Real Time- Polymerase Chain Reaction (RT-PCR) Test to Diagnose Brucellosis in Sheep in ANBAR Province

Sabaa mohammad nazoa, Bashar Sadeq Noomi¹,Ayad Hameed Ebraheem²

1-Department of Microbiology College of Veterinary Medicine, University of Tikrit.

2-Department of Anatomy and histology, College of Veterinary Medicine University of Tikrit.

DOI:

Received     accepted:

Abstract

The aims of the study were to determine incidence ratio of brucellosis in aborted ewes in Anbar governorate using real time PCR technique and bacterial culture and evaluating the efficiency of bacterial culture. 50 samples of stomach contents of an aborted fetus and vaginal swabs from aborted ewes were collected. The incidence ratio of brucellosis in ewes in Anbar governorate were 72% in PCR test However in bacterial culture were 46%. The positive prediction value and Negative prediction value and  Sensitivity and Specificity  of bacterial culture were (100%, 51.8 %,63%,100%) respectively when use Real time PCR as an assessment test. Conclusions: Anbar province is endemic with brucellosis, especially in sheep. The Real Time PCR is a fast, easy and accurate test in detecting even small numbers of bacteria in a sample, Significant superiority of polymerase chain reaction technology over bacterial culture.

Key words: Brucellosis in Anbar, ewes, Real time PCR, Bacterial culture.

Introduction

Brucellosis is one of zoonotic and infectious disease that spread all over the world especially in the Middle East and cause by facultative intracellular bacteria called Brucella (1). This bacterium is a coccobacillary in shape and Gram- negative, non-motile, non-spore forming, non-capsulated, aerobic This genus contains ten species incuding (Brucella.melitensis, Brucella abortus, Brucella suis, Brucella canis, Brucella ovis, Brucella neotomae, Brucella microti, Brucella ceti, Brucella pinnipedialis) (2) French agency for food, environmental and occupational health and safety (3). The disease can infect wide range of living organisms including human and animal (small ruminant, cattle, camels, dog, cat, equine, swine, aquatic creatures, reindeer, weeds) Especially those who are sexually mature (4, 5).  Huge economic losses cause by abortion of pregnant animals, low milk production, permanent and temporary sterility, Losses in calves and lambs breeding stations, as well as the cost of treatment and control and slaughter programs (6).

The most important signs of Brucellosis in adult female are abortion in the third trimester of pregnancy, especially in small ruminants, in addition to endometritis and placentitis and in male causing epididymitis or orchitis and Hygromas especially in ram and wither fistula in horse (7, 6). The disease is transmitted from animal to human through direct contact with infected animals, or by eating food contaminated with bacteria, most cases occur by ingesting unpasteurized milk or contaminated products (8). The diagnosis of Brucellosis depends on two types of tests: first, direct tests: which reveal the presence of the pathogen and indirect tests: which detect the presence of immune responses (9,10). Indirect serological tests have many disadvantages, including obtaining false positive results (11). Therefore, the polymerase chain reaction and bacterial culture were used for the purpose of accurate isolation. Bacterial isolation is the most confirmatory of the diagnosis, because it reveals the presence of the pathogen in the very early stages of the disease, so it is the gold standard for diagnosis (12). Brucella colonies appear on Brucella media after 4 to 7 days of incubation at a temperature of 37 degrees and are in the form of white to pale round pearly colonies (13). One of the disadvantages of this method is that it takes a long time (14). Polymerase Chain reaction (PCR)It is an easy method to replicate or create copies of the DNA sequence using enzymes or primers outside the body (15). The main purpose of using this technique is to make rapid copies of millions to billions copies of a specific and specific part of the DNA, this allows scientists to take a very small sample of DNA and amplify it into a large amount enough to study (16).

Material and Methods

Sample collection: Abomasa contents of aborted fetuses as well as vaginal swabs collected from 50 aborted ewes in in Anbar province.

 Bacterial culture:Brucella Agar base (HIMEDIA- Indian) and Selective Supplement were used and add 5% sterile serum for bovine or horses also to media. Use The culture loop for cultured stomach contents, while the vaginal swabs were cultured by Planning the swab on the surface of the petri dish. Each sample was cultured on two petri dish of Brucella culture Agar, the first dish was incubated aerobically (17). While the second dish was incubated anaerobically in the presence of 5% of CO₂ (Carbon dioxide) using aerobic conditions (jar candle) and the dishes were Incubate For a period of less than 7 days (17).

Real Time- Polymerase Chain Reaction (RT-PCR): The Kit Extraction DNA™ gSYNC used genomic DNA extraction Manufactured by Biotech Geneaid.( use by the manufacturer's instructions), used a v1 Detection Kit Brucella from the Bosphorus company Turkish bosphore and contain: Mix Master PCR( DNA Poymerase ,PCR Buufer,Mix of dNTP) ,Interna Control ,Postive control,DH₂O.and use by manufacturer's instructions ( The stage of preparing the reaction mixture: making sure that all components of the kit are melted before use and the mixture was prepared using the materials shown in the following table (1):

Figure 1: Aborted ewe fetus

Table (1): material that use polymerase chain reaction and his quantities

Table (2): Primer used

Table (3): Programming the Real time PCR instrument

Result and Discussion

Bacterial Culture

The results of bacterial culture of samples from aborted fetuses and vaginal swabs from 50 aborted ewes showed that Brucella bacteria were isolated from 23 samples with an isolation rate of 46%. When the polymerase chain reaction test was performed on 50 different samples of stomach contents of an aborted fetus or vaginal swabs of aborted ewes using Real Time PCR device, positive results were obtained by 72% (36 sample from 50). All isolates, when culture on Brucella agar after 5-7 days of aerobic and anaerobic incubation at 37 °C, gave colonies with smooth appearance, convex, pale to pearly white color colonies. The result reached by this study Close to the result obtained   by Al-abdali , Were was the result 40% in sheep using bacterial isolation (18). Also, the percentage obtained from this study was close to the percentage reached by (19), which is 42% in sheep and goats using bacterial isolation in Iraq. While the percentage obtained increased from the percentage recorded by (20) Which reach 31% when bacterial isolation from the contents of aborted sheep embryos was used. And also, about the percentage recorded by )21(, which is 33%, where bacterial isolation was used from the contents of aborted fetuses of sheep in Iraq (Mosul). Real time Polymerase Chain reaction (RT-PCR), Since there was no previous study in Anbar province, Real Time PCR was adopted to diagnose Brucellosis, so the results of this study were compared with other regions, where some research gave results that reached 100% when using this test to detect Brucella in aborted sheep and goats (22). (23) indicated that the detection rate of Brucella bacteria using the polymerase chain reaction was 57.1%. The percentage of isolation of Brucella by using the polymerase chain reaction was 66.6% in sheep (24, 25) found that the rate of Brucellosis isolate was 48.1% when using the polymerase chain reaction technique to diagnose brucellosis among sheep and goat abortions in Jordan. The difference in the percentage obtained in the study from the percentages recorded in other studies is due to the geographical distribution, the different places, the methods of control and public awareness in the places where the studies were carried out, as well as the fact that the samples were taken at different times, which is not a condition in conjunction with the occurrence of abortion.

Finding of present study showed that 72% of the samples gave positive results for the detection of Brucellosis by using the Real time PCR Technique in Anbar province, and it is normal to obtain such results because when the samples were taken, it was found that there was a great complaint by the owners about repeated abortions, especially for the herds owners in large numbers. It was also noticed that the breeders were ignorant of the way to get rid of aborted fetuses, as they do not follow healthy methods to get rid of aborted fetuses, as well as the absence of health awareness in the animal trade, especially rams, where most owners export rams to other areas without conducting tests on them. It also shows the absence of control programs, as it was found from the history of the case that most of the herds are not vaccinated against Brucella. All these reasons would increase the risk of disease spreading among the herd easily.

 Compare between bacterial culture and RT-PCR. When comparing the results of bacterial with the results of the PCR, it was found that 36 positive samples for the PCR test were 23 positive samples for bacterial isolation and 13 negative samples for bacterial isolation. While the 14 negative cases of PCR, none of them were positive for bacterial isolation, and all of them were Negative for bacterial isolation. This difference may be due when using the polymerase chain reaction as an assessment tool is that these bacteria are opportunistic and may also be intracellular. Also, the polymerase device detects very small quantities of bacteria and also dead that the bacterial culture fails to detect.

positive prediction value = 23 / 23+0 = 100% 

Negative prediction value = 14 / 14+13 = 51.8 %

Sensitivity = 23 / 23 + 13 = 63%

Specificity = 14 /14 + 0 = 100%

Figure, 2: Colony of Brucella on Brucella agar showing the pearly white colony(

Real time Polymerase Chain reaction (RT-PCR)

Figure, 3: Real-time polymerization reaction curves for Brucella genus amplification (Sample that Cross the threshold in FAM are Shown with Ct are POSTIVE and sample that not break the threshold are shown as No C. these sample are considered negative)

(^o= Positive Control).

Table 4. Number of positive samples.

 Table (5) shows the analysis of these results

References 

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استخدام اختبار تفاعل البلمرة المتسلسل فی الوقت الحقیقی لتشخیص الحمى المالطیة فی الأغنام فی محافظة الأنبار

سبأ محمد نزوع‘بشار صادق نومی ¹ ‘أیاد حامد ابراهیم²

1-فرع الأحیاء المجهریة الدقیقة کلیة البیطریة الطب، جامعة تکریت.

2-فرع التشریح والأنسجة کلیة الطب البیطری جامعة تکریت.

الخلاصة

هدفت الدراسة إلى تحدید نسبة الإصابة بمرض البروسیلا فی النعاج المجهضة فی محافظة الأنبار بتقنیة تفاعل البلمرة المتسلسل فی الوقت الحقیقی (RT PCR) والزرع البکتیری وتقییم کفاءة الاستزراع البکتیری عنداستخدام تفاعل البلمرة المتسلسل فی الوقت الحقیقی (RTPCR) کاختبار تقییم.  تم جمع محتویات المعدة من الاجنة المجهضة والمسحات المهبلیة من 50 نعجة مجهضة. وکانت نسبة الإصابة بمرض البروسیلا فی النعاج فی محافظة الأنبار 72٪ فی اختبار PCR و46٪ فی الزرع البکتیری.کانت قیمة التنبؤ الإیجابی وقیمة التنبؤ السلبی وحساسیة وخصوصیة الزرع البکتیری (100٪ ، 51.8٪ ، 63٪ ، 100٪) على التوالی .الاستنتاجات: محافظة الأنبار متوطنة فی داء البروسیلا ، خاصة فی الأغنام ، اختبار تفاعل البلمرة المتسلسل (RTPCR) هو اختبار سریع وسهل ودقیق فی الکشف حتى عن أعداد صغیرة من البکتیریا فی العینة ، تفوق کبیر لتکنولوجیا تفاعل البلمرة المتسلسل على  تقنیة الزرع البکتیری .

الکلمات المفتاحیة: داء البروسیلا فی الأنبار ، النعاج ، تفاعل البلمرة  المتسلسل فی الوقت الحقیقی ، الزرع البکتیری .