DIAGNOSIS AND EPIDEMIOLOGY OF BRUCLLOSIS IN CATTLE OF PABNA
& MYMENSINGH DISTRICTS OF BANGLADESH
Md. Siddiqur Rahman, Md. Khairul Azad, Md. Shamim Ahasan, Amitavo
Chakrabartty, Laila Akther
Department of Medicine, Faculty of Veterinary Science, Bangladesh Agricultural University, Mymensingh
2202, Bangladesh
ABSTRACT
Brucellosis is a zoonotic disease caused by different species of the genus Brucella that are pathogenic for a
wide variety of animals and human beings and it is an economically important disease in Bangladesh. In
Bangladesh, approximately 80% of people live in villages, and rural income is largely dependent on
livestock; the people are in close contact with livestock on a daily basis. 6.5% of national income and 3.5%
gross domestic product (GDP) come from livestock. There are an estimated 23.4 million cattle, 1.86 million
buffalo, 33.5 million goats, 1.1 million sheep in Bangladesh. Diseases are one of the major constraints of
livestock development in Bangladesh and after tuberculosis, brucellosis is the most important bacterial
disease of livestock in Bangladesh. This study was undertaken to study the diagnosis and epidemiology of
brucellosis in cattle of Pabna & Mymensingh districts in Bangladesh. A total of 260 cattle sera samples were
collected. Among the sera samples 120 sera samples were collected from Pabna & 140 sera samples were
collected from Mymensingh. The epidemiological data were collected by questionnaire; RBT & SAT were
used as a screening test & I–ELISA as confirmatory test. The overall 2.3% seroprevalence of brucellosis was
found in this study. In Pabna the prevalence of brucellosis was 2.5% and in Mymensingh the prevalence of
brucellosis was 2.14%. In female the prevalence of brucellosis was higher (2.67%) compared to the
prevalence (1.8%) of brucellosis in male. It is observed that, a higher prevalence of Brucella was found in
female than male, natural breeding than artificial one and aged animal than young, also in aborted animal
compared with non aborted animal. The result showed that female animal had more possibilities to get
infection of Brucella than cow served with artificial incrimination. Necessary protection should be needed as
it is a zoonotic disease. Further studies for isolation, identification and typing of Brucella are recommended.
INTRODUCTION
Brucellosis is a zoonotic disease caused by different species of the genus Brucella that are pathogenic for a
wide variety of animals and human beings (Mathur, 1971). In animals, the brucellosis mainly affects
reproduction and fertility, reduces the survival of newborns, and reduce milk yield. Mortality of adult
animals is insignificant (Sewell & 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 the most widespread zoonosis worldwide (Mustafa & Nicoletti, 1995).
The first description of an outbreak of undulant fever caused by B. abortus involved college students who
drank raw cow milk in the dormitory (Hugh-Jones, 2000). Brucellosis has been an emerging disease since
the discovery of B. melitensis as the cause of Malta fever in the spleen of a fatal human case on the island of
Malta in 1886 and isolated by David Bruce one year later in 1887 and B. abortus isolated from the aborted
cattle by Bernard Bang, in 1897 (Nielsen and Duncan, 1990; Hugh-Jones, 2000; Rahman, 2004).
Brucellosis in human beings is caused by the 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, and the organism can be chronically located in the supramammary lymphatic
nodes and mammary glands of 80% of infected animals. Thus they continue to secrete the Brucella organism
in their body fluids (Cordes and Carter, 1979; Redkar et al., 2001).
The higher prevalence of brucellosis in cows of better managed farms and estimated of human brucellosis as
12.8% in herders and agricultural workers and 21.6% in goat farmers (Rahman et al., 1983). The
seroprevalence of brucellosis in cattle was 2.4%-18.4% whiles the herd-level seroprevalence in cattle was
62.5% in Bangladesh (Rahman et al., 2006).The seroprevalence of brucellosis was 4.5% in cattle and 6% in
human. (Azimun et, al., 2007). The seroprevalence of brucellosis was 5% in cattle (Rahman et al., 2010).
Enzyme Linked Immunosorbent Assay (ELISA) has been evaluated for many years for the detection of
serum antibody to brucellosis in domestic animals. It has gained popularity over recent years as an
alternative to other serological tests. ELISA for diagnosis of brucellosis has several advantages compared
with other tests.
1) It is a direct method of identification of specific antibody and therefore has little chances of obtaining
false positive reactions.
2) ELISA is more sensitive test than the agglutination test and thus has the potential to detect infected
animals.
3) The antibody enzyme conjugate employed has light chain reactivity and thus is able to detect all classes of
antibody. A combined determination of all classes of antibody allows accurate serological diagnosis at any
stages o disease.
4) ELISA results provide an epidemiological tool for best of knowledge, an ELISA for the diagnosis of
brucellosis materials are not practicing.
Therefore, the present study was designed to adopt on ELISA for the detection of antibodies to Brucella in
Pabna and Mymensingh district of Bangladesh with the following objectives.
I. Detection of brucellosis in cattle by I-ELISA in Pabna and Mymensingh district of Bangladesh.
II.
To identify the risk factor & distribution of brucellosis in cattle in Pabna and Mymensingh
district of Bangladesh.
MATERIALS AND METHODS
The study was conducted in the Department of Medicine, Faculty of Veterinary Science, Bangladesh
Agricultural University, Mymensingh.
Sources of samplesA total of 260 blood sera samples were collected from cattle of Pabna and Mymensingh. Among cattle sera
samples, 120 were collected from Pabna, and 140 samples were collected from Mymensingh. In Pabna the
samples were collected from Upazila livestock office Bhangoora, Shordarpara, Kashipur and Gojatola & in
Mymensingh the samples were collected from BAU Vet. Clinic, Sasmore, Sutiakhali and Digharkanda.The
study recorded some clinical, epidemiological and reproductive information. The questionnaire based data
on age, gender, breed, area, client’s complaint, pregnancy status, types of housing and breeding, number of
animals in herds, disease history, reproductive problems such as abnormal uterine discharge, abortion or
previous abortion, repeat breeding in cows and reproductive diseases in bulls were recorded.
After collection of blood samples, all the blood samples were processed for sera preparation and were tested
with Rose Bengal Test (RBT) and Slow Agglutination test for screening and Indirect Enzyme Linked
Immunosorbent Assay (I-ELISA) was used for confirmatory diagnosis.
Blood and sera samples collection
With the help of the owner and the attendant at first the animal were controlled. Then the site of blood
collection at jugular furrow was soaked with tincture of iodine or alcohol. About 4-7 ml of blood was
collected from jugular vein of each cattle with the help of sterile disposable syringe and needle and was kept
undisturbed on a tray for at least 30 minute at room temperature in a slightly inclined position to facilitate
clotting and separation of serum. After this period, the clotted blood samples with sera are transferred to
refrigerator at 40C and kept overnight. Then the blood samples with sera were centrifuged at 3000 rpm for 10
min. After centrifugation a clear sera were found. Later on, the sera were aliquated into sterilized labeled
eppendorf tube and stored at -20ºc for until used.
Serological study
The serological test for the diagnosis of brucellosis in cattle was performed by Rose Bengal Test (RBT),
Slow Agglutination Test (screening test), and indirect Enzyme Linked Immunosorbent Assay (i-ELISA) for
confirmatory diagnosis.
RESULTS
A total of 260 sera samples from cattle were collected from Pabna and Mymensingh. The sera was tested by
Rose Bengal Test, Slow Agglutination Test & Indirect Enzyme Linked Immunosorbent Assay (I- ELISA)
and the results has shown in Table 1, 2, 3, 4, and 9.
Table 1. Overall seroprevalence of brucellosis in cattle
Total number of sera samples collected and
tested
Total number of
positive cases
Percentage of positive cases
260
6
(2.31%)
The overall seroprevalence of brucellosis in cattle has been shown in table 1. The overall seroprevalence was
found 2.31% in the cattle of Pabna and Mymensingh district on the basis of I-ELISA.
Table 2. Rose Bengal Test (RBT) result in cattle
Total number of sera
samples collected and tested
Number of positive
reactor by RBT
Number of negative
reactor by RBT
Percentage of positive
cases by RBT
260
11
249
(4.23%)
The seropositive rate of brucellosis by RBT has been presented in Table 2 and it was shown that out of 260
cattle were 11 positive to RBT with a prevalence of 4.23%.
Table 3. Slow Agglutination Test (SAT) result in cattle
Total number of sera
samples collected and tested
Number of positive
reactor by SAT
Number of negative
reactor by SAT
Percentage of positive
cases by SAT
260
8
252
(3.07%)
The seropositive rate of brucellosis by SAT has been presented in Table 3 and it was shown that 8 (out of
260) cattle were sensitive to SAT with a prevalence of 3.07%.
Table 4. Seropositive result in cattle by I-ELISA
Total number of sera
samples
collected and tested
Number of positive
reactor by
I- ELISA
Number of negative
reactor by
IELISA
Percentage of positive
reactor
by I- ELISA
260
6
254
(2.31%)
The seropositive result in cattle by I- ELISA has been shown in table 3 and 6 out of 260 cattle sera were
shown positive to indirect ELISA and the prevalence was 2.31%.
Table 5. Comparison of the serological test result
Total number of sera
samples tested
Total number and % of positive reactor
RBT
SAT
I- ELISA
260
11 (4.23%)
8 (3.07%)
6 (2.31%)
The comparison of the serological test result has been shown in Table 5 and the highest prevalence was
found in RBT in cattle and it was 4.23%. RBT appeared more sensitive compared to SAT and I-ELISA.
Table 6. Age related seroprevalence of brucellosis based on RBT, SAT, & I-ELISA
Species
Age of animals
Cattle
< 2 years
≥ 2-4 years
> 4 years.
Number of sera samples
collected and tested
60
80
120
Number and % of positive reactor
RBT
SAT
I-ELISA
2(3.33%)
1(1.67%)
1(1.67%)
3(3.75%)
2(2.5%)
1(1.25%)
6 (5.0%)
5(4.17%)
4(3.33%)
Age related seroprevalence of brucellosis based on RBT, SAT and I-ELISA was shown in Table 5. The
higher prevalence was seen in cattle aged over 4 years.
Table 7. Sex related seroprevalence of brucellosis based on RBT, SAT & I- ELISA in cattle
Number and % of positive reactor
Number of sera samples collected and
tested
RBT
SAT
I- ELISA
Male
110
4(3.64%)
3(2.73%)
2(1.82%)
Female
150
7(4.67%)
5(3.33%)
4(2.67%)
Sex related seroprevalence of brucellosis based on RBT, SAT and I- ELISA in cattle shown in Table 6. The
Sex of animal
higher prevalence was seen in female than male.
Table 8. Abortion related seroprevalence of brucellosis based on RBT, SAT and. I-ELISA in cattle
Number of sera samples
collected and tested
History of abortion
20
No previous abortion.
240
** = Significant at 1% level of probability (p<0.01)
Types of condition
Number and % of positive reactor
RBT
SAT
I- ELISA
3(15%)
2(10%)
2(10%)
8(3.33%)
6(2.25%)
4(1.67%)
Abortion related seroprevalence of brucellosis based on SAT and I- ELISA in cattle shown in Table 8. The
higher prevalence was seen in aborted cattle compared with non abortus cattle.
Table 9. Breed related seroprevalence of brucellosis based on RBT, SAT and I-ELISA in cattle
Number and % of positive reactor
RBT
SAT
I- ELISA
Types of condition
Number of sera samples
collected and tested
Breed by AI(cross breed)
110
4(3.64%)
3(2.73%)
2(1.81%)
Natural breeding
150
7(4.67%)
5(3.33%)
4(2.67%)
Breeding related seroprevalence of brucellosis based on RBT, SAT and I-ELISA in cattle shown in Table 9
and more positive case was found in cattle bred by natural breeding than artificial insemination.
Table 10. Locations related seroprevalence of brucellosis based on RBT, SAT and I- ELISA in cattle
Location
Number of sera samples collected
and tested
Pabna
Mymensingh
120
140
Number and % of positive reactor
RBT
SAT
I- ELISA
6(5%)
4(3.33%)
3(2.5%)
5(3.57%)
4(2.86%)
3(2.14%)
Area related seroprevalence of brucellosis based on RBT, SAT and I- ELISA has been shown in Table 10.
The higher prevalence was seen in Pabna compared to Mymensingh.
DISCUSSION
Brucellosis is an important zoonotic disease and serological surveillance is essential to know the status of
the disease in Bangladesh. Many countries have eradicated Brucella abortus from cattle. Each year half a
million cases of brucellosis are reported worldwide but according to WHO, these numbers greatly
underestimate the true prevalence. The importance of brucellosis is not known precisely, but it can have a
considerable impact on human and animal health, as well as on socioeconomic impacts, especially in which
rural income relies largely on livestock breeding and dairy products (Islam et al., 1983). The objective of
the study were optimized of RBT, SAT and ELISA to detect Brucella antibody at Pabna and Mymensingh
district.
A total of 260 cattle sera samples were collected to study serodiagnoses of brucellosis in cattle. The
overall seroprevalence of brucellosis in cattle was 2.3% which is similar compare to the seroprevalence of
brucellosis, 2.33% (300) reported by (Amin, 2003) and 2% (250) reported by (Amin et al., 2004). But this
finding is in with Rahman et al., (2006) who reported animal-level seroprevalence of brucellosis
in cattle is 2.4%-18.4% while the herd-level seroprevalence in cattle is 62.5%.
Cattle aged more than 4 years of age had insignificantly higher prevalence (3.33%) than that aged
below 4 years. The findings did not correlate with the observation of Sarumath et al., (2003).Amin, 2003
and Amin et al., 2004). It may be considered that the higher prevalence of brucellosis among older cattle
might be due to maturity with the advance age. However, the older animals supposed to be more infected,
because of more contact with infectious agents and sometimes from malnutrition during pregnancy. But
there was no significant association statically between age group and the prevalence when sera sample
tested with SAT and I-ELISA. Sergeant (1994) also found that there was no apperent association
between age serological status, or age and the prevalence. But Ghani et al., (1998) stated that several
epidemiological factors, such as age , sex, breed, location, herd size and living condition can influence
the sero prevalence of brucellosis.
The result of RBT shown that the prevalence of brucellosis in cattle was higher in female (4.67%).
Similarly results of I-ELISA showed the higher prevelance in female 2.67% than male 1.82%.This finding
was similar to the findings recorded by Sharma et al., (2003).
The prevalence of brucellosis in cattle bred by natural breeding was (4.67%) by RBT, (3.33%) by SAT and
(2.67%) by I-ELISA and the prevalence was (3.64%) by RBT, (2.73%) by SAT and (1.81%) by I-ELISA in
case of artificial insemination. The findings of present investigation corresponded with the findings of
Sarumathi et al., (2003).
In this study, the highest prevalence of brucellosis in cattle was found in Pabna district especially in female
4.7%, 3.33% and 2.67% as detected by RBT, SAT and ELISA compared to the prevalence of 3.57%, 2.86%,
2.14% as detected by RBT, SAT and ELISA at Mymensingh. During this study some clotted blood were
seen in some serum sample, but it did not influence the test result.
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