preparation, poor cooking techniques, and temperature misuse, all contribute to human bacterial infection especially salmonellosis (Ehuwa et al., 2021). Typhoid fever, the most dangerous type of salmonellosis for humans, is still a big issue in the developing nations, largely because of the absence of sanitary and food safety standards (Crump and Mintz, 2010; Akil and Ahmad, 2019). Non-typhoid Salmonella is thought to be the cause of 93.8 million cases of mortality worldwide each year (Antunes et al., 2016). Eighty-three millions of these illnesses are thought to be foodborne (Majowicz et al., 2010).
Because of the high rate of crosscontamination during slaughter and processing, there are estimated risk levels for Salmonella outbreaks associated with these procedures in the broiler production process. These actions are thought to have a 12%, and a 33.5% chance of causing an outbreak, respectively (Akil and Ahmad, 2019). In addition, the main problem is that Salmonella infections in healthy chickens can occur without any outward signs of illness, which is why many farmers do not realize that their flocks are suffered from salmonellosis (Beam et al., 2013). Therefore, it is required to identify Salmonella infection in broiler flocks before slaughter (healthy chicken).
Furthermore, due to the significant costs associated with illness prevention, treatment, and control efforts, the rising frequency of salmonellosis has become a burden for most developing countries (Lee et al., 2015). Additionally, due to the advent of novel serotypes and antibiotic resistance, researchers and society are now more aware of the wide variance in Salmonella serovars and the frequently shifting patterns in salmonellosis (Antunes et al., 2016). Salmonella has developed an antimicrobial resistance, mostly as a result of improper use of antibiotics in the poultry sector (Sorour et al., 2023) and meat (Oludairo et al., 2022). Therefore, this study was aimed to identify the incidence of Salmonella in Kuwait's breeding flocks; to determine the most prevalent serovars in the breeding flocks; to describe the seasonal variation in the occurrence of Salmonella, and to determine which antibiotics are the most effective against various Salmonella serotypes.

MATERIALS AND METHODS Sampling
A total of 2064 chicken carcasses were collected from ten broiler flocks between January and December 2017 in Kuwait. After that, 4128 samples (2064 from liver; 2064 from intestine, and caecum) were collected from the same chicken. The age and number of birds were described in the table (1). All the samples were collected and transmitted in ice, sent to the Laboratories for Diagnosis Animal Diseases and Research immediately after death for isolation and identification of Salmonella species.

Ethical approval
The ethical approval for this research design was not required.

Isolation and biochemical identification of Salmonella species
According to ISO 6579-2002, Salmonella from chicken and its relevant organs were isolated (ISO 6579, 2002). Chicken liver and intestinal samples were collected in an antiseptic environment as possible. Pre-enriching samples in buffered peptone water at a sample to broth ratio of 1:10 was followed by an 18-hour incubation period at 37°C. The pre-enriched broth was then utilized as an inoculum in Rappaport-Vasiliadis (RV) (EMD Chemicals Inc., Darmstadt, Germany), and incubated for 24 hours at 42°C. Rambach Agar (RA), Brilliance Salmonella Agar (BSA), and Xylose Lysine Deoxycholate Agar (XLD) (Difco, Sparks, NV, USA), which they utilized as selective agars, each received one loop of RV. The selective agar plates were then incubated for 24 hours at 37°C. The samples with red colonies (with or without black centers) on XLD, RA, and BSA were purified and placed through biochemical tests using the Analytical Profile Index (API-20E, BioMérieux, Inc., France).

Serological test
A slide agglutination test utilizing commercial antisera (SSI Diagnostica, Hillerod, Denmark) was used to serotype all biochemically verified Salmonella isolates based on somatic (O) and flagellar (H) antigens (Popoff et al., 2004).

Statistical analysis
The SAS Institute Inc., North Carolina, USA, JMP Pro16.1 software was used to perform descriptive and inferential statistics. The Chi-square test was used to examine the frequency of Salmonella species isolates and the seasonal patterns in order to determine the presence or abscent significant relationship. The results were significant at P < 0.05.

According
to the morphological characterization, all S. enterica serovars isolated in the current study were motile, Gram-negative short rods or bacilli with red colonies, some of which had black centers on XLD, red colonies on RA, and purple colonies on BSA. All the isolates were negative for oxidase, urease, and indole, but positive for Lysine decarboxylase, methyl red, fermented mannitol, and glucose, and produced hydrogen sulfide (H 2 S). Table (2) showed that Salmonella species was isolated from 2064 (16.2%) dead broilers at different ages, where the age of 0-7 days had the lowest number of isolates. Within the same column, frequencies with various letters are significantly different (P < 0.05).

DISCUSSION
One of the most common foodborne infections in humans is salmonellosis. The constant threat to human health arises from the ongoing presence of Salmonella spp. in animals raised for food and the increasing resistance to antimicrobial drugs. The chicken sector suffers significant financial losses due to Salmonella. Salmonella infections in poultry flocks cause considerable direct losses for poultry producers. Young chicken may experience growth retardation, or even death as a result of infections they acquire either horizontally in the hatchery or vertically from their parents. For chicken breeders, preventing the spread of Salmonella to the offspring or to the people can be costly (Sohail et al., 2021). In the current study, the broilers that prepared for slaughter was examined for the presence of Salmonella.
Although chickens may be infected with Salmonella at any age (Friedman et al., 2003), the present results revealed that 14-30 days old broilers (41.74%) were more susceptible to Salmonellosis. This variation in susceptibility may be due to the presence or absence of the chicken innate immunity which is the first barrier against any infection (Cheema et al., 2003).
Regarding the seasonal variation, out results were in agreement with the study in Japan, by Ishihara et al., (2020) who found that Salmonella spp. was higher during the winter and spring than the other seasons. However, our findings disagreed with Zdragas et al., (2012) who reported that there was seasonal variation of Salmonella isolation. Its peak was recorded in summer than winter. Additionally, depending on the age of the chicken, the air temperature in broiler houses must be strictly regulated. In other words, it's possible that Salmonellosis spread outside or inside the broiler houses is not the cause of the higher risk of Salmonella isolation from chicken in the winter and spring. On the other hand, the management practices, such those impacting ventilator or the time frame for opening window ' s coverings in chicken houses, should be adjusted according to the season (Ishihara et al., 2020). The types and numbers of Salmonella carriers into broiler flocks, such as hygienic insects or wild animals, would fluctuate based on the temperature of the air outside the broiler houses.
The broilers aged 7 -14 days had the highest isolation rate of S. Enteritidis (68.1%), whereas no S. Munchen could be isolated from the broilers aged 14 -30. However, the prevalence of S. enteritidis was estimated to be 6.3% in Uruguay as mentioned by Betancor et al., (2010). Previous investigations have shown that raw chicken meat can contain Salmonella enterica as documented by Amin and Abd El-Rahman, (2015); Soguilon-Del Rosario and Rivera, (2015). Salmonella Enteritidis and S. Typhimurium are introduced independently from other Salmonella sero-types for two reasons: first, because the epidemiology of these bacteria differs from other Salmonella spp., second, because they are the most frequently sero-types linked to this human disease in the majority of countries as summarized by Bangtrakulnonth et al., (2004).
Antimicrobials susceptibility testing showed that 88% to 60% of the isolates were sensitive to Amoxicillin Clavulanic acid drug. These results disagree with Rodrigues, et al., (2017) who found that 66.7% of the isolated Salmonella in West-Center regions of Brazil was resistance to Amoxicillin Clavulanic acid. The use of Beta-lactams in animal feed as a growth performance additive, in addition, to the miss use of antibiotics in poultry farms may lead to the presence of Beta-lactamas resistant Salmonella. In this study, Salmonella isolates showed resistance to more than 3 classes of antibiotics, and these results agreed with Elkenany et al., (2019) and Zhang et al., (2018).

CONCLUSION
The findings of the current study revealed that S. enterica infection causes a persistent enteric infection in chickens older than 7 days without manifesting clinical signs and serves as a source of contamination for carcasses at the point of slaughter and entry into the human food supply. The seasons had noticeable impact on the rate of Salmonella isolation. The two most prevalent and isolated serotypes from the broilers were S. Enteritidis and S. Typhimurium. The study also found a higher incidence of Salmonella that is resistant to antibiotics. It requires immediate action to reduce the use of antibiotics and to take biosecurity precautions that would help to prevent the spread of Antimicrobials resistant Salmonella. Due to the spread of these strains in broiler farms, specific biocontrol measures were necessary to control Salmonellosis. These actions can prevent the negative effects of antibiotics and guarantee the environmental and food safety generated from animals.