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Research Article
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Detection of Coxiella burnetii in Poultry Egg samples in Iran Using Nested PCR Assay |
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Ebrahim Rahimi
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Abbas Doosti
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ABSTRACT
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Q-fever is a widespread zoonosis caused by the obligate intracellular microorganism Coxiella burnetii. The epidemiology of Q-fever in Iran is essentially unknown. For this reason, this study for the presence of C. burnetii in hen, duck, goose, quail and ostrich sell egg samples in Isfahan, Gellan and Mazandaran provinces, Iran was conducted. In the present study, 369 hen, duck, goose, quail and ostrich sell egg samples from 15 villages of Isfahan, Gellan and Mazandaran provinces, Iran were tested for C. burnetii using a nested PCR assay. In total, 2 of 130 (1.5%) hen egg samples were positive; the positive samples originated from 1 of 15 (6.6%) villages. Eight of 104 (7.7%) duck egg samples from 2 villages were positive for C. burnetii. As for 34 gooses, 70 quail and 31 ostrich egg samples; they were all tested negative for C. burnetii. To our knowledge, this study is the first report of direct identification of C. burnetii using PCR in hen and duck egg samples in Iran. Further intensive prevalence studies on Coxiella infection and on possible dangers of poultry products will be needed to elucidate the epidemiology of Q-fever in Iran.
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Received: August 01, 2011;
Accepted: November 30, 2011;
Published: January 14, 2012
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INTRODUCTION
Q-fever, a zoonosis caused by the obligate intracellular Coxiella burnetii
is endemic throughout the world. The microorganism affects arthropods, birds
and pets, domestic and wild mammals as well as humans (Maurin
and Raoult, 1999; Parker et al., 2006). In
human, infection is often asymptomatic and in its mild form can be mistaken
for other flu-like illnesses. Acute Q-fever presents mainly as atypical pneumonia
or hepatitis (Arricau-Bouvery and Rodolakis, 2005) and
chronic infection, however, is a severe disease that requires prolonged antibiotic
therapy, because the infection can result in endocarditic (Zhang
et al., 1998). Q-fever is essentially an airborne disease. Infections
occur after inhalation of aerosols generated from infected animal placentes,
body fluids or contaminated dust resulting from contaminated manure and desiccation
of infected body fluids (Arricau-Bouvery and Rodolakis,
2005; Fretz et al., 2007). Since, birds are
part of the host spectrum of C. burnetii, infected domestic poultry can
transmit the agent to humans by contaminated raw eggs or through formites (Hirai
and To, 1998).
Various methods have been used to identify C. burnetii infected animals
and food samples including microscopic examination, isolation in laboratory
animals, immunodiagnostic tests and Polymerase Chain Reaction (PCR) assays.
Microscopic examination has poor diagnostic specificity and sensitivity (Arricau-Bouvery
and Rodolakis, 2005; Kazar, 2005) and bacteriological
isolation is time-consuming and requires confined level-3 laboratories. Recently,
PCR assay has become a very useful method for the detection of C. burnetii
DNA in biological samples (Uwatoko et al., 1996;
Lorenz et al., 1998; Berri
et al., 2003; Ongor et al., 2004;
Guatteo et al., 2006).
The epidemiology of Q-fever in Iran is essentially unknown and to the authors
knowledge, the prevalence rate of C. burnetii in poultry eggs in Iran
has never been reported. The objective of the present study was to determine
the prevalence rate of C. burnetii in chicken, duck, goose, quail and
ostrich sell egg samples in Isfahan, Gellan and Mazandaran provinces, Iran using
a nested PCR assay.
MATERIALS AND METHODS Collection of samples: Overall, 369 chicken (n = 110), duck (n = 104), goose (n = 34), quail (n = 70) and ostrich (n = 31) sell egg samples from 15 villages of Isfahan, Gellan and Mazandaran provinces, Iran from September 2009 to September 2010 were randomly selected. The samples were immediately transported to the laboratory in a cooler with ice packs and were processed within an hour of collection.
PCR detection of C. burnetii: C. burnetii was isolated
from sell egg samples as described previously by Fretz et
al. (2007). Purification of DNA was achieved using a genomic DNA purification
kit (Fermentas, GmbH, Germany) according to the manufacturers instruction
and the total DNA was measured at 260 nm optical density according to the method
described by Sambrook and Russell (2001).
All oligonucleotide primers were obtained from a commercial source (Cinna Gen,
Iran). The nested PCR assay used to screen for C. burnetii was designed
from the nucleotide sequence of the com1 gene encoding a 27-KD outer
membrane protein (OMP) as previously described (Zhang et
al., 1998) and the amplification was carried according to the method
described elsewhere (Fretz et al., 2007). For
the nested PCR assay with primers OMP1-OMP2 and OMP3-OMP4, the first amplification
was performed in a total volume of 25 μL containing 5 μL of DNA sample,
0.5 mM MgCl2, 0.2 mM (each) dNTPs, 1 μM primer OMP1, 1 μM
primer OMP2 and 0.5 U/reaction of Taq DNA polymerase (Roche Applied Science,
Germany).
The PCR assay was performed at 94°C for 4 min and then for 30 cycles of 94°C for 1 min, 56°C for 1 min and 72°C for 1 min in a DNA thermal cycler (Master Cycler Gradiant, Eppendrof, Germany). In the second amplification, the reaction was performed in a total volume of 25 μL containing 2 μL of DNA sample, 0.5 mM MgCl2, 0.2 mM (each) dNTPs, 0.8 μM primer OMP3, 0.8 μM primer OMP4 and 0.5 U/reaction of Taq DNA polymerase. The PCR assay was performed at 95°C for 4 min and then for 30 cycles of 94°C for 1 min, 57°C for 1 min and 72°C for 1 min. The PCR-amplified products (OMP1-OMP2: 501 bp; OMP3-OMP4: 438 bp) were examined by electrophoresis in a 1.5% agarose gel, stained with a 1% solution of ethidium bromide and examined under UV illumination. In the present study, C. burnetii DNA (Serial Number: 3154; Genekam Biotechnology AG, Germany) and DNase free water were used as the positive and negative controls, respectively. Statistical analysis: Data were transferred to a microsoft excel spreadsheet (Microsoft Corp., Redmond, WA, USA). Using SPSS 16.0 statistical software (SPSS Inc., Chicago, IL, USA), a Pearson chi-square test and Fisher's exact two-tailed test analysis was performed and differences were considered significant at values of p<0.05.
Table 1: |
Prevalence of Coxiella burnetii in chicken, duck, goose,
quail and ostrich egg samples in Isfahan, Gellan and Mazandaran provinces,
Iran |
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RESULTS AND DISCUSSION In the present study, 369 chicken, duck, goose, quail and ostrich egg samples from 15 villages of Isfahan, Gellan and Mazandaran provinces, Iran were tested for C. burnetii using a nested PCR assay. In total, 2 of 130 (1.5%) hen egg samples were positive; the positive samples originated from 1 of 15 (6.6%) villages. Eight of 104 (7.7%) duck egg samples from 2 villages were positive for C. burnetii. As for 34 gooses, 70 quail and 31 ostrich egg samples; they were all tested negative for C. burnetii (Table 1). No significant differences in the prevalence rates (p>0.05) were observed between egg samples isolated in Isfahan, Gellan and Mazandaran provinces.
The prevalence of C. burnetii in hen and duck egg samples observed in
this study (1.5% and 7.7%, respectively) is similar to a recent report in Japan
that showed a prevalence of C. burnetii of 4.2% in shell eggs and 17.6%
in mayonnaise samples using a PCR assay (Tatsumi et al.,
2006). In a study conducted in the Switzerland, all 504 sell egg samples
were negative for C. burnetii using a nested PCR assay (Fretz
et al., 2007). In another study conducted in Japan, all of 200 chicken
egg samples collected from supermarkets in Tokyo were negative by the nested
PCR method (Hirai et al., 2005). The discrepancy
between the different studies might be explained by the fact that the analyzed
eggs were produced in different regions.
The findings of the present study are limited to PCR-based detection of C. burnetii DNA in poultry egg samples, so we are unable to speculate on the viability of organisms in egg samples, or on the sensitivity and specificity of the nested PCR assay compared to other diagnostic methods. The results of this investigation suggest that Q-fever should be considered as a possible case of unexplained fever in Iran, particularly among veterinary peoples and farmers. CONCLUSION Although no extensive prevalence study was undertaken, our results indicate that Coxiella burnetii infection appears to be relatively frequent in domestic duck eggs in Iran. Therefore, the results of this study indicate that domestic duck eggs are important sources of C. burnetii infection in Iran. To our knowledge, this study is the first report of direct identification of C. burnetii using PCR in chicken, duck, goose, quail and ostrich sell egg samples and the first report of direct identification of C. burnetii in chicken and duck sell egg samples in Iran. Further intensive prevalence studies on Coxiella infection in poultry foodstuffs will be needed to elucidate the epidemiology of Q-fever in Iran. ACKNOWLEDGMENTS The authors would like to thank Dr. Hassan Momtaz, Majid Riahi and Manouchehr Momeni for the sincere help in performing technical parts of the project. We are also grateful to Zahra Alijantabar, Morteza Mosavian and Mohamad Husain Safarian for assistance with sampling.
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REFERENCES |
1: Arricau-Bouvery, N. and A. Rodolakis, 2005. Is Q Fever an emerging or re-emerging zoonosis? Vet. Res., 36: 327-349. CrossRef | PubMed | Direct Link |
2: Berri, M., N. Arricau-Bouvery and A. Rodolakis, 2003. PCR-Based Detection of Coxiella burnetii from Clinical Samples. In: Methods in Molecular Biology, Sachse, K. and J. Frey (Eds.). Humana Press Inc., Totowa, NJ., USA., pp: 153-161.
3: Fretz, R., W. Schaeren, M. Tanner and A. Baumgartner, 2007. Screening of various foodstuffs for occurrence of Coxiella burnetii in Switzerland. Int. J. Food Microbiol., 116: 414-418. CrossRef | Direct Link |
4: Guatteo, R., F. Beaudeau, M. Berri, A. Rodolakis, A. Joly and H. Seegers, 2006. Shedding routs of Coxiella burnetii in dairy cows: Implications for detection and control. Vet. Res., 37: 827-833. CrossRef | PubMed | Direct Link |
5: Hirai, K. and H. To, 1998. Advances in the understanding of Coxiella burnetii infection in Japan. J. Vet. Med. Sci., 60: 781-790. CrossRef | PubMed | Direct Link |
6: Kazar, J., 2005. Coxiella burnetii infection. Ann. N. Y. Acad. Sci., 1063: 105-114. CrossRef | PubMed | Direct Link |
7: Lorenz, H., C. Jager, H. Willems and G. Baljer, 1998. PCR detection of Coxiella burnetii from different clinical specimens, especially bovine milk, on the basis of DNA preparation with slica matrix. Applied Environ. Microbiol., 64: 4234-4237. Direct Link |
8: Maurin, M. and D. Raoult, 1999. Q Fever. Clin. Microbiol. Rev., 12: 518-553. Direct Link |
9: Ongor, H., B. Cetinkaya, M. Karahan, M.N. Acik, H. Bulut and A. Muz, 2004. Detection of Coxiella burnetii by immunomagnetic separation-PCR in the milk of sheep in Turkey. Vet. Rec., 154: 570-572. PubMed | Direct Link |
10: Parker, N.R., J.H. Barralet and A.M. Bell, 2006. Q fever. Lancet, 367: 679-688. CrossRef | Direct Link |
11: Sambrook, J. and D.W. Russell, 2001. Molecular Cloning: A Laboratory Manual. 3rd Edn., Cold Spring Harbor Laboratory Press, New York, USA., ISBN-13: 9780879695774.
12: Uwatoko, K., M. Sunairi, A. Yamamoto, M. Nakajima and K. Yamaura, 1996. Rapid and efficient method to eliminate substances inhibitory to the polymerase chain reaction from animal fecal samples. Vet. Microbiol., 52: 73-79. CrossRef | Direct Link |
13: Tatsumi, N., A. Baumgartner, Y. Qiao, I. Yamamoto and K. Yamaguchi, 2006. Detection of Coxiella burnetii in Market Chicken Eggs and Mayonnaise. In: Century of Rickettsiology: Emerging, Reemerging Rickettsioses, Molecular Diagnostics and Emerging Veterinary Rickettsioses, Hechemy, K.E., J.A. Oteo, D.A. Raoult, D.J. Silverman and J.R. Blanco (Eds.). 1st Edn. Wiley-Blackwell Publishing Co., USA., ISBN-13: 978-1573316392, pp: 502-505.
14: Zhang, G.Q., S.V. Nguyen, H. To, M. Ogawa and A. Hotta et al., 1998. Clinical evaluation of a new PCR assay for detection of Coxiella burnetii in human serum samples. J. Clin. Microbiol., 36: 77-80. PubMed | Direct Link |
15: Hirai, H., Z. Pang, D. Bao, T. Miyazaki and L. Li et al., 2005. Cbln1 is essential for synaptic integrity Nat. Neurosci., 8: 1534-1541. CrossRef | PubMed | Direct Link |
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