Brucellosis is an important public health problem in many developing
countries. The disease is found globally, but is more common in the Mediterranean
basin, the Arabian Peninsula, the Indian subcontinent and in parts of
Mexico and Central and South America (Young et al., 2000). In the
Islamic Republic of Iran, brucellosis represents a major health problem
and continues to be reported with increasing frequency from various parts
of the country (Panahi, 2000). Incidence of brucellosis in Khorasan province
in the northeastern part of Iran is considered high in comparison with
other infected provinces. However, according to data derived from active
surveillance during 2001-2005, the incidence was between 120-400 per 100
000 people (Karimi, 2000). According to that active case finding, most
of cases were among farmers, slaughterers and butchers or had an occupational
risk factor (Karimi, 2000). Furthermore, one large study from 1986 disclosed
that approximately 7.4% of cows were infected (Panahi et al., 2000).
As 83% of cases in the country are in individuals less than 40 years old
(Panahi et al., 2000), the importance of occupational exposure
especially during adolescence and young adulthood can not be overemphasized
(Young et al., 1998).
Diagnosis is confounded largely because of a non-specific clinical picture,
so diagnosis is only made certainty when Brucella species are recovered
from blood, bone marrow or other sites (Young, 2000;
Young, 1998). Although most laboratories now employ rapid isolation techniques
(BACTEC, Dupont isolator, polymerase chain reaction methods etc), these
techniques are not available in most developing countries and conventional
methods of isolation are too slow to use routinely for diagnosis (Young,
2000; Young, 1998; Sifuentes, 1997; Dabdoob and Abdulla, 2000). Therefore,
in the absence of bacteriologic confirmation, a presumptive diagnosis
can be made on the basis of a single high or rising titer of specific
antibodies (Young, 2000; Young, 1998). A variety of serological tests
has been applied to diagnosis of brucellosis, of which serum agglutination
test (SAT) is the most widely used (Dabdoob and Abdulla, 2000; Hurtado,
2001, Al-Sekait, 1999). Evaluation of various enzyme-linked immunosorbent
assays (ELISA) for IgG and IgM has shown that these techniques are generally
more sensitive and specific than conventional tests (Hurtado, 2001; Gad
El-Rab and Kambal, 1998), but these techniques are also not generally
available for routine use in developing countries, particularly in rural
areas. As the serum level of antibodies in high-risk and general populations
were examined in our study, serum levels were investigated in order to
avoid reducing the sensitivity of the SAT through the routine application
of a predetermined titer (1:160) (Gad El-Rab, 1998) and because no single
titre of Brucella sp. antibodies is always ‘diagnostic’
(Young, 2000). This enabled us to define a cut-off level that can be used
as a simple and rapid diagnostic test in infected areas (Al-Sekait, 1999).
Materials and Methods
In this cross-sectional study, the rate of Brucella sp. seropositivity
was investigated from September 2005 to July 2006 in urban and rural regions
of Khorasan Razavi province, Islamic Republic of Iran. The study population
consisted of 10 groups: 120 slaughterhouse staff, 140 local butchers,
100 veterinarians and their assistants, 50 domestic animals seller, 70
local milkmaids, 50 animal husbandry staffs, 220 animal owners, 100 shepherds,
80 milk collectors, 20 staff of pasteurization manufactories and 100 people
for control group from the general population. Whole of subjects are healthy
people which have a contact with domestic animals in selling, slaughtering
process or their products such as meat, diary and wool .The patients or
suspects were excluded from the study. The control group selected from
general population which have the following criteria:
||Whom had no any contact with domestic animals.
||And did not consumption of unpasteurized diaries.
||And no previously history of Brucellosis and diagnosed at present.
After randomized sampling, all groups were met at their workplaces and
after a brief explanation about the aims of the project voluntarily agreed
to participate. Those individuals with previous history of brucellosis
or compatible signs or symptoms such as night sweating, prolonged fever,
fatigue, anorexia, weight loss, headache or artheralgia at any time during
the preceding two years were excluded. Also, after physical examination,
those individuals with positive evidence of lymphadenopathy, hepatomegaly
or splenomegaly were excluded from the study. As a result, 99 individuals
including 20 butchers and 25 slaughterers were either excluded or chose
not to participate.
A total of 908 asymptomatic people participated in this study (118 women
and 789 men). Each completed a questionnaire in which data about sex,
age, education and job, consumption of unpasteurized dairy products or
raw meat, direct contact with domestic animals or handling of parturient
domestics or placental membranes were recorded. General knowledge regarding
the routes of transmission of the disease was also questioned. For each
case, a blood sample (5 cc) was obtained by veno puncture. After agglutination
process and centrifugation for serum separation, all samples were analyzed
by the SAT, Combs Wright and 2-mercaptoethanol (2 ME) titres.
Chi-squared and T student tests were used as statistical methods in order
to determine the correlation of epidemiological variables with serologic
The SAT and Combs Wright used for all ten groups in which were positive
for slaughterhouse staff 65.3%, local butchers 30%, veterinarians and
their assistants 31.8%, domestic animals seller 35.4%, local milkmaids
28.6%, animal husbandry staff 23.9%, animal owners, 33.3% shepherds 33.3%,
milk collectors 16%, staff of pasteurization manufactories 8%. Overall,
30.3% of all participants were positive, with varying degrees of positivity.
Moreover, 2ME test among those, whom had more than1:20 of SAT titre in
mentioned groups were positive 66.7, 60, 25, 50, 30.5, 33.3 and 51.9%,
respectively. Meanwhile, 2ME test was 43.7% positive among people with
positive SAT more than 1:20, while, 50% of them had titer more than 1:20,
21% of them had 1:40 titre and 25% had 1:80 titre of SAT. There were only
2 people with titre more than 1;80 of SAT . More details have shown in
Most SAT determination was found 20.4% in veterinary technicians at 1:20
titre. But illegal and local slaughters had the most 2 ME positive test
among people in which had SAT positive test at this titre (p<0.01).
No higher titre was found in the general population.
|| Serum levels of SAT according to occupation of subjects
|Values expressed as percent and number
|| Serum levels of 2 ME titre according to occupation
|Values expressed as percent and number
||Serum levels of Combs Wright titre according to occupation
|Values expressed as percent and number
|| Serum levels of Combs Wright and Wright titre according
to occupation of subjects
|Values expressed as percent and number
There was also a strong correlation between occupational status and positive
SAT titre 1:40 (p = 0.001). Lack of literacy and ignorance of routes of
transmission were significantly correlated with the rate of seropositivity
by 2 ME (with any titre) and SAT titre 1:80 (p = 0.025 and p = 0.016,
Recreational and occupational exposure to Brucella sp. has been
recognized as an important risk factor even in areas in which brucellosis
has been disappearing (Al-Sekait, 1999; Dajani et al., 1989; Al-Ballaa,
1995). The aim of this study was to determine chance of contamination
for healthy people in which at risk of Brucella infection and their serum
antibodies titre cut off points of this infection antibodies.
This study showed that the positive SAT titre base is between 1:20-1:40
among people in which are at risk in endemic areas. There was a significant
difference in the incidence of seropositivity to Brucella sp. between
men and women. This recent finding may be due to the gender dependent
for these occupations. Overall, SAT titre 1:40 could be used as a cut-off
point in the general population in this region. Furthermore, there were
the same results for 2 ME titres (i.e., 1:20-1:40). However, this cut
off value may be acceptable titres, in this regions.
Rates of seropositivity among high-risk occupations vary greatly in various
countries. For example, 35.7% of abattoir workers in Saudi Arabia were
seropositive in one study (Al-Sekait, 1999). In Lebanon, 1.7% of persons
in high-risk occupations were sero-positive based on SAT titre 1:80 (Araj
and Azzam, 1996). In northern Jordan, the rate of seropositivity among
high-risk people was reported to be 8.2% (Abo-Shehada et al., 1996).
Approximately 14% of asymptomatic, ‘at risk’ individuals screened
in northern India were seropositive for Brucella sp. (Handa, 1998).
These great differences may be due to cultural variations, especially
poor hygiene practices employed by persons in high-risk occupations in
various countries. This study also showed that the educational status
have a main role for utilization of protective instruments and conducting
of prophylactic processes.
Seroprevalence rates among the general population also vary greatly in
the Middle East (Dabdoob and Abdulla, 2000; Al-Sekait, 1999; Dajani et
al., 1989; Al-Ballaa, 1995; Mousa, 1998; Luhi, 1998; Al- Shamahy,
1997; Idris, 1993). In a similar study in southern Saudi Arabia, 4900
subjects were randomly selected in a house-to-house survey. Investigations
included interview, clinical examination and blood sampling for antibody
titre determination by a microplate agglutination test. Standard tube
agglutination and 2 ME tests further analyzed reactive sera. A significant
proportion of the population (19.2%) in the southern region had serological
evidence of exposure to Brucella sp. antigen (Al-Ballaa, 1995).
A more recent study using SAT in various regions of Saudi Arabia (Al-Sekait,
1999) found the seroprevalence rate of brucellosis to be 20% in the northern
region, 19% in the southern region and 11.6% in the western region. These
rates of seropositivity were much lower than the rates reported from our
study. Surprisingly, in the Republic of Yemen, a nearby country in the
Arabian Peninsula, the rate of serologically positive samples was reported
to range from 0% to 0.8% (Al-Shamahy, 1997). In Oman, the frequency of
serologically positive sera in six locales ranged between 0 and 2% (Idris,
1993). In Iraq, based on rose Bengal screening test and SAT, approximately
6% of healthy randomly selected subjects were seropositive (Dabdoob, 2000).
Data from Kuwait reported seroprevalence rates of approximately 12% (Al-Sekait,
1999). Higher seroprevalence rates have been reported in sub-Saharan countries,
with percentages of 18% in Uganda and 13% in Nigeria (Al-Sekait, 1999).
In a cross sectional study that has performed in Shiraz, one of southern
cities of Iran, the cut off points for titre of SAT and 2 ME were 1:80
and 1:20, respectively among high risk people (Karimi, 2003). This difference
with our study may be due to the less prevalence of infection in that
These data demonstrate the importance of regional variations of the disease
in the endemic areas. However, high prevalence of lower titre seropositivity
in this study may be a significant marker for probably and previous exposure,
although only one investigated risk factor (direct contact with placental
membranes of domestic animals) was significantly related to SAT titres
of 1:40 (p = 0.05). A history of such contact should be considered when
interpreting SAT titres in endemic areas. There was also a strong relation
between illiteracy and ignorance of routes of transmission and positive
titres 1:40 (for 2 ME, p = 0.005 and SAT, p = 0.01).
In the absence of an efficient and effective method for control of the
disease, an educational programme, especially one regarding the routes
of transmission, would be a cost-effective method for prevention and control.
This is particularly so in this area as fresh white cheese, a popular
food consumed daily by many people, is usually produced from unpasteurized
sheep or goat milk.
Conclusion: A single titre of SAT 1:40 in the presence of 2 ME
titre 1:20 can be diagnostic in the general population in this area of
the country. Nonetheless, serological studies in high-risk individuals
should be interpreted cautiously and confirmed only after a four-fold
rising of titres or through bacteriological confirmation. Although the
rising titre of antibodies to Brucella sp. is the most reliable
serological method for accurate diagnosis, it is not always possible to
postpone the diagnostic or therapeutic process.
The authors wish to thank the Mashhad University of Medical Sciences
for its financial support and M. Hazareh moghadam, A. Yousof nezhad for
their cooperation in this study.