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Serological, Clinical, and Epidemiological Profile of Human Brucellosis in Rural India

Background: Brucellosis is an important but neglected zoonotic disease in India. Due to frequent animal contact, high prevalence of this disease, though expected in rural population, has not been much studied. Aim: The study was carried out to determine serological, clinical, and epidemiological profile including associated risk factors for human brucellosis in rural India. Materials and Methods: In this cross-sectional study, serum samples from 1,733 individuals residing in rural areas were screened for the presence of anti-brucellar antibodies by Rose Bengal Plate test (RBPT), Serum Agglutination test (SAT), and 2-Mercaptoethanol test (2-ME). Clinical symptoms, epidemiological data including risk factors and knowledge about brucellosis were evaluated by personal interview using a structured questionnaire. Results: Of the 1,733 individuals, 998 had direct contact with animals, whereas 735 had no direct contact. The overall positivity rates by RBPT, SAT, and 2-ME test were 10.50% (182), 7.32% (127), and 5.88% (102), respectively. Clinical symptoms resembling brucellosis were seen in 151 (8.71%) subjects. Animal contact especially during milking, parturition/abortion was the major risk factor, followed by raw milk ingestion. None of the participant knew about brucellosis. Conclusion: Regular surveillance of the disease with awareness programs emphasizing prevention and control are needed.


Introduction
Brucellosis is the commonest zoonotic disease of worldwide distribution. (1,2) In animals, it presents as a chronic infection that persists for life and Brucellae are shed in large numbers in milk, urine, and products of pregnancy. In humans, it is mainly seen in the individuals who come in contact with animals directly. (3)(4)(5) It remains a significant threat to human health in India, especially in rural areas. The rural population is primarily engaged in agriculture for which cattle are used. To supplement the income, small ruminants especially goats and sheep are reared. These animals, most of the times, are kept in the yards of houses or even brought inside. Children adopt these animals as pets. Due to inordinate exposure to animals and their products and ignorance regarding zoonotic diseases, high prevalence of brucellosis, though expected, is not much studied in India.
The purpose of this study was to determine the prevalence of antibrucellar antibodies among the rural population, to study the association between the epidemiological data and seropositivity as well as to evaluate the clinical symptoms, risk factors, knowledge, attitude, and practice levels regarding brucellosis in rural population. in this area was not known. The villages from this catchment area were selected randomly, one by one, to fulfill the desired minimum sample size. It was planned to select the villages till the village wise cumulative total of studied subjects was less than the minimum sample size. The random selection of village was terminated when the cumulative total was equal or more than the desired minimum sample size.
General health check-up camps were conducted in these randomly selected villages. Brucellosis awareness program was one of the objective purposes behind organization of these camps. This cross-sectional health camp-based study was approved by the Institutional Ethical Committee.
The reported average prevalence rate of brucellosis in high risk population is found to be 8.5%. ( Subjects eligible to participate in the study were identified as per following inclusion-exclusion criteria.

Inclusion criteria
Individuals residing in the study villages for more than 1 year irrespective of symptoms of brucellosis.

Exclusion criteria
Individuals staying in the study villages for less than 1 year were excluded.
Consent from each study subject, eligible and willing to participate in the study, was obtained.
All the participants were interviewed with a pre-designed questionnaire regarding age, sex, occupation, contact with animal/animal products, type of animal, duration of contact, raw milk ingestion, knowledge regarding animal and human brucellosis as a disease, its transmission, clinical symptoms, prevention, etc.
The questionnaires were completed with the assistance of a trained person, in the local language. Depending on the nature of animal contact the individuals residing in rural areas were grouped into two groups as: Group I directly exposed (DE) and group II indirectly exposed (IE). Individuals in group I had regular direct contact with animals either at home or work place and those in group II had animals in the neighborhood, i.e., indirect contact. About 3 ml of blood sample was collected from each individual with or without symptoms. Serological study was done using the Rose Bengal Plate test (RBPT), Serum Agglutination test (SAT), and 2-Mercaptoethanol test (2-ME). Antigens for RBPT and SAT tests were procured from Indian Veterinary Research Institute, Izatnagar, U.P. and Institute of Animal Health and Veterinary Biologicals, Hebbal, Bangalore.
The tests were performed according to manufacturer's guidelines. For 2-ME test, the dilution of serum was made in 0.85% saline containing 0.1M 2-ME in place of phenol saline. (8) Test results were noted after 20 ± 2 hours of incubation at 37°C in the water bath. For each serum, sample titers were noted after comparing the tubes in the test series with the antigen control tubes for degree of opacity of the supernatant fluid. The tests were considered positive if the SAT and 2-ME titers were ≥160 IU and ≥80 IU, respectively.
Repeat serological test was performed for the individuals with symptoms resembling brucellosis but negative tube tests titers and also in the asymptomatic individuals with positive tube tests. The results were evaluated for seroprevalence, clinical symptoms, and risk factors regarding brucellosis. The data was analyzed using GraphPad InStat designed by GraphPad Software Inc.

Results
Of the 1,733 subjects screened, 998 were grouped in group I and 735 in group II. Positive reaction by RBPT was noted in 179 individuals in group I and three in group II. Titers ranging from 40 to 5120 IU by SAT and from 40 to 2560 IU by 2-ME test were noted in 170 and 119 individuals, respectively, in group I. In group II, SAT and 2-ME titers ranged between 1280-2560 IU and 640-1280 IU, respectively [ Table 1].
Fever, joint pain, low backache, fatigue, headache, sleep disturbance (insomnia and night sweats) were the reported symptoms. One hundred and fortyeight (14.82%) individuals in group I had the clinical symptoms of which 99 had significant SAT and 2-ME titers. In group II, only three individuals had symptoms and all of them had significant SAT as well as 2-ME test titers [ Table 2].
All the three individuals of group II with significant SAT and 2-ME titers complained of fever, joint pain, low backache, fatigue, and headache. As the activity of brucellosis is indicated by presence of significant 2-ME titers, the results of 2-ME test were considered for further evaluation. (9) Of the 1,733 subjects only 3 had heard about brucellosis as an animal disease, and no one was aware about its transmission to humans. As knowledge regarding human brucellosis was lacking, attitude toward preventive practices was also not observed. Tables 3 and 4.

Discussion
Brucellosis in India was recognized early in the previous century and since then has been reported from almost all the states. The serological studies have reported prevalence rate of 5% in cattle, 3% in buffaloes, 8.23% in sheep, and 4.43% in goats. (10,11) A wide variation in the prevalence of human brucellosis ranging from 0.8 to 26.6% has been reported by various authors. (7,(12)(13)(14)(15)(16)(17) In the present study, seroprevalence documented was 13.3% and 9.9% by SAT and 2-ME tests, respectively, in group I and 0.4% in group II. Totally, 153 individuals complained of health problems like fatigue, headache, insomnia, joint pain, fever, low backache. Among the individuals who complained of symptoms 102 had significant SAT as well as 2-ME titers suggestive of an active brucellosis.   In the remaining 51 symptomatic subjects, 19 showed significant SAT but insignificant 2-ME titers and 32 showed insignificant titers both by SAT and 2-ME tests. Six individuals had no symptoms but had significant SAT titers. Repeat serological test for 51 symptomatic and 6 asymptomatic individuals did not show any increase in titers on twice, fortnightly follow-up. The significant SAT titers in these 25 individuals could be due to repeated exposure to antigenic stimuli or sub clinical infection; consistent with the observations of other authors. (16,18,19) Fever, joint pain, low backache, fatigue, weight loss, headache, and sleep disturbance (insomnia and night sweats) were the reported symptoms. Noteworthy association (P < 0.0001) could be established between fever, joint pain, low backache, and fatigue and significant tube test titers, whereas no association was found between weight loss, headache, and sleep disturbance.  (20)(21)(22)(23) Children and young adults were most commonly affected in this study. Our results are in line with earlier workers. (5,24) Among the subjects with significant tube agglutination test titers, maximum (26.47%) cases were in the pediatric age-group; this might be due to the fact that all these children regularly played with goats and sheep. Mean age for males was found to be 27.05 ± 18.04 and for females 30.656 ± 15.55 years. Youngest patient in this study was 1.4 years and the eldest 74 years. Higher seropositivity in males than in females has been reported by earlier workers. (25)(26)(27)(28) However, in this study, no significant difference in seropositivity and gender was noted. Level of academic education also did not influence brucella seropositivity as reported by Yohannes et al. (17) Noteworthy difference was established between seroprevalence and the occupation in group I, with maximum positivity among shepherds. Raw milk ingestion, close contact with animals, exposure to their products during milking and parturition were the major risk factors and goat was the main animal to which these subjects were exposed.
None of the individuals in group II had come in contact with animals or animal products directly, but most of them had consumed raw milk regularly, hence for group II raw milk ingestion was the major risk factor.
Regarding the knowledge about brucellosis, of the 1,733 subjects, only 3 had heard about the disease but none knew about the modes of transmission or clinical manifestations.

Conclusions
Human brucellosis can be quite common in rural India. Close contact with goats and sheep, and raw milk ingestion are the major risk factors. It is also concluded that knowledge attitude and practice (KAP) levels regarding brucellosis are too poor and there is no association between academic education and KAP levels. Regular surveillance of the disease in animals is needed to control animal brucellosis. Efforts are needed to educate rural population, regarding the disease, modes of transmission, clinical symptoms, risk factors, and preventive measures to decrease the incidence of human brucellosis.
6. Mathur TN. Brucella strains isolated from cows, buffaloes, goats, sheep and human beings at Karnal: Their significance