Campylobacter coli in young chicks using two different cloacal swab techniques.

Campylobacter coli in young chicks using two different cloacal swab techniques. CHAPTER ONE 1.0 Introduction Campylobacter coli is a gram negative, thermophilic, obligated microerophilic bacteria that is usbiquitous in temperature enviroments. It colonize the intestional mucosa of most warm blooded hosts, individual all food producing animals and human (Skirrow, 1994). However, the favoured environment appears to be the intestine of all avains as a commensal organism, with the possible exception of ostriches (Hald, et al., 2000). Campylobacter generally colonize avains as a commensal organism, with the possible exception of ostriches (Verwoerd, 2001). In contrast, in humans, particularly in children and in adults in industrialized countries, infection is associated with cute enteritis (Tauxe, 1992). It is generally assumed that campylobacter contaminate poultry meat during processing, surving throughout the food chain supply to constitute a risk to human health (Tauxe, 1992). The campylobacter genus consist of a large and diverse group of bacteria currently comprising 25 species, two provisional species and eight subspecies. Members of the campylobacter genus are morphologically diverse, including spiral, curved or rod shaped. These bacteria are nutritionally fastidious (required complex nutritional environments) and grow under strictly anaerobic or microaerobic conditions. Members of the campylobacter genus naturally colonize humans, other mammals, birds, reptiles and shelffish. The most well known member is C. jejuni, a leading cause of bacterial gastroenteritis in humansworld wide. C. coli which is closely related to C. jejuni and C. coli have been recognized as important pathogens in human and animal (Moore et al., 2005). As a result, these campylobacter spp have been coined ‘emerging campylobacter spp’ and include C. concisus, C. laris, C. upsaliensis and C. ureolyticus. However, not all species are currently considered to be emerging pathogens, as they are either newly identified or little is known regarding their clinical relevance and pathogenic potential. Recognition of the emergence of certain campylobacter spp in gastrointestinal disease is largely owing to the improved ability to isolate and detect these bacteria in the intestinal tract, facilitated by the advent of molecular techniques, the use of innovative isolation methodologies, and an improved understanding of the nutritional requirement and growth conditions for these organism. Subsequently, increasing number of research studies are now trying to elucidate how emerging campylobacter sppinitate disease. The major routes of transmission in humans are consumption of contaminated or under cooked meat (especially poultry), unpasteurized milk or dairy products, and untreated water. People can also be infected by contact with infected animal or feaces. Campylobacteriosis in human ranges from mild to severe, but most cases are self limiting. Although complications are uncommon,C. jejuni is a major triggering event for guillain-Barre syndrome. C. fetus is an opportunistic human pathogen and mainly causes systemic infection in people with compromised immune system. Antibiotic resistance in campylobacter spp is a serious problem world wide, particulary for fluoroquinolones and tetracyclines. In addition, campylobacteriosis may be acquired by direct contact with infected human shedders in the family environment. Nosocomial infection occurs and cases of congenital transmission are rarely documented. Campylobacteriosis in children is often acquired from immature diarrhoeic pets (Burnenset al., 1992). In the context of international trade, the ubiquitous nature of campylobacter jejuni and the multiple reservoirs and sources of infection miltigate against impleding trade on the basis of contamination. Establishing an import barrieragainst poultry or red meat contaminated with campylobacter would be unjustified. Inroking sanitary and phytosanitary measures would be blantantly protectionist and inconsistent with the rules of the world trade organization (Lim et al., 1992). In chickens, campylobacter is a commensal organism and is a common contaminant of raw poultry product (Koroliket al., 1998; Stas 1999; Young et al., 1999). The entry of campylobacter into broilers before harvesting for slaughter remains unclear. Potential sources of campylobacter include contaminated water (stern et al., 2002), spread from animal and insect reservoirs (Gregory et al., 1997), vertical transmission through broiler breeder flocks (Cox et al., 2002), contamination within the hatcheries (Hiettet al., 2002), and horizontal transmission from broiler to broiler. Any 1 or a combination of these routes may play a role in the colonization of campylobacter in broilers. Broilers breeders and hatchery positive sample remain a controversial subject with regard to campylobacter colonization. Numerous studies have suggested that campylobacter is rarely found in broiler chicks until 2 to 4 weeks of age (Evans and Sayers, 2000; Shreeveet al., 2000; Stern et al., 2001). One explanation was that campylobacter was present in viable but non-culturable forms in water, which could potentially play a role in the ability to detect campylobacter through traditional culture methods (Stern et al., 1994; Ziprinet al., 1999). Furthermore, the lack of sensitivity and reliability of drag swabs to detect low levels of campylobacter contamination of a flock may explain these results as well. Recent evidence has suggested that broilers may become contaminated with campylobacter through broiler breeders and fertile eggs. On skirrow or other blood-containing agars, characteristics campylobacter colonies are slightly pink, round, convex, smooth and shiny, with a regular edge. On charcoal-based media such as MCCDA, the characteristics colonies are greyish, flat and moistened, with a tendency to spread, and may have a metallic sheen. Campylobacter spp require microaerobic conditions consisting of (5% O2, 10% CO2 and 85% N2) (Oie, 2008) OIE Terrestrial manual chapter). They neither ferment nor oxidase carbohydrates. Energy is obtained from amino acid or tricarboxylic acid cycle intermediate, not carbohydrates. Some species, particularly C. jejuni, C. coli and C. lari, are thermophilic, grow optimally at 420C (Garrityet al., 2005). The application of sequence-based typing schemes, such as multilocus sequence typing (MLST), to classify and characteisecampylobacter isolate has results in major advances in the understanding of campylobacter ecology and epidemiology (Colleset al., 2012). MLST generates data in form of nucleotide sequence or allelic profiles that are electronically portable, comparable and can be shared via pubically accessible online database (Jolleyet al., 2004). Despite over 30 years of research, campylobacteriosis is the most prevalent bacterial cause of food borne infection in many countries including in the EU and the USA (Bolton, 2015). As earlier stressed that poultry species are important reservoirs for the transmission of campylobacter species and their high body temperature provides an optimal environment for the growth of the organism (Noormohamedet al., 2014). It is important to lence genes and their capacity for survival in poultry meat and hence their contribution to the incidence of campylobacteriosis (Abu et al., 2016) and the large genetic diversity of campylobacter must be considered in epidermiological evaluations and microbial risk assessments of campylobacter in poultry (Alter et al., 2011; Vidal et al., 2016). As a first step, colonization of the intestine require the ability to move into the mucus layer covering the intestinal cells. Campylobacter motility is conferred by the polar flagella, which together with their ‘cork-screw’ shape allow them to efficiently penetrate this mucus barrier (Szymanski et al., 1995; Haag et al., 2012). The most important uinclence factor that has been studied and well characterized in campylobacter spp was the flagellin, which is encoded by the fla agene (Hermanset al., 2011). The global regulator, carbon starvation regulator (CSrA) gene, has been well characterized in several bacterial genera and is know to regulate a number of independent pathways via a post transcriptional mechanism, but remains relatively uncharacterized in the genus campylobacter (Thompson, 2012). Many of vinilence genetic factor connected with campylobacter invasiveness are placed on the PVir plasmid for example, Vir BII gene that encodes the IV secretory system protein. It has been showed that strains with mutation in the VirBII sequence have much lower adhesion and penetration ability in vitro in compansion to original strains, as well as lower pathogenicity 0069n viro the plasmid gene Vir BII (Bacon et al., 2000). One of the most important genes responsible for campylobacter invasion is the Cia B (Campylobacter invasive antigen B) gene which is known to be involved in the translocation of campylobacter into host ells for the purpose of host cell invasion and also plays a significant role in cecal colonization in chicken (Croinin et al., 2012). Although numerous farm-based studies have been conducted in the past decades, the sources of flock infection, mode of transmission, and the host and environmental factor affecting the spread of campylobacter on poultry farms are still poorly understood [Saline et al., 2002]. There has been a major debated on whether vertical or horizontal transmission is responsible for the introduction of campylobacter into chicken flocks. For years, the prevalling theory has been that horizontal transmission form the environment is the major source of C. coli infection for broiler flocks, and that vertical (egg-borne) transmission from is unlikely. Potential sources of flock infection include used litter, unlikely. Potential sources of flock infection include used litter, untreated drinking water, other farm animals, domestic pets, wildlife species, house flies insects farm equipment and workers and transport vehicles (Kazooalaet al., 1990; Vande-Giessen et al., 1992). However, none of these suspected source has been conclusively identified as the formal source of infection for broiler farms. In many cases, it was difficult to determine which of the event (the flocks infection or environmental contamination) occurred first as no study plan was included to monitor the direction of campylobacter transmission. Circumstarital evidence has accumulated suggesting that vertical transmission of C. jejuni form breeder flocks to the broiler farms via the egg may occur. Earlier studies showed that, even when at low level C. jejuni could be isolated from both outer (Doyler, 1984) and inner (Shankeret al., 1986) shell surface of eggs laid by naturally infected commercial layers or broilers breeders. Shane et al., (1986) isolated the organism from both interior surface of egg shell and egg content after swabbing faeces containing C. jejuni onto the egg surface. following experimental infection of eggs with C. jejuni by either temperature differential method or inoculation of egg albumen via direct injection (Clark and Bueschkens, 1985). Shankeret al., (1986) recovered organism from both the contents of unhatched eggs and from newly hatched chicks. Investigation using sensitive molecular detection methods demonstrated the presence of campylobacter DNA in embryos and newly hatched chicks (Chumaet al., 1994; 1957) and in hatcheries (Hicetteet al., 2002). Campylobacters are readily defected in the faeces of colonized birds, under experimental condition, in-vivo passage organism can exhibit an enhancement of colonization potential of at least 1,000 fold in most strains (Ringoret al., 1994). The presumable reflects the up regulation of bacterial factor, important for colonization. As chicken are coprophagic, faecal shedding is presumably an important factor in the dissemination of organism around large broiler flock once the first bird become colonized. Certainly colonization is detected form bird-to-bird transmission within the flocks is extremely rapid, and the majority of the birds are colonized within few days (Miflin 2001; Templeton et al., 2002). These colonization kinetics indicates the measurements with in flock prevalence at slaughter and are likely to be unsuccessful, which relevant to quantitative risk assessment model currently under development (Hartnetet al., 2001). Interestingly, epidemiological investigation of commercial flocks indicates that naturally acquired flock colonization is age dependent. Newly hatched chicks appears to be free of campylobacter. This negativity persist until at least 10 days of ages (which is called the lag phase) the most flocks become infected after 2 to 3 weeks of replacement of chicks into a broiler house (Evan et al., 2000). Colonized chickens usually shows no observable clinical symptoms of infections even when young chicks are exposed to high close experimental condition. Therefore, this study will determine Campylobacter coli in young chicks using two different cloacal swab techniques. Aim: to determine Campylobacter coli in young chicks using two different cloacal swab techniques. Objectives 1.1 Statement of Problems Campylobacter is a commonly reported bacterium causing foodborne illnesses and has become a world wide concern. A significant portion of human diarrheal infection is attributed to under cooked poultry and poultry products. Campylobacter has a natural growth temperature of 370C to 420C. Similar to the internal body temperature of live poultry, allowing the organism to become a commensal inhabitant of the intestinal tract. The high prevalence of campylobacter on poultry meat and derived products is of significant to consumers. 1.2 Limitation Campylobacter is the parental (named genus) of any other species of the bacteria that exist different host in which they habour. This project is limited to the coli species and the host (Broiler chicken). Objectives The first objective of this project is to carryout an experiment by rearing a day old-chick for 12 weeks (i.e to maturity stage) by feeding them at interval with a commercial feed in a well image pen for the small period of time. There are several method scientist has used in determine campylobacter coli in sample collected from the different part of the chicken based on different principles, techniques such as multi-locus sequence typing (MLST), polymerase chain reaction (PCR), pulsed field gel electrophoresis (PFGE), random amplification of polymorphic DNA (RAPD) etc. the second objective of this project is to determine the presence of campylobacter in young chicks using swab and culture based method. The swab will be collected form the clocal of the birds at interval (i.e 1 week) and cultured in a selective media which will be incubated at 41.50C for 44 hours under microaerobic condition. Colony collected will under microscope. The last of objective of this project is to determine the level at which the bacteria colonized the birds by calculating the bacteria load using serial dilution.