MOLECULAR EPIDEMIOLOGY, RISK FACTORS AND ASSESSMENT OF ANTIBIOTIC RESISTANCE OF SALMONELLAE IN COMMERCIAL LAYER FARMS IN SIX SELECTED STATES IN NIGERIA
MOLECULAR EPIDEMIOLOGY, RISK FACTORS AND ASSESSMENT OF ANTIBIOTIC RESISTANCE OF SALMONELLAE IN COMMERCIAL LAYER FARMS IN SIX SELECTED STATES IN NIGERIA
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Date
2016-08
Authors
FAGBAMILA, Idowu Oluwabunmi
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Abstract
Poultry salmonellosis remains a major constraint to poultry production in all parts of
Nigeria with farmers still experiencing great losses caused by Salmonella infection
despite huge amounts of resources expended on vaccination and medication. Relatively
few African countries report surveillance data on Salmonella and as such, very limited
information is available on Salmonella isolation for this continent. A cross sectional
study was carried out to determine the prevalence, risk factors and the circulating
Salmonella serovars in commercial layer farms in Nigeria. The study also identified the
different antibiotic resistance patterns seen in the Salmonella species and assessed the
mechanisms of their resistance and determined the degree of relatedness within the
different serovars isolated. In all, 358 farms were visited in the six selected States (Imo,
Edo, Ogun, Kaduna, Plateau and Bauchi) and 1790 samples comprising of litter, dust,
faeces, feed and water were collected and tested for Salmonella according to ISO
6579:2002; Annex D protocol. Samples were cultured for Salmonella after pre-enriching
in buffered peptone water, enriched in Rappaport Vassiliadis, Mueller Kauffmann
tetrathionate broth and Modified semi solid Rappaport Vassiliadis before plating on
Brilliance Salmonella agar, Bismuth sulphite agar, and Xylose lysine deoxycholate agar.
Biochemical tests were conducted and suspected isolates serotyped and phage-typed.
Minimum inhibitory concentration (MIC) was conducted on 200 isolates using
commercial Sensititre plate. Polymerase chain reaction (PCR) and sequencing were
conducted on the resistant isolates to detect resistance genes and point mutation (s).
Pulsed field gel electrophoresis (PFGE) was conducted on 110 isolates comprising 8
different serovars while Multilocus variable number tandem repeat analysis (MLVA)
was conducted only on 5 Salmonella Typhimurium isolates. The study established a
Salmonella farm prevalence of 43.6% and sample prevalence of 13.4% with the highest
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isolation rate of 16.8% from faecal samples. Sixty three serovars were isolated with S.
Kentucky being the most prevalent (16.73%) and widely distributed in 5 of the 6
selected states (83.3%) with Ogun State having the highest isolation rate (65.5% and
21.8% for farm and sample prevalence respectively). Using multivariable logistic
regression model, type of housing (AOR = 2.84; 95% CI: 1.00 - 8.03), age of farmer
(Adjusted odd ratio (AOR) = 2.79; 95% CI 1.33 - 5.86), crate type (AOR = 1.74; 95%
CI 1.05 - 2.88) and poultry association of Nigeria (PAN) membership (AOR = 1.67;
95% CI 1.07 - 2.62) were found to increase risk of Salmonella occurrence on farms.
Farm fencing (AOR = 0.46; 95% CI: 0.29 - 0.73) and staff having protective clothing on
the farm (AOR = 0.54; 95% CI: 0.33 - 0.89) reduced the risk of Salmonella. Of the 200
isolates for which the MIC of 14 antimicrobial agents were determined, 95 (47.5%) had
various resistance profiles. The predominant resistance pattern was GenSulTetCipNal.
Resistance to conventional antibiotics (AMP =19.6%, GEN =47.4%, TET =75.3% and
SUL =75.3%) was high but all the isolates were susceptible to Cefotaxime, Ceftazidime
and Meropenem. Resistance to ciprofloxacin and Nalidixic acid was also high (89.7%
and 77.3%) in most of the isolates and all the S. Kentucky were resistant to both
antibiotics. Resistance genes detected included tetA (tetracycline), sul1, sul2, sul3
(sulphamethoxazole), catA1, cmlA1, floR (chloramphenicol), dfrA5-14 (Trimethoprim),
aac(3)-le (gentamicin), tem1, tem30, tem57 (ampicillin) and qnrA, qnrB, qnrS
(quinolones). Mutations were detected on the gyrA gene (Ser83→Phe, Asp87→Gly,
Ser83→Tyr, Leu55→Phe), parC gene (Thr57→Ser) and pmrA gene (Thr89→Ser,
Val93→Gly). The comparison of PFGE profiles obtained from 110 isolates identified
PFGE cluster patterns for each of the eight serovars investigated with significant ranges
of genetic similarities. The MLVA result showed that two S. Typhimurium isolates had
the same allelic genes STTR9 and STTR3 but differed in the rest of the allelic genes
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while the other three isolates which were 100% genetically related had identical allelic
genes STTR9, STTR5 and STTR3 but lack allelic genes STTR6 and STTR10. This
study found S. Kentucky, S. Poona and S. Larochelle as major serovars associated with
poultry and environmental contamination in Nigeria. In addition, this study reported the
occurrence of rare/ infrequent serovars (e.g. S. Elizabethville, S. Larochelle, S. Carno
and S. Schwarzengrund) which can be of global importance because of travels,
transhumance, and trade in animals and food products. This study also highlighted
occurrence of resistance to important antimicrobials like fluoroquinolones. As
antimicrobials are easily obtainable over the counter, there is need for authorities in
Nigeria to regulate antimicrobial use in agriculture and in clinics to limit the spread of
multidrug-resistant Salmonella among humans and animals.
Description
A THESIS SUBMITTED TO THE SCHOOL OF POST GRADUATE STUDIES,
AHMADU BELLO UNIVERSITY, ZARIA, IN PARTIAL FULFILMENT OF
THE REQUIREMENTS FOR THE AWARD OF THE DEGREE OF DOCTOR
OF PHILOSOPHY IN VETERINARY PUBLIC HEALTH AND PREVENTIVE
MEDICINE.
DEPARTMENT OF VETERINARY PUBLIC HEALTH AND PREVENTIVE
MEDICINE, AHMADU BELLO UNIVERSITY, ZARIA, NIGERIA
Keywords
MOLECULAR EPIDEMIOLOGY,, RISK,, ASSESSMENT,, ANTIBIOTIC RESISTANCE,, SALMONELLAE,, COMMERCIAL LAYER FARMS,, SIX SELECTED STATES,, NIGERIA