Background: Campylobacter species are gram negative and flagellated bacteria under the genus Campylobacter, family Campylobacteriaceae. These pathogens cause zoonotic infections among human and animal populations. This study was undertaken between December 2006 and May 2007 to determine prevalence, risk factors and genetic diversity of thermophilic Campylobacter isolates from children less than 5 years and chickens in Morogoro Municipality, Tanzania. Methods: The Skirrow’s protocol was used for isolation and identification of Campylobacter from 268 human stool specimens and 419 chicken cloacal swabs. Patient biodata and risk factors associated with human infection were also collected. Genetic diversity of Campylobacter isolates was determined by a RAPD-PCR technique using OPA 11 primer (5′-CAA TCG CCG T-3′). Phylogenetic analysis and band pattern comparison were done by Bionumerics software and visual inspection. Results: Stool samples from 268 children and 419 cloacal swabs from chickens were analyzed. Prevalence of thermophilic Campylobacters in children was 19% with higher isolation frequency (p = 0.046) in males (23.5%) than females (13.8%). Campylobacter jejuni (78.4%) was more isolated (p = 0.000) than C. coli (19.6%) and 2% were unidentified isolates. In chickens, the prevalence was 42.5% with higher isolation rate (p = 0.000) of C. jejuni (87%) than C. coli (13%). Campylobacters were more frequently recovered (p = 0.000) from indigenous/ local chickens (75.0%) followed by cockerels (52.2%), broilers (50.0%) and lowest in layers (22.7%). Keeping chickens without other domestic animals concurrently (p = 0.000), chicken types (p = 0.000) and flock size (p = 0.007) were risk factors for infection in chickens. One hundred and fifty two (152) thermophillic Campylobacter isolates were genotyped by RAPD-PCR of which 114 were C. jejuni (74 from chickens and 40 humans) and 38 C. coli (28 from chickens and 10 humans). Comparison of Campylobacter isolates from children and chickens revealed high diversity with only 6.1% of C. jejuni and 5.3% of C. coli being 100% genetically similar. Conclusions: This study has recorded high prevalence of thermophilic Campylobacter in children less than 5 years and chickens in Morogoro municipality. The observed genetic similarity among few C. jejuni and C. coli isolates from children and chicken suggests existence of cross transmission of these pathogens between children under 5 years and chickens.
This cross-sectional study was conducted in Morogoro Municipality, Tanzania (37°4’E; 4°49’S and altitude of 487–600 m above sea level) between December 2006 and May 2007. Aim of the current study was to determine the prevalence, risk factors for infection and genetic relatedness among thermophilic Campylobacter isolates from children below 5 years of age and chickens. Sample sizes were calculated using the formula n = Z 2 p (1-p)/ d 2 [23] where: n is sample size; Z is the multiplier from the normal distribution, p is the expected prevalence and d is the desired absolute precision. The expected prevalence of campylobacter infection (p) used for sample size estimation was p = 20% for humans [24] and p = 70% for chickens [25]. Other values (Z, d, and CI) were kept constant. With Z value of 1.96 at 95% confidence interval (CI) and desired precision (d) of 0.05, the calculated minimum sample sizes (n) were 250 and 330 for humans and chickens, respectively. Children under 5 years of age attending Outpatient Department (OPD) at Morogoro Regional and Mazimbu Hospitals, Mafiga, Madizini, Usangi and Upendo health facilities were enrolled in this study. Children that were admitted, hospitalized and those under antibiotic therapy were excluded to avoid confounding effects on the bacterial isolation. Human stool samples were collected in clean sterile 10 ml-plastic containers by parents/guardians and submitted to the medical laboratory technicians. The samples were aseptically transferred into sterile 10 ml-universal bottles containing 5 ml of Campylobacter enrichment broth and stored at 4 °C before and during shipment to the laboratory. Biodata and the possible risk factors associated with human infection (age, sex, keeping chickens, keeping other animals and boiling or treating drinking water) were recorded. History of the study children experiencing a gastrointestinal disorder characterized by passing out loose and watery stool at least three times a day in the past 2 weeks and consistency of the stool samples were the criteria used to categorize the patients as diarrhoeic or non-diarrhoeic. Chicken cloacal swab samples were collected from 22 chicken flocks/farms located in various areas within Morogoro Municipality. After collection, the swabs were put into universal bottles containing 5 ml of Campylobacter Enrichment Broth (Oxoid Ltd, Basingstoke, Hampshire, England). These samples were placed on ice blocks in a cool box at approximate temperature of 4 °C and transported to the laboratory within 2 h. Study chicken populations included indigenous/local chickens, broilers, layers and cockerels. Categories of chickens sampled are in line with types preferably kept by majority of farmers/livestock keepers in Morogoro municipality, Tanzania. These were: indigenous/local chickens are of mixed sexes and free ranging while broilers are chickens of mixed sexes kept for meat production. On the other hand, layers are all females kept for egg production and cockerels are all males kept for dual purposes namely meat and reproduction. The chickens were samples from 22 different flocks/farms located in various areas within Morogoro municipality. Among the indigenous/local chickens were those at the Morogoro Central Market ready for sale. For convenience, the flocks were classified as small when number of chickens of the chickens was categorized as 1 to 199, medium (200–299) and large (300 and above up to 7000). Age groups of the chickens were assigned as 0–4, 5–9, 10–14, 15–19 and 20 weeks and above (20+). It is worthwhile to note that age of 49 chickens could not be ascertained due to lack of proper record keeping and these were excluded in age related analysis. In addition, chickens less than 3 weeks of age and those under treatment with antibiotics were excluded to avoid confounding effects of the maternal immunity and negative growth of Campylobacter on the media, respectively. For convenience, flocks with 199 chickens and less were classified as small, those with 200–299 chickens as medium and flocks/farms with 300 and/ more as large. Information on chicken types, flock size, age and keeping of other animals was obtained by field observations and confirmed in interviews with the owners. Campylobacter species are relatively slow-growing, fastidious bacteria that require specialized culture conditions; hence, they grow best under reduced oxygen tension on nutritional basal media supplemented with 5–10% blood. In the laboratory, human stool samples and chicken cloacal swabs were aseptically inoculated in 10 ml-universal bottles containing 5 ml of Campylobacter Enrichment Broth (Lab M, International Diagnostics Group, plc, Lancashire, UK). The bottles were incubated at 37 °C for 24 h in an incubator (Heraeus B5050, Germany). Thereafter, one loopful of the enriched human or chicken samples was plated onto modified cefoperazone charcoal deoxychocolate agar (mCCDA) (Oxoid Ltd, Basingstoke, Hampshire, England) supplemented with CCDA selective supplement (Oxoid Ltd, Basingstoke, Hampshire, England). The plates were put in an anaerobic jar (Coldstream Engeneering Ltd, 18–10, Arista, Sweden) with microaerophilic environments generated by a lighted candle and then in the incubator (Memmert, Germany) at 43 °C for 48 h. Bacterial colonies suspected to be thermophilic campylobacter species based on growth at 43 °C and colony morphology were subjected to further examination by microscopy using Gram’s staining, motility and biochemical tests using Skirrow’s protocol as previously described [3]. Confirmed thermophilic Campylobacter isolates were sub-cultured on mCCDA (Oxoid Ltd, Basingstoke, Hampshire, England) and three loopfuls of 48-h old colonies were harvested and transferred into cryogenic vials (Nalgene®, Nalge Nunc Int. Corp, USA) containing 1 ml of brain heart infusion broth (Oxoid Ltd, Basingstoke, Hampshire, England) with 20–30% glycerol (v/v). The vials were incubated at 37 °C for 24 h, initially stored at -20 °C for 24 h and then transferred to -80 °C until when further analysis by RAPD-PCR genotyping was performed. A total of 152 Campylobacter isolates were genotyped by RAPD-PCR using OPA 11 primer (5′-CAA TCG CCG T-3′) as described by Miwa et al. [7] and their genetic relatedness compared. Of these, 74 C. jejuni and 28 C. coli were isolated from chickens and 50 (40 C. jejuni and 10 C. coli) from humans. The DNA templates were prepared as described by Miwa et al. [7]. The RAPD reaction mixture consisted of 50 mM KCl, 10 mM Tris-HCl (pH 8.4 at 25 °C), 2.5 mM MgCl2, 0.1% Triton X-100, a 200 μM concentration of each deoxynucleoside triphosphate, 0.3 μM of the primer, 2.5U of Taq DNA polymerase (Invitrogen), 2.5 μl of the template DNA, and sterile nuclease-free water to a final volume of 25 μl. The 25 μl of reaction mixture was cycled in a Mastercycler (Eppendorf®, Germany) through the following temperature profile: an initial denaturation step at 94 °C for 1 min; 45 cycles of 94 °C for 1 min, 36 °C for 1 min, and 72 °C for 2 min; and a final elongation step at 72 °C for 5 min. The PCR products were held at 4 °C until when electrophoresis was performed. Five microliter of amplified DNA fragments were electrophoresed alongside 3 μl of 1-kb ladder (Promega, Madison, USA) through 1% (w/v) agarose gels (Molecular grade – low EEO, Whitehead Scientific (Pty) Ltd) in 1X TBE buffer (0.45 M Tris, 0.44 M Boric acid and 0.01 M EDTA) (SIGMA®, Sigma Chemical Co., St Louis, USA). The agarose gels were electrophoresed at 60 V for 90 min, stained with ethidium bromide (Promega, Madison, USA) 0.005% (v/v) and photographed using a computerized image capturing machine, Kodak 4000®. Data were stored in a Microsoft Office Access database and analyzed using Epi-Info software [26]. Comparison of dichotomous variables was done using Chi-square (χ 2) test at a critical probability of 0.05 and 95% confidence interval. Gel images were imported to BioNumerics version 4.61 computer software (Applied Maths) for analysis and dendrogram production. Pairwise comparisons were accomplished using the Dice similarity coefficient, and the dendrograms were created using the unweighted pair group method using a geometric average (UPGMA). For the whole dataset, the most appropriate optimization and position tolerance settings, as determined by the software, were 0 and 1%, respectively. As the gel images could not be normalized, visual inspection was done to determine similarity of the RAPD profiles and their proportions expressed as percentage of the total number of C. jejuni and C. coli isolates analyzed.
N/A
DIMA AI Care
