Prevalence and diagnostic test comparison of brucellosis in cattle in Pabna and Mymensingh districts of Bangladesh.
Brucellosis is a zoonotic disease caused by different species of the genus Brucella that are pathogenic for a wide variety of animals and humans. In animals, brucellosis mainly affects reproduction and fertility, reduces the survival of newborns and milk yield. Mortality in adult animals is insignificant (Sewell and Brocklesby, 1990). According to the Food and Agricultural Organization (FAO), the World Health Organization (WHO) and the World Organization of Animal Health (OIE), brucellosis is considered to be the most widespread zoonosis worldwide (Mustafa and Nicoletti, 1993).
Although it has been eradicated in many developed countries in Europe, Australia, Canada, Israel, Japan and New Zealand (Geering et al., 1995) in some other areas it has emerged as a major zoonotic disease in sheep and goats. The importance of brucellosis was primarily due to its public health significance and economic loss to the animal industry (WHO, 1971). Bangladesh has been reported as an endemic country for brucellosis because of a considerable number of human and animal populations are exposed to the infection each year.
Brucellosis in humans is caused by exposure to livestock and livestock products. Infection can result from direct contact with infected animals and can also be transmitted to consumers through raw milk and milk products. Brucellosis spreads between animals in a herd and the disease is a systemic infection that can involve many organs and tissues. Once the acute period of the disease is over, symptoms of brucellosis are mostly not pathognomonic, however, the organism can be located in the supramammary lymph nodes and mammary glands of 80% of infected animals. Thus they continue to secrete Brucella in their body fluids (Redkar et al., 2001).
Under the name Malta fever, the disease (now called brucellosis) first came to the attention of British medical officers in the 1850s in Malta during the Crimean war. The causal relationship between the organism and disease was first established in 1887 by Dr. David Bruce (Wilkinson and Lise, 1993). Brucellosis is endemic worldwide including Bangladesh (Das et al., 2008) but often a neglected disease.
It causes a great economic loss to the livestock industries through abortion, infertility, birth of weak and dead offspring, increased calving interval and reduction of milk yield (Rahman et al, 2006).
In Bangladesh it was first reported in cattle in 1967 (Mia and Islam, 1967), and in humans in 1983 (Rahman et al., 1983). Islam et al. (1983) estimated the annual economic loss in Bangladesh due to bovine brucellosis in indigenous cows as 720,000 EUR (total) and 12000 EUR per 1000 cross-bred cows and a total of 276000000 EUR in cross-bred cows. Recently brucellosis has been reported in cattle, buffalo, sheep, goat and pig in different regions of Bangladesh (Rahman et al., 2011a, 2011b; Rahman et al., 2010; Nahar and Ahmed, 2009; Uddin et al., 2007a, 2007b; Rahman et al., 2006; Amin et al., 2004). The diagnosis of brucellosis is confirmed by isolation of Brucella spp. by bacteriological culture or by the detection of an immune response by serological test to its antigens (Orduna et al., 2000). But the diagnosis of brucellosis based exclusively on Brucella isolation presents several drawbacks. The slow growth of Brucella may delay diagnosis for more than 7 days and also, the sensitivity is often low, ranging from 50 to 90% depending on disease stage, Brucella spp., culture medium, quantity of bacteria and culture technique employed (Gotuzzo et al., 1986). Hence, the serological tests are important for diagnosis of brucellosis.
Serological test like the rose Bengal test (RBT), slow agglutination test (SAT), mercaptoethanol test, enzyme linked immunosorbent assay (ELISA) and complement fixation test (Islam et al., 1983) are generally used for the detection of Brucella infection in animals. Enzyme linked immunosorbent assay (ELISA) has been evaluated for many years for the detection of serum antibody to Brucella in domestic animals. It has gained popularity over recent years as an alternative to other serological tests because it has several advantages compared with other tests such as, (a) it is direct method of identification of specific antibody, (b) it is more sensitive test than the slow aggluti-nation test, (c) the antibody enzyme conjugate employed has light chain reactivity and thus able to detect all classes of antibody. Despite having these advantages, there has been limited use of ELISA for the diagnosis of brucellosis in Bangladesh. Therefore, the present study was designed to diagnose brucellosis by adopting I-ELISA as well as RBT and SAT to detect antibodies to Brucella organism and to identify the risk factor and distribution of brucellosis in cattle in Pabna and Mymensingh districts of Bangladesh for prevention and control.
Materials and Methods
A total of 260 blood sera samples were collected from cattle of Pabna and Mymensingh districts of Bangladesh. Among cattle sera samples, 120 were collected from Bhangoora, Shordarpara, Kashipur and Gojatola of Pabna and 140 samples were collected from BAU Veterinary Clinic area, Sasmore, Sutiakhali and Digharkanda of Mymensingh during the period from May to December, 2011 (Table 1). Sampling was carried out as multistage sampling with the farm being selected first in the study area and then cattle randomly selected within each farm. The sampling frame within each farm was the list of all cattle on farm record. The sampling unit consisted of the animals selected from the list of all cattle within each farm using computer generated random numbers. The questionnaire based data on age, sex, breeding strategy, pregnancy status, area, history of abortion in cows were recorded.
Blood and sera samples collection. Animals were restrained with the help of the owner. Then the site of blood collection at the jugular furrow was soaked with iodine or alcohol. About 5-7 mL of blood was collected from the jugular vein of each cattle using a sterile disposable syringe and needle and was kept undisturbed on a tray for at least 1 h at room temperature in a slightly inclined position to facilitate clotting and separation of serum. After this period, the clotted blood samples with sera were transferred to a refrigerator and were kept overnight at 4[degrees]C. Then the blood samples with sera were centrifuged at 3000 rpm for 10 min. Later on, the sera were aliquated into sterilised labeled Eppendorf tube and stored at -20[degrees]C until used.
Serological study. The serological test for the diagnosis ofbrucellosis in cattle was performed by rose Bengal test (RBT), slow agglutination test (SAT) for screening and indirect enzyme linked immunosorbent assay (I-ELISA) for confirmatory diagnosis.
Rose Bengal plate test. Rose Bengal test (RBT) was performed according to the procedure as described by OIE (2004) which is being routinely used and described previously by Uddin et al. (2007a, 2007b) using B. abortus antigen (obtained from Dae Sung Microbiological Lab, South Korea). The serum samples and B. abortus antigen were kept 1 h at room temperature before starting the test. Thirty uL of each serum to be tested was placed on a glass plate circled approximately 2 cm in diameter. Then the vial of antigens was shacked gently and 30uL of antigen was put beside each of the sera. The antigens and the serum were mixed on the plate with a stirrer and spread over the entire area enclosed by the circle. Then the plate was placed on a mechanical rotator as 80-100 rpm for 4 min and the reading was noted immediately. Any agglutination or precipitation was considered as positive, whereas, no reaction (negative) indicated the absence of Brucella antigen in the sera.
Slow agglutination test (SAT). SAT was carried out with EDTA as described by Garin et al. (1985). The SAW (synbiotics, concentrated suspension of B. abortus, Weybridge, stain 99) antigen was diluted (1 mL antigen with 19 mL SAT buffer solution). The SAT buffer was prepared by adding 0.93 g EDTA (5 mM, Triplex[R]) to 500 mL PBS, where PBS was prepared by adding 5 tablets of PBS (Dulbecco-A, Oxoid, UK) to 500 mL distilled water. Briefly, the slow agglutination test was performed in flat bottom 96 well micro plates. At first for each test serum, a row of 3 wells of the 96 well micro plates was selected to make double dilution of the sera. 168 [micro]L of SAW buffer was pipetted in first well and 100 [micro]L in the 2nd well and 3rd well, respectively, of the micro plate. Then 32 [micro]L of serum was added in 1st well (dilution 1/6.25) after well mixing of the serum and PBS EDTA in the 1st well and 100 [micro]L was taken from this well and was placed in the second well (1/12.5). 100 [micro]L from the 2nd well was transferred into the 3rd well and finally 100 [micro]L of liquid in excess was discarded from 3rd well. Note that, all wells contained 100 [micro]L. Then in each well 100 [micro]L of standardized SAW antigen was added. This gives the serial serum dilution of 1/12.5, 1/25, 1/50. The plate was then incubated at 37[degrees]C for 24 h (+/-4 h) for reading. After 24 h, the agglutination reaction was observed by using a magnifying mirror against illumination source. Notably, for every group of samples tested, a positive control serum was included. Reading was taken on the basis of this protocol and the standardization was performed (75% agglutination of the OIEISS). The results were interpreted according to instruction of Veterinary Agrochemical Research Centre (Groeseleaberg 99, 1180 Brussels, Belgium).
Indirect enzyme linked immunosorbent assay (I-ELISA). All the samples found to be positive in RBT were further confirmed using I-ELISA. The assay was performed according to the protocol provided by the manufacturer's instructions (Svanova Biotech AB, art No.10-2700-10, SE-751 83 Uppsala, Sweden).
All reagents supplied by the manufacturer company were equilibrated to room temperature 18 to 25[degrees]C (64 to 77[degrees]F) before use. An amount of 100 [micro]L of sample dilution buffer was added to each well that would be used for serum samples and serum controls. After that 4 [micro]L of positive control serum (reagent A) and 4 [micro]L of negative control serum (reagent B) was added, respectively, to selected wells coated with B. abortus antigen. For confirmation purposes it was run the control sera in duplicates. The plate was shaken thoroughly and sealed then incubated at 37[degrees]C (98.6[degrees]F) for l h. The plate was rinsed 3 times with PBS-Tween buffer and filled up the wells at each rinse, emptied the plate and tapped thoroughly to remove all remains of the fluid. Then 100 [micro]L of HRP conjugate was added to each well and incubated at 37[degrees]C (98.6[degrees]F) for 1 h. The plate was rinsed again according to the previous way. Then 100 [micro]L substrate solution was added to each well and incubated for 10 min at room temperature 18 to 25[degrees]C (64 to 77 [degrees]F). Begin timing after the first well was filled. The reaction was stopped by adding 50 [micro]L of stop solution to each well and mixed thoroughly. The stop solution was added in the same order as the substrate solution was added. The optical density (OD) of the controls and samples was measured at 450 nm in a micro plate photometer. The OD was measured within 15 min after the addition of stop solution to prevent fluctuation in OD values. Any change in colour observed by naked eye indicated positive reaction.
Statistical analysis. The questionnaire-based data was processed in Microsoft Excel and analysed in SPSS. The z-test for proportions was used to compare the results between the serum tests. The z-test for proportions was done to find out the significant differences in the prevalence of Brucella based on the result of RBT in terms of age, sex, history of abortion, breeding strategies and study area.
Results and Discussion
A total of 260 sera samples from cattle were collected from Pabna and Mymensingh, 120 were collected from cattle of Pabna district and 140 from cattle of Mymensingh district (Table 1). The sera were tested by rose Bengal test (RBT), slow agglutination test (SAT) and indirect enzyme linked immunosorbent assay (I-ELISA) and the results are shown in Table 2 and 3. The overall prevalence of Brucella was found to be 4.23%, 3.07% and 2.31% by RBT, SAT and I-ELISA, respectively. It was shown that out of 260 cattle sera examined by RBT, 11 cattle sera showed positive reaction to RBT with a prevalence of 4.23%; 8 were positive to SAT with a prevalence of 3.07% and 6 to indirect ELISA with the prevalence of 2.31% (Table 2). The comparison of the serological test result revealed the highest prevalence in RBT than SAT and I-ELISA. But the prevalence of Brucella determined by RBT, SAT and I-ELISA did not differ significantly at 5% level of significance (P value = 0.456).
Out of 260 cattle, 110 were male and 150 were female. The prevalence of Brucella in female was 4.67%, 3.33% and 2.67% in RBT, SAT and I-ELISA, respectively, and in male 3.64%, 2.73% and 1.82% in RBT, SAT and ELISA, respectively. The prevalence of Brucella was higher in female than male (Table 3) which was not statistically significant (P value=0.683).
In the present study, higher prevalence of 5.0%, 4.17% and 3.33% by RBT, SAT and I-ELISA, respectively was reported in cattle of more than 4 years old in comparison to 3.75%, 2.5% and 1.25% in age group of [greater than or equal to] 2-4 years and 3.33%, 1.67% and 1.67% in the age group of < 2 years by RBT, SAT and I-ELISA, respectively (Table 3). The difference among different age groups of cattle was statistically insignificant (P value = 0.884).
A higher prevalence of Brucella was found in cattle with history of abortion that was 15%, 10% and 10% than without history of abortion that was 3.33%, 2.25% and 1.67% by RBT, SAT and I-ELISA, respectively (Table 3). There exists a statistically significant difference between prevalence of Brucella in cattle with history of abortion than without history of abortion (P value = 0.013).
In this study, the prevalence of Brucella in indigenous cattle was 4.67%, 3.33% and 2.67% in RBT, SAT and I-ELISA, respectively, while cross-bred cattle had a prevalence of 3.64%, 2.73% and 1.81% in RBT, SAT and I-ELISA, respectively (Table 3). This difference was not statistically significant (P value = 0.683).
In this study, the highest prevalence of Brucella in cattle was found in Pabna district especially in female 5%, 3.33% and 2.5% compared to the prevalence of 3.57%, 2.86%, 2.14% in Mymensingh district as detected by RBT, SAT and ELISA, respectively. The difference in prevalence of Brucella in cattle between the two areas was not statistically significant (P value=0.568).
The overall seroprevalence of Brucella in cattle was 2.3% which is similar with the reports of Amin et al. (2004) and Rahman et al. (2006) who reported that the prevalence was 2.33%, 2% and 2.4%, respectively. The prevalence is lower than the finding of Nahar and Ahmed (2009) who reported 4.5% prevalence. This variation in the prevalence may be due to variation in the age, breed, sex, pregnancy status of the animal, study area, hygienic condition, breeding techniques, herd size, reproductive diseases and diagnostic tests applied (Kebede et al., 2008).
The comparison of the serological test result revealed a higher prevalence in RBT when compared to SAT and I-ELISA. The RBT showed more positive reaction to Brucella as compared to SAT and I-ELISA. Higher sensitivity and specificity of RBT was also reported by Muktaderul et al. (2011) and Muma et al. (2007), while Chakraborty et al. (2000) reported lower sensitivity but higher specificity of RBT.
The prevalence of Brucella was higher in female than male. This finding was similar to the findings recorded by Sharma et al. (2003). This may be due to presence of allantoic factors including erythritol, possibly steroid hormones and other substances in the female reproductive tract, especially in the gravid uterus which stimulate the growth of most of the Brucellae (Radolf, 1994). In the present study, higher prevalence was reported in cattle of more than 4 years old than in age group of [greater than or equal to] 2-4 years and in the age group of < 2 years. This finding is coincided with the finding of Kazi et al. (2005). In contrast to the findings of the present study, Rahman et al. (2011a) reported the prevalence of brucellosis in the cows aged 2.5-4 years as 2 59%, while, in the cows over four years of age as 4 35%. Similarly, Amin et al. (2004) reported 2.3% and 4% prevalence in the < 4 and > 4 years age group, respectively. Age wise prevalence has also been studied by Abubakar et al. (2010) who showed that the incidence of brucellosis increased with age, and the incidence is high in sexually mature animals. The older animals are more susceptible to brucellosis due to more contact with infectious agents. Aged females suffering from malnutrition during pregnancy are more likely to be infected. Sergeant (1994) also found that there was no apparent association between age and serological status, or age and the prevalence. Ghani et al. (1998) and Uddin et al. (2007a, 2007b) stated that several factors such as age, sex, breed, location, herd size and living condition influence the seroprevalence of Brucella. It appears that the higher prevalence of brucellosis among older cows might be related to maturity with the advancing age. Thereby, the organism may have propagated to remain either as latent infection or it may cause clinical manifestation of the disease (Amin et al., 2005).
A higher prevalence of Brucella was found in cattle with history of abortion than without history of abortion. The present finding is in agreement with Rahman et al. (2006) who reported brucellosis to be higher in cattle with a history of abortion (15%) as compared to those with a history of returns to service (145%). In this study, the prevalence of Brucella in indigenous cattle was slightly more than cross-bred cattle. The difference between the two groups was not statistically significant. Akbarmeher and Ghiyamirad (2011) reported that there exists differences in the prevalence of brucellosis in different breeds, but not statistically significant. In this study, the highest prevalence of brucellosis in cattle was found in Pabna district especially in female compared to the prevalence of Brucella in Mymensingh district.
Definitive diagnosis of brucellosis can be accomplished only through the direct demonstration and identification of the causative agent(s) by culture and isolation procedures (Orduna et al., 2000). But culture requires level 3 biological safety cabinet as the chance of laboratory personnel to be infected is high. Due to the lacking of laboratory facilities causative agent could not be isolated by culture. Further study is required for definitive diagnosis of brucellosis by highly sophisticated techniques like culture, PCR etc.
Authors are grateful to Svanova Biotech AB, art No.10-2700-10, SE-751 83 Uppsala, Sweden and Dr. A.K.M.A. Rahman, Department of Medicine, BAU for supplying the ELISA and SAT kit, respectively.
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Md. Siddiqur Rahman (a)*, Md. Khairul Azad (a), Md. Shamim Ahasan (a), Roma Rani Sarker (a), Amitavo Chakrabartty (a), Laila Akter (b) and Mohammad Jasim Uddin (a)
(a) Department of Medicine, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh 2202, Bangladesh
(b) Department of Livestock Services (DLS), Framgate, Dhaka, Bangladesh
(received June 1, 2012; revised March 1, 2013; accepted March 21, 2013)
* Author for correspondence; E-mail: firstname.lastname@example.org
Table 1. Collection of serum samples from cattle in Pabna and Mymensingh districts Area/location No. of cattle samples Pabna district 120 Mymensingh district 140 Total 260 Table 2. Overall seroprevalence of brucellosis in cattle based on RBT, SAT and I-ELISA Total no. No. of positive of samples reactors collected and tested by RBT by SAT by I-ELISA 260 11 8 6 Total no. Percentage of Level of of samples positive reactors significance collected and tested by RBT by SAT by I-ELISA 260 4.23% 3.07% 2.31% NS NS = not significant at 5% level of significance (P value = 0.456). Table 3. Demographic factors related seroprevalence of brucellosis in cattle based on RBT, SAT and I-ELISA No. of sera No. and % of positive reactors samples collected and tested by RBT 95% CI by SAT Age < 2 years 60 2(3.33%) 1.21-7.87% 1(1.67%) [greater than 80 3(3.75%) 0.41-7.91% 2(2.5%) or equal to] 2-4 years > 4 years. 120 6 (5.0%) 1.1-8.9% 5(4.17%) Sex Male 110 4(3.64%) 0.14-7.14% 3(2.73%) Female 150 7(4.67%) 1.29-8.05% 5(3.33%) History of abortion Yes 20 3(15%) 0.65-30.65% 2(10%) No 240 8(3.33%) 1.06-5.6% 6(2.25%) Breeding Breed by AI 110 4(3.64%) 0.14-7.14% 3(2.73%) (Cross breed) Natural breeding 150 7(4.67%) 1.29-8.05% 5(3.33%) (Indigenous) Location/Area Pabna 120 6(5%) 1.1-8.9% 4(3.33%) Mymensingh 140 5(3.57%) 0.5-6.64% 4(2.86%) No. of sera No. and % of positive reactors samples collected and tested 95% CI by I-ELISA 95% CI Age < 2 years 1.57-4.91% 1(1.67%) 1.57-4.91% [greater than 0.92-5.92% 1(1.25%) 1.18-3.68% or equal to] 2-4 years > 4 years. 0.59-7.75% 4(3.33%) 0.12-6.54% Sex Male 0.32-5.78% 2(1.82%) 0.68-4.32% Female 0.46-6.2% 4(2.67%) 0.09-5.25% History of abortion Yes 3.15-23.15% 2(10%) 3.15-23.15% No 0.37-4.13% 4(1.67%) 0.05-3.29% Breeding Breed by AI 0.32-5.78% 2(1.81%) 0.68-4.31% (Cross breed) Natural breeding 0.48-6.2% 4(2.67%) 0.1-5.25% (Indigenous) Location/Area Pabna 0.12-6.54% 3(2.5%) 0.29-5.29% Mymensingh 0.2-5.62% 3(2.14%) 0.26-4.54% No. of sera #Level of samples significance collected and tested Age < 2 years NS [greater than or equal to] 2-4 years > 4 years. Sex Male NS Female History of abortion Yes * No Breeding Breed by AI NS (Cross breed) Natural breeding (Indigenous) Location/Area Pabna NS Mymensingh #Level of significance determined based on the results of RBT; NS = not significant; * = significant at 5% level of significance (P value=0.013); 95% CI = 95% confidence interval.
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|Author:||Rahman, Siddiqur; Azad, Khairul; Ahasan, Shamim; Sarker, Roma Rani; Chakrabartty, Amitavo; Akter, La|
|Publication:||Pakistan Journal of Scientific and Industrial Research Series B: Biological Sciences|
|Date:||Nov 1, 2013|
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