Evaluation of PCR, ELISA, and Culture Methods for the Diagnosis of Animal Brucellosis Al-Bayatti and Amina Diyala Agricultural Sciences Journal , 1( 1 ) ; 1 – 17 ,2009 . 1 Evaluation of PCR, ELISA, and Culture Methods for the Diagnosis of Animal Brucellosis . Sahar A. Al-Bayatti* and Amina N. Al-Thwani** *Veterinary Laboratory and Research Department, General Veterinary Company, Ministry of Agriculture. **Genetic Engineering and Biotechnology Institute for Post Graduate Studies, University of Baghdad. ABSTRACT The present study was undertaken to evaluate polymerase chain reaction (PCR) technique in the diagnosis of brucellosis in comparison with the enzyme linked immunosorbent assay (ELISA) and microbiological culture techniques in animal’s blood and aborted fetuses. Thirty animal blood samples were used 20 of them were rose bengal plate test (RBPT) positive and which were subdivided into two groups (11) vaccinated and (9) not-vaccinated, and 10 negative controls. ِ All these samples were examined by PCR using primer pair to amplify a 223-bp region within a gene coding for 31-kDa Brucella antigen, ELISA and culture. Five aborted fetuses were also included in this study these were examined by PCR using the same mentioned pair of primers and culture only. The results of the 20 RBPT +ve blood samples revealed that 13 and 7 were also positive by PCR and ELISA representing 65% and 35% sensitivity, none was positive by culture. There was no positive case among the control group by all the tests which included in this study so its specificity were 100%. The overall agreement between PCR and ELISA and PCR and culture were 73.33% and 56.66% respectively. From the five aborted fetuses which included in this study 4 were positive for Brucella infection by PCR and 3 of the 4 were also positive by culture, one was negative by both. The overall agreement between PCR and culture was high and reached 80%. On the basis of Al-Bayatti and Amina Diyala Agricultural Sciences Journal , 1( 1 ) ; 1 – 17 ,2009 . 2 biochemical characteristics results the two isolates which were from sheep fetuses were Brucella melitensis and one Brucella abortus isolates from aborted buffalo fetus. Due to many advantages, like speed, safety, high sensitivity and specificity, PCR is recommended to use in the diagnosis of animal brucellosis but its results need more evaluation in the vaccinated animal. INTRODUCTION The term “brucellosis” is applied to a group of closely related infectious diseases, all caused by bacterial pathogens in the genus Brucella. Manifestation of the disease may be ranged from abortion in the infected female’s to orchitis or epididymitis in the male or even death. Characteristically all Brucella species establish persistent infection in the mononuclear phagocyte system of the natural host species. Brucellosis causes major economic losses to the agriculture industry and the causative agent is classified as a category B pathogen by the Centers for Disease Control and Prevention (CDC ,2003). Most of the countries that are faced with the economic losses and public health issues caused by animal brucellosis have governmental programs for the eradication or control of the disease. Accurate diagnostic procedures are critical for the success of these programs (Nicoletti, 1982). Because of their potential to detect very small numbers of organisms, PCR –based assays have been applied recently to diagnose many infectious diseases. However there are only a few reports on the use of PCR for the diagnosis of animal brucellosis from blood samples and less from aborted fetuses. Moreover, the advantages of such techniques over the ELISA and culture have not yet been clearly established especially on vaccinated animals. Al-Bayatti and Amina Diyala Agricultural Sciences Journal , 1( 1 ) ; 1 – 17 ,2009 . 3 The aim of this study was to evaluate the new methods of PCR-based assay in the diagnosis of animal brucellosis in compare with ELISA and culture. MATERIALS AND METHODS Collection and treatments of samples Thirty blood samples were collected, in fact that twenty were taken from animals which shows evidence of brucellosis (some are with history of abortion and other RBPT+ve) and ten apparently healthy controls. Five ml blood sample volume were withdrawn from each animal, 2 ml of it was injected into prepared sterile trypticase soy broth with 2% sodium citrate and incubated for 48 hours at 37ºc, then it was sub cultured on duplicate agar plates one of them incubated aerobically and the other in an microaerophilic atmosphere (containing 5-10% carbon dioxide), Brucella spp. were identified using the standard methods advised by Alton et al. (1988). The rest of the blood samples were divided as follows: - one ml was used for DNA extraction, using the commercial purification system (Wizard Genomic DNA Purification Kit, Promega, Madison, WI) according to the manufacturer’s instruction for DNA purification from blood. The rest of two ml injected into plane tubes and used for serum collection for serology tests. The serum then submitted to RBPT as described by Morgan , (1967) then used for ELISA, using the kit provided by the (NOVATEC-Germany) company and according to their instruction. Five aborted fetuses were also included in this study; two of them were aborted sheep fetuses the other two were aborted buffalo fetuses while one was aborted horse fetus, all were examined by direct culture on trypticase-soy- agar and using their stomach contents, which were withdrawed by a sterile syringe. The specimens were cultured on duplicate agar plates and incubated one Al-Bayatti and Amina Diyala Agricultural Sciences Journal , 1( 1 ) ; 1 – 17 ,2009 . 4 in air and the other in an atmosphere containing an Added 5-10% of carbon dioxide, as advised by Alton et al. (1975). Using the same (Wizard Genomic DNA Purification Kit) DNA was extracted from the stomach contents of the aborted fetuses, then the DNA which was extracted from the blood and from the stomach contents of the aborted fetuses submitted to PCR amplification presses. PCR assay DNA amplification system provided by Promega-company, USA and genus specific B4/B5 primer pair designed by Baily to amplify a 223-bp region within agene coding for 31-kDa membrane protein specific to the genus Brucella (Baily et al. 1992). Primers name Sequences B4 5'-TGGCTCGGTTGCCAATATCAA-3' B5 5'-CGCGCTTGCCTTTCAGGTCTG-3' The reaction mixture contained 5µl of 10X PCR buffer, 3.5µl of 25 mM MgCl2 , 1.5µl of the dNTP mix (10mM each), 5µl of B4 primer of 10 pmol/µl 6µl of B5 primer of 8 pmol/µl, 0.5µl of 5υ/µl Taq DNA polymerase and 10 µl of sample DNA in a volume of 50µl. The reaction was performed in a thermal cycler (Techgene, Cambridge Ltd. UK). The cycling condition were an initial denaturation at 95ºc for 5 min, template denaturation at 94ºc for 1 min, annealing at 55ºc for 1 min, and primer extension at 72ºc for 1 min, for a total of 40 cycles, with a final extension at 72ºc for 7 min. Al-Bayatti and Amina Diyala Agricultural Sciences Journal , 1( 1 ) ; 1 – 17 ,2009 . 5 Ten µl of the amplified products were mixed with 3µl of loading buffer and analyzed by electrophoresis in 2% agarose gel stained with 0.5µg/ml ethidium bromide, at 100v for 25 min, in 0.5x TBE buffer, then visualized under UV light using ultraviolet transelumenater(USA). DNA ladder (100-1000 bp) was used. A sample was considered positive for Brucella spp. when a specific fragment of 223 bp was detected in the gel (Baily et al.,1992). Statistical analysis The percentages of positive, specificity, sensitivity and the overall agreement between PCR and ELISA and PCR and culture were calculated (Showman , 1986). RESULTS AND DISCUSION 1- Animal's blood samples: The results of ELISA, PCR and culture detection in animal's blood are shown in Table (1). RBPT results were positive in all the animals included in this table, the ELISA results were positive in only 7 of the infected animals groups which represent 35% sensitivity, but it was negative in the entire 10 control group, representing 100% specificity. The infected animals group was subdivided into two groups a-vaccinated, in which the percentage of positive results by ELISA was 54.5% higher than that of the second b-non vaccinated group which was 11.1%. Table (2). PCR was positive in 13 out of 20 infected animals, Table (1), and thus represent 65% sensitivity while it was negative in the control group representing a100% specificity. Figure (1) shows the results of some animal's blood PCR amplification process. The percentage of PCR positive in the vaccinated group which was 81.8% higher than that of the non-vaccinated group which was 44.4% ( the same effect previously noticed in ELISA), Table (2). Al-Bayatti and Amina Diyala Agricultural Sciences Journal , 1( 1 ) ; 1 – 17 ,2009 . 6 The results of PCR which compared to that of ELISA showed that the PCR results were identical to that of ELISA only in the control (RBPT-ve) group, while it differ in the infected (RBPT+ve) as shown in Table (3), which revealed that from the 13 positive cases in PCR only 7 were also positive by ELISA with a 46.15% identical rate and from the 7 PCR negative 6 were also negative by ELISA with a 85.71% identical rate. There were no positive cases by culture among both the infected and the control animal groups, Tables (1 & 2). Table 1. ELISA, PCR and culture results of animals infected with brucellosis. Sample No. ELISA * titer PCR Culture animal's Kind vaccine state 1 23.7 Neg. Neg. Goat Vaccinated 2 16.8 Pos. Neg. Goat Vaccinated 3 2.8 Pos. Neg. Goat Vaccinated 4 1.1 Pos. Neg. Goat Vaccinated 5 0.9 Pos. Neg. Goat Vaccinated 6 1.1 Pos. Neg. Goat Vaccinated 7 1.2 Neg. Neg. Sheep not- Vaccinated 8 0.9 Neg. Neg. Cattle not- Vaccinated 9 1 Pos. Neg. Buffalo not- Vaccinated 10 15.7 Pos. Neg. Buffalo not- Vaccinated 11 1.1 Pos. Neg. Buffalo not- Vaccinated 12 1.8 Neg. Neg. Goat not- Vaccinated 13 2.2 Neg. Neg. Cattle not- Vaccinated 14 12.5 Pos. Neg. Goat Vaccinated 15 37.4 Pos. Neg. Sheep Vaccinated 16 2.3 Pos. Neg. Cattle not- Vaccinated 17 2.7 Neg. Neg. Cattle not- Vaccinated 18 23.8 Pos. Neg. Sheep Vaccinated 19 18.6 Pos. Neg. Sheep Vaccinated 20 8.2 Neg. Neg. Sheep Vaccinated *A titer < 9 is considered negative (Neg.) while > 11 positive (Pos.) 9-11 grey zone. Table 2. Characteristics of infected animals and control groups and the percentages of the tests results. Groups No. of Samples ELISA PCR Culture Pos. Neg . % Pos. Neg. % Pos . Neg . % Al-Bayatti and Amina Diyala Agricultural Sciences Journal , 1( 1 ) ; 1 – 17 ,2009 . 7 RBPT (+ve) infected animals vaccinated 11 6 5 54.5 9 2 81.8 0 11 0 Not-vac. 9 1 8 11.1 4 5 44.4 0 9 0 Sumtotal 20 7 13 35 13 7 65 0 20 0 control RBPT(-ve) 10 0 10 0 0 10 0 0 10 0 When the total results of both the sensitivity and specificity of each tests was analyzed statistically it was found that PCR was the only efficient test in this group of animal samples. The figures (3) and (4) illustrate PCR evaluation results in animal samples. Table 3. Comparison of animals PCR results with ELISA and Culture. characteristic state of PCR No. of Sampl es ELISA Culture Pos. Neg . Identical ly% Pos. Neg. Identica lly% PCR+ve in infected groups v a c c in a te d 9 5 4 55.5 0 9 0 Not- vac. 4 1 3 25 0 4 0 sum total 13 6 7 46.15 0 13 0 PCR-ve in infected groups v a c c in a te d 2 1 1 50 0 2 100 Not- vac. 5 0 5 100 0 5 100 sum total 7 1 6 85.71 0 7 100 Al-Bayatti and Amina Diyala Agricultural Sciences Journal , 1( 1 ) ; 1 – 17 ,2009 . 8 control RBPT -ve 10 0 10 100 0 10 100 From Table (3) the overall agreement (overall agreement: is the proportional similarity of the results of both tests) between the PCR and each of ELISA and Culture were calculated as following: PCR and ELISA= %33.73100 30 1066   PCR and culture %66.56100 30 107    Fig. 1. Results of PCR detection. The amplified fragments were separated by electrophoresis on a 2% agarose gel, stained with ethidium bromide at 100 volts for 25 min. photographed under UV light. First lane is marker, 100-bp ladder. 2- Aborted fetuses. The results of PCR detection and microbiological culture are shown in Table (4). Three samples were positive by both PCR and culture; one was positive by PCR and negative by culture, while one negative by both PCR and culture, the percentage of overall agreement between them reached 80% as Al-Bayatti and Amina Diyala Agricultural Sciences Journal , 1( 1 ) ; 1 – 17 ,2009 . 9 shown in figure (4).From the three aborted fetuses which were positive by culture 2 were aborted sheep fetuses and one was buffalo's aborted fetus. Brucella organisms first recognized in smears obtained from fetal membranes stained with modified Ziehl-Neelsen stain, which stained red against a blue background, figure (2), while Brucella culture recognized then on the basis of colonial morphology and slide agglutination test, subsequent examination by routine bacteriological methods confirmed the genus Brucella, so the obtained isolates were Gram-negative, coccobacilli, arranged singly, in pairs, short chain and with small groups, negative for haemolysis on blood agar, and it does not grow nor lactose- fermenting on MacConkey agar, negative for nitrate reduction and indol production, while they were positive for oxidase and catalase tests. The colony morphology and the bacteriological and biochemical characteristics of the isolates revealed that the isolates which obtained from the aborted sheep fetuses differ from that which obtained from aborted buffalo fetus in that they grew well without CO2 requirement and they were negative for H2S production, while the isolate obtained from the buffalo fetus required the CO2 to grow and was able to produce H2S, beside the other differences like the host preference and size. On the basis of biochemical results, the isolates from the sheep fetuses were diagnosed as Brucella melitensis while the isolate from the buffalo fetus was Brucella abortus. Only one case was positive by PCR and negative by culture and was from aborted horse fetus. Table 4. The results of PCR and culture in aborted animal's fetuses. Animal's Kind No. of Samples PCR Culture Identically % Pos. Neg. Pos. Neg. Sheep fetuses 2 2 - 2 - 100 Buffalo 2 1 1 1 1 100 Al-Bayatti and Amina Diyala Agricultural Sciences Journal , 1( 1 ) ; 1 – 17 ,2009 . 10 fetuses Horse fetus 1 1 - - 1 0 80% is the overall agreement between the PCR and Culture. Fig. 2. Fetal membranes stained with Ziehl-Neelsen stain (100X). Notice the round red clumps within the tissue cells. 65% 35% 0 20 40 60 80 100 (RBPT+ve) PCR ELISA Culture Al-Bayatti and Amina Diyala Agricultural Sciences Journal , 1( 1 ) ; 1 – 17 ,2009 . 11 Fig. 3. Chart illustrate the percentages of positive according to PCR, ELISA and culture. Fig. 4. Percentages of overall agreements between the PCR and each of ELISA and culture. DISCUSSION Brucellosis in animals is a serious economic disease. Losses due to abortions, stillbirths, irregular breeding, and loss of milk production are economic consequences, so accurate diagnosis is required to achieve success in disease control. There are probably more procedures to diagnose brucellosis in animals than any other disease. Yet, problem remains such as diagnosing incubative or latent infections and differentiating residual vicinal titers. As none of the available serological test conferred unequivocal specificity, new means of diagnosis depends on genetic tools were adopted. So in this study an evaluation of PCR-based assay capability in diagnosing animal brucellosis was undertaken. Twenty animal blood samples which were positive by RBPT selected for subsequent tests by ELISA, microbiological culture and PCR. They were subdivided into two groups. 11 vaccinated in which ELISA was positive in 6 of them in the percentage 54.5% and the other 9 not vaccinated group in which ELISA was positive in only one of them in the percentage 11.1%. Seven was the 73.33% 80% 56.66% 0 10 20 30 40 50 60 70 80 90 Animals blood /Aborted fetuses ELISA Culture Al-Bayatti and Amina Diyala Agricultural Sciences Journal , 1( 1 ) ; 1 – 17 ,2009 . 12 total number of positive by ELISA. Thus, its sensitivity was 35% and as none of the control group was positive; its specificity reached 100%. ELISA failed to detect 13 RBPT positive samples, 6 of them were also negative by PCR and culture, thus they are truly negative while 7 were positive by PCR which mean that they may be in the early stage of infection when IgG is under the level of detection by ELISA or it maybe false PCR results due to the high sensitivity of the test which lead it to detect the gene of the vaccine strain (there is no study on the vaccination of Brucella and its effects on the PCR results to compare the results with). Anyhow no single test appears to be free from demerits, this is the reason why many workers carried out comparative studies to determine the efficacy of different tests (Nielsen, 2002), and this is what was concluded by Gall and Nielsen, (2004) after reviewing various serological tests, (although they found that ELISA was more sensitive), besides Guarino et al., (2000) during their study on buffalo noticed that there were 5 samples negative by ELISA but positive by PCR. Also Gupta et al. ,(2006) in their study on goat found that PCR was able to detect 12 sample which was negative by ELISA. However, many others revealed that ELISA is of higher sensitivity among other serological tests (Kerby et al.,1997 ; OIE, 2004 ; Kanani, 2007 ; Tanmay,2007). PCR was able to detect 13 out of 20 RBPT positive animal cases, 6 of them were positive also by ELISA, while one of the ELISA positive was negative by PCR, which may be considered as false negative PCR result according to Navarro et al. ,(2004) who mentioned that if there are clinical and serological finding to support the presence of brucellosis in patients with negative blood culture and negative PCR should considered as false negative PCR and blood culture results, and this is were in the chronic granulomatous stage of infection when the DNA is under the threshold of detection (10-100fg) by the specific primers B4/B5. Al-Bayatti and Amina Diyala Agricultural Sciences Journal , 1( 1 ) ; 1 – 17 ,2009 . 13 PCR-based assay was more sensitive, 65% in comparison with ELISA and culture, while its specificity reached 100%. Other researchers also conclude that PCR was more sensitive in comparison with ELISA and other conventional serological tests (Guarino et al. ,2000 ; Gupta et al. ,2006 ; Amin et al., 2001 ; Kanani , 2007 ; Tanmay, 2007). Of all infected and control blood samples, none was positive by culture, this may be due to that most of them were vaccinated. Anyhow Guarino et al. (2000) also could not detect Brucella or any other organisms in buffalos blood of 44 samples which included in their study while they were able to detect 13 of them positive by PCR. Some researchers tried to overcome some of the PCR and the ELISA techniques limitations in diagnosis of brucellosis by the adoption of a combination between them (Morata et al. ,2003). Aborted fetuses due to brucellosis showed nonspecific lesions. In view of the considerable problems related to direct diagnosis of brucellosis in aborted fetuses, one of the main objectives of the present study was to evaluate the PCR using the same specific primers used in all the study as a tool for the detection of Brucella spp. in aborted fetuses. Taking the microbiological culture as a standard for comparison in this group, three samples were positive by PCR and culture, one was positive by PCR and negative by culture and one was negative by both PCR and culture, considering the one that was negative by culture is not a guarantee of the absence of Brucella infection. This makes PCR more sensitive than culture while the specificity was 100% and identical to culture. The results of the present study confirmed the results of Scarcelli et al., (2004) who analyzed samples obtained from 67 aborted bovine fetuses by means of bacteriological methods and PCR and also found that the samples that were positive by PCR (34/67) more than that of culture (26/67), so he concluded that Al-Bayatti and Amina Diyala Agricultural Sciences Journal , 1( 1 ) ; 1 – 17 ,2009 . 14 PCR was more sensitive than culture. Other researchers have observed the same divergences between PCR and culture results (Fekete et al.,1992; Cetinkaya et al.,1999 ; Marques et al. ,2001) while Leyla et al. ,(2003) who detected Brucella by PCR and culture from fetal stomach contents found 38 of 39 culture positive were also positive by PCR, which was negative in all of the culture negative samples. So PCR sensitivity and specificity were determined as 97.4 and 100% respectively. References Alton, G.G.,L.M. Jones and D.E. Pietz. 1975. Laboratory techniques in brucellosis 2 nd . Ed. Geneva. Monograph. Series No. 55. ch. 2 p. 113. Alton, G.G.,L.M. Jones, R.D. Angus and J.M. Verger. 1988. Techniques for the brucellosis laboratory. 1 st ed. INRA. Paris. Ch.2 p. 114. Amin, A.,M. Hamdy and A. Ibrahim. 2001. Detection of Brucella melitensis in semen using the polymeras chain reaction assay. Vet. Microbiol 83: 37- 44. Baily, G.G.,J.B. Krahn, B.S. Drasar and N.G. Stoker. 1992. Detection of Brucella melitensis and Brucella abortus by DNA amplification. J. Tropical Med. Hyg. (95) 4 : 271-275. Cetinkaya, B.,H. Ongor,A. Muz. 1999. Detection of Brucella species DNA in the stomach content of aborted sheep foetuses by PCR. J. Vet. Rec. 144: 239-240. CDC, CDC-PHEPR Biological Diseases/ Agents. 2003. CDC. ( http://www.cdc.gov/ oh/ohs/biosefty/imprtper. Htm,Internet). Fekete, A.,J. Bantle and S. Halling. 1992a . Detection of Brucella by polymerase chain reaction in bovine fetal and maternal tissue. J. Vet. Diagn. Invest. 4: 79-83. Gall, D. and K. Nielsen. 2004. Serological diagnosis of bovine brucellosis: a review of test performance and cost comparison. Rev. Sci. Tech. off. Int. Epiz. 23: 989-1002. Guarino, A.,L. Serpe, G. Fusco, A. Scaramuzzo and P. Gallo. 2000. Detection of Brucella species in buffalo whole blood by gene-specific PCR. Vet. Rec.. 147: 634-636. Gupta, V.,R. Kumari, D. Verma, K. Singh, S.V. Singh and V. Vihan. 2006. Detection of Brucella melitensis from goat tissue employing PCR. Indian. J. of Anim. Sci. 76: 793-795. Kanani, A. N. 2007. Serological, cultural and molecular detection of Brucella http://www.cdc.gov/ Al-Bayatti and Amina Diyala Agricultural Sciences Journal , 1( 1 ) ; 1 – 17 ,2009 . 15 infection in breeding bulls. A Ph. D. Thesis. College of Vet. Med. Anands agricultural Univ. Gujarat, India. Kerby, P. J.,J.L. Quiroga, J.J. McGrane and D.A. Stagg. 1997. Field evaluation of an indirect ELISA for detection of brucellosis in lowland Bolivia. Trop. Anim. Helth. Prod. 29: 65-72. Leyla, G.,G. Kadri and O. Umran. 2003. Comparison of polymerase chain reaction and bacteriological culture for the diagnosis of sheep brucellosis using aborted fetus samples. Vet. Microbiol. 93: 53-61. Marques, O.,C. Alves and L. Prestes. 2001. Detection of Brucella DNA from aborted bovine foetuses by polymerase chain reaction. Aust. Vet. J. (79) No. 7 p. 500-501. Morata, P.,M. Queipo-Ortuno,J. Reguera, M. Garcia-Ordonez, A. Cardenas and J. Colmenero. 2003. Development and Evaluation of a PCR-Enzyme- Linked Immunosorbent Assay for Diagnosis of Human Brucellosis. J. Clin. Micro. (41), No. 1 p: 144-148. Morgan, W.J. 1967. The Serological Diagnosis of Bovine Brucellosis. Vet. Rec. (80) 21: 621-620. Navarro, E.,M. Casao and J. Solera. 2004. Diagnosis of human brucellosis using PCR. Expert Reu. Mol. Diagn. 4(1) p. 115-123. Nicoletti, P. 1982. Diagnosis and Vaccination for the control of brucellosis in the Near East. FAO, Ani. Prod. and Hlth paper. Series No. 38, Rome. Nielsen, K. 2002. Diagnosis of brucellosis by serology. Vet. Microbiol. 90: 447- 459. OIE (World Organization for Animal Health) .2004. Bovine brucellosis, Section 2.3. In OIE Manual of standards for diagnostic tests and Vaccines, 5 th Ed. OIE, Paris. Scarcelli, E.,R. Piatti, M. Cardoso, S. Miyashiro, F. Campos,S. Teixeira, V. Castro and M. Genovez. 2004. Detection of bacterial agents by isolation and identification and multiplex PCR in aborted bovine fetuses. Revista Brasileira de Reproducao Animal 28: 23-27. Showman, A. 1986. The Biological Statistic book. Al-Mosul Univ. Press. Tanmay, P. 2007. Serological, Cultural and molecular detection of Brucella infection in bovine including quantification in milk by real time PCR, Msc. thesis College of Veterinary Science and Animal Husbandry. Anand Agricultural Univ. India. Al-Bayatti and Amina Diyala Agricultural Sciences Journal , 1( 1 ) ; 1 – 17 ,2009 . 16 تقييم كفاءة اختبارات PCR داء البروسيالتاالليزا والزرع الجرثومي في تشخيص, .في الحيوانات **آمنه نعمه الثويني* سحر احمد البياتي وزارة الزراعه/ الشركه العامه للبٌطره/ قسم المختبرات والبحوث البٌطرٌه * وزارة التعلٌم العالً والبحث العلمً / جامعة بغداد/ معهد الهندسة الوراثٌة والتقنٌات االحٌائٌه للدراسات العلٌا** الخالصة PCR( polymerase chainاستهدفت الدراسة الحالٌة تقٌٌم الفحص المعتمد على تقنٌة reaction) بالمقارنة مع كل من فحصً االلٌزا(enzyme linked immunosorbant assay ) .والزرع الجرثومً فً تشخٌص داء البروسٌال فً دم الحٌوانات واجنتها المجهضه RBPT+ve))Rose] موجبه لفحص الروزبنكال20 عٌنة دم لحٌوانات منها 30تم أستخدام Bengal Plate Test ]والثانٌه (11) والتً بدورها تم تقسٌمها الى مجموعتٌن األولى ملقحه بعدد PCRكل هذه العٌنات تم فحصها بالأللٌزا وال. سٌطرة سالبه للفحص المذكور10و (9)غٌرملقحه بعدد زوج قاعدي مشفرلمستضد البروسٌال ذو 223باستخدام زوج البادئات المخصص بمضاعفة جٌن بوزن وباستخدام نفس البادئات المشار PCRاجنة مجهضة اختبرت بالـ 5 كٌلودالتون كما تم أستخدام 31وزن . الٌها والزرع الجرثومً فقط تم تأكٌد األصابة بالبروسٌال فً عٌنات دم الحٌوانات العشرٌن والتً كانت موجبة لفحص حسب أختبار األلٌزا وهً بهذا تمثل نسب حساسٌة بلغت 7 وPCR حسب اختبار13 الروزبنكال فً كما ان . لم ٌتم الحصول على اي عزل جرثومً من هذه العٌنات. على التوال35ً% و %65 األختبارات الثالثة كانت سالبة فً جمٌع افراد مجموعة السٌطرة بهذا تكون الخصوصٌة لكل منهم و % 73.33 والزرع الجرثومً بلغت PCR واأللٌزا و PCR نسبة التوافق الكلٌة بٌن %100. . على التوال%56.66ً اربعة منها اظهرت نتٌجة موجبة لفحص , خمسة اجنة مجهضة تم شمولها فً هذه الدراسة PCRًواحدة كانت سالبة لكال االختبارٌن. وثالثة من هذه األربعة كانت اٌضا موجبة للزرع الجرثوم . على اساس نتائج % . 80 والزرع الجرثومً كانت األعلى وقد بلغت PCRنسبة التوافق الكلٌة بٌن Al-Bayatti and Amina Diyala Agricultural Sciences Journal , 1( 1 ) ; 1 – 17 ,2009 . 17 العزلتٌن اللتٌن تم الحصول علٌهما من اجنة األغنام المجهضة كانتا من نوع , األختبارات الباٌوكٌمٌائٌة Brucella melitensis بٌنما العزلة التً تم الحصول علٌها من جنٌن الجاموس كانت من نوع Brucella abortus . والمتمثله فً السرعه واالمان والحساسٌه والخصوصٌه PCR بلنظر للفوائد العدٌده لل االان قدرته على تمٌٌز الحٌوانات المصابه من الملقحه غٌر . العالٌتٌن نوصً باستخدامه فً التشخٌص . دقٌقه وتحتاج مزٌد من الدراسه