Prevalence of Campylobacter species in fecal samples of pigs and humans from Zuru Kebbi State , Nigeria

Aim: The study was carried out to survey and determine the prevalence of Campylobacter species among pigs and humans within the pig rearing areas of Zuru Kebbi State, Nigeria. Materials and Methods: A cross-sectional study was conducted among household pigs and humans between September, 2013 and February, 2014. Isolation and characterization of Campylobacter species were performed using standard culture isolation techniques and biochemical characterization. A total of 450 fecal samples comprised of 300 from pigs and 150 from humans was collected and analyzed. Results: Prevalence of Campylobacter were 278 (92.67%) and 94 (62.67%) for pigs and humans respectively. The most encountered Campylobacter species in both cases was Campylobacter coli (276 [74.19]) followed by Campylobacter jejuni (62 [16.66]). The least isolated species in pigs was Campylobacter hyointestinalis 5 (1.8%) while Campylobacter lari 2 (2.13%) was least isolated in humans. C. lari was not found in pigs. No significant association (p>0.05) existed between Campylobacter isolates and the age and sex of both pigs and humans in this study. Conclusion: Both pigs and humans within the pig rearing areas of Zuru have been shown to harbor Campylobacter species and this might be due to extensive system of pig farming with indiscriminate defecation by pigs coupled with unhygienic disposal of human wastes in the environment and poor personal hygiene.


Introduction
Campylobacter, Gram-negative, small curved spiral rods of 0.2-0.5 μm long with a single unsheathed polar flagellum [1], sometimes present in large numbers in the small intestine of most animals including pigs.They are the causative agents of campylobacteriosis in animal and human which are of public health importance, especially Campylobacter enteritis due to Campylobacter jejuni and Campylobacter coli [2].Due to increasing incidence, expanding spectrum of infections, potential of HIV-related deaths due to Campylobacter, and the availability of a complete genome sequence of C. jejuni NCTC11168, interest in campylobacteriosis research and control in developing countries is growing [3].Campylobacteriosis is known as the most common cause of severe form of bacterial diarrhea worldwide [4] and the most frequently reported cause of gastrointestinal infection in man exceeding cases of salmonellosis and shigellosis [5].This infection occurs mainly in infants, elderly people and patients with underlying disease.The disease in humans is accompanied by fever, bloody diarrhea, headache and abdominal pain, also nausea and vomiting may occur.In immunocompromised individuals it may spread to the blood stream and become life-threatening [6].
It is widely assumed that campylobacteriosis is primarily a food-borne disease in which food of animal origin such as poultry and pork play a major role [2] and of which wild and domestic animals, particularly birds and pigs serve as major reservoirs [6].It has been estimated that 500 cells of C. jejuni can cause human illness [2].This means that even very small number of Campylobacter cells in water or food may be a potential health hazard.The classical clinical signs of campylobacteriosis in pigs include, sporadic abortion in sows and birth of weak or dead piglets [7,8], mild and sometimes creamy diarrhoea in piglets that last for several days which often leads to death.The resultant effect of the observed signs often leads to a decrease in productivity and subsequent economic losses.
The unhygienic and poor nature of the farrowing pens in many developing countries [9,10], as observed in the study area, the semi-intensive system of pig rearing in the study area can facilitate the spread of Campylobacter among pigs and from poultry to pigs, since poultry birds are allowed to roam freely in search of feeds.Eating of unhygienically or insufficiently cooked pork, carcass contamination during slaughter, and roaming-pets that bring infection home contributes greatly as a source of Campylobacter infection in humans [11].Furthermore, there is close contact and interactions between pigs, humans and other animal species in the study area, coupled with the fact that there is no baseline information on the prevalence of Campylobacter species in the study area, then study of this kind becomes imperative.

Ethical approval
Ethical clearance as approved by the Research Ethical Committee of the Faculty of Veterinary Medicine, Usmanu Danfodiyo University Sokoto was obtained prior to this study.

Sampling sites
The study was carried out within the pig rearing areas of Zuru Kebbi State, Nigeria, between September, 2013 and February, 2014.Zuru lies in the southern part of Kebbi State on the latitude 11°5'-11°56'N and longitude 4°3'-5°25'E.The areas lie within Sudan savannah and has mean annual rainfall of 1000 mm [12].The human population is estimated at 165,547 with land area of 653 km 2 [13].People in the study area engage in crop farming, hunting, blacksmithing, livestock rearing such as cattle, sheep, goats, and pigs.Pigs are raised semi-intensively and serve as a source of income for the households [14].

Sample size determination
There is lack of data on the prevalence of Campylobacter species from the study area.Therefore, sample size for the pig population was calculated at 20% prevalence [15] and a prevalence of 5% as obtained by Coker et al. [3] in Lagos State, Nigeria was used for humans at 95% confidence interval, with a desired precision of 5% using n = t 2 × P e×p (1−P e×p )/d 2 [16].n=Sample size, t=1.96 (S.E), P=Prevalence, d=Level of precision (5%).
A minimum of 245 and 72.9 sample sizes was derived for pigs and humans respectively, and were also respectively adjusted to 300 and 150 so as to increase detection chances.Thus, a total of 450 fecal samples was collected from both pigs and humans.

Sample collection and transportation
A convenient sampling technique was employed to collect fecal samples from pigs and humans.Fecal samples were collected using sterile swab sticks per rectum from restrained pigs.Fecal sample from volunteer pig farmers and their household members were also collected using sterile swab sticks within the first 4 h of defecation.Age was determined by dentition [17] coupled with information from pig owners and sex was determined by physical examination of external genitalia.Both age and sex were recorded at the point of collection, and fecal samples collected were transferred into bijou bottles containing Amies transport (pre-enrichment) media.The samples were transported in an ice-packed container within 3-4 h of collection to the Veterinary Public Health Laboratory, Usmanu Danfodiyo University, Sokoto, Nigeria.

Sample processing and isolation
The fecal sample was plated onto cefoperazone charcoal deoxycholate agar, using the swab stick with fecal matter and incubated at 42°C for 48 h under microaerophilic condition generated by a gas generating pack (Campygen CN25, Oxoid).

Phenotypic identification of Campylobacter spp.
The plates were examined for typical Campylobacter colonies, characterized by greyish, creamy/white, flat, moistened colonies with tendencies to spread.All the distinct colonies found were gram-stained, and different biochemical tests such as catalase, oxidase, hippurate hydrolysis and H 2 S production in triple sugar iron agar tests were performed.The isolates were identified to species level using the standard Campylobacter phenotypic identification tests as recommended by Atabay and Corry [18].C. jejuni among the confirmed isolates was identified using the hippurate hydrolysis test [19].A small amount of pure culture was inoculated in 0.4 ml of 1% sodium hippurate (1 g of sodium hippurate and 99 ml of distilled water) in a tube.The tube was caped and incubated for 2 h at 37°C.Then, 0.2 ml of 2% ninhydrin solution was added and re-incubated for further 15 min at 37°C.The development of a purple-violet color identified the isolate as C. jejuni.
Antibiotic sensitivity test was determined using the disc diffusion method as described by Bauer et al. [20].The antibiotic discs used were; nalixidic acid (30 mg), cephalothin (30 mg) and metronidazole (30 mg).

Statistical analysis
The results obtained were presented in tables and percentages.Chi-square (χ²-test) was used to test for any significant association between Campylobacter isolates from pigs and humans feces with some epidemiological variables such as age and sex.

1).
As shown in Table-2, the highest number of samples (160 [53.33%]) were collected from the male pigs with a total of 144 (90.0%) positive for Campylobacter species.However, female pigs have the higher percentage frequency (95.71%) than the males.With respect to age of the pigs sampled, adult pigs have a higher percentage (137 [94.48%]) of positive samples than the young pigs (Table -3).There was no significant association (p>0.05) between the isolates, age and sex of the pigs.
For humans, the highest number of samples (72 [48.0%]) were collected from people between the ages of 10 and 29 years with 44 (61.11%) positive samples, while the lowest number (34 [22.66%]) of samples collected were obtained from people who are 30 years and above, however with highest positive samples of 25 (73.53%) as shown in Table-4.In relation to the sex of the people, higher isolates, 46 (64.79%) were obtained from the female.Furthermore, there was no significant association between the isolates and ages and sex of the people (Table -5).

Discussions
The presence of Campylobacter species in pigs and human fecal samples has been established in this study.The prevalence of 92.66% from pigs is consistent with previous studies that reported a 90% [10] and 99% [21] prevalence in swine fecal material.This corroborate the fact that Campylobacter are swine intestinal commensals [22], as Campylobacter has been found to be isolated from almost all the fecal sample of pigs and only on rare occasions were Campylobacter not isolated and the reasons may be due to collection, transportation, storage and isolation techniques [23].Also, contrary to an isolation rate of 5-20% reported by Coker et al. [3] in Lagos State, Nigeria, a higher prevalence of 62.66% was recorded from human fecal sample in this study.Coker's work was a hospital laboratory-based screening of pathogens from patients presented with diarrhea.Diarrhea in human could be of many causes [24].However, humans sampled in this study were those who reared pigs in their houses and as such in very high proximity with pigs and this could have resulted in the difference in prevalence.
The most encountered Campylobacter species in this study is C. coli, both from pigs and humans.This agreed with previous reports [23], that C. coli is more adapted to swine intestine [1,25,26] and swine production environment and have been isolated from pigs on farm up to 100% of fecal samples [27].This could also suggest that C. coli is a normal gut inhabitant of pigs, and that it exists in large amounts [22], or it is also possible that C. coli are more easily detected in pigs' feces than any other Campylobacter species [7,21].A higher prevalence of C. coli isolated from human fecal sample contrasts previous studies where C. jejuni was predominant species isolated from humans [3].The humans sampled in this study might have been infected with more of the C. coli from the pigs reared within their area of habitation.Also, C. jejuni is more isolated from clinical cases [1].
Prevalence of C. jejuni was also high when compared with previous studies [23] in which it was rarely Both age and sex had no significant effect on the prevalence of Campylobacter species both in pigs and humans.However, in humans, contrary to previous works where higher isolate were gotten from children <10 years old and the elderly [3], higher isolates were observed between the ages of 10 and 29 years in this study.This might be because in the study area, people from this age group usually have more contact with pigs and other domestic animals such as poultry, than other age groups.
In conclusion, most pigs and humans found in the pig rearing areas of Zuru Kebbi State harbored Campylobacter species at a very high prevalence of 92.67% and 62.67% from pigs and humans respectively.We, therefore recommended that pig farming should be intensive and sited far away from human dwellings, with maximum biosecurity and bio-safety activities being put in place.Hands should be thoroughly and frequently washed with soaps and water after contact with pigs, pets and other farm animals.Also, the use of molecular techniques may increase the diagnostic efficiency of Campylobacter species in both humans and pigs from the study area.

Table - 1
: Percentage isolation of Campylobacter species in pigs' and humans' fecal samples from Zuru Kebbi State.Sex distribution of Campylobacter isolates in pigs fecal samples from Zuru Kebbi State.

Table - 3
: Age distribution of Campylobacter isolates in pigs fecal samples from Zuru Kebbi State.

Table - 5
[7]28]distribution of Campylobacter isolates in human fecal samples from Zuru Kebbi State.The higher rate of isolation in pigs here might be due to contamination from humans and other animal species such as poultry and wild birds.Other species: C. upsaliensis and C. hyointestinalis had a low isolation rate, this could be due to the fact they are less frequently encountered in pigs and man.The lower isolation rate of these species of Campylobacter may be attributed to the incubation temperatures of 42°C used in this study which optimized the growth of thermophilic Campylobacters: C. coli and C. jejuni, which are of public health importance[8,28].C. lari was not isolated in the fecal sample of pigs; this is not strange as C. lari is not usually associated with pigs[7].