brucellosis is a worldwide bacterial zoonosis posing hazards to the public health and causing economic losses for livestock production industry [ 13 ] .
animal brucellosis is mainly characterized by reproductive involvement resulting in abortion and infertility , whereas human brucellosis is a febrile illness known as undulant fever which can lead to chronic debilitating complications [ 3 , 4 ] .
the disease results from facultative intracellular bacteria belonging to the genus brucella which are gram - negative , nonspore - forming and noncapsulated coccobacilli [ 3 , 5 , 6 ] . currently , the genus consists of 10 species classified based on their host preferences and phenotypic differences [ 2 , 7 ] .
three species are also divided into biovars which are b. abortus , b. melitensis , and b. suis with 7 , 3 , and 5 biovars , respectively . in sheep and goats
, brucellosis is primarily due to b. melitensis which is the most pathogenic species for human beings , responsible for the main proportion of human cases in endemic regions including mediterranean and middle east countries [ 1 , 3 , 810 ] .
humans catch the disease via direct contact with infected animals , their vaginal discharges , and aborted fetuses ; dealing with pure cultures ; and consumption of unpasteurized contaminated milk and milk products [ 3 , 6 , 11 ] .
the latter is the major way through which b. melitensis is transmitted to people in endemic areas [ 1 , 5 ] .  
identification of b. melitensis biovars involved in small ruminant brucellosis is a critical component of epidemiological surveys required for designing proper preventive and control strategies . in iran , due to the existence of other related factors including illegal livestock import from neighboring countries where b. melitensis is also prevalent and uncontrolled animal movements ,
it was considered necessary to study changes in the epidemiologic distribution of different biovars in order to determine introduction of new biovars and evaluate the success of ongoing control measures .
therefore , the present study was performed to renew our knowledge about the current prevalence status of b. melitensis biovars causing abortion in sheep and goats in iran .
the present cross - sectional study was carried out from 2007 to 2009 . according to iranian veterinary organization classification
, all country provinces were divided into 3 categories with low ( < 2% ) , intermediate ( 2 - 3% ) , and high ( > 3% ) brucellosis prevalence .
this classification is based on a seroepidemiological survey carried out in 2003 using conventional rose bengal , serum agglutination , and 2-mercaptoethanol tests .
two provinces were then selected from each category based on geography and availability of a central laboratory in the provincial veterinary general office with skilled personnel for bacterial isolation .
the selected provinces were kerman and markazi ; ilam and khorasan razavi ; chehar - mahal bakhtiari and mazandaran from low , intermediate , and high prevalence categories , respectively .
all small ruminant aborted fetuses referred to the central laboratory of the provincial veterinary general office during the study period were cultured for brucella isolation .
brucella isolates were sent to brucellosis department of razi vaccine and serum research institute to be identified at species and biovar levels .
specimens obtained from liver , lungs , spleen , and fetal stomach content of referred aborted fetuses were cultured on brucella agar ( bd , usa ) containing 5% ( v / v ) inactivated horse serum , 2% ( w / v ) dextrose , and brucella selective supplement ( oxoid , uk ) according to the manufacturer 's instruction .
tissue specimens were first decontaminated using ethanol and flame and then homogenized in sterile normal saline by means of a grinder . for each specimen ,
two sets of 3 agar plates were inoculated and incubated at 37c with one set in the air having 10% co2 .
inoculated plates were monitored every 2 days to detect any colonial growth , and in the case of no colony observation , they were kept at most for 35 days .
suspected colonies based on their morphological characteristics were subcultured on brucella agar slopes for further evaluation and biovar identification .
brucella isolates , confirmed by microscopic characterization using gram stain and results of catalase , oxidase , and urease tests , were examined for biotyping according to the standard methods described by alton et al . .
briefly , biovars were identified based on agglutination with a and m monospecific antisera , co2 requirement for growth , h2s production , lysis by tbilisi ( tb ) and berkeley ( bk2 ) phages , and growth on media containing 20  g / ml basic fuchsin and thionin dyes [ 1215 ] .
in the present survey , a total number of 851 aborted sheep and goat fetuses were cultured microbiologically and 265 brucellae were isolated .
biotyping of these 265 isolates showed that 246 ( 92.8% ) were b. melitensis biovar 1 .
eighteen isolates ( 6.8% ) were identified as b. melitensis biovar 2 ; and interestingly , one isolate ( 0.4% ) , which was obtained from mazandaran province , was determined as b. abortus biovar 3 .  
table 1 illustrates numbers of brucella isolates attained in different provinces and results of their biotyping . 

b. melitensis biovar 3 was isolated in none of the six provinces during the study period , and b. melitensis biovar 2 was only isolated in two provinces which are khorasan razavi and kerman belonging to intermediate and low prevalence categories , respectively .
small ruminant brucellosis is still a major animal and public health burden in many parts of the world particularly in the middle east and mediterranean region [ 1 , 6 , 8 , 10 ] .
sheep and goats are primarily infected by b. melitensis as its preferential hosts [ 8 , 18 ] .
regarding the public health , b. melitensis is the most important zoonotic pathogen amongst the brucella spp .
[ 1 , 3 , 5 ] , accounting for the vast majority of human cases all around the world .
in endemic areas , it is transmitted to people mostly through consumption of unpasteurized milk and milk products from sheep and goats [ 3 , 5 , 19 ] . 

b. melitensis is divided into three biovars which are differentiated using conventional laboratory methods such as agglutination with a and m monospecific antisera and lysis by brucella phages [ 12 , 13 ] .
determination of biovars involved in animal brucellosis is an important step for epidemiologic characterization of the disease in any country and a preliminary requirement for designing control and eradication programs .
additionally , owing to the ability of brucellae to adapt to new environments and its re - emergence [ 3 , 5 , 6 ] , revealing changes in epidemiologic features of brucella species / biovars can help to unravel complexity of interactions between the organism , animals , and humans . in iran , the first b. melitensis isolation from an aborted sheep fetus was reported in 1950 [ 16 , 17 ] .
thereafter it has been widely isolated in different parts of the country mainly from sheep and goats but also occasionally from cattle , camel , and sheepdogs [ 17 , 20 ] .
all biovars of b. melitensis exist in iran among which biovar 1 is known as the most widespread .  
table 2 summarizes biotyping results of iranian b. melitensis isolates during two periods of time from 1971 to 2000 .
immunization of host species with b. melitensis strain rev.1 has been the main strategy for the control of small ruminant brucellosis in iran since 1960s . during 19832003 ,
a test - and - slaughter campaign was also conducted in adult sheep and goats using rose bengal , serum agglutination , and 2-mercaptoethanol tests , while vaccination was limited to young animals . from 2003 ,
control program has been based on mass vaccination using full doses ( containing 13 10 bacteria per dose ) of rev.1 vaccine in lambs and kids at 47 months of age , and its reduced doses ( containing 0.52 10 bacteria per dose ) in adult female animals .
in addition to the mass vaccination , other measures including public education , promotion of sanitary husbandry practices , and microbiological evaluation of abortion outbreaks have been implemented . as a result ,
the number of new human cases reported annually dropped from 39 in 2005 - 2006 to 15.9 in 2010 - 2011 per one hundred thousand people in the country ( unpublished data ) .
our survey showed that b. melitensis biovar 1 is the most frequent cause of clinical brucellosis in small ruminant populations of the provinces included .
this finding is in agreement with the results of the previous studies in the country [ 16 , 17 ] .
isolation of b. abortus biovar 3 , which is an enzootic etiologic agent of bovine brucellosis [ 17 , 21 , 22 ] , from an aborted sheep fetus in our study suggests the possibility of cross - species transmission of the pathogen from cattle .
b. abortus has been sporadically identified and reported as a causative agent for sheep brucellosis in iran [ 17 , 21 ] .
are present , and cattle and small ruminants are raised in close contact , transmission to nonpreferred hosts may occur [ 17 , 21 , 23 ] .
this should be taken into account while implementing control measures . whereas the estimation of disease prevalence was not the aim of this study
, the results showed that in spite of implementing vaccination and other control measures for years , small ruminant brucellosis in clinical form still persists in various parts of the country .
nevertheless , there is a need for country - wide investigations covering the whole target population including traditionally and nomadically reared flocks   to ascertain geographical distribution of different biovars region by region .
it will also help trace potential outbreaks , especially in provinces neighboring western and eastern borders .
for this purpose , using geographic information system ( gis ) will be helpful in analyzing interactions between animal and human brucellosis .
the present study revealed that b. melitensis biovar 1 remains the most prevalent cause of clinical form of small ruminant brucellosis in various provinces of iran .
however , more studies are required to determine current status of the disease throughout the country and the evaluation of its impact on human health .
further control and prevention measures can be implemented to curtail the human and animal disease incidence to a greater extent .
 to determine the most prevalent biovar responsible for brucellosis in sheep and goat populations of iran , a cross - sectional study was carried out over 2 years in six provinces selected based on geography and disease prevalence . 
 specimens obtained from referred aborted sheep and goat fetuses were cultured on brucella selective media for microbiological isolation . 
 brucellae were isolated from 265 fetuses and examined for biovar identification using standard microbiological methods . 
 results showed that 246 isolates ( 92.8% ) were b. melitensis biovar 1 , 18 isolates ( 6.8% ) were b. melitensis biovar 2 , and , interestingly , one isolate ( 0.4% ) obtained from mazandaran province was b. abortus biovar 3 . in this study , 
 b. melitensis biovar 3 was isolated in none of the selected provinces , and all isolates from 3 provinces ( i.e. , chehar - mahal bakhtiari , markazi , and ilam ) were identified only as b. melitensis biovar 1 . in conclusion , we found that b. melitensis biovar 1 remains the most prevalent cause of small ruminant brucellosis in various provinces of iran . 
1. Introduction
2. Materials and Methods
3. Results
4. Discussion
5. Conclusion