Comparison of Milk Ring Test; Serum Plate Agglutination Test and Polymerase Chain Reaction for the Detection of Bovine Brucellosis

Shamim Saleha^1*, Abdul Basit¹, Kashif Rahim¹, Muhammad Shahid², Mirza Ali Khan²

*Corresponding author: shamimsaleha@yahoo.com

ARTICLE CITATION: Saleha S, Basit A, Rahim K, Shahid M and Khan MA (2014). Comparison of milk ring test; serum plate agglutination test and polymerase chain reaction for the detection of bovine brucellosis. Res. j. vet. pract. 2(1): 5 – 8.
Received: 2013–11–10, Revised: 2013–12–01, Accepted: 2013–12–02
The electronic version of this article is the complete one and can be found online at (http://dx.doi.org/10.14737/journal.rjvp/2014/2.1.5.8) which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited

Abstract

In the following study, comparative efficacy of Milk Ring Test (MRT), Serum Plate Agglutination Test (SPAT) was determined by calculating the sensitivities and specificities for detection of Brucellosis in bovines from district Peshawar of Khyber PakhtunKhwa, Pakistan. Considering Polymerase Chain Reaction (PCR) as a gold standard diagnostic test, MRT showed very low sensitivity (4.8%), while its specificity was 90.9 %, likewise, SPAT also showed low sensitivity (41 %), while its specificity was 66.7% in bovines. Statistically, high specificities and low sensitivities of MRT and SPAT in bovines recommended the poor efficacy of these tests, when used individually in contrast to PCR. As PCR is more reliable test, while considering its sensitivity for antigen detection as compare to antibody detection, therefore, PCR may be used as a routine screening test in clinical practices in farms animals for detection of Brucellosis.

INTRODUCTION

Brucellosis is considered the most contagious and zoonotic bacterial infection of livestock worldwide (Munir et al., 2010). Brucella abortus and Brucella melitensis are the principal cause of brucellosis in bovines (Radostits et al., 2000; Karaca et al., 2007). These brucella species also cause brucellosis in humans (Gul et al., 2007). In animals, brucellosis frequently results in abortion, birth of weak calves, death of young stock, infertility in males and reduced milk yield in females (Maadi et al., 2011; Abubakar et al., 2011).

There is neither a single diagnostic test available by which a bacterium can be identified as Brucella nor any signs and symptoms of brucellosis are specific, therefore, a combination of cultural, serological and molecular methods is necessary for accurate diagnosis. However, all these methods have serious limitations (Poester et al., 2010; Abubakar et al., 2011). Accurate diagnosis of brucellosis requires Brucella isolation and detection in the laboratory, which is impractical for regular screening of large populations and could not be used as criteria for control and eradication of disease. Serological tests are relatively easy to perform and provide a practical advantage in detecting the prevalence of Brucella infection (Abubakar et al., 2011). The use of the Polymerase Chain Reaction (PCR) to identify Brucella DNA at genus, species and even biovar levels has becoming extended to improve diagnostic tests (Poester et al., 2010; Chimana et al., 2010).

In diagnostic laboratories, sera usually are screened with any simple serological test of high sensitivity. For milk samples, Brucella milk ring test is used for screening and monitoring the dairy herds at regular intervals (Chimana et al., 2010). Rarely, the clinical samples after initial screening by conventional methods are subjected to PCR for confirmation of disease (Abubakar et al., 2011).

In the agriculture based economy of Pakistan livestock accounts for 37.5 percent of agricultural value–addition and about 9.4% of its GDP. Brucellosis infection has a considerable impact on human and animal health as well as on socioeconomic factors where rural income relies mainly on land cultivation and domestic animals farming and people usually live in very close proximity with their livestock (Maadi et al., 2011; Shafee et al., 2011). People practice mixed crop–livestock farms where usually breed 90% buffaloes and 10% cattle, for dairy products and milk production (Afzal and Naqvi, 2004).

In Pakistan, the incidence of brucellosis is increasing day by day, particularly in large dairy herds. Several studies have reported the high incidence rates of brucellosis in livestock farms in government and private sector in different districts and provinces of Pakistan (Abubakar et al., 2010; Hamidullah et al., 2009; Iftikhar et al., 2008; Mukhtar and Kokab, 2008; Omer et al., 2010; Rabab et al., 2000; Shafee et al., 2011). Brucellosis can be controlled in Pakistan by routine screening of domestic livestock and animal vaccination programs (Abubakar et al., 2011). In Pakistan, veterinarians mostly rely on the conventional screening tests due to the lack of more specific diagnostic facilities and financial limitations. Rose Bengal Precipitation Test (RBPT), Serum Plate Agglutination Test (SPAT) and Milk Ring Test (MRT) are the most commonly performed tests at both government and private livestock laboratories in Pakistan (Asif et al., 2009; Gul et al., 2007).

This study was carried to evaluate Milk Ring Test, Serum Plate Agglutination Test and Polymerase chain reaction (PCR) for the detection of brucellosis in milk and serum samples of bovines, collected from private farms located in the district Peshawar of Khyber PakhtunKhwa region of Pakistan.

MATERIALS AND METHODS

In this study, 142 milk and blood samples from bovines were collected (70 cattle and 72 buffaloes) using the simple random sampling from different private farms in district Peshawar of Khyber PakhtunKhwa region in Pakistan.

Initial Screening Tests on Milk and Serum Samples of Bovines
The serum samples were subjected to SPAT for screening Brucella antibodies as described by (Alton et al., 1975). The results of agglutination were recorded. A titer of 1:80 or above was considered positive for brucella infection. Milk ring test was conducted on milk samples as described by (Alton et al., 1988). The positive samples were differentiated on the basis of blue ring present on the top of milk after overnight reaction.

DNA Extraction and PCR
DNA was extracted from same serum and milk samples by using DNA isolation kit (Shanghai ZJ Bio– Tech Co., Ltd. China), then PCR assay was performed in a total reaction volume of 50μl (Shanghai ZJ Bio– Tech Co., Ltd. China), according to supplier’s manual. This kit contains a reaction mix for the specific amplification of Brucella DNA. The amplification was performed in a DNA thermal cycler (Multi–gene Labnet international Inc.USA). Initial denaturation was carried out at 94 °C for 2 minutes, and then for 35 cycles the sample DNA was denatured at 93 °C for 15 seconds, annealed at 55°C for 30 seconds, and extended at 72°C for 30 seconds. The final incubation was done at 72 °C for 10 minutes. For positive controls, DNA extracted from Brucella abortus and Brucella melitensis strains, obtained from veterinary research institute, Peshawar was used. However, for negative control, distilled water was used. The PCR products were resolved and analyzed by using 1.5% of agarose gel electrophoresis and photographed on UV photo–documentation system (Multi–gene Labnet international Inc.USA) .The clear bands of Brucella species DNA were considered as positive results.

STATISTICAL ANALYSIS

Considering PCR as a gold standard diagnostic test, relative sensitivity, specificity, positive and negative predictive values of RBPT and STAT were calculated by using the MedCalc Software (Version 12.3.0).

RESULTS

The prevalence rate of Brucellosis detected in cattle and buffaloes in district Peshawar, using MRT, SPAT and PCR are given in Table 1. The overall prevalence rate detected by SPAT (38.7%) was greater than MRT (8.4%) when a total of 142 milk and serum samples of bovines were tested by MRT and SPAT. Moreover, serum samples were found more positive by PCR than milk samples. But PCR gave more positive results than MRT and less positive results than SPAT (Table 1).

It was also found that 0.7 % milk samples were positive to both MRT and PCR and 77.5 % milk samples were negative to both tests. Similarly, 27.5% serum samples were positive to both SPAT and PCR and 22.5 % serum samples were negative to both tests (Table 2).