Molecular Prevalence and Risk Factors of Campylobacter Infection in Puppies in the Nairobi Metropolitan Region, Kenya

Campylobacter species are widely distributed pathogens; however, data on its epidemiology in puppies remain scanty, especially in Kenya. A cross-sectional study was conducted in the Nairobi Metropolitan Region to determine molecular prevalence and associated risk factors of Campylobacter species infection in puppies. A total of 260 rectal swabs were collected from puppies from breeding kennels, shelters, and the University of Nairobi Veterinary Teaching and Referral Hospital. The samples were subjected to polymerase chain reaction (PCR) assays for identification of Campylobacter species. Data on potential risk factors associated with puppy exposure were collected using a semistructured questionnaire. Multivariable mixed effects logistic regression analyses were performed with kennels as random effects. Campylobacter species were detected in 64 of the 260 sampled puppies yielding an overall prevalence of 24.6%. Multivariable results showed that puppies from shelters, puppies from kennels that are washed daily, puppies with a recent history of vomiting, and those treated with antibiotics in the past month were significantly associated with the presence of Campylobacter species. Being a kenneled puppy and having had concurrent bacterial infections were identified as protective factors. This study provides molecular evidence of puppy exposure to Campylobacter species which could have impact on puppy health and highlights the need to develop awareness and management strategies to potentially reduce the risk of transmitting this pathogen among puppies, to humans, and other animals.


Introduction
Campylobacteriosis, caused by thermophilic bacteria of the genus Campylobacter, is a signifcant zoonotic gastrointestinal disease afecting humans and animals, including dogs, globally [1][2][3][4]. Te vast majority of Campylobacter infections in humans are attributable to the consumption of contaminated or undercooked poultry [2,5,6], contaminated water [7], or raw milk [1]. Close contact with pets has also been identifed as a signifcant source of human Campylobacter species infections [8,9] with dogs, particularly puppies (less than one year), serving as potential reservoirs of Campylobacter infection for their owners, with infants and young children having a higher risk of infection [10].
Tough the detection of Campylobacter species is generally performed using the conventional culture method, it is time-consuming and labor-intensive, due to the fastidious nature of the species [23]. Hence, molecular-based assays, like polymerase chain reaction (PCR) and sequencing enable rapid and precise detection [1,24,25].
Despite reports of puppies serving as essential reservoirs for Campylobacter pathogens, current data on Campylobacter species epidemiology in Kenyan puppies are limited. Terefore, this study aimed at determining the molecular prevalence and associated risk factors of Campylobacter species in puppies in the Nairobi Metropolitan Region, Kenya.

Ethical Approval.
Tis study was approved by the Biosecurity, Animal Use, and Ethics Committee (BAUEC) of the Faculty of Veterinary Medicine, University of Nairobi, Kenya (FVM BAUEC/2019/237). Verbal consent was sought from breeders, kennel managers, and puppy owners prior to sampling.

Study Area and Design.
Study areas and design have been previously described [26] (Figure 1). In brief, this study was a cross-sectional study undertaken between January 2021 and August 2021 in breeding kennels, shelters, and the University of Nairobi Veterinary Teaching and Referral Hospital in the Nairobi Metropolitan Region, Kenya. Tese facilities were selected purposefully based on the high populations of puppies (less than one year) and the diversity of puppy breeds and management practices. Puppies in these facilities were randomly selected and sampled.

Sample Collection.
Te sampling methods have been previously described [26] (Figure 1). In brief, in order to determine potential risk factors associated with Campylobacter species infection, a detailed questionnaire was administered to collect puppy-level factors (age, breed, sex, vaccination status, and deworming status) and management factors (type of food, type of housing, kennel hygiene, and environmental hygiene). Prior to sampling, each puppy was assigned a body condition score (BCS) in accordance with the Waltham Size, Health, and Physical Examination (SHAPE) Score ™ which contains seven scores from A (underweight) to G (obese) based on the presence and amount of subcutaneous and abdominal fat [27] (https://www.slideshare.net/ WalthamCPN/waltham-pocket-book-of-healthy-weightmaintenance-for-cats-and-dogs-71137293). Te Canine Infammatory Bowel Disease Activity Index (CIBDAI) clinical scoring system by Jergens et al. [28] was used for the assessment of the puppies' general health status concerning gastrointestinal infection. Te numerical index assesses the severity of illness based on the presence and frequency of six cardinal signs of gastrointestinal infection. Based on the total cumulative scores, the infection was classifed as follows: clinically insignifcant (0 to 3), mild (4 to 5), moderate (6 to 8), or severe (9 or greater). A total of 260 rectal swabs were then collected from the puppies: breeding kennels (n = 210), shelters (n = 6), and veterinary hospital (n = 44).

Polymerase Chain Reaction (PCR) Analysis for Identifcation of Genus Campylobacter.
Te isolates utilized in this study were obtained from a previous study on culture prevalence in puppies in Kenya [26] (Figure 1). Campylobacter isolates were isolated on mCCDA (Oxoid, CM0935) and identifed by biochemical tests (oxidase and catalase tests). Genomic DNA was obtained from these presumptive Campylobacter species isolates using the boiling method as described by Wang et al. [29]. Polymerase chain reaction (PCR) amplifcations were performed using a thermal cycler (Bio-Rad T100 ™ Termal cycler). To confrm members of the genus Campylobacter, primers (C412GF 5′-GGATGA CACTTTTCGGAGC-3′ and C1228R 5′-CATTGTAGC ACGTGTGTC-3′) [30] targeting the 16S rRNA gene were used. Te polymerase chain reaction was performed in a total volume of 12.5 μl containing mastermix of 6.25 μl and 0.25 μl each of forward and reverse primers, 5 μl of DNA template, and 0.75 μl of sterile distilled water.
Te thermocycling conditions used were initial denaturation at 95°C for 15 minutes, followed by 25 cycles each of denaturation of 95°C for 30 seconds, annealing at 58°C for 1.5 minutes, extension at 72°C for 1 minute, and fnal heating at 72°C for 7 minutes. Samples were held at 4°C prior to analysis.
Controls were used for all PCR assays, and 10 μl of amplifed products was identifed by electrophoresis in a 1.5% (weight/volume) agarose gel in 1X Tris-Borate-EDTA (TBE) bufer, subsequently stained with ethidium bromide and ran for 30-45 minutes at 200 V, and visualized by UVilluminator (UVP GelMax 125 Imager, USA). Te sizes of the amplicons were determined using 100 bp molecular ladder. Specifc amplifed fragments expected were of size 816 bp which corresponded to the Campylobacter genus.
Te unit of observation corresponded to an individual sample, and each sample represented an individual puppy. If Campylobacter was detected by PCR in a sample, the puppy was considered infected.

Data Entry and Analysis.
Questionnaire data and PCR results were entered into Microsoft Excel version 2016 (Redmond, WA, USA) before being exported to STATA 17.0 (StataCorp LLC, USA) for analysis. Campylobacter species prevalence and other demographic parameters were computed using descriptive statistics. Te chi-square test was used to compare Campylobacter species carriage ratios between diferent categorical groups. Potential factors associated with Campylobacter species carriage in puppies were investigated using univariable logistic regression analysis. Covariates were retained in the model if statistically signifcant at p ≤ 0.2 using a backward stepwise elimination procedure. All variables that showed an association with the outcome variable in the univariable logistic regression analysis (p < 0.05) were considered in the fnal mixed efects logistic regression analysis. Potential clustering of puppies within kennels was controlled by including kennels as a random efect in the modeling. Model ft was assessed by checking for multicollinearity, overall goodness of ft of the model, infuential data points, and outliers.

Molecular Prevalence of Campylobacter Species Infection in Puppies in the Nairobi Metropolitan Region, Kenya.
Polymerase chain reaction was used to identify Campylobacter species isolates obtained from a previous study [26]. Tis was done by targeting the 16S rRNA gene specifc to Campylobacter species which produced a specifc band corresponding to the expected size of 816 bp ( Figure 2). Te results from PCR analysis revealed a molecular prevalence of 24.6% (64/260).

Prevalence of Campylobacter Species Infection Based on the Canine Infammatory Bowel Disease Activity Index (CIBDAI) Clinical Scoring System.
Fifty-four out of 260 puppies exhibited one or more of the six cardinal signs of gastrointestinal infection used to assess the degree of illness.
Polymerase chain reaction identifed Campylobacter species in 36.4% and 13.7% of the puppies whose infection status was classifed as severe and clinically insignifcant, respectively ( Table 2).

Discussion
Te study of zoonotic diseases such as campylobacteriosis is necessary due to the increasing number of people keeping dogs in their homes. Given that dogs, especially puppies, can be reservoirs of pathogenic Campylobacter, it is necessary to increase information about the epidemiology of this disease in these animals. Polymerase chain reaction (PCR) detected 64 Campylobacter species (24.6%, 64/260) by targeting the 16S rRNA gene specifc for these microorganisms. Tis proportion was within the range of 8.58% to 75.7% reported in studies done in the past fve years [15,[31][32][33]. Te relatively high prevalence of thermophilic Campylobacter species observed in this study among puppies is a cause for concern, as their feces contaminate the environment and may serve as a source of infection for humans, particularly children.
Tough clinical manifestations of gastroenteritis include diarrhea and vomiting [34], this study found no signifcant association between diarrhea occurrence and Campylobacter-positive status, a fnding that is in agreement with previous research [35,36]. However, this study found a statistically signifcant association between vomiting in puppies and the isolation of Campylobacter species. Tis fnding contradicts those of Verma et al. [11], who found no correlation between Campylobacter species infection and the incidence of vomiting in dogs. Findings of this study, however, concur with those of Guest et al. [37], who found a link between gastrointestinal signs and Campylobacter species infection in puppies.
Shelter-housed puppies were at a higher risk for Campylobacter species carriage. Tese fndings are in agreement with those of previous studies [36,38]. Campylobacter species carriage is more prevalent among puppies who share a habitat with other puppies such as in shelters [12,39,40]. Tis may be due to the fact that puppies are from multiple sources and the stress of comingling predisposes them to stress and vices such as coprophagia which may lead to the ingestion of these bacteria, resulting in further infection, shedding in feces, and contamination of the environment [41]. Te puppies may also roll in the feces, contaminating their fur [42] and further spreading the bacteria to surfaces with which they come into contact.
It is recognized that kennel hygiene is a potential risk factor for Campylobacter species carriage in dogs [43]. In this study, the daily washing of kennels was a highly signifcant risk factor for Campylobacter species carriage in the studied puppies. Despite the fact that daily washing of kennels improves hygiene, Campylobacter species are sensitive to desiccation and do not survive in dry environments [44], and thus daily washing increases their survivability in kennels as well as increases the chance of contamination of water sources [45], allowing water to be a vehicle for dissemination [46]. Puppies may also lick the residue water, resulting in pathogen ingestion.
Treatment with antibiotics in the past month was greatly signifcant with the risk of Campylobacter species carriage which was in agreement with a study by Leonard et al. [18]. Tis could be due to the inappropriate use of antibiotics in the treatment of other systemic infections with campylobacteriosis co-infection, thus promoting the emergence of antimicrobial resistant strains of Campylobacter species.

Conclusion
Tis study has shown that puppies in Kenya carry Campylobacter species which can be transmitted to humans and other animals through contaminated environmental sources. Polymerase chain reaction (PCR) should be regarded as indispensable in clinical and epidemiological research. It is important to develop awareness and management strategies to potentially reduce the risk of transmitting this pathogen from puppies to humans and other animals.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.

Conflicts of Interest
Te authors declare that they have no conficts of interest.