The present study was designed to determine the prevalence of thermophilic campylobacters in poultry raised at farms and their living environment.
Prevalence of thermophilic campylobacters
The overall Campylobacter comprising poultry faeces (n=346) and environmental samples(n=199) was recorded as 12.29% (67/545). Other findings reported from the studies conducted in broiler flocks have also reported almost similar overall prevalence. Chokboonmongkol et al. (23) reported 11.2% Campylobacter spp. prevalence in broiler flocks from Thailand while another study from Ecuador reported 12.4% prevalence of Campylobacter in broiler flocks (24). In India, limited studies have been done on Campylobacter prevalence in poultry. These studies have revealed prevalence ranging from 13.54-21.8% (13.54%,21; 15.89%,19; 21.8%,25 14.28%,26 and 20%, 27). However, a much higher prevalence of Campylobacter as high as 72.2% from cloacal swab samples has also been documented from poultry by Vaz et al. (28). Another study by Ingresa et al. (29) reported 71.4% prevalence of Campylobacter in poultry caecal samples and 69.1% for poultry faecal samples.
Faecal prevalence of Campylobacter was 18.2% (63/346).Detection of campylobacters in poultry faeces poses a significant risk for contamination of chicken meat. The organisms frequently colonize the bird’s intestine and shed in large numbers through faeces. Faecal shedding of Campylobacter spp. is a source of infection to other birds in the flock. Bacteria present in faeces can contaminate feed and water supply of the same flock. Moreover, there is a risk of Campylobacter transmission to their flocks by means of frequent human movement.
Only 4 isolates could be recovered from 199 environmental samples with a prevalence of 2.01%, which included 3 isolates from water and 1 isolate from litter sample However, Vaz et al. (28) recorded much higher 63.8% Campylobacter prevalence in litter samples from Brazilian broiler flocks. Similarly, Lisa et al. (30) reported 64.3%, 64.3% and 45.7% Campylobacter prevalence in soil, compost, and processed waste water respectively. Presence of campylobacters in environment is significant as campylobacters are able to form biofilms as a survival mechanism outside the host (31). Detection of Campylobacter spp. from water samples is important because all the birds in a flock drink water from the same waterer which aid in furthur spread within a flock. Also, capability to form a biofilm poses the risk of its presence in cold water inspite of chemical treatment (32).
Feed and manure samples of our study did not reveal any presence of Campylobacter spp. However, these sources cannot be neglected as a source of infection. Zero prevalence of Campylobacter in manure and feed samples could be due to less number of samples processed.
Interestingly, in this study majority of the isolates were identified as C. coli (76.11%) and only 23.88% isolates were C. jejuni. (Table4). C. jejuni is considered to be the predominant species colonizing poultry (33,34). Many studies ( 35,36,37) report the dominance of C.jejuni over C.coli in poultry. In India, Chattopadhyay et al. (38) and Rajendran et al. (39) also showed that C. jejuni were more frequent than C. coli in poultry faecal samples. However, in accordance to our study, many other authors have reported C.coli dominance. Pergola et al. (40) reported 70.71% prevalence of C. coli and 17.14% C. jejuni from cloacal swab samples. Monika (21) and 19) also reported higher C.coli presence of 75% and 67.44%,respectively of the total isolates recovered from poultry faeces of Uttarakhand. Also, Wieczorek et al(41) in their retrospective study of five-years on prevalence and antimicrobial resistance of Campylobacter from poultry carcasses in Poland also found C.coli as a dominant species over C.jejuni In our opinion, the initial dominance of a species and further spread due to improper control measures can decide the higher presence of a species. Better colonization ability of either of the two species in poultry intestine and persistence in outside environment may decide the dominance.
No C. lari and C. upsaliensis were detected in this study. However, C. lari isolation from poultry is reported by some authors. Very few studies support the presence of C.lari in poultry isolates. Pillai (42) and 25 isolated 2 and a single isolate of C.lari from chicken samples in Bangalore and Bareillly respectively. Oyarzabal and Hussain (43) are of the opinion that, with the development of DNA based methods for the identification of isolates; C. lari has not been reported for more than 10 years in the United States, which suggests that previous reports may have been misidentifications from the traditional biochemical tests which were used for species confirmation. Further studies on poultry using molecular diagnostic techniques would answer the same. Acke et al (44) reported that dogs are the main reservoirs for C. upsaliensis which could probably be the reason for non-isolation of this organism in our study.
No previous data on Campylobacter prevalence in poultry farms is available for selected locations except for Pantnagar and Haldwani. Probably Isolation of Campylobacter from the locations except the two (Pantnagar and Haldwani) has not been reported so far. Poultry farms at Pantnagar screened before have reported the prevalence rates of 16 % (45), 11.66 % (46) and 13.54 % (21). However, a lower prevalence of 6.9 % (19) and 5.34 % (47) also has been reported from Pantnagar. Rawat et al (20) reported 4.17 % Campylobacter prevalence in faecal samples of broilers collected from an organized farm of Pantnagar.
Prevalence of virulence genes
Total 48 Campylobacter isolates including 39 C. coli and 9 C. jejuni were included for virulence gene detection using PCR. Nineteen isolates (n=19) could not be revived and thus were not included in the virulence gene analysis. The genes associated with bacterial motility (flaA) and adhesion to epithelial cells (ca dF),were present in all (100%)the isolates.These genes are known to be conserved in Campylobacter spp. (48,49) and play a key role in the development of Campylobacter infection. The cdtB (97.9%) was second most prevalent gene. This gene along with cdtA and cdtC cytoxin gene has the ability to interfere with the division and differentiation of the intestinal crypt cells, ,thus has an important role in diarrhoea. This combination has been recorded with a prevalence of 96.6–97.6% in positive strains (50) which is in accordance with our study. It also suggests that the three genes( cdtA, cdtB and cdtC) should be included together in future studies for assessing toxic property.
The cgtB gene was found in 22.9% of the positive Campylobacter spp. isolates. Not much data is available on the presence of this gene in the campylobacters though this gene, as wlaN, also codes for a β-1,3-galactosyltransferase enzyme that is required for the production of sialylated lipooligosaccharide responsible for Guillain-Barré syndrome (GBS)(51)
Other gene ciaB exhibited in 12.5% isolates. This gene is important for Campylobacter survival in the intestinal tract. The product of the ciaB marker, which play a role both in the intestinal invasiveness and in colonization of the epithelial cells (52), was identified in campylobacters by other authors also in a lower percentag (53,54).The presence of this gene is significant as it helps the organisms to overcome the stress conditions presented by the intestine and cause disease. Additionally, expression of ciaB has been observed to reduce under nutritional stress (55).
None of the Campylobacter isolate harboured wlaN gene. Many studies conducted on C. jejuni and C.coli have reported total absence of this gene (48,56,57).However, Kim et al.(58) identified the wlaN gene among 100% of 63 human and in 78.6% of 42 animal C. jejuni isolated tested in Korea. The product of the wlaN gene is also thought to be involved in development of of Guillain–Barre' syndrome after C. jejuni infection (49,58,59).
In our study, C.jejuni (cadF(100%), flaA(100%), ciaB(66.66%), cdtB(88.88%) and cgtB(33.33%) ) possessed more number of virulent genes than C.coli (cadF and flaA(100%), cdtB(100%) and cgtB(20.51%)). Moreover, ciaB gene presence ( responsible for both epithelial and intestinal mucosal invasion) only in C.jejuni isolates may suggest this species dominance over C.coli in being more pathogenic (60) and a cause for regulars diarrhoeal cases in humans (7). The virulent profile of C. jejuni (59, 61) showed that the greatest potential of this species over the other in causing clinical cases in humans (81.1%) (62) is due to the properties of invasion, colonization and toxin production which are essential to elicit its pathogenesis. In contrast, C. Coli shows its priority is to ensure the survival through mechanisms (63).
Either of the virulence genes except wlaN were found in both faecal and environmental (water(n=3) and litter(n=1)) samples. This indicates potential risk to consumers..
Virulence genes cadF and flaA were detected in all isolates (100%) recovered from all four farms. Pant (45) recorded 100% prevalence of flaA and cadF genes in the isolates recovered from diverse sources collected from Udham Singh Nagar district. The presence of virulence genes such as cdtA and cdtB have been reported (46,64) who screened the sources from Pantnagar and nearby areas. Campylobacter isolates of the same region were also shown to express wlaN, iam, ciaB and dnaJ virulent genes (47).
High frequency of detection of virulence genes cadF (100%), flaA (100%) and cdtB (97.9%) in Campylobacter species in farms is a matter of concern. Casabonne et al. (65) studied the prevalence of seven virulence and toxin genes, i.e. flaA, cadF, ciaB, cdtB, cgtB, docC and wlaN from the diarrhic patients. He found all the isolates were positive for flaA, cadF and cdtB genes (100%) and 40.0%, 23.3%, 20.0% and 6.7% were positive for ciaB, docC, wlaN and cgtC, respectively. Wieczorek and Osek (66) showed the presence of cadF and flaA gene in 100% of the isolates obtained from poultry and human. Talukder et al. (57) studied pathogenic genes namely flaA, cadF, pldA, ciaB, cdtA, cdtB, cdtC and wlaN in 40 C. jejuni and 5 C. coli strains isolated from diarrheal patients in Bangladesh and found 100% prevalence of flaA, cadFand pldA genes. The detection rates of ciaB, cdtA, cdtB, cdtC and wlaN genes were reported as 95%, 97.5%, 97.5%, 97.5% and 7.5% respectively.
Phenotypic Antimicrobial Susceptibility
Forty two isolates (11 C. jejuni and 31 C. coli) were revived for the phenotypic antimicrobial susceptibility. Campylobacter isolates exhibited highest frequency of resistance to cefoxitin (97.61%) followed by ciprofloxacin (64.28 %), nalidixic acid (33.33 %), ampicillin (28.5%) and ceftriaxone (14.28%) (Fig. 19). Two isolates (4.76%) were resistant to tetracycline. However, only one isolate showed resistance to clindamycin, sulfafurazole and erythromycin. All isolates (n=42) were susceptible to levofloxacin and gentamicin (Table 14).
The antibiotic resistance profile in this study was almost identical to the findings of Rajagunalan (19) who observed C. jejuni to be 100% sensitive to gentamicin, ampicillin and erythromycin and 100% resistant to cephalothin and co-trimoxazole. Narvaez et al. (67) reported that 71.4% of Campylobacter isolates had sensitivity against nalidixic acid followed by tetracycline (48.1%), ciprofloxacin (5.5%), azithromycin (1.78%) and erythromycin (1.78%). All isolates were susceptible to clindamycin, florfenicol, gentamicin and telithromycin and tetracycline resistance was attributable to the presence of the tet(O) gene. Kashoma et al. (68) reported Campylobacter isolates with resistance to ampicillin (63%), ciprofloxacin (9.3%), erythromycin (53.7%), gentamicin (0%), streptomycin (35.2%), and tetracycline (18.5%), azithromycin (42.6%), nalidixic acid (64.8%), chloramphenicol (13%) and tylosin (90.2%) respectively. The variation in the antimicrobial sensitivity pattern of the Campylobacter isolates has been reported earlier.
Ten isolates of 41 (n=10, 23.80%) were multidrug resistant (MDR) exhibiting resistance to at least 3 or more antimicrobial classes. Only one isolate (ID.C4) was pan-susceptible. Higher resistance to β-lactam antimicrobials was detected in our study such as cefoxitin, ceftriaxone and ampicillin. Resistance to ampicillin (28.5%), a “critically important antimicrobial”, crucial in human medicine is alarming, since it limits our options to treat critical human infections. Resistance was also detected against tetracycline (n=2) and clindamycin (n=1); antibiotics classified as “highly important” in human medicine according to WHO. Clinical management of Campylobacter infection becomes more difficult because of increasing development of resistance against antibiotics.
Of 16 different AMR combinations, 8 resistance patterns were MDR represented by 10 isolates. The most common MDR patterns were AMP CX CIP TE (n=2) and AMP CX CIP (n=2). Resistance pattern AMP CX CIP TE (n=2) had faecal origin and was identified from two separate locations,viz Haldwani and Pantnagar farm 2. Another MDR pattern AMP CX CIP (n=2) also had faecal origin. However, this pattern was identified from Bazpur and Bindukhatta farms. Distribution of antimicrobial resistance patterns across sample types and farm location is detailed in (Table 17). Most number of AMR patterns were detected from Bazpur farm (n=7), followed by Pantnagar farm 2 (n=6) and Bindukhatta farm (n=5). Four AMR patterns per farm were detected from Haldwani, Pantnagar farm 1 and Jawaharnagar farm. Significant diversity in the AMR patterns was detected across different farms and sample types. This may conclude the presence of genotypic diversity among the isolates circulating across locations and within a single location.
Genotypic Characterization of AMR Determinants
Out of 41 isolates showing phenotypic resistance, 29 isolates showed presence of at least one resistance genes targeted (blaOXA-61, tet(O), cmeB and ermB). Most prevalent resistance gene combination was blaOXA-61+ cmeB, which was detected in 11 isolates. A variety of antimicrobial resistance genes (ARGs) conferring resistance to various classes of antibiotics detected in this study is a matter of concern because these antibiotics are frequently used in human medicine and also these resistant determinants can be transferred to susceptible bacterial population by horizontal gene transfer (HGT). Nesme and Simonet (69) reported that soil is prone to genetic exchange by means of horizontal gene transfer between ecologically distinct lineages present in other ecosystems. Kashoma et al. (68) reported antimicrobial resistance genes blaOXA-61 (52.6%), cmeB (26.3%), tet(O) (26.3%) and aph-3-1 (5.3%) in Campylobacter isolates.
Risk factor analysis
Out of 11 risk parameters tested, only feeding of branded feed was found to be significantly associated with Campylobacter colonization of the examined broiler flocks (p-value 0.0047). In a similar study, Hald et al. (70) reported that 35% Campylobacter positive flocks used purchased wheat. Authors further reported that farmers who purchased wheat from a feedstuff dealer (p value 0.026) had a higher risk of Campylobacter infections in their broiler flocks compared to farmers who fed home-grown wheat. Various studies have been conducted to determine potential risk factors for Campylobacter infection in poultry farms (70,71,72,73,74). Cardinale (75) reported that an elevated risk of Campylobacter infection at poultry farms was associated with several factors namely presence of other animals (mainly laying hens, cattle and sheep) in the farm, farm staff not wearing proper work clothing while working in poultry houses, un-cemented poultry-house floors and the use of cartons that transport chicks from the hatchery to the farm as feed plates (rather than specifically designed feed plates). However, thorough cleaning and disinfection of poultry-house surroundings and manure disposal outside the farm were associated with decreased flock risk. In our study, the strength of association of risk factors with the prevalence of Campylobacter organism could be better identified with more number of samples screened at much larger number of farm locations.