Brucellosis is named after Sir David Bruce, who in 1886 isolated the causative agent from a soldier in Malta where the disease caused considerable morbidity and mortality among British military personnel. During the 19th century, brucellosis was thus known as Malta or Mediterranean fever (Charters, 1980). Brucellosis infection is caused by species of the bacterial genus Brucella (Morgan & MacKinnon, 1979; Halling & Young, 1994). There are six different species of Brucella, whereby Brucella abortus is the predominant species infecting cattle (Morgan & MacKinnon, 1979). Apart from cattle, goats, sheep, pigs, buffaloes, camels, reindeer and, less frequently, other mammals are affected by brucellosis (Charters, 1980).It is characterized by abortion, with excretion of the organisms in uterine discharge and in milk. Major economic losses result from abortion, loss of calves, and reduced milk yield in females and infertility in males (WHO, 1971). It is a zoonotic infection and a serious threat to public health. The Brucella may enter the body through digestive tract, lungs or mucosal layers and intact skin. Then it may spread through blood and the lymphatic system to any other organ where it infects the tissues and causes localized infection (Lapaque et al., 2005). Although exact incidence of the brucellosis in bovines in Pakistan is not known but has been reported to vary from 3.25 to 4.4 percent in different areas of Pakistan (Naeem et al., 1990). Brucellosis is one of the world's major zoonotic problems. Though it has been eradicated in many developed countries in Europe, Australia, Canada, Israel, Japan and New Zealand (Geering et al., 1995).
CURRENTLY DESCRIBED BRUCELLA SPECIES AND HOST ASSOCIATIONS
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1-6, (7), 9a
Pigs, hares, reindeer, rodentsb
Newly Described Species
a The status of biovar 7 is under review as the reference strain is believed to represent a mixed culture. Biovar 8 was suspended by the Subcommittee on the Taxonomy of the Genus Brucella in 1978 (Corbel and Banai, 2005).b Host preference is biovar dependant.
In general, brucellosis can be found in any season of a year. The epidemic peak occurs from February to July (Fig. 1) and is closely related to the months associated with delivery and abortion in animals (Shang et al., 2002).
Fig. 1 Monthwise Prevalence of Brucellosis in China from 1950 to 1999 (Shang et al., 2002).
PREVALENCE OF BRUCELLOSIS IN LIVESTOCK IN DIFFERENT COUNTRIES
Br. melitensis biovar 3
Hamoda and Montaser (1998)
Montasser et al. (1999)
Sharma et al. (1979)
Zowghi et al. (1990)
Zowghi and Ebadi (1988)
Ahmed and Munir (1995a & b)
Ajmal et al. (1989)
Silva et al. (2000)
IMPACT OF BRUCELLOSIS ON CATTLE PRODUCTION
In infected cattle population's brucellosis might lead to a lower calving rate due to temporary infertility and/or abortion, resulting in a decreased milk production cows, increased replacement costs as well as lowered sale value of infected cows (Nuru & Schnurrenberg, 1975). General economic losses, however, go far beyond the financial losses suffered by cattle producers alone. Not only cattle but also other species might be affected by brucellosis, including humans. Chukwu (1987) summarised the economic losses of brucellosis to be:
Always on Time
Marked to Standard
1) Losses due to abortion in the affected animal population.
2) Diminished milk production, Brucella mastitis and contamination of milk.
3) Cull and condemnation of infected animals due to breeding failure.
4) Endangering animal export trade of a nation.
5) Human brucellosis causing reduced work capacity through sickness of the affected people.
6) Government costs on research and eradication schemes.
7) Losses of financial investments.
Testing of livestock for brucellosis is done by culture and serology or by testing milk samples (Nielsen, 2002).The main serological test used for diagnosis of brucellosis is the Rose Bengal Plate Test (RBPT), which has very high (>99%) sensitivity but low specificity (Barroso et al., 2002). As a result, the positive predictive value of this test is low and a positive result is required to be confirmed by some other more specific test like serum agglutination test (SAT) and ELISA. However, the negative predictive value of RBPT is high as it excludes active brucellosis with a high degree of certainty. The sensitivity and specificity of the SAT test are 95.6 and 100.0%, respectively, while that specificity of the ELISA is 45.6% (Memish et al., 2002). Milk ring test is based on agglutination of antibodies present in the milk sample with an antigen employed. (Alton et al. 1975)
BRUCELLOSIS IN VARIOUS SPECIES BASED ON SAT AND RBPT (%)
Omer et al. (2000)
Welsh and Dirato (2004)
Jackson et al. (2004)
Dhand et al. (2005)
Acosta et al. (2006)
Cadmus et al. (2006)
SEROPREVALENCE OF BRUCELLOSIS IN CATTLE AND BUFFALOES AT GOVERNMENT AND PRIVATE FARMS IN PUNJAB PAKISTAN.( Nasir et al 2004)
No of samples
Government livestock farms
Private livestock farm
1-The use of live, attenuated rough strain B. abortus RB51
It is produced from a rifampicin-resistant mutant of B. abortus strain 2308 and denominated B. abortus strain RB51 (Schurig et al., 1991). ''R'' standing for ''rough'' and ''B'' for Brucella; 51 does not stand for the number of passages which were necessary to select strain RB51, it refers to an internal laboratory nomenclature used at the time it was derived. Strain RB51 is attenuated as indicated by studies carried out in mice, guinea pigs, goats and cattle, from all of which it is cleared in a relatively short time. Furthermore, it has no, or highly reduced, abortifacient characteristics (Schurig et al., 1991; Palmer et al., 1996;Roop et al., 1991). When used in single vaccination protocols its protective effect in cattle is similar to that induced by strain 19 (Cheville et al., 1992, 1996a,b). Current experiments underway in the field under both high and low brucellosis prevalence indicate that immunity induced by strain RB51 (at least 1 year after vaccination) is similar to or better than that induced by strain 19 (Lord et al., 1998a). The strain can be isolated using a selective medium (Hornsby et al., 2000) and identified by molecular typing (Vemulapalli et al., 1996).
The basic premise of DNA vaccines involves the introduction of gene(s) encoding protein antigens responsible for stimulating a protective immune response (Robinson, 1997). The gene(s) is on a plasmid vector that has the ability to replicate in prokaryotes without expressing the protein but it has the ability to replicate and express the protective antigen in the immunized eukaryote. These DNA vaccines are useful for targeting the in vivo expressed gene(s) of intracellular pathogens like Brucella, even though they can also be used for the expression of toxins and protective antigens from other organism (Gurunathan et al., 2000). In the field of Brucella vaccines, there have been a limited number of studies utilizing this DNA technology. The majority of the effort has been in the area of small animal models. It remains to be shown that the method will be transferable to farm animals. Several investigators have shown that DNA-based vaccines induce long-lived humoral and cellular immune response against a variety of antigens from bacteria, virus and parasites (Gurunathan et al., 2000). This has not been true in the case of Brucella antigens as seen from the limited numbers of publications. Once an animal is injected with the DNA vaccines, the cells harboring the genes of interest could continue to express antigens for extended periods of time, thereby inducing a strong immune response conferring longlived immunity associated with memory cells. Al-Mariri et al. (2001) using DNA vaccines encoding the bacterioferritin or p39 (a putative periplasmic binding protein) of Brucella were able to induce typical Th1- dominated immune response in BALB/c mice as demonstrated by the presence of interferon-gamma (INFg) and immunoglobulin G isotype. They also demonstrated that only the p39 gene (injected intramuscularly) was able to induce a moderate level of protection against wild type B. abortus 544 challenges. They also demonstrated that just purified protein p39 adjuvanted with CpG oligodeoxynucleotide (ODN) was able to induce a significant level of protection against a Brucella challenge. Recently, (Velikovsky et al. (2002) have shown that B. abortus lumazine synthase (BLS) gene in a pCDNA3-based vaccine was able to induce a significant level of antigen specific immune response in BALB/c mice. It induced a humoral response with IgG2a as the dominant isotype as well as INFg which is basically a Th1 response. They also demonstrated that repeated vaccination induces a significant level of protection against a B.abortus 544 challenge in a BALB/c mouse model. The immune response to individual antigens, including those of Brucella spp. (Newman et al., 1996; Qureshi et al., 1996), is governed by the genetic background of an individual.
ZOONOTIC RISK AND GENETIC DIVERSITY
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Members of the genus Brucella represent some of the world's major zoonotic pathogens responsible for enormous economic losses and considerable human morbidity (Godfroid et al., 2005; Pappas et al., 2006a). There is considerable circumstantial evidence of the significance of brucellosis over human history (Evans, 1950; Hoover and Friedlander, 1997; Capasso, 2002). As biosafety level three pathogens some Brucella are also considered bioterrorist threats (Kaufmann et al., 1997; Kortepeter and Parker, 1999; Pappas et al., 2006b) reflecting previous weaponisation (Greenfield et al., 2002) and the fact that the organism is highly infectious, can be readily aerosolized and outbreaks might be difficult to detect due to non-specific symptoms associated with infection. Brucella species are characterised by extremely high levels of nucleotide similarity but varywidely inhost tropisms, microbial and disease phenotypes and pathogenicity. For many years molecular studies and the development of molecular typing tools were hampered by this lack of diversity. However, gradual progress was made in identifying useful markers and tools (VizcaÄ±Â´no et al., 2000; Morenoet al., 2002; Bricker, 2002) Human infections present in various forms with the most common symptoms being fever, malaise, sweats and lymphadenopathy but may also lead to the development of severe complications such as endocarditis, meningoencephalitis, arthritis, spondylitis, orchitis and psychological disturbance (Young, 2005; Corbel and Banai, 2005).
Measuring genetic diversity within a population is of particular importance for bacterial pathogens as it can reflect differences in virulence, antibiotic susceptibility and other phenotypes important for treatment and control of disease and knowledge of the population structure can shed light on the epidemiology, evolution and emergence of pathogenic organisms. There have been very few classical bacterial population genetic studies applied to Brucella to date. Such studies were, until recently, carried out using multilocus enzyme electrophoresis (MLEE) the gold standard approach for studying the population genetics and global molecular epidemiology of bacteria (Boerlin, 1997) MLEE indexes diversity by assessing electrophoretic variants of enzymes. Application of this approach to Brucella simply confirmed the apparent lack of genetic diversity with 99 Brucella isolates found to correspond to only six electrophoretic types with some of these containing multiple species (GaÂ´ndara et al., 2001). The first Brucella genomes became available in 2002 when the publication of the B. melitensis 16 M genome was followed a few months later by B. suis 1330 (DelVecchio et al., 2002; Paulsen et al.,2002). In light of the extreme homogeneity between the classical Brucella species early attempts to identify useful epidemiological markers and to understand the phylogenetics and inter-species relationships of the group advanced only slowly. However, it is now clear that the classical taxonomy based on host specificity and phenotype and which predates molecular characterisation represents an astonishingly accurate picture of genetic relationships. While it has been debated for many years whether the degree of differentiation merits species status it is now becoming apparent that Brucella species are reproductively isolated and (with the exception of B. suis/canis) represent monophyletic lineages separated by long branch lengths (Whatmore et al., 2007; Foster et al., 2009).
Brucellosis is mainly caused by Brucella abortus in bovines which results in great effect on economy, reduced milk production, abortions in last trimester, long calving interval. In Pakistan incidence is increases day by day due to unawareness. Brucellosis is also a greater Zoonotic risk for human being, especially for veterinarians. It is diagnose by different tests e.g. Milk Ring Test (MRT), Rose Bengal Plate Test (RBPT), Serum Agglutination Test (SAT) and ELISA. Brucella is also a bioterrorist. Brucellosis is control by vaccinations. RB51 vaccine is used. Now a day's DNA vaccines are used. Brucellosis eradication program is needed in Pakistan by the help of government, international organizations like OIE, FAO. To prevent the spreading of the disease to neighboring countries.