AKAN  M

BABACAN O

MÜŞTAK HK

TORUN E

ÖNCEL T

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SummaryMycoplasma bovis is one of the most pathogenic agents in the Mycoplasma species that cause disease in cattle. In particular, young calves at less than 4 months of age are a considerable risk from pneumonia caused by M. bovis. In this study, we investigated M. bovis from tracheal swabs and blood sera of cattle which showed respiratory symptoms. A total of 127 tracheal swab samples were collected from seven different farms in Turkey. In addition, a total 254 acute and convelance sera were collected from the same cattle at intervals 15 days. The materials were collected from cattle between 3-12 months of age that reported respiratory problems such as broncho-pneumonia with coughing, depression, lethargy and fever. Mycoplasma bovis was investigated in tracheal swab samples and sera collected from the cattle by using PCR and ELISA respectively. The PCR results showed that M. bovis infections were positive in 4 different farms. The rates ranged from 5.3% (1/19) to 37.5% (6/16). Out of the 127 cattle examined, 45 (35.4%) were positive for M. bovis antibodies, while 82 (64.6%) were found to be negative. All PCR positive cattle were also found to be positive by ELISA. However by using ELISA, M. bovis infections were positive in all farms and the ELISA positive rates ranged from 20% (2/10) to 68.8% (11/16). Considering these results, in especially chronic infections, ELISA is a more useful method than PCR to detect M. bovis infection.Keywords: Cattle, ELISA, Mycoplasma bovis, PCRSığırlarda Mycoplasma bovis Infeksiyonunun ELISA ve PCR ile TeşhisiÖzet Mycoplasma bovis, Mycoplasma etkenleri içerisinde sığırlarda infeksiyona neden olan en patojen etkenlerden biridir. Özellikle, 4 aylık yaşın altındaki genç buzağılarda, M. bovis’in neden olduğu pnömonilerde artan bir risk bulunmaktadır. Bu çalışmada solunum sistemi infeksiyonu semptomları gösteren sığırların trachea svapları ve kan serumlarından M. bovis infeksiyonunun teşhisi ve M. bovis teşhisi için serolojik ve moleküler metodların karşılaştırılması amaçlanmıştır. Türkiye’de bulunan 7 farklı çiflikten gönderilen 127 tracheal svap örneği ile 15 gün arayla aynı sığırlardan alınan 254 adet akut ve konvelesans serum örneği PCR ve ELISA yöntemleriyle incelendi. Bu örnekler 3-12 aylık yaşlar da olan ve bronkopnömoni, öksürük, depresyon, halsizlik ve ateş gibi solunum sistemi infeksiyonu semptomu gösteren sığırlardan toplandı. Tracheal svap örneklerinin PCR sonuçlarına göre 4 farklı çiftik M. bovis infeksiyonu yönünden pozitif bulundu. Oranlar %5.3 (1/19) ile %37.5 (6/16) arasında bulundu. Mycoplasma bovis antikorları yönünden incelen 127 sığıra ait serumlarda, 45 (%35.4) adeti pozitif olarak saptandı; 82 (%64.6) serum ise negatif olarak saptandı. PCR’da pozitif olarak saptanan tüm sığırlar ELISA yöntemiyle de pozitif olarak saptandı. M. bovis infeksiyonu tüm çiftliklerde pozitif olarak saptandı ve ELISA oranları %20 (2/10) ile %68 (11/16) arasında değişkenlik gösterdi. Bu sonuçlar göz önüne alındığında, özellikle kronik infeksiyonlarda M. bovis infeksiyonunun teşhisinde ELISA’nın, PCR yöntemine göre daha uygun bir yöntem olduğu sonucuna varıldı.Anahtar sözcükler: ELISA, Mycoplasma bovis, PCR, SığırDiagnosis of Mycoplasma bovis Infection in Cattle by ELISA and PCR            Mehmet AKAN 1              Orkun BABACAN 2    Ebru TORUN 1 	 											Hamit	Kaan	MÜŞTAK	1     Taner ÖNCEL 3123Ankara University, Faculty of Veterinary Medicine, Department of Microbiology, TR-06100 Dışkapı, Ankara - TURKEYMinistry of Food, Agriculture and Animal Husbandry, Giresun Food Control Laboratory TR-28000 Giresun - TURKEYPfizer İlaçları Ltd. Şti., Animal Health Department, Bostançikmazı, 55, TR-34347 Ortakoy, İstanbul - TURKEYMakale Kodu (Article Code): KVFD-2013-9959Mycoplasma bovis is one of the most pathogenic agents in the Mycoplasma species that cause disease in cattle. Mycoplasma bovis-associated pneumonia occurs in cattle, including dairy and beef calves, beef cattle after arrival at a feedlot, and adults at any age [1]. Mycoplasma bovis is a particularly important cause of calf pneumonias [2,3]. Especially young calves under 4 months of age are at increased risk for pneumonia caused by M. bovis [2]. INTRODUCTION İletişim (Correspondence) +90 454 2150097 orkun_babacan@hotmail.comJournal Home-Page: http://vetdergi.kafkas.edu.tronline SubmiSSion: http://vetdergikafkas.org RESEARCH ARTICLEKafkas Univ Vet Fak Derg20 (2): 249-252, 2014DOI: 10.9775/kvfd.2013.9959250Diagnosis of Mycoplasma bovis ...Mycoplasma bovis infections can be explained as chronic and polymicrobial [1-5]. Animals can be infected via the respiratory system [2]. Respiratory tract and nasal secretions are important for epidemiology of infection [1,6]. Beside this, the importance of aerosols in calf-to-calf transmission of M. bovis is unknown but recently Maunsell et al.[1] reported that M. bovis has been isolated from air in shed containing diseased calves and calves may be experimentally infected by inhalation of M. bovis. Infected cattle spread M. bovis to enviroment through respiratory sectertions for many months as reservoir [4].The severity of pneumonia could be varried depending on the maintenance and environmental conditions. The effects of antibiotics and vaccines are not known to have negligible effects in calves [3]. Control of infection is difficult and economic loses is imminent [7,8]. The first condition is to ensure a high level of hygiene in the prevention of M. bovis infection. Because of non-specific clinical manifestations, and a wide range of variation in the epizootiology and pathogenesis, all M. bovis infections are have to be had specific diagnostic and control procedures [7]. Mycoplasma bovis infections could be diagnosed by bacteriological culture and serological methods [2,6,9,10]. Nevertheless, these methods are time consuming and false-negative results could be common [9]. Serological methods are less time consuming than the cultural methods and also more samples can be investigated. Recently, new molecular identification methods were improved and used in diagnosis of M. bovis infections worldwide by several authors [11-13]. PCR is much shorter in comparison to the conventional culture method for the identification of M. bovis infection [12].In Turkey there are few reports about the M. bovis infections in cattle [14]. In this study we investigated M. bovis from tracheal swabs and blood sera of cattle that showed respiratory symptoms for the situation of M. bovis in Turkey. Also we aimed to compare the efficiency of molecular and serological methods for detection of M. bovis infections. MATERIAL and METHODSA total of 127 tracheal swab samples and 254 acute and convelance sera (15 day intervals) were collected from 6-12 months age cattle located in seven different geographically distinct farms in Turkey that had respiratory problems such as broncho-pneumonia with symptoms coughing, depression, lethargy and fever (Table 1). All farms were beef farms and the capacities were between about 100 and 14.000 cattle. All the samples were transported to the laboratory in cold chain and were stored at –20ºC.Molecular Identification of Mycoplasma bovisDNA extraction was performed by the boiling method from directly swab samples [14,15]. The swab samples were analyzed by PCR using M. bovis spesific primers derived from the mb-mp81 gene, as described by Foddai et al.[13,14]. Mycoplasma bovis spesific primers were used to amplify 447 bp of mb-mp gene of M. bovis (mb-mp1F: 5-TAT TGG ATC AAC TGC TGG AT-3; mb-mp1R: 5-AGA TGC TCC ACT TAT CTT AG-3). Amplification was performed in a total reaction volume of 50 μl, containing 5 μl 10x PCR buffer, 5 μl 25 mM MgCl2, 250 μM of each dNTP, 1.25 U Taq DNA polymerase, 20 pmol of each primer and 25 ng of template DNA. The reaction conditions were as follows: denaturation at 94°C for 1 min, annealing at 54°C for 1 min and extension at 72°C for 1 min for 30 cycles, followed by a final extension step at 72°C for 10 min. The amplified products were detected by staining with 10 mg/ml ethidium bromide after electrophoresis at 80 V for 2 h in 2% agarose gels [14]. The results were screened from agarose gel by the molecular imaging system (Gene Genius, Syngene, England). Mycoplasma bovis DNA, which is used as positive control in PCR tests, was obtained from Prof. Dr. Burhan ÇETİNKAYA from Fırat University Veterinary Faculty Department of Microbiology Elazığ/Turkey.ELISABio-X M. bovis ELISA kit (BIO K 260, Belgium) was used in the serological analysis. The test was carried out according to the manufacturer’s instructions. After the test, the absorbance values were read at 450 nm with a Titertec Multiscan MS plate reader.RESULTSThe PCR results showes that M. bovis infections were positive in 4 different farms. The rates ranged from 5.3% (1/19) to 37.5% (6/16). The overall percentage, with a mean of a 12.6% (16/127) (Table 2, Fig. 1).The ELISA results showed in Table 3. Out of the 127 cattle examined, 45 (35.4%) were positive for M. bovis antibodies, while 82 (64.6%) were found to be negative. All PCR positive cattle were also found positive by the ELISA. Mycoplasma bovis infections were positive in all farms and the ELISA positive rates ranged from 20% (2/10) to 68.8% (11/16). The Table 1. Origins of sera and swap samplesTablo 1. Serum ve svap örneklerinin orijinleriFarmNumber of SamplesAge (Months)Swabs SeraFarm 1 16 32 6-12Farm 2 10 20 6-12Farm 3 16 32 6-12Farm 4 19 38 3-12Farm 5 10 20 6-12Farm 6 27 54 6-12Farm 7 29 58 6-12251AKAN, BABACAN, TORUNMÜŞTAK, ÖNCELoverall percentage was found positive as 35.4 % (45/127) (Table 3).DISCUSSIONThis research showed that M. bovis infection is a common respiratory problem in cattle in Turkey. Mycoplasma bovis infections are causing various economic loses such as treatment, laboratory diagnosis and product for dairy and beef cattles. In Turkey, Karahan et al.[14] were investigated a total of 148 samples (3 lungs, 4 eye swabs, 51 nasal swabs and 90 milk samples) from three different farms in Eastern Turkey. They found 23% (34/148) samples to be positive. These samples were 3 lung, 12 to 51 nasal swabs and 19 to 90 milk. Authors concluded that M. bovis was relatively common in eastern region of Turkey especially in cattle with both mastitis and respiratory problems. In this study, the PCR results shows that 12.6% (16/127) positive samples were detected and ELISA results showed 35.4% (45/127) positive rates. In this study, we found approximately same detection rates and this finding supports the Karahan et al.[14]. PCR was reported to have effective specificity and sensitivity in diagnosis of M. bovis infections [11]. Previous studies have shown that several species can be detected via two-stage nested PCR. However, in these procedures it is need to be looked carefully to their sensitivity characteristics. Sung et al.[12], reported that the optimization of primer sequences and the reaction conditions presented hereenhanced the sensitivity and ensured the high degree of specificity of the two-stage nested PCR. It was revealed that the specificity of the method presented their study was sufficient for the discrimination of mycoplasma contamination from other probable contaminants, including E. coli, S. aureus, and budding yeasts. It was showed in this study that the use of PCR makes the identification of M. bovis infection much shorter comparing to the conventional culture method. The other method for detection of M. bovis is serology. Serological tests shows increasing antibody titres ten to fourteen days after the onset of clinical symptoms. Consequently, the pathogen can not be detected during the incubation period [7,9]. Sachse et al.[7] reported that the authors developed an ELISA for M. bovis antibodies using whole-cell antigen of the agent for solid-phase coating. The assay has proved sufficiently specific and the sensitivity of detection (105-106 cfu/ml) was 100 times greater than Table 2. PCR findings of tracheal swab samplesTablo 2. Tracheal svap örneklerine ait PCR bulgularıFarm Number of Samples Positive Numbers (%)Farm 1 16 6 (37.5)Farm 2 10 0Farm 3 16 0Farm 4 19 1 (5.3)Farm 5 10 3 (30)Farm 6 27 0Farm 7 29 6 (20.7)Total 127 16 (12.6)Fig 1. Agarose gel electrophoresis of PCR. Line 1, molecular weight markers (Fermentas); Line 2, positive control; L3-L20, swab samplesŞekil 1. PCR sonucu elde edilen agaroz jel elektro-forez görüntüsü. Sıra 1, moleküler ağırlık marker’ı (Fermentas); Sıra 2, pozitif kontrol; L3-L20, svap örnekleriTable 3. ELISA results in comparison with PCR findingsTablo 3. PCR bulguları ile karşılaştırmalı ELISA sonuçlarıFarm Number of Cattle PCR Positive (%)ELISA Positive Total (%) ++ +++ ++++Farm 1 16 6 (37.5) 11 (68.8) 0 5 6Farm 2 10 0 2 (20) 2 0 0Farm 3 16 0 9 (56.2) 5 4 0Farm 4 19 1 (5.3) 4 (21.1) 1 2 1Farm 5 10 3 (30) 4 (40) 1 0 3Farm 6 27 0 7 (25.9) 6 1 0Farm 7 29 6 (20.7) 9 (31) 0 3 6Total 127 16 (12.6) 45 (35.4) 15 14 16252Diagnosis of Mycoplasma bovis ...with other serological methods [7]. The critical problem in these methods is the serological cross reactions between Mycoplasma strains.Comparison of the PCR and the ELISA results in this study showed that positive rates in PCR were less then the ELISA in all farms. The results can be explained as sampling: It must be taken into consideration with respiratory sampling that M. bovis can be better recovered from broncho-alveolar lavages than nasal swabs, although this method is much more diffucult [8]. All animals in these farms were treated with different antibiotics. PCR results could be affected negatively due to regular treatment with antibiotics at a high dosage (mycoplasma cell numbers in materials may be less then detectable limits). Antibodies to M. bovis persist for several months and can be detected easily with the ELISA. Because of the lack of cell wall in M. bovis, certain groups of antibiotics do not effective [2]. These antibiotics are used to treat for the secondary bacterial infections but often ineffective to treat Mycoplasma infections [4]. Because of the difficulties of the treatment with antibiotics, vaccine is important for M. bovis infections. One experimental vaccine study in calf reported that a single dose of vaccine prepared from saponised M. bovis cell can provide effective control against mycoplasma induced calf pneumonia. Calves tested for 6 months after immunisation had high level of humoral immunity [4]. No vaccine is currently used against M. bovis infection in Turkey.Considering these results, the ELISA was found to be more useful method than PCR to detect M. bovis infection because of the persistence of M. bovis antibodies especially in chronic infections and the results also induce a strong need for the effective vaccine development for Mycoplasma infections in Turkey.AcknowledgementsAuthors thanked to Prof.Dr. Burhan ÇETİNKAYA for supplied M. bovis DNA, which is used as positive control in PCR tests.REFERENCES1. Maunsel FP, Woolums AR, Francoz D, Rosenbuch RF, Step DL, Wilson DJ, Janzen ED: Mycoplasma bovis infections in cattle. J Vet Intern Med, 25, 772-783, 2011.2. Nicholas RAJ: Recent developments in the diagnosis and control of mycoplasma infections in cattle. WBC Congress, Quebec, Canada, 2004. www.ivis.org3. Soehnlen NK, Aydin A, Murthy KS, Lengerich EJ, Hattel AL, Houser BA, Fenton GD, Lysczek HR, Burns CM, Townsend AM, Brooks JW, Wolfgang DR, Jayarao BM: Epidemiology of Mycoplasma bovis in Pennsylvania veal calves. J Dairy Sci, 95, 247-254, 2012.4. Nicholas RAJ, Ayling RD: Mycoplasma bovis: Disease, diagnosis, and control. Res Vet Sci, 74, 105-112, 2003.5. Arcangioli MA, Duet A, Meyer G, Dernburg A, Bezille P, Poumarat F, Le Grand D: The role of Mycoplasma bovis in bovine respiratory disease outbreaks in veal calf feedlots. Vet J, 177, 89-93, 2008.6. Caswell JL, Archambault M: Mycoplasma bovis pneumonia in cattle. Anim Health Res Rev, 8 (2): 161-186, 2008.7. Sachse K, Pfützner H, Hotzel H, Demuth B, Heller M, Berthold E: Comparison of various diagnostic methods for the detection of Mycoplasma bovis. Rev Sci Tech Off Int Epiz, 12 (2): 571-580, 1993.8. Thomas A, Dizier I, Trolin A, Mainil J, Linden A: Comparision of procedures of isolating pulmonary Mycoplasmas in cattle. Vet Res Commun, 26 (5): 333-339, 2002.9. Ghadersohi A, Coelen RJ, Hirst RG: Development of a specific DNA probe and PCR for the detection of Mycoplasma bovis. Vet Microbiol, 56, 87-98, 1997.10. Fulton RW: Bovine respiratory diseases research (1983- 2009). Anim Health Res Rev, 10 (2): 131-139, 2009.11. Hayman B, Hirst R: Development of a semi-nested PCR for the improved detection of Mycoplasma bovis from bovine milk and mucosal samples. Vet Microbiol, 91, 91-100, 2003.12. Sung H, Kang SH, Bae YJ, Hong JT, Chung CB, Lee CK, Song S: PCR-based detection of Mycoplasma species. J Microbiol, 44, 42-49, 2006.13. Foddai A, Idini G, Fusco M, Rosa N, de la Fe C, Zinellu S, Corona L, Tola S: Rapid differential diagnosis of Mycoplasma agalactiae and Mycoplasma bovis based on a multiplex-PCR and a PCR-RFLP. Mol Cell Probes, 19, 207-212, 2005.14. Karahan M, Kalin R, Atil E, Çetinkaya B: Detection of Mycoplasma bovis in cattle with mastitis and respiratory problems in eastern Turkey. Vet Rec, 166, 827-829, 2010.15. Ongor H, Kalın R, Karahan M, Cetinkaya B, McAuliffe L, Nicholas RAJ: Isolation of Mycoplasma bovis from broiler chickens in Turkey. Avian Pathol, 37 (6): 587-588, 2008.

Diagnosis of mycoplasma bovis infection in cattle by elisa and pcr

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Diagnosis of mycoplasma bovis infection in cattle by elisa and pcr

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