J. Commun. Dis. 44(2) 2012 : 91-95
Sero-prevalence of Brucellosis in Occupationally Exposed Human beings of Himachal Pradesh (India) Shalmali*, Panda Ashok Kumar*, Chahota Rajesh** (Received for publication April 2011)
Abstract The chief objective of respective study was to investigate the seroprevalence of brucellosis among occupationally exposed human beings in Himachal Pradesh. A total of 165 serum samples that were obtained from human beings from various regions of the state were screened through a battery of serological tests which included RBPT, STAT, 2-MET, dot-ELISA and indirect-ELISA. 165 of human sera samples included 42 from veterinarians, 40 shepherds, 35 livestock owners, 20 workers at veterinary hospitals/clinics, 16 abattoir workers and 12 veterinary pharmacists. The overall seroprevalence of brucellosis among occupationally exposed human beings was observed to be 6.66% showing highest in abattoir workers (18.75%) followed by pharmacists (8.33%), veterinarians (7.14%), and livestock owners (5.71%) and shepherds (5.00%). In humans it is prevalent as an occult infection or under diagnosed disease, especially; in case of abattoir workers the highest seropositivity for brucella agglutinins was observed. IndirectELISA and Dot-ELISA proved best in the diagnosis of brucellosis. Keywords: Brucellosis, serodiagnosis of Brucella agglutinins, occupationally exposed persons, Himachal Pradesh.
* Dept of Veterinary Public Health and Epidemiology, ** Department of Veterinary Microbiology Dr. G C Negi College of Veterinary and Animal Sciences (DGCN-COVAS), Chaudhary Sarvan Kumar Himachal Pradesh Krishi Vishvavidyalaya-CSKHPKV, Palampur-176062 Himachal Pradesh. Correspondence to: Dr. Shalmali, D/o Dr. Chander Paul Kashyap, Ward-8, Ghuggar Haar, Palampur176061, Distt-Kangra (H.P.) Phone: 09418421247, E-mail:
[email protected]
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INTRODUCTION Brucellosis is a major zoonotic disease that still is one of major Veterinary, Public Health and Economic concerns globally. It is an important infectious disease, which remains under diagnosed in India. The disease is caused by gram negative, coccobacilli, belonging to genus Brucella. The transmission of Brucella infection occurs by consumption of raw/unpasteurised milk & milk products and uncooked meat, direct contact with infected animal, its secretions and infectious aborted foetus/retained placenta or infected lochia, or during slaughtering of an infected animal. The people involved in animal husbandry practices and veterinary services are at high risk of infection because of the occupational exposure. A few recent serological studies have revealed the prevalence and importance of brucellosis as human disease problem in India. In Himachal Pradesh, since animal husbandry is a major source of livelihood in hilly terrain, there is constant occupational exposure and no extensive serological study has been undertaken in the state to find out the reach ability of Brucella in human population. Therefore, the proposed serological investigation was carried out for assessing the prevalence of brucellosis in animals and occupationally exposed humans in the state and formulation of its control and prevention strategies. The respective study also evaluated the efficacy of different serological tests for diagnosis of animal and human brucellosis. In nutshell, the chief objective of the study was to know about the status of Brucellosis among human beings especially, occupationally exposed persons
along with the evaluation of efficacy of the serological tests employed in the study. MATERIALS AND METHODS Sample collection and Sample processing: Aseptic collection of blood was done from humans (veterinarians 42, shepherds 40, livestock owners 35, and workers at veterinary hospitals 20, pharmacists 12 and abattoir workers 16) through intravenous route. Serum was obtained by blood clot method and later on preserved by keeping under deep refrigeration. Tests employed: Rose Bengal Plate Test (RBPT) The RBPT was performed according to the method described by Alton et al. (1975).1 0.03 ml of RBPT antigen was placed next to the equal volume of serum on a clean glass slide. The serum and antigen were combined and mixed well. The slide was kept for 4 min. A positive test was indicated by formation of agglutinate. Standard Tube Agglutination Test (STAT) The STAT was performed according to procedure described by Alton et al. (1975) or OIE, Manual, (2004) employing two fold serial dilution of the test serum.1 A titre value of 80 IU per ml and above is considered positive for brucellosis. Mercaptoethanol Test (2-MET) Mercaptoethanol (tube agglutination) test (MET) is to be performed as per the procedure described by Alton et al. (1975).1 The dilution of serum was made in 2-mercaptoethanol solution. A 0.1 ml per litre Mercaptoethanol solution in normal saline was used. The technique of serum dilution, Brucella plain antigen and incubation procedure were the same as described for STAT.
Sero-prevalence of brucellosis in occupationally exposed human beings of Himachal Pradesh (India)
Martin et al. (1987).5 McNemar's chi square (÷2) test for the significance of difference and kappa value (ê) calculation for the agreement of the tests was carried out with the obtained results from the sero-diagnostic tests.
Enzyme Linked Immunosorbent Assays (ELISAs). Enzyme Linked Immunosorbent Assay (ELISA) was performed as per procedure described by Hudson and Hay (1991) or OIE, Manual, (2004).2,3 ELISA value =
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RESULTS
OD of test sample
The present study revealed the overall seroprevalence of brucellosis to be 6.67 percent in human beings. Seroprevalence of brucellosis in human sera samples by RBPT, STAT, 2MET, Dot-ELISA and Indirect ELISA was given as 3.03%, 4.24%, 3.56%, 7.78% and 6.67%, respectively, Table-1 Higher seropositivity for Brucella agglutinins was observed in abattoir workers followed by para-veterinary staff, veterinarians, livestock owners and shepherds as shown in the Table-2.
OD of negative control
ELISA value of more than 2 was taken as positive test result. Dot-ELISA is to be performed according to the procedure described by Batra et al. (1989).4 A positive test was indicated by appearance of a brown dot against white background. Statistical analysis: Statistical analysis of the data was done according to the methods described by
Table 1 : Seropositivity given by various tests performed on human sera samples Human serum samples Test employed
RBPT
STAT
2-MET
Dot-ELISA
Indirect-ELISA
Total positive
5
7
6
13
11
% positive
3.0
4.2
3.6
7.88
6.67
Table 2 : Occupation wise seroprevalence of brucellosis among humans Occupation
Samples examined
Samples confirmed positive
Percent Seroprevalence
Veterinarian
42
3
7.142
Pharmacist
12
1
8.33
Workers at vet. hospitals/ dispensaries
20
0
0
Abattoir worker
16
3
18.75
Shepherd
40
2
5.00
Livestock owner
35
2
5.71
Total
165
11
6.66
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Table 3 : Comparative evaluation of different serological tests for detection of Brucella antibodies in serum samples of human beings Human serum samples Titre / Test employed
RBPT
STAT
2-MET
Dot-ELISA
Indirect-ELISA
Not detectable
-
-
-
1
1
1:40
-
-
-
2
1
1:80
-
-
-
3
2
1:160
2
3
2
3
3
1:320
3
4
4
4
4
Total
5
7
6
13
11
% Positive
3.03
4.24
3.63
7.88
6.67
*M = 0.88 %
*H 3.79
Among the battery of serodiagnostic tests (Dot-ELISA, indirect-ELISA, STAT, 2-MET and RBPT) employed in the present study, Dot- ELISA proved to be quick and a bit sensitive to Indirect-ELISA [yielded maximum number of seropositives i.e.7.78 per cent] (Table-3). Brucella reactors with positive STAT titre value were detected positive by both enzyme immunoassays. Indirect-ELISA came out to be the second most sensitive test (6.67 per cent). Since DotELISA is more of a qualitative assay; the confirmation for the positive samples for brucellosis in the present study was based on Indirect-ELISA. RBPT detected the least number of seropositives, however all the seropositives detected by this method, were also positive by the other tests employed in all the species. The results of STAT and 2-MET were analysed and 0.88 percent higher prevalence was found in STAT, which indicated the difference between active and past infection (Table 3 *M). Also, samples with STAT titre
2.53
value equal or higher than 160 IU in human beings, were found positive by ELISAs. Moreover, 7.78 and 6.67 percent samples were found positive by Dot-ELISA and IndirectELISA respectively, among the STAT negative samples (Table 3 *H). Hence, among all the serodiagnostic tests conducted, enzyme immunoassays (Dot-ELISA and Indirect-ELISA) were found to be comparatively efficient. DISCUSSIONS Brucellosis in animals has been well established by innumerable studies. Complementing this study on human brucellosis, studies on human brucellosis in other states of the India have also pointed out the significant prevalence of the disease in human population. Thakur et al. (2002) found 4.82 per cent Brucella-positive human sera samples, with 23.07 per cent seropositivity among veterinarians during a sero-epidemiological study on human brucellosis.6 Hussain et al. (2004) conducted a study on human population in the regions of Assam and found
Sero-prevalence of brucellosis in occupationally exposed human beings of Himachal Pradesh (India)
5.77 per cent seropositive cases for brucellosis.7 In the present study abattoir workers came out to be highly seropositive among occupationally exposed human beings, who may be justified as the slaughtering practices in this area are not purely scientific/hygienic and they do have an intimate contact with the animals and their products during the slaughtering process. However, none of the samples collected from workers at regional veterinary hospitals /dispensaries revealed positivity for Brucella agglutinins. This whole aspect requires further exploration through studies conducted for a reasonably long period of time along with, more coverage on the sample size for much better epidemiological records. Comparative study of the serodiagnostic tests for brucellosis revealed the immunoassays as the most efficient diagnostic tests. Likewise, numerous other studies have found the two tests to be highly efficient in the detection of brucellosis in various species of animals, like; Saramurthi et al. (2003) compared indirect-ELISA with RBPT and STAT for screening bovine brucellosis and found indirect-ELISA to be much more specific.8 Later on, Asghar et al. (2005) reported in his study that ELISA had comparable sensitivity and specificity to RBPT as it detected more positive cases than RBPT. 9 Moreover, high seropositivity exclusively given by ELISAs could only be best ascribed in their nature being primary binding assays which can detect more than 1/100th of the antibodies to those detected by various secondary binding assays (Tizard, 1982).10 Reliable diagnostic methods are the cornerstones for the success of any surveillance programme. Since, the individual test sometimes do bear variable efficacy, It is therefore, essentially recommended that a
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battery of serodiagnostic tests, which are quick, safe and easy to perform, should be applied to screen a given population in order to ensure detection of brucellosis. ACKNOWLEDGMENTS IVRI (Regional) – Palampur; and National Centre for Disease Control, Delhi. REFERENCES 1.
Alton GG, Jones LM and Peitz DE. 1975. Laboratory techniques in Brucellosis. Monograph series (WHO). Second Edition, 55: 145-148. 2. Hudson JD and Hay FC. 1991. Practical Immunology. 3rd edition, 44. Oxford Blackwell publications, London. 3. OIE, Manual of Diagnostic Tests and Vaccines for Terrestrial Animals, 5th edition, 2004. Part 2. Section 2.3, Chapter 2.3.1. Bovine brucellosis. 4. Batra HV, Chand P, Ganju I and Mukherjee P. Dot-ELISA for detection of bovine brucellosis. Research in Veterinary Sciences. 1989; 46(2): 143-146. 5. Martin SW, Meek AH and Willeberg. 1987. Veterinary Epidemiology: Principles and methods. (pp: 343). Iowa state university press, Ames. 6. Thakur SD and Thapliyal DC. Seroprevalence of brucellosis in man. Journal of Communicable Diseases 2002; 34(2): 106. 7. Hussain SA, Rahman H and Pal D. 2004. Brucellosis in occupationally exposed persons. Journal of Veterinary Public Health. 2004; 2(1/2): 73-74. 8. Sarumathi C, Reddy TV, Sreedevi B and Rao, UVNM. Comparison of Avidin-Biotin ELISA with RBPT and STAT for screening of bovine brucellosis. Indian Veterinary Journal. 2003; 80(11): 1106-1108. 9. Asghar A, Hassanain O and Abbas B. 2005. Seroprevalence of brucellosis in sheep slaughtered during rituals. Indian Veterinary Journal. 2005; 82 (7): 801-802. 10. Tizard I. 1982. Serological assays. Journal of American Veterinary Medicine Association. 1982; 181: 1162-1165.
