Asian Journal of Animal and Veterinary Advances

Year: 2023 | Volume: 18 | Issue: 2 | Page No.: 74-82
DOI: 10.3923/ajava.2023.74.82

Epidemiological Investigation of Campylobacter spp. Colonization in Broiler Farms, Mirsharai, Chattogram

Tahia Ahmed Logno, Keya Ghosh, Md. Sirazul Islam, Tridip Das and Md. Ahasanul Haque

Abstract: Background and Objective: Being significant zoonotic bacteria, Campylobacter spp. does not spread from broiler to human only via consumption of contaminated meat but also through the handling of live broilers and during the preparation of contaminated meat and meat products. This baseline survey was conducted to estimate the prevalence of Campylobacter spp. colonization and its associated risk factors in broilers of Mirsharai Upazila. Materials and Methods: From October to December 2019, pooled cloacal swab samples were collected from broiler farms. Standard bacteriological and molecular techniques were followed to isolate and identify Campylobacter spp. A standardized questionnaire was used to collect epidemiological data. Risk factors have been considered at the farm level. Results: Of 20 pooled cloacal swab samples from 20 broiler farms the prevalence of Campylobacter spp. was estimated to be 45% (95% CI-23.1-68.5). In risk factor analysis, the factors significantly associated with Campylobacter colonization were shed number, flock size, water supply, floor type, litter type, farmer’s experience and use of distinct cloth or separate footwear while entering the farms. Most of the farms had a 2.5-5% mortality rate and coccidiosis and necrotic enteritis were the most frequent causes of death. Farmers used a wide range of antibiotics mainly for growth promoters or prevention purposes rather than therapeutic purposes. Conclusion: The study gathered evidence of the presence of Campylobacter spp. colonization in broiler flocks and identified the factors that could aid in the development of effective strategies for managing Campylobacter colonization in chickens to prevent campylobacteriosis in humans via broilers.

Keywords: prevalence, Broiler, risk factors, cloacal swab, farms, Campylobacter spp. and mortality rate

INTRODUCTION

Campylobacteriosis, caused by Campylobacter spp. is a Gram-negative, non-spore-forming, S-shaped, or spiral bacteria. Currently, there are 17 species of Campylobacter with six subspecies. However, the most commonly reported species are C. jejuni, C. coli, C. lari, C. fetus and C. upsaliensis¹. The primary habitat of Campylobacter species is in the intestinal tract of warm-blooded animals. Campylobacter jejuni and Campylobacter coli are most commonly found in humans. Campylobacter lari can cause recurrent diarrhoea in children. Campylobacter fetus is found in cattle and sheep as well as an opportunistic pathogen in humans. Campylobacter upsaliensis is found in dogs and cat². Furthermore, in the case of poultry, C. jejuni and C. coli are the most common³. Campylobacter has no detrimental effect on the intestinal health of the chicken. Hence, bird growth is not affected following natural exposure⁴. The primary clinical sign caused by Campylobacter in humans is acute diarrhea⁵. Thrombophlebitis, endocarditis, neonatal sepsis and pneumonia are also reported⁶. Acute colitis and acute appendicitis are found in some cases⁷. Guillain-Barre Syndrome⁸ and Miller-Fisher Syndrome⁹ are major post-infection complications. Farm animals are the primary cause of campylobacteriosis since they are the major reservoir ofCampylobacter species¹⁰.

Most outbreaks of campylobacteriosis are caused by the consumption of contaminated poultry meats and poultry products¹¹. Poultry meats and their products cause about 60-80% of the global campylobacteriosis cases¹². The risk of transmission is greater from broiler chickens because of the high level of consumption. Campylobacter spreads from birds to humans via consumption of contaminated meat and by handling live birds (broiler and layer) and during the preparation of contaminated meat and meat products¹³. Campylobacter infection has risen more in developed and developing countries over the last ten years. The overall prevalence of Campylobacter colonization in broiler meat across Europe was 37.4%¹⁴. In the Asian context, the prevalence of Campylobacter was reported to be 35.1% in Vietnam¹⁵ 67% in Sri Lanka¹⁶ and 38.6% in India¹⁷. In Bangladesh, 54-75% prevalence was reported in the case of broiler meat and 26-78% in cloacal swab sample^18-21.

Risk factors for Campylobacter colonization vary depending on farming practices, geographical location and climatic conditions^20,21. However, some factors are more or less considerable in every farm level Campylobacter colonization. Age of shed^21-22, disinfection of shed surroundings²³, the interval between new batch^21-22, flock size²⁴, age of birds^21,25, the experience of farmers²⁶ and introduction of new birds in flock²⁷ are some of the important risk factors related to Campylobacter colonization in the broiler. Major farm strategies to prevent and reduce Campylobacter colonization comprise biosecurity measures which are must not only for Campylobacter but also for other diseases²⁸. Vaccination against campylobacteriosis was a partial success since an effective vaccine against campylobacteriosis is still challenging²⁹. Host genetic selection, given the significant difference in Campylobacter susceptibility, was found in different chicken lines³⁰ and antimicrobial alternatives such as bacteriophage and bacteriocin treatment to reduce or eliminate Campylobacter from colonized chicken³¹.

Food-borne zoonotic pathogens of poultry are vital because they are connected to humans directly. Campylobacter is a significant food-borne pathogen⁹ that is not well studied in Bangladesh. Considering the public health significance of Campylobacter and the limited scale of studies on Campylobacter being conducted previously at local and national levels in Bangladesh, the present study was conducted to estimate the proportionate prevalence of Campylobacter colonization, identify potential risk factors associated, observe the mortality rate and causes of mortality in farms and observe the usage of antibiotics in farms and awareness of farmers regarding antibiotics.

MATERIALS AND METHODS

Ethical approval: Oral consent from the farm owners was taken during sample and epidemiological data collection.

Study area: Mirsharai is one of the largest and oldest Upazila (sub-district) of Chattogram. It is located in the Southwestern part of Chattogram (22°39 and 22°59 N and 91°26 and 91°38 E), Bangladesh. Most people depend on agriculture for their livelihood³². With 1.4 million poultry, there are around 600 poultry farms, including 517 broiler farms, 12-layer farms and two breeder farms (Upazila Livestock Office, Mirsharai, Chattogram 2019).

Sample and epidemiological data collection: From October-December 2019 (three months), of 517 broiler farms, 20 farms, each consisting of at least 500 birds, were randomly selected for the present study. Before sampling the birds and recording epidemiological information, there was necessary verbal permission from the individual farmer. Regardless of flock size, five birds per farm were randomly sampled. Accordingly, 100 birds were brought under-sampling. Cloacal swabs were obtained from birds by inserting swab sticks into the vent (until faecal contamination) and pooling five swab samples according to individual farm into a 15 mL falcon tube containing 7 mL buffer peptone water with a unique identifying number. Collected samples were then transferred through a cooling box maintaining 4°C and stored at -20°C until laboratory diagnosis.

A pre-tested structured questionnaire was used to record epidemiological data at the farm level through face-to-face interviews and physical observation. Data included several houses, type of floor, water supply, litter materials, amount of litter materials used, number of flocks per year, number of employees, use of footwear and distinct cloth, foot bath facility, flock size, age of birds, number of dead birds per flock, all-in all-out system, disinfection of farm before restock, house empty for >14 days before restock, information on vaccination and age of vaccination, usage of antibiotics and duration of usages along with farmers demographic information.

Bacteriological culture: Samples were inoculated on Campylobacter agar base (Oxoid^™ Ltd., Basingstoke, UK) containing selective growth supplement (Oxoid^™ Ltd., Basingstoke, UK) and 10% (v/v) defibrinated sheep blood by streaking method. The plate was then incubated at 42°C in an anaerobic jar (Oxoid^™ AnaeroGen^™ 2.5 L) under microaerophilic conditions with a CO₂ sachet (Thermo Scientific^™ Oxoid AnaeroGen 2.5 L sachet, 10 % CO₂, 95 % humidity) for 72 hrs²¹. Dewdrops like colonies were found on the Campylobacter positive agar plate. Campylobacter isolates were stored in 300 μL glycerin (50%) and 700 μL Brain Heart Infusion Broth at -20°C until further testing.

Molecular detection: The DNA extracts of all Campylobacter positive isolates were performed using the boiling-lysis method³³. The DNA extracts were stored in a -20°C freezer for conducting the conventional PCR. The DNA concentration obtained from each pooled sample was measured at 260 nm using a Qubit 4 fluorometer (Thermo Fisher Scientific, Waltham, Massachusetts, US). DNA extracts were then evaluated by a published multiplex PCR test to determine lpx gene³⁴. The followings were the primer sequences: lpxAF9625 (5’-TGCGTCCTGGAGATAGGC-3’), lpxA C. coli (5’-AGACAAATAAGAGAGAATCAG-3’) and lpxA C. jejuni (5’-ACAACT TGGTGACGATGTTGTA-3’) (forward primers) and lpxARKK2m (5’-CAATCATGDGCDATATGASAATAHGCCAT-3’) (reverse primer). The 20 μL reaction mixture constituted 10 μL New England Biolabs 2X Master Mix (containing Taq DNA Polymerase, dNTPs, MgCl2, KCI and stabilizers), 0.5 μL each forward and reverse primer and 2 μL of DNA extract template 6 μL Nuclease free water. The thermal cycling included 95°C for 5 min followed by 94°C for 1 min (denaturation), 52°C for 1 min (annealing), 72°C for 1 min (extension) for 35 cycles and with final extension, 72°C for 5 min. The PCR products on the 1% agarose gel were visualized through ethidium bromide staining. About 100 bp DNA marker (New England Biolabs, Massachusetts, US) was used as a standard molecular ladder.

Data entry and statistical evaluation: All data obtained were entered into Microsoft office excel-2007, USA (MS excel 2007). Data were cleaned, sorted and coded in MS Excel 2007 before exporting to STATA-14 (StataCorp,4905, Lakeway Drive, College Station, Texas77845, USA) for descriptive and univariable statistical analysis. The proportionate prevalence of Campylobacter colonization was calculated using the number of Campylobacter positive farms divided by the total number of farms. Frequency distribution of Campylobacter spp. was presented according to categories of each selected factor: Number of houses, litter amount, slaughter age, vaccination, vaccination age, dead birds per flock, all-in all-out system, empty for >14 days between flocks, disinfection before re-stock, antibiotic use and duration of usage). Fisher’s exact test was performed to assess associations between the categorized response variable of Campylobacter colonization and the selected independent variables. The results were expressed in frequency number, percentage and p-value.

RESULTS

Farm characteristics and farmers’ demography: According to the findings of the present study, the educational status of most of the farmers had below SSC (60%). Only 10% of the farmers had graduated. As 70% of farms had a flock size of 500-2000. Most of the farmers (70%) had experience in farming for less than four years and 30% of farmers had experience in farming for more than five years. Poultry sheds were made of mud floors (80%) and tin-made ceilings (70%). A total of 80% of farms used sawdust as litter. Most of the farmers responded that they don’t have distinct cloth for entering the farm, however, 25% of farmers use separate footwear. A total of 30% of farms had foot bath facilities having potassium permanganate. Almost all farmers (90%) reported that they follow the “all-in-all-out” system and keep the house empty for 14 days before restocking (Table 1).

Farm prevalence of Campylobacter and its distribution: The proportionate prevalence of Campylobacter was greater in farms containing multiple sheds (61.5%, 95% CI: 36-88.9%), small (500) to medium flock (1000) size (70%), water supplied with deep tube wells (66.7%), floor made of mud (68.8%), sawdust litter (56.3%) and no use of distinct cloth (57.9%) or separate footwear (60%) while entering the farm than that of the counterpart of each variable (Table 2).

Mortality status within each farm: According to the responses of farmers, the average maximum mortality per flock over a year was recorded as up to 0-2.5% mortality in 15% of farms, 2.5-5% mortality in 60% of farms and >5% mortality in 25% farms. Reported causes of mortality were Newcastle disease (30%), necrotic enteritis (40%), coccidiosis (40%), infectious bursal disease (1.45%) and avian influenza (1.45%) (Table 3).

The pattern of antimicrobial usage: According to farmers’ responses on antimicrobial usage over six months (July to December, 2019), the farmers used multiple antimicrobials for different purposes. Among antimicrobial usage, amoxicillin, doxycycline and sulfur drugs (sulfaclozine, sulfadimidine and sulfadimethoxine) were frequently used (15% each), followed by a combination of amoxicillin with enrofloxacin or ciprofloxacin (10%) and ciprofloxacin, cloxacillin and oxytetracycline (5% each). According to WHO classification (WHO, 2019) reserve group of antimicrobials was used in 2 farms, whereas the watch group of antimicrobials was used in 18 farms (Table 4).

DISCUSSION

Poultry intestines provide a suitable environment for Campylobacter colonization, increasing the risk of human campylobacteriosis from consuming contaminated poultry meat, which is a major public health concern³¹. Humans can get infections in various ways, but studies indicated that broilers are the most important source of infection since a poultry intestine constitutes a favourable environment for Campylobacter colonization³⁵. The present study aimed to estimate the proportionate farm-level prevalence of Campylobacter in broiler poultry at Mirsharai in Chattogram, to know the distribution of Campylobacter by different factors and describe overall mortality and antimicrobial usage pattern.

The overall farm-level prevalence of Campylobacter in Mirsharai was 45% which corresponds to the latest findings²⁰ (40.5%). However, variable Campylobacter farm prevalence was reported at 4.9% and 100%²¹. Reasons for prevalence vary due to the rearing system, farm management, biosecurity and hygiene^36-39.

Like the present study, an increased risk of Campylobacter was associated with an increasing number of sheds on a farm^36-40. Several houses in the same premise may lead to an increased prevalence of Campylobacter colonization through the introduction of bacteria into the sheds possibly because of increased movement of personnel^20-21. Many studies reported higher Campylobacter prevalence with an increasing number of flock sizes which does not support the finding of the present study^27,41. Some earlier studies however found no link between flock size and Campylobacter occurrence^36,42. A larger flock might give more chances of Campylobacter colonization because of the large volume of water, food and litter as well as the larger movement of personnel. The effect of small flock size on Campylobacter status in the present study might be due to the specific production system and management of the farm³¹.

In previously reported studies, the source of water supply did not influence the Campylobacter colonization³⁸, though the present study found some influence of the source of water supply on Campylobacter occurrence. Farms, where water is supplied from a tube well, tend to have more Campylobacter prevalence than those where water is supplied from a deep well, but this explanation has not been tested in the present study. The usage of untreated groundwater was identified as a risk factor in a prior study, which is consistent with the current conclusion⁴³. Depth of underground water level might affect this factor. This possibility is still not much explored and needs further study.

The use of rice husk as litter material was previously reported to increase the level of prevalence of Campylobacter²⁰, but the present study did not identify such a connection with Campylobacter occurrence. Using sawdust as litter material can cause respiratory problems resulting in decreased body immunity of birds and thus sawdust might play a role in Campylobacter colonization.

Unlike urban farmers, the social-economic status of farmers in countryside areas is generally poor⁴⁴. They start farming with low investment. Hence, the sheds are not well built. Most of the houses were mud-made floors. In the present study, floor type was found with a significant influence on Campylobacter occurrence.

Farming experience is an important factor in the occurrence of Campylobacter. Better farm hygiene and biosecurity along with personnel training can reduce Campylobacter occurrence²⁶. And experienced farmers tend to be more compliant in these matters⁴⁵. Similarly, the present study found less Campylobacter occurrence in the farms which were established before 2014 (more than 5 years of experience). Although a few earlier studies found no real effect of biosecurity measures such as the use of separate footwear or distinct cloth on Campylobacter occurrence^38,46. Many other studies determined the significant effect of such bio-security measures on Campylobacter occurrence^21,36,47. The present study found an influence of using separate footwear and cloth on Campylobacter occurrence where usage of separate footwear and cloth reduced Campylobacter occurrence. That might be because changing shoes and clothes before entering the farm prevents environmental contamination from the outside of the farm.

Farmers under the present investigation maintained 14 days intervals between batches and practised an “All-in all-out system” which is indicative of biosecurity practices. However, other factors might have contributed to the occurrence of Campylobacter.

According to farmers’ responses, most of the farms (60%) had a 2.5-5% mortality rate in a production cycle which is considered expectable⁴⁸. The most frequent causes of mortality were Coccidiosis and Necrotic Enteritis (40%) which generally occurred because of their endemicity. Though vaccines against ND and IBD are commonly practised in broiler poultry farms available, death occurred due to ND and IBD which might be due to the failure of the vaccine. A wide range of antibiotics was used in the studied farms. A reserve group of antimicrobials was reported to be used by a couple of farmers though most of the farms used a watch group of antimicrobials. It was discovered in 1950 that adding antibiotics to the diet of animals at the sub-therapeutic level may increase the rate of growth of the animal³¹. Since growth rate is the most important for broiler production in the present study farmers used antibiotics as a growth promoter. Farmers are not educated about the proper use of antibiotics and are also not aware of antimicrobial resistance. Hence this widespread use of antibiotics has led to an increase in antibiotic resistance. Moreover, it was found that farmers used Reserve group antibiotics. So, farmers should be trained about antibiotics with the risk and dangers of their misuse. Government officials as well as various non-government organizations should come forward in this regard.

CONCLUSION

The overall farm level Campylobacter prevalence was quite high. The common occurrence of Campylobacter in farms having multiple sheds, small to medium flock size (500-1000), water supplied with deep tube wells, mud floor, use sawdust litter. A wide range of antibiotics was used in the studied farms. A reserve group of antimicrobials was used in 2 farms which should be banned. However, most of the farms used watch groups of antimicrobials. Mortality due to Necrotic Enteritis, Coccidiosis and Newcastle Disease was observed. As the prevalence is high, improved farm hygiene and biosecurity measures should be practised. Farms should be built with more caution with cemented floors and with a pure water supply. Wider usage of antibiotics with reserve and watch groups should be prevented. The use of reserve group antibiotics should be stopped. For the aforementioned aspects, farmers’ education and awareness would be of utmost importance.

SIGNIFICANCE STATEMENT

The colonization of Campylobacter spp. in cloacal samples of broiler chickens from field conditions indicates that the prevalence of the organism in broilers is common. This organism act as a reservoir for future campylobacteriosis in humans. As Campylobacter can spread from broiler to human by several routes, control of colonization in the primary broiler production is believed to have the greatest public health benefit. We have gathered evidence of the presence of Campylobacter spp. colonization in broiler flocks at Mirsharai, Chattogram through this baseline survey. The further extended study might provide useful information to formulate a national control program.

ACKNOWLEDGMENTS

The authors would like to express their gratitude to Dr. Shemol Chandra Poddar, Dr. Jayita Basu and all the staff of Upazilla Veterinary Hospital, Mirsharai for their cordial co-operations and time during the research period. Special thanks to Dr. Sharmin Chowdhury, Professor, Department of Pathology and Parasitology, Chattogram Veterinary and Animal Sciences University for her kindful facilitation during work in the lab.

REFERENCES