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Bovine and Caprine Brucellosis Detected by Milk Indirect ELISA and Milk Ring Test in Islamabad Capital Territory, Pakistan


Bovine and Caprine Brucellosis Detected by Milk Indirect ELISA and Milk Ring Test in Islamabad Capital Territory, Pakistan

Mudussar Nawaz1, Iahtasham Khan2, Muhammad Shakeell*, Arfan Yousaf1, Zahid Naseer1, Munibullah1, Ali Zohaib3, Riaz Hussain4 and Qudrat Ullah5

1Department of Clinical Studies, Faculty of Veterinary and Animal Sciences, Pir Mehr Ali Shah Arid Agriculture University, Rawalpindi, Pakistan

2Section of Epidemiology and Public Health, Department of Clinical Sciences, University of Veterinary and Animal Sciences, Lahore Sub-Campus Jhang, Pakistan

3Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China 

4University College of Veterinary and Animal Sciences, The Islamia Univeristy of Bahawalpur, Bahawalpur, Pakistan

5Department of Clinical Sciences, Faculty of Veterinary and Animal Sciences, Gomal University, Dera Ismail Khan, Pakistan


Brucella abortus and Brucella melitensis are important zoonotic pathogens. Brucellosis is an occupational hazard for veterinarians and animal handlers. The current work aimed to determine the prevalence of brucellosis in buffaloes and goats of Islamabad Capital Territory, Pakistan. A total of 341 milk samples from buffaloes (n=180) and goats (n=161) were screened by (Milk Ring Test) MRT and milk i-ELISA for anti-Brucella antibodies. Higher prevalence was found in buffaloes (5.6% and 16.1%) than in goat (4.97% and 1.9%) both through MRT and i-ELISA respectively. Commercial production and old age were important risk factors for spread of brucellosis both in goats and buffaloes. A history of abortion was significantly associated (P<0.05) with Brucella positive MRT and milk i-ELISA tests. In contrast, history of stillbirth had no significant association with anti-Brucella antibody titers. In conclusion, brucellosis is prevalent in Islamabad Capital Territory.

Article Information

Received 06 September 2019

Revised 11 December 2019

Accepted 27 December 2019

Available online 30 December 2020

Authors’ Contribution

IK, RH and AY conceived and designed the study. MN, MS and ZN collected samples, performed the experimental work and analyzed the data. AZ, MU, ZN, QU, and MS wrote and critically reviewed the manuscript.

Key words

MRT, Milk i-ELISA, Brucella, Abortion, Prevalence


* Corresponding author:

0030-9923/2021/0001-0391 $ 9.00/0

Copyright 2021 Zoological Society of Pakistan

Brucellosis is a highly contagious zoonotic disease caused by intracellular, facultative and Gram-negative bacteria of the genus Brucella (B.). Almost all species of animals can be infected including humans. B. abortus (mainly infecting bovines), B. melitensis (mainly infecting sheep and goats) and B. suis (mainly infecting pigs) have got great attention due to their potential zoonotic importance (Blasco and Molina-Flores, 2011; Godfroid et al., 2011). Brucella infection causes huge economic losses by decreased milk production, retention of fetal membranes, weak offspring, low fertility rate, metritis, arthritis, epididymitis, orchitis and abortion (Wadood et al., 2009; Nicoletti, 2010; Ali et al., 2017). Impact of brucellosis is more obvious in rural areas where sale of dairy products and production of livestock are the main sources of income and humans are more prone to infection due to direct contact with infected animals and their products. There are various predisposing factors that are directly linked with spread of the disease such as vaccination status, age of animals, parity number, insemination method or purpose of rearing (Aulakh et al., 2008; Abubakar et al., 2010; Ali et al., 2017; Khan et al., 2018).

Brucellosis can be diagnosed by different tests including Rose Bengal Plate Test (RBPT), Serum Agglutination Test (SAT), Complement Fixation Test (CFT) and Milk Ring Test (MRT) from suspected serum and milk samples (Alton et al., 1988; Samartino et al., 1999). Enzyme Linked Immunosorbent Assays (ELISAs) have also been standardized for serum and milk samples and are used for confirmation of clinical disease (Shafee et al., 2011; Abubakar et al., 2012). Polymerase Chain Reaction (PCR) is a latest technique used for detection of Brucella DNA in different species. MRT is an economical assay and easy to perform. False-positive results are reported from colostrum samples, samples from animals vaccinated in less than four months, animals with subclinical or clinical mastitis or soon after parturition (Nielsen, 2002; OIE, 2009). In the current study, MRT and milk i-ELISA tests were used for screening of herds. The aim of the present study was to determine the seroprevalence of brucellosis and identify different risk factors related to Brucella infection in buffaloes and goats in Islamabad Capital Territory (ICT).


Materials and methods

The present study was carried out in the peri-urban area of ICT, Pakistan during the period February-May, 2017. Islamabad lies in a hilly area having a great diversity of fauna and flora. The area is located in north-eastern Pakistan with an elevation of 575 m between the northern part of the Punjab and the western part of Azad Kashmir.

A total of 341 milk samples (5 ml) were collected from buffaloes (Nili-Ravi) and goats (Teddy, Beetal and crossbred) according to standard procedures (OIE, 2009). Initial milk streams were discarded before the actual milk samples were collected. The samples were transported to and stored at 4°C at Veterinary Research Laboratory, Pir Mehr Ali Shah Arid Agriculture University Rawalpindi. Samples were analyzed within 6 hours by Milk Ring Test (MRT). All screened animals were reported to be unvaccinated.

In the present study, parameters like education level and main occupation of the farmer, type of husbandry, vaccination and deworming status were also documented. The samples were taken during routine visits and the owners were informed and asked for participation in the study.

For Milk Ring Test the MRT antigen was purchased from Veterinary Research Institute (VRI), Lahore, Pakistan. Milk samples were analyzed as per manufacturer’s recommendations. Briefly, 30-50µl of homogenized MRT antigen was mixed with 1ml milk sample in a test tube at room temperature and was well shaken. This antigen-milk mixture was incubated at 37 °C for 1 hour and observed for presence of a violet/bluish colored ring over the milk fraction. Presence of ring indicated positive reaction of anti-Brucella antibodies.

For Milk Indirect ELISA the milk i-ELISA kit (ID Vet, France) was used for detection of anti-Brucella antibodies as per manufacturer’s instructions. Briefly, all milk samples were centrifuged at 5,000 g for 5 mins. A volume of 100 µl skimmed milk was collected under the cream and transferred to a well of the iELISA plate and incubated at 21 °C for 45 mins. After washing a (3×2) volume of 100 µl of conjugate was added and incubated again at 21 ºC for 30mins. After (3×2) washing 100 µl of substrate was added and incubated at 21ºC for 15mins. Finally, 100 µl of stop solution was added to each well and optical density was measured at 450 nm.



A total of 4.97% (8/161) goat samples and 5.6% (10/180) of buffalo milk samples were found positive by MRT. iELISA showed 1.9% (3/161) and 16.1% (29/180) positive results for goat and buffalo samples, respectively.


Table I. Comparative seroprevalence of MRT and milk iELISA in buffaloes and goats.

Animal species






4.97% (8/161)

1.9% (3/161)



5.6% (10/180)

16.1% (29/180)



5.27% (18/341)

9.38% (32/341)


History of abortion was found to be significantly associated with a seropositive to MRT and iELISA results (Table II). Still birth was not found significantly associated with positive results (Table III).


Table II. Association of abortion with brucellosis.







MRT +ve























P-value<0.05 was considered significantly associate; P-value <0.01 was considered highly significant; P-value >0.01 was consider non-significant.


Table III. Association of still birth with brucellosis.







MRT +ve

Normal birth











Stillbirth absent





0.221 NS






P-value <0.05 was considered significantly associate. P-value <0.01 was considered highly significant. P-value >0.01 was consider non-significant.


Age [Young stock (goats<6months and buffaloes<24months) compared to old stock (goats>6months and buffaloes>24months)] and purpose of rearing (domestic use or business purpose) of animals showed significant association with seropositivity by iELISA. Old stock was more prone to be seropositive than young stock. The animals kept for business purpose were more often seropositive as compared to the domestic purpose animals. Farm type (home vicinity and away from home) and unit of animals/farm (1-8, 9-15 and >15) did not show apparent influence on prevalence of anti-Brucella antibodies (Table IV).


Table IV. Relationship of different factors with positive results of the milk iELISA.



Positive samples




Young stock




Old stock


Farm type

House vicinity




Away from house


Animal unit

1 to 8




9 to 15




Rearing purpose








P-value<0.05 was considered significantly associated.



Milk ring test (MRT) and milk indirect ELISA are used to screen anti-Brucella antibodies in buffaloes and goat milk. Higher prevalence was reported for buffaloes compared to goats with both the tests. Milk iELISA was found to be highly sensitive for the diagnosis of bovine brucellosis (98.2%) (Kattar et al., 2007). Milk iELISA could be an effective screening test because it can detect lower titers of anti-Brucella antibodies especially during early infection (Guarino et al., 2001). A previous report describes similar prevalence of anti-Brucella antibodies in cattle and buffaloes using MRT and milk iELISA (Shafee et al., 2011).

Several studies reported that milk ELISA is more sensitive than MRT for the diagnosis of brucellosis in farm animals (Kerkhofs et al., 1990; Kang’ethe et al., 2000; Vanzini et al., 2001). Variation of sensitivity and specificity of serological tests may be influenced by external environment (season or temperature), disease endemicity, vaccination status of animal and herd and presence of any other gram-negative bacterium that might cross-react due to similar epitopes with Brucella (Greiner and Gardner, 2000; Mainar-Jaime et al., 2005; Matope et al., 2011; Khan et al., 2018).

Abortion and Brucella seropositivity showed significant association. It can be speculated that relevant number of abortions in buffaloes or goats were caused by brucellosis. Thus brucellosis is to be considered a threat for other animals as well as humans. Another study conducted in the same area also reported that abortion is risk factor for brucellosis. Relationship of stillbirth was recorded only in goats; however, there is no available data in buffaloes. Results showed non-significant association between brucella seropositivity and stillbirth in goats.

In the present study, age and rearing purpose of animals were found to be the main risk factors for brucellosis. Similar findings were recorded earlier where association of age with presence of brucella infection was observed (Mohammed et al., 2011; Ali et al., 2017; Mohamand et al., 2014; Khan et al., 2018). Higher seroprevalence of brucellosis in animals kept for commercial purposes might be due to frequent selling and buying events, exposure of purchased to infected animals in markets, no screening before sale or purchase time because of fear of business loss among people in peri-urban areas.



In conclusion, the overall prevalence of brucellosis was 4.97% and 1.9% in goats and 5.6% and 16.1% in buffaloes through MRT and i-ELISA respectively in Islamabad Capital Territory. Goats older than 6 months, buffaloes older than 2 years and animals kept for business purpose were at high risk for brucellosis.



Financial support from German Federal Foreign Office through Friedrich-Loeffler-Institute (FLI), Jena, Germany, via Project # je-0113, titled “Brucellosis in Pakistan” is highly appreciated.

Statement of conflict of interest

The authors have declared no conflict of interests regarding the publication of this article.


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