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International Journal of Science, Environment ISSN 2278-3687 (O) and Technology, Vol. 2, No 5, 2013, 877 – 885
PREVALENCE OF YERSINIA ENTEROCOLITICA IN HUMAN
POPULATION AND TRANSMISSION VEHICLES IN ANAMBRA
*Amasiani, R.N.1, Agbo, I.C.2, Ezeifeka, G.O.3 and Chah, K.F.2
2Department of Veterinary Pathology and Microbiology, University of Nigeria, Nsukka.
3Department of Veterinary Microbiology and Parasitology,
Michael Okpara University of Agriculture, Umudike
E-mail: email@example.com (*Corresponding Author)
Abstract: Two hundred and ninety-two stool samples from diarrheic and non-diarrheic patients and 208 samples from different transmission vehicles such as ice-cream, sachet water, stream water, borehole water, rain water, well water and beef suya from the six health zones of Anambra State were screened for Yersinia enterocolitica. The isolation method used was cold enrichment using phosphate buffered saline prior to subculturing onto cefsulodin-irgasan-novobiocin (CIN) agar. Pathogenicity testing and antimicrobial susceptibility testing on recovered isolate were performed. Of the 292 human faecal samples screened, 75 (25.7%) were positive for Y. enterocolitica, while 37 (17.8%) of the 208 possible transmission vehicles analyzed yielded growth of Y. enterocolitica. The prevalence of Y. enterocolitica infection was highest among individuals aged between 1 – 10 years (36.1%) while those 41 – 50 years and 71 – 80 years did not harbor the organisms. None of the isolates was resistant to ciprofloxacin, ofloxacin, gentamicin and tetracycline while 97.3% were resistant to amoxicillin-clavulanic acid. All isolates were resistant to co-trimoxazole and amoxicillin. Keywords: Yersinia enterocolitica, human stool, transmission vehicle, Nigeria.
Yersinia enterocolitica is a pleomorphic Gram-negative bacillus that belongs to the
family Enterobacteriaceae. It is a well described enteric pathogen with distinctive clinical
manifestations, a range of outcomes and predilection for children (Bottone, 1999). Although
such infection occurs at all ages, the majority of patients are usually less than 5 years old
(Koehler et al., 2006). The spectrum of the disease ranges from asymptomatic to life
threatening sepsis especially in infants.
The incidence of human infection caused by Y. enterocolitica has increased tremendously
with reports coming from several parts of the globe (Schlundt, 2002; Bottone, 1999). In
Nigeria it has been incriminated in human clinical cases and has been isolated from
Received Aug 11, 2013 * Published October 2, 2013 * www.ijset.net
878 Amasiani, R.N., Agbo, I.C., Ezeifeka, G.O. and Chah, K.F.
apparently healthy animals (Okwori et al., 2009). Strains of Y. enterocolitica have been
isolated from a wide variety of foods as well as from animals and environmental sources such
as water (Sandery et al., 1996), milk and milk products (Okwori et al., 2009), vegetables
(Cocolin and Comi), meats (Fredriksson-Ahomaa et al. 2006, Grahek-Ogden et al., 2007),
animals and human beings (Fredriksson-Ahomaa et al., 2000, Fredriksson-Ahomaa et al.
Most cases of Y. enterocolitica infection are sporadic but reports have documented large
outbreaks centred on a single contaminated source (Zartash, 2009). Yersinia enterocolitica is
associated with acute diarrhea, terminal ileitis, mesenteric lymphadenitis and pseudo-
appendicitis (De Berardis et al., 2004). Y. enterocolitica has caused high rate of morbidity
and mortality globally among children as a result of poor hygiene and lack of access to
portable water. Its incidence has been reported in different parts of West Africa including
In Nigeria, much attention has not been given to the routine isolation of this pathogenic
organism which also is a major cause of acute gastroenteritis in patients especially children.
There is paucity of information on the distribution and pattern of occurrence of the organism
in our environment. There is need to identify the risk factors that predispose to the occurrence
of the organism in various clinical hosts and reservoirs. Therefore, the objective of this study
was to determine the prevalence and risk factors of Y. enterocolitica in human population in
This study was conducted in Anambra State, Nigeria. The State is divided into six health
zones. Two hospitals from each health zone were selected using simple random technique.
Isolation and identification of Y. enterocolitica
A total of 500 samples, comprised of 292 feacal samples from diarrheic and non diarrheic
hospital patients and 208 samples from transmission vehicles such as milk and ice-cream,
water (stream, boreholes, well water, rain water and sachet water), (beef suya were randomly
collected from the six zones in Anambra State. Two grams of each faecal sample were
suspended in 10 ml of phosphate buffered saline (PBS) (pH 7.2), vortexed for 30 s and
subjected to cold enrichment by incubating at 40C for 21 days. A unit volume of each
transmission vehicle (except beef suya) was added to 9 ml of PBS (pH 7.2), mixed
Prevalence of Yersinia Enterocolitica in Human Population ………. 879
thoroughly and also subjected to cold enrichment. For suya samples, 2 g of each sample was
pounded in a mortar then suspended in 9 ml of PBS (pH 7.2), mixed thoroughly and similarly
incubated. After cold enrichment a loopful of each sample was streaked on Cefsulodin-
Irgasan-Novobiocin (CIN) agar (Oxoid, Basingstoke, United Kingdom). Inoculated plates
were incubated at 250C for 24-48 hrs. Two to three colonies with deep red/purple centres and
sharp edges surrounded by a translucent boarder (“bull eye”) were selected and purified on
fresh CIN agar for Gram staining and motility test (Cheesebrough, 2004). Presumption
Yersinia colonies were biochemically confirmed by catalase, coagulase, Indole, Methyl-red,
Voges- Proskauer (IMViC) and sugar (rhamnose, sucrose and sorbitol) fermentation tests
Swiss albino mice were used to determine the invasive ability of Y. enterocolitica. The effects
on mice mimic the major pathological feature of human disease. The mice were infected by
oral administration (peroral test), intraperitoneal and eye inoculation (Sereny test) as
Per-oral test: A colony of the test organism was suspended in 1ml of normal saline. Three
Swiss albino mice were deprived of drinking water for more than 12 hrs and were allowed to
drink the above suspension. Death of two or more mice was considered a positive per-oral
result according to Ali and Shareef (1991).
Intra-peritoneal test: A colony of the test organism that were grown at 25 0C for 24 – 48hrs
on CIN agar was suspended in 1ml normal saline. 0.1 ml of the suspension was injected intra-
peritoneally into each of three albino mice. Death of the mice between 2 – 7 days was
considered a positive result according to Ali and Shareef (1991).
Sereny test: The eyes of three albino mice were infected with one loopful of the organism
that was suspended in 1ml of normal saline. Development of conjunctivitis gives a positive
sereny test as described by Ali and Shareef (1991).
The sensitivity profile of each isolate to seven antibiotics (ciprofloxacin [10 µg], ofloxacin
[10 µg], gentamicin [10 µg] tetracycline [25 µg], amoxicillin-clavulanic acid [25 µg],
amoxicillin [25 µg] and cotrimoxazole [25 µg]) was performed using the standard disc
diffusion method (Konemman et al., 2006). The diameter of inhibition zones produced by the
different antibiotics against each test organism was measured using a meter rule (CLSI,
880 Amasiani, R.N., Agbo, I.C., Ezeifeka, G.O. and Chah, K.F.
The prevalence and distribution of Y. enterocolitica in humans and transmission vehicles in
Anambra State is shown in Table 1. Of the 292 human faecal samples screened, 75 (25.7%)
were positive for Y. enterocolitica, while 37 (17.8%) of the 208 possible transmission
vehicles analyzed yielded growth of Y. enterocolitica. Beef suya (63.3%), stream water
(38.5%) and ice-cream (26.8%) were the transmission vehicles found to be contaminated by
Y. enterocolitica (Table 1). The prevalence of Y. enterocolitica infection was highest among
individuals aged between 1 – 10 years (36.1%) while those 41 – 50 years and 71 – 80 years
Out of the 75 isolates from human samples 50 (66.7%) caused death in mice 2 – 7 days
following intra-peritoneal or oral administration as well as conjunctivitis in the Sereny test.
Forty-seven of the 50 isolates pathogenic to mice were isolated from diarrheic patients. None
of 37 isolates from transmission vehicles was pathogenic to mice.
The antibacterial resistance profile of the isolates is presented in Table 3. None of the isolates
was resistant to ciprofloxacin, ofloxacin, gentamicin and tetracycline while 97.3% were
resistant to amoxicillin-clavulanic acid. All isolates were resistant to cotrimoxazole and
The prevalence of Y. enterocolitica (36.1%) recorded among children between 1 – 10 years of
age in this study is similar to the findings of Omoigberale and Abiodun (2002) who
documented a prevalence rate of 32.8% among diarrheic children in Benin, Nigeria. The
isolation rate in the present study, however, differed from 7.5% and 1.4% reported in the
same age group by Okwori et al. (2007) in Jos and Onyemelukwe (1993) in Enugu
respectively. Nigeria had generally reported low prevalence of Y. enterocolitica infection
compared to other parts of the world. In Southeast, United States of America (between 1988
– 1991) 77.6% of infections in children aged 12 months and younger were caused by Y.
enterocolitica, making the organism the second most common cause of bacterial
gastrointestinal infection in children (Metchock et al., 1991)
The high prevalence of Y. enterocolitica infection as seen amongst children 1 – 10 years of
age could be due to impaired or compromised immunity, social and sanitary habits as
documented in a similar finding by Lal et al. (2003). In this study, it was observed that
Prevalence of Yersinia Enterocolitica in Human Population ………. 881
prevalence of Y. enterocolitica decreased as the age increased. This may result from
improved personal hygiene as the individuals attain adulthood.
Yersinia enterocolitica is ubiquitous in the natural environment and may have originated
from water, soil, and food contamination. It was not isolated from industrial ice cream but
rather from non-industrial ice cream. Francis et al., (1980) found out that Y. enterocolitica
does not survive pasteurization. However, other worker reported that viable Y. enterocolitica
may persist after pasteurization if the organism is present in large numbers (Ackers et al.,
2000, Salmah and Shareef, 1991). These finding may explain the absence of Y. enterocolitica
from industrial ice cream. The absence of Y. enterocolitica in the sachet water, borehole
water and rain water could be due to boiling during processing of sachet water, chlorination
of sachet and borehole water and lack of exposure of the three to any subsequent animal or
As mentioned previously, Y. enterocolitica is an ubiquitous microorganism and may be
expected to be found in many foods. In the present investigation 37 Y. enterocolitica isolates
were obtained from ice cream, stream water, well water and beef ‘suya’. The prevalence rate
of 25.7% indicates that the probable source of human infection was through these
transmission vehicles. In addition, Y. enterocolitica has been isolated from samples of
ingredients used in the production of ice-cream such as cream, egg and pasteurized milk
(Tacket et al, 1994). Norma and Ana, (2000) also reported an outbreak of yersiniosis
associated with consumption of contaminated water and food.
The effect in mice mimics the major pathological features of human disease by causing
deaths of mice within 2-7 days in intraperitoneal and oral administration and conjunctivitis in
sereny test (Aulisio et al., 1983). Une (1977) demonstrated that pathogenic strains of Y.
enterocolitica were able to penetrate cultured HeLa cells, whilst non-pathogenic strains could
not. Recovery of the test isolates from vital organs, stool sample and pus from the eyes of the
infected mice used for animal model proves the invasiveness of the organism, hence its
In this study, antimicrobial profile of Y. enterocolitica to different antibiotics showed that all
the isolates were susceptible to ciprofloxacin, ofloxacin, gentamicin and tetracycline. Only
three isolates were susceptible to amoxicillin-clavulanic acid. Strains isolated by Okwori et
al. (2007), in Jos, Nigeria were susceptible to ciprofloxacin, floxavid and streptomycin.
In conclusion, this study has shown that Y. enterocolitica is present and active in human
population and some transmission vehicles in Anambra State, Nigeria. However it may go
882 Amasiani, R.N., Agbo, I.C., Ezeifeka, G.O. and Chah, K.F.
undetected because of ineffective isolation procedures or because of its similarity to other
enterobacteria with respect to biochemical reactions. Efforts should be made to include the
isolation of this organism in our routine laboratory exercises to reduce death that might occur
when the organism is implicated in human disease.
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884 Amasiani, R.N., Agbo, I.C., Ezeifeka, G.O. and Chah, K.F.
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Table 1: Prevalence of Y. enterocolitica in humans and transmission vehicles in Anambra
Prevalence of Yersinia Enterocolitica in Human Population ………. 885
Table 2: Percentage distribution of Y. enterocolitica isolates among different age groups
Table 3: Resistance profile of Y. enterocolitica isolates (n=112) to common antibacterial
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