Isolation and Characterization of Campylobacter spp. from Domestic Animals and Poultry in South of Iran

MATERIALS AND METHODS

Sampling sites and samples collection: Fecal samples from domestic animals and poultry were collected in south of Iran (Fars and Boshehr states) within six months during 2006.

In all 455 fecal samples were collected from healthy domestic animals (cow, horse and camel) and Poultry from different farms in Fars and Boshehr states, Iran. The samples were collected from each animal using sterile stick and polyethylene bag and transferred to the laboratory within one hour of sampling. The samples were subjected to detection of Campylobacter immediately upon arrival in the laboratory. Time of sampling varied from 8 am to 5 pm during the animal grazing and feeding.

Sample processing and isolation: The preT-KB method was used for isolation of campylobacters (Baserisalehi et al., 2004). One gram of the collected feacal samples were emulsified in sterile phosphate-buffered saline (pH = 7.0, 0.1 M) at 10% (w/v) concentration. The suspension was centrifuged at 8500 rpm for 10 min, followed by holding at room temperature. After 10-15 min a loopful of supernatant was withdrawn and spread onto the KB medium. The plates were incubated at 37°C for 48 h under microaerophilic conditions and examined daily for 5 days.

Phenotypic identification of Campylobacter spp.: All presumptive campylobacters subjected to Gram staining, oxidase and catalase tests, microscopic examination of wet mount under dark field and phase contrast microscope.

Table 1:	Biotypes of thermophilic Campylobacter spp.
* Based on Lior scheme (1984); + Positive; - Negative

The isolates exhibiting characteristic motility of Campylobacter were characterized using standard Campylobacter phenotypic identification tests recommended by Atabay and Corry (1997). These tests included H₂S by lead acetate strip, nitrate reduction, growth in 1% glycine and 3.5% NaCl, growth at different temperatures (25, 37 and 42°C), hippurate hydrolysis, indoxyl acetate hydrolysis, urease production, resistance to nalidixic acid (30 μg) and cephalothin (30 μg).

Additional tests for identification of campylobacters were alkaline phosphotase production and Glucose fermentation.

Biotyping of thermophilic Campylobacter spp.: Biotyping of thermophilic Campylobacter isolates was carried out using Lior scheme (1984). According to the biotyping scheme, Camp. jejuni, Camp. coli and Camp. lari were divided into seven biotypes based on three tests viz., hippurate, rapid H₂S and DNase. Camp. jejuni comprise four biotypes, Camp. coli two biotypes and Camp. lari one biotype (Table 1). Therefore, in order to know occurrence of different biotypes of thermophilic Campylobacter, the thermophilic isolates from fecal samples were biotyped using hydrolysis of hippurate, rapid production of H₂S and deoxyribonuclease enzyme production (DNase) tests.

RESULTS

Isolation frequencies of campylobacters from fecal samples: Fecal samples were collected from a total of 455 cow, horse, camel and poultry and analyzed for detection of Campylobacter. Of all, 85 samples were positive for Campylobacter. Therefore, it can be concluded that approximately one-fifth of the samples (18.7%) were harboured Campylobacter. The results obtained from the present study indicated that Campylobacter occurred with different levels in almost all of the sources of investigation. As seen in the Table 2, the frequency of occurrence of Campylobacter in poultry was relatively high and in camel was relatively low. Therefore, it can be concluded that cow, horse and poultry are reservoir of campylobacters hence; these bacteria may enter the environment of investigation through the feces of domestic animals and poultry with different levels.

Table 2:	Isolation rates of Campylobacter spp. from feces of domestic animals and poultry

Occurrence of catalase positive and negative campylobacters in domestic animals and poultry: A total of 85 Campylobacter isolates from different sources were tested for catalase production. The results indicated that majority of the Campylobacter isolates from different sources other than camel were catalase positive. However, catalase negative Campylobacter strains were not recovered from horse, but all isolated strains of Campylobacter from camel were catalase negative (Table 3).

Identification and biotyping of catalase positive thermophilic campylobacters: In all 85 catalase positive and negative campylobacters were subjected to tests recommended by Atabay and Corry (1997). As shown in Table 4 frequency of occurrence of Camp. jejuni in all sources was high followed by unidentified Campylobacter whereas, isolation frequencies of Camp. lari and Camp. coli was found to be similar. Although, Camp. jejuni and Camp. coli were isolated from all sources other than camel, Camp. lari was detected neither camel nor horse. However, all Campylobacter isolates from camel were catalase negative, isolation rates of them from horse was nil. In addition, the characterization of catalase negative campylobacters isolates indicated that all of the catalase negative Campylobacter isolates were Campylobacter sputorum while, high isolation rate was recorded Campylobacter sputorum var sputorum and low isolation rate was recorded Campylobacter sputorum var bubulus (Table 4).

Biotyping: The catalase positive Campylobacter spp. were biotyped by the extended scheme of Lior (1984). The results indicated that most of the Campylobacter biotypes existed in all of the sources other than camel. This result illustrated that the frequency of occurrence of Camp. lari biotype I in the area of investigation was relatively high followed by Camp. jejuni and Camp. coli biotypes I. Therefore, the present work clearly showed that the area of investigation was harboured different biotypes of Campylobacter.

As shown in Table 5 the highest isolation rate of Camp. jejuni biotype I was relatively recorded among poultry and cows, while isolation rate of Campylobacter biotypes I and III among horse was similar. Furthermore, Camp. jejuni biotypes II and III, IV were not recovered from cow and poultry, respectively. Although rate of existence of Camp. coli biotype I in cow and horse was relatively high, frequency of occurrence of Camp. coli biotype I and II in poultry was similar. Besides, our data illustrated that Camp. lari biotype I was detected from poultry as well as cow, while it was absent in the other sources. The results obtained from this study demonstrated however, untypable Campylobacter was detected from fecal samples of cow and horse, but it was not detected from fecal samples of poultry.

Table 3:	Occurance of catalase positive and catalase negative *Campylobacter in fecal samples
*Weak catalase positive campylobacters were placed in the catalase negative campylobacters category

Table 4:	Prevalence of existing Campylobacter spp. in fecal samples of domestic animals and poultry
The Figures in the parentheses represent percentage of isolates of each species/group; *Number of Campylobacter isolates

Table 5:	Occurrence of biotypes* of catalase positive thermophilic Campylobacter spp. in environmental samples
*Biotying according to Lior scheme (1984); The figures in the parentheses represent percentage of isolates of each biotype

In general, frequency of occurrence of Camp. jejuni biotype I (20%) was relatively high, while, biotype II (2%) was relatively low. Amongst Camp. coli isolates, biotype I (20%) existed in most frequently. Although, Camp. lari and untypable Campylobacter were not fitted for typing according to Lior scheme, our observations indicated these bacteria existed in this geographical area.

DISCUSSION

Infection with campylobacters is established zoonoses and the organisms can be transmitted to human being via food (meat and milk), water and through contact with farm animals and pets. A number of potential risk factors associated with Campylobacter infection include inadequately cooked chicken, domestic pets such as cat and dogs, raw milk, untreated water, poor food hygiene and handling practices (McMahon and Mahmood, 1993). In order to ascertain the likely sources of Campylobacter it is necessary to characterize strains, which are commonly isolated from food chain and environment and to identify these strains in the human infections.

Campylobacters after entry into the environment use its particular characteristics viz., unique metabolism along with complete citric acid cycle, complex and highly branched respiratory chain and great regulatory functions enable them to survive and colonize a number of environments in addition to the mammalian or avian gut (Kelly, 2001).

Poultry, especially broiler chickens, are some of the most important sources of Campylobacter infection in humans and the water supply has been shown to be a prominent factor in colonization of campylobacters in chickens (Kapperud et al., 1993). In addition, distribution of Campylobacter species in chicken and lamb was similar to that seen in humans, suggesting that both of these food sources play a significant role in human infection.

Based on foregoing evidence, domestic animal and poultry could be considered as a link between natural habitat of campylobacters and human being. Therefore, to determine possibility of dissemination of campylobacters and estimate their frequency of occurrence in domestic animals and poultry the present study was conducted to isolate Campylobacter spp. from fecal samples of domestic animals and poultry. Then Thermopilic Campylobacter isolates were subjected for biotyping using Lior schemes. The results obtained from the present study indicated that all sources of survey were contaminated with different levels of campylobacters. According to our observations the major vehicle of campylobacters in this area was relatively poultry and minor vehicle was camel. Several studies parallel to our finding have shown that poultry is a major source of Campylobacter and chicken meat is predominantly associated with Campylobacter infection in man (Harrios et al., 1986; Humphery et al., 1993).

As seen in the Table 2 and 3 frequency of occurrence of catalase positive campylobacters as well as catalase negative campylobacters in poultry was relatively high. Besides, catalase positive campylobacters and catalase negative campylobacters were not isolated from fecal samples of camel and horse, respectively.

However, Campylobacter were isolated from all sources of investigation, but isolation frequencies of them in camel were rare. To find out the reason concerning to low frequency of occurrence of campylobacters in camel, it must be noted that camels are adapted to harsh environments such as desert, whereas campylobacters are very sensitive to adverse environmental conditions viz., dryness and low water activity. Besides, methanogenic bacteria are common residents of the digestive tracts of ruminant animals such as cattle, sheep, buffalo, camel and goats (Johnson and Johnson, 1995), whereas population of these bacteria in intestinal tracts of camels due to their diet is not as much as the other ruminant animals. Hence, low populations of metanogenic bacteria in the rumen of camel cause accumulation of H₂ and therefore survival of sensitive bacteria such as campylobacters quickly affected by high concentration of H₂. Hence, based on our finding camels could not be considered as a vehicle of campylobacters.

The Lior schemes were used for biotyping of Campylobacter strains isolated in this study. Of the 50 strains of Thermophilic Campylobacter tested, 20 strains of Campylobacter coli and Campylobacter jejuni were belonged to biotype I.

The most prevalent of Campylobacter isolates biotypes I was associated with Campylobacter lari and less prevalent was related to Campylobacter jejuni biotype II followed by untypable Campylobacter. Therefore, based on foregoing evidence probably most of campylobacteriosis in this geographical area is related to Thermophilic Campylobacter biotypeI. Mégraud and his colleagues (1987) after biotyping of Campylobacter strains isolates using Lior schemes reported Campylobacter coli and Campylobacter jejuni biotype I was the most prevalent (48.2%) of campylobacters in the France. Although, their geographical area of investigation was differ to our investigated area, their result was parallel to our finding indicating high frequency of occurrence of Campylobacter biotype I in both areas.

In addition, the results obtained in this study have clearly illustrated that some species of the catalase negative campylobactes existed in the environment of this geographical area. However, Campylobacter species other than Thermophilic catalase positive campylobacters named non-pathogenic Campylobacter, but recently several studies reported that these organisms also have potential of causing illness in man (Ie Roux and Lastovica, 1998; Amisu et al., 2001).

Overall, although, Coker et al. (2000) and Raji et al. (2000) stated chicken, goat, sheep and pig are major vehicle of Camp. jejuni and Camp. coli in developing countries, we believed that climate and relative humidity affected the population of campylobacters in the environment. Therefore population of campylobacters in the environment is depended on the weather status of the countries. On the other hand, existence of campylobacters in the intestinal tract of animals depended on their diet and intestinal tracts conditions.

ACKNOWLEDGMENT

I am indebted to my colleagues Miss Roeintan, Miss Mohammad Zadeh and Mr. Sheaah for their significant contributions in the research program.