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Human health is inextricably linked to animal health and production. This link between human and animal populations, and with the surrounding environment, is particularly close in developing countries like Pakistan where animals provide transportation, draught power, fuel and clothing as well as proteins (meat, eggs and milk). In both developing and industrialized countries, however, this can lead to a serious risk to public health with severe economic consequences, because number of diseases (known as zoonoses) are transmitted from animals to humans. This risk is high among the individuals who are in contact with the animals because of their occupation.
Brucellosis is one of the five common bacterial zoonoses in the world caused by organisms belonging to the genus Brucella, which are gram-negative, non-sporing, facultative intracellular bacteria (Corbel, 1997). The genus Brucella consists of six species according to antigenic variation and host preference. These are Brucella abortus (cattle), B. melitensis (sheep and goats), B. suis (pigs), B. canis (dogs), B. ovis (sheep) and B. neotome (wood rat). Recently, Brucella has been discovered from a variety of marine mammals including cetaceans (e.g. dolphins), seals and otters (Jahans et al., 1997).
Brucellosis in animals and humans is endemic in many developing countries including Pakistan. Control of brucellosis in animals is a pre-requisite for the prevention of this disease in human beings. Recently, Brucella melitensis has been declared by the Centers for Disease Control and Prevention to be one of the three bioterrorist agents due to the expense required for the treatment of human brucellosis patients. Also, the economic and agricultural loss caused by bovine brucellosis emphasizes the financial impact of brucellosis in the society. Human brucellosis is significant public health problem in an agricultural country like Pakistan, where the vast majority of the population is involved in land cultivation and livestock farming. Currently, B. abortus RB51 strain to immunize cattle and B. melitensis Rev.1 strain to immunize goats and sheep are used in many countries. However, these genetically undefined strains still induce abortion and persistent infection, raising questions of safety and efficacy (Corbel, 1997). Human vaccine against brucellosis doesnot exist.
Bang's disease, Infectious abortion, Malta fever, Mediterranean fever and Undulant fever are synonyms for brucellosis. Humans contract brucellosis through contact with infected animals or from consumption of unpasteurized dairy products prepared from the milk of infected animals. In most host species, the disease primarily affects the reproductive system. In animals, the disease is characterized by abortion, retained placenta, orchitis, epididymitis and arthritis with the excretion of organism in the uterine discharge, milk and semen. In humans, the disease is associated with protean manifestations and characteristically recurrent febrile episodes that led to the description of this disease as 'undulant fever'. Other nonspecific symptoms in humans are chills, profuse sweating, headache, arthralgia, myalgia, leg and back pain, malaise, fatigue, weight loss, inattention and depression. Fatalities are not common, though the syndrome can last for a few weeks to a year, even with treatment (Young, 1994).
BrucellosisÂ is a zoonosis transmitted to humans from infected animals. A type of fever characterized by fairly regular remissions or intermissions has been recognized along the Mediterranean littoral since the time of Hippocrates in 450 B.C. Much later in the 19th century, the disease was found to affect British armed forces and the local population of Malta. J A. Marston, an assistant surgeon of the British Medical Department working in the Mediterranean in 1861, first described the symptoms of brucellosis in himself as "gastric remittent fever" (Marston, 1861). The cause of this disease was obscure until 1887 when Sir David Bruce - a Scottish physician reported numerous small coccal organisms in stained sections of spleen from a fatally infected soldier and isolated and identified organism in culture from spleen tissue of four other British soldiers stationed at Malta (Bruce, 1887). This organism, which he designated Micrococcus melitensis, produced a remittent fever in inoculated monkeys. One animal died from the infection and the organism was recovered in pure culture from the liver and spleen. The organism derived its species name from Melita (honey), the Roman name for the Isle of Malta. Hughes ML, in a monograph in 1897, portrayed the findings in people in greater detail, emphasizing "undulant fever" and suggested the name undulant fever (Hughes, 1887).
Wright and Smith in 1897 detected antibodies to M. melitensis in human and animal sera through agglutination test, which unravelled the zoonotic potential of the disease (Wright and Smith, 1897). Later, Zammit an young Maltese physician working with Mediterranean Fever Commission in 1905 confirmed it by isolating the organism from the milk and urine of goats (Zammit, 1905).Thus he concluded that the goat was the reservoir of M. melitensis and the consumption of the raw milk and cheese infects man.
In the same year that Hughes monograph appeared, Bang in Denmark isolated a gram negative rod from cattle, which had aborted. The third member of the group, which is also bacillary in shape, was recovered from the foetus of aborted swine by Traum in 1914 in the United States of America and implicated as an agent of brucellosis in man by Huddleson in 1943. In 1918, Alice Evans an American bacteriologist published reports which contained convincing evidence that M. melitensis from goats and a gram-negative rod from cows could not be differentiated morphologically or by their cultural and biochemical reactions but there were antigenic differences which could be shown by agglutination absorption test. Meyer and Shaw (Meyer and Shaw, 1920) further confirmed Evan's observations and suggested the generic name Â Brucella in honour of
Sir David Bruce. The possible pathogenicity of B. abortus to man was suggested by Evan in 1918 and confirmed by others. In 1956, Buddle and Boyce discovered B. ovis , the cause of epididymitis in rams. In 1957, Stoenner and Lackman isolated B. neotomae from desert wood rat in Utah in USA. In 1968, Carmicheal and Bruner discovered B. canis as the cause of an epidemic of abortions in beagles. Human infections due to B. canis have been reported (Lucero et al., 2005). Two new Brucella species, provisionally called
B. pinnipediae and B. cetaceae, have been isolated from marine hosts within the past few years (Ross, et al., 1996). There are three reports in the literature of humans infected with marine mammal strains of Brucella; one infection occurred in a research laboratory worker after occupational exposure (Brew et al., 1999), and the other two were community-acquired infections (McDonald et al., 2006).
The taxonomy of Brucella species is still unclear and unresolved. Based on 16S rRNA gene sequences, Brucellae are categorised as Î±-2 proteobacteria and have close phylogenetic relationships with Agrobacterium, Rickettsia, Rhizobium and Rhodobacter (Moreno et al., 1990). Brucellae have been classified according to differences in pathogenicity and host preference, into six species: B. melitensis, B. abortus, B. suis,
B. ovis, B. canis and B. neotomae (Jahans et al., 1997).
In fact Verger and colleagues used DNA-DNA hybridization studies to investigate 51 Brucella strains of all species and found them to be identical (Verger et al., 1985). With these results, they proposed that all species should be considered as biovars of B. melitensis. However, because of the differences in the animal reservoirs and in the severity of clinical disease associated with the different species, this proposal has not been widely accepted.
Table-1 summarizes the taxonomic characteristics of Brucella species.
The scientific classification of Brucella is as under;
Class Alpha proteobacteria
Species B. abortus, B. melitensis, B. suis, B. canis, , B. ovis, B. neotome
Human virulence *
+ + + +
cows, camels buffaloes,
+ + to + + +
Carmichael and Bruner, 1968.
Stoenner and Lackman, 1957.
B.pinnipediae and B.cetaceae (provisional)
Minke whales, dolphins, seals
Ewalt and Ross, 1994.
*virulence is graded on a scale from no virulence (-) to the highest degree of virulence (+ + + +).
Table 1: Taxonomy of Brucella species (Pappas et al., 2005).
Cell morphology and culture characteristics:
Brucellae are small gram negative cocci or coccobacilli or short rods measuring about 0.5 to 1.5Âµm in length by 0.5 to 0.7Âµm in width. They can occur as single cells, in pairs or in short chains. They are non spore forming, uncapsulated and aflagellate and, therefore, are nonmotile (Corbel et al., 1984).
The metabolism of brucellae is mainly oxidative and energy is produced by utilization of amino acids and carbohydrate substrates. For many strains i-erythritol is the preferred energy source. They grow under aerobic conditions at an optimal temperature of 37 °C, with many strains requiring supplemental CO2 for growth. The optimum pH for growth ranges between 6.6 and 7.4. Growth usually results in alkalization of the medium. All strains lose viability at 56 °C, however temperatures above 85 °C may be required to insure complete killing of Brucella. They are sensitive to a wide variety of disinfectants including formaldehyde , hypochlorite, iodophores and phenols.
On Serum dextrose agar (SDA) colonies appear transparent, raised, and convex with an entire edge and have smooth, shiny surface (Corbel et al., 1984). On primary isolation using SDA, Brucella colonies are rarely seen prior to 48 hours. At 48 hours, colonies are approximately 0.5-1.0mm in diameter. Colony variants can be classified under four morphological categories: smooth, rough, smooth-rough intermediate and mucoid. This classification is based on characteristics of bacterium' lipopolysaccharide (LPS). At the microscopic level, smooth organisms have LPS molecules containing a polysaccharide O-side chain made from a homopolymer of perosamine on their surface, while rough organisms lack this chain on their LPS (Moreno et al., 1984). Rough colonies are usually less transparent than smooth variants. They have a more dull, granular surface and appear matte white, yellowish white/buff, or brown in colour. Mucoid colonies are similar to rough colonies except that they have a sticky glutinous texture.
In terms of antibiotic susceptibility, nearly all strains of Brucella are susceptible in vitro to gentamycin, tetracycline (and its derivatives), and rifampicin. Additionally, many strains are also susceptible to ampicillin, chloramphenicol, erythromycin, kanamycin, streptomycin and sulfamethoxisole/trimethoprim. Susceptibility to antibiotics can differ among species, biovars and even strains. These differences can aid in identification of specific strains of Brucella.
Fig. 1: Colonies of B. melitensis.
Fig. 2: Brucella are gram -ve in their staining morphology.
The genome of Brucella contains two circular chromosomes of 2.1 and 1.5 Mb, respectively (figure 3 & 4). Both replicons encode essential metabolic and replicative functions and therefore are chromosomes, not plasmids (Jumas-Bilak et al., 1995). Natural plasmids have not been detected in Brucella, although transformation has been effected by wide host range plasmids following conjugative transfer or electroporation (Rigby and Fraser, 1989).
The complete genomic sequence of B. melitensis, B. abortus and B. suis has been achieved recently (DelVecchio et al., 2002). The average size of the Brucella genome is 2.37 x 109 daltons, with a DNA G + C content of 58-59mol% (De Ley, et al., 1987). All types show > 95% homology in DNA-DNA pairing studies, justifying the nomination of Brucella as a monospecific genus. Restriction fragment patterns produced by infrequently cutting endonucleases support the differentiation of the nomen species (Allardet-Servent et al., 1988).
Restriction endonuclease analysis has generally been unsuccessful for strain differentiation, but polymerase chain amplification of selected sequences followed by restriction analysis has provided evidence of polymorphism in a number of genes including omp 2, dnaK, htr and ery (the erythrulose-1-phosphate dehydrogenase gene) (Sangari et al., 1994). The omp 2 gene is believed to determine dye sensitivity, one of the traditional typing methods for biotype differentiation. Its polymorphism and the capacity for post-translational modification of its product may explain the tendency for variation in dye sensitivity patterns even within species and have been used as the basis for a genetic classification of Brucella (Ficht et al., 1996).
A substantial number of antigenic components of Brucella have been characterized. However, the antigen that dominates the antibody response is the lipopolysaccharide (LPS). LPS of rough strains (R-LPS) is similar to LPS of smooth strains (S-LPS) except that the O-chain is either absent or reduced to a few residues. Strong cross-reactions in both the agglutination and complement fixation tests have been reported between smooth species of Brucella and Â YersiniaÂ enterocolitica O:9, Â EscherichiaÂ hermanni, Escherichia coli O:157, Â SalmonellaÂ O:30, Stenotrophomonas maltophila and Vibrio cholerae O:19 (Perry and Bundle, 1990). These have been attributed to similarities on the O-specific side chains of the lipopolysaccharide molecule of the organisms. Numerous outer and inner membrane, cytoplasmic and periplasmic protein antigens have also been characterized. Some are recognized by the immune system during infection and are potentially useful in diagnostic tests (Goldbaum et al., 1993). Omp25 is an outer membrane structural protein that is highly conserved in all Brucellae. It is associated with both lipopolysaccharide and peptidoglycan. Recently, ribosomal proteins have emerged as immunologically important components since they confer protection against challenge with Brucella on account of both antibody and cell mediated responses (Corbel, 1976). One such example is L7/L12. This elicits delayed hypersensitivity response as component of brucellins (Bachrach et al., 1994) and as fusion proteins, which has been shown to stimulate protective response (Oliveira et al., 1996). Hence this appears to have potential as candidate vaccine component.
The basis for the virulence of Brucella can be attributed to the ability of these bacteria to escape the host defense mechanisms and to survive and replicate within the host cells.
Brucella organisms are capable of invading and residing in professional phagocytes (Baldwin and Winter, 1994), such as macrophages, as well as non-phagocytic cells (Detilleux, 1990). The mechanism of attachment and entry into these cells by Brucella has yet to be clearly elucidated. Virulence mechanisms identified so far to be associated with the ability to reside within phagocytic and /or non-phagocytic cells are as follows:
Ability to inhibit phagolysosomal fusion.
Degranulation and activation of the myelo-peroxidase-halide system.
Production of tumor necrosis factor (Caron et al., 1994).
In both phagocytic and non-phagocytic cells, Brucella has the ability to replicate within membrane-bound compartments (Pizarro-Cerda et al., 1998). In non-phagocytic cells, such as HeLa cells, virulent B. abortus 2308 has been documented to replicate in the endoplasmic reticulum by utilizing the autophagic machinery of the HeLa cell (Pizarro-Cerda et al., 1998). In professinal phagocytes, the membrane-bound compartment within which virulent Brucella organisms can proliferate is the phagosome. By some unknown mechanism, Brucella is able to block phagolysosome fusion (Frenchick et al., 1985). It is now thought that the production of adenine and guanine monophosphate can inhibit phagolysosome fusion (Corbel, 1997). The ability to produce these compounds is therefore considered a virulence factor of Brucella. In contrast, attenuated strains of Brucella are unable to prevent such fusion and are thereby destroyed by the lysosomal contents (Pizarro-Cerda et al., 1998).
Research on intracellular survival and replication of Brucella within professional phagocytes has mainly focused on macrophages. Survival within macrophages is apparently associated with the production of many different proteins. These proteins tend to be stress-induced proteins such as heat shock or acid-induced proteins. They include the 17, 24, 28, 60 and 62 kDa proteins (Lin and Ficht, 1995). Two of these proteins, the 17 and 28 kDa proteins, seem to be induced only during intracellular cohabitation of Brucella with macrophages.
Another stress-induced protein, HtrA, has been involved in inducing a granulomatous reaction and reduced levels of infection during the early phase of infection. However, this does not result in reduced levels in the later phases of infection.
Two other types of proteins that have been put forth as possible virulence factors are siderophores and Cu-Zn superoxide dismutase (Cu-Zn SOD). Iron sequestering by siderophores may be an integral virulence factor in intracellular survival of Brucella species. Low levels of iron in vivo aid the host's ability to restrict microbial growth (Corbel, 1997). Cu-Zn SOD may have a significant role in the early phase of intracellular infection, but contradictory results have been reported (Tatum, 1992).
Recently, a two-component regulatory system has been discovered in B. abortus. The Bvr (Brucella virulence related proteins) system consists of a regulatory (BvrR) and a sensory protein (BvrS). This regulatory system, BvrR-BvrS, may play a critical role in the ability of B. abortus to invade and multiply within the cells (Sola-Landa, 1998).
Non-protein components of Brucella may also contribute to its ability to survive within cells. One such cellular component is lipopolysaccharide (LPS). Smooth Brucella organisms are better able to survive intracellularly than do there rough counterparts. Therefore, smooth lipopolysaccharide (S-LPS) probably plays a significant role in pathogenesis.
B. abortus S-LPS is 100 times less potent than that of E.coli (Goldstein et al., 1992) and Salmonella (Freer et al., 1996) in inducing TNFÎ± from macrophages as well as oxidative metabolism and lysozyme release by human neutrophils. This feature of S-LPS has been proposed to contribute to the survival of B. abortus within phagocytic cells. In addition, Brucella S-LPS is not susceptible to the actions of polycationic molecules, suggesting that smooth Brucella can resist the cationic bactericidal peptides of the phagocytes (Martinez de Tejada et al., 1995). S-LPS has also been found to confer antiphagocytic properties to Brucella and does not activate the alternate pathway of complement cascade.
The epidemiology of brucellosis is complex and it changes from time to time. Wide host range and resistance of Brucellae to environment and host immune system facilitate its survival in the populations. Worldwide, brucellosis remains a major source of disease in humans and domesticated animals. The disease is endemic especially in countries of the Mediterranean basin, the Arabian Gulf, the Indian subcontinent and parts of Mexico and Central and South America. Human brucellosis is found to have significant presence in rural/nomadic communities where people live in close association with animals. Worldwide, reported incidence of human brucellosis in endemic disease areas varies widely, from <0.01 to >200 per 100,000 population (Boschiroli et al., 2001). The true incidence of human brucellosis however, is unknown for most countries including Pakistan. It has been estimated that the true incidence may be 25 times higher than the reported incidence due to misdiagnosis and underreporting. It has been shown that the incidence of human brucellosis is significantly high where ovine/caprine brucellosis caused by B. melitensis is endemic (WHO, 1997). Sheep and goats and their products remain the main source of infection, but B. melitensis in cattle has emerged as an important problem in some southern European countries, Israel, Kuwait and Saudi Arabia. B. melitensis infection is particularly problematic because B. abortus vaccines do not protect effectively against B. melitensis infection; the B. melitensis Rev.1 vaccine has not been fully evaluated for use in cattle. Despite vaccine campaigns with Rev.1 strain,
B. melitensis remains the principal cause of human brucellosis worldwide. Screening of household members of an index case is important epidemiological step since this picks up additional unrecognised cases (Mantur et al., 2006).This must be taken into account by the family clinicians caring for these patients, so that timely diagnosis and provision of therapy occur, resulting in lower morbidity. The recent isolation of distinctive strains of Brucella from marine mammals (Ross et al., 1996) as well as humans (McDonald et al., 2006) has extended the ecological range of human brucellosis. Because new strains may emerge and existing types adapt to changing social and agricultural practices, the picture remains incomplete.
It is a well-characterized occupational disease in shepherds, abattoir workers, veterinarians, dairy industry professionals and personnel in microbiologic laboratories. Males are affected more commonly than females (Mantur et al., 2006), which may be due to risk of occupational exposure. Human brucellosis affects all age groups.
Fig. 5: World wide incidence of human brucellosis (Pappas et al., 2006)
Table 2: Global incidence of human brucellosis (Pappas et al., 2006).
Annual cases per million of population
Annual cases per million of population
Central and South America
No data, possibly endemic
No data, possibly endemic
Endemic, no data available
Modes of transmission of Brucella:
Brucella organisms enter the human body through several routes. These routes vary according to the endemic nature of the disease and the presence or absence of control and eradication programs.
Followings are the main routes of entry of Brucella organisms.
Oral route: Ingestion of unpasteurized infected milk and its products is one of the most common modes of transmission of disease in endemic countries (Busch and Parker, 1972).
Respiratory route: The risk of spread of Brucella organisms through inhalation depends on the traditions of animal husbandry in endemic countries. In rural areas, most of the farmers keep the animals in their houses. It is the main route of transmission in laboratory workers handling the Brucella cultures (Young, 2005).
Cutaneous route: Skin abrasions or accidental skin puncture during meat processing is the route of entry of Brucella organisms among abattoir workers (Flynn, 1983). Farmers and veterinarians involved in the process of delivering infected animals, with ungloved hands, also develop local skin lesions.
Conjunctival route: Accidental entry of brucella organisms or splashing of live Brucella vaccine into the eyes during vaccination is a well-known route of entry among veterinarians (Williams, 1982).
Blood transfusion: Transmission of Brucella by blood transfusion from infected individuals, with subsequent development of disease in recipient has been well documented (Wood, 1955).
Sexual transmission: Brucella species have been cultured from human semen and a sexual link has been demonstrated (Wyatt, 1996).
Transplacental transmission: Brucella organisms can cross the placental barrier in pregnant women with active disease, causing abortion of fetus or brucellosis to the newborn (Madkour et al., 1996).
Transmission through breast milk: A nursing mother with brucellosis may transmit the organisms to her infant through breast milk (Al-Abdely et al., 1996). .
Fig. 6: Sources of transmission of brucellosis.
Clinical manifestations in Brucellosis:
Brucellosis is a systemic disease that can involve any organ or system of the body. The onset of systems generally occurs within 2-3 weeks after exposure. The cardinal manifestation of human brucellosis is a fluctuating pattern of fever, due to which it is also called as undulant fever. Among other nonspecific symptoms are chills, profuse sweating, headache, arthralgia, myalgia, leg and back pain, malaise, fatigue, weight loss, inattention and depression. Fatalities are not common, though the syndrome can last for a few weeks to a year, even with treatment (Young, 1994). Most of the patients with brucellosis have complaints referable to multiple organs.
1. Osteoarticular Brucellosis:
Osteoarticular brucellosis involving bones and joints are the most common complication of brucellosis, occurring in 20-60% cases. Sacrolitis is found in most patients with back pain (Ariza et al., 1993).
2. Hepatic Brucellosis:
The liver is mostly involved in brucellosis but often without giving symptoms. Hepatomegaly is present in 20-30 % of cases. Brucellar hepatitis resolves completely with therapy (Ariza et al., 2001).
3. Gastrointestinal Brucellosis:
This complication may mimic typhoid fever. Acute ileitis and colitis were reported in patients infected with B. melitensis, and in endemic areas, pancreatits has also been observed (Madkour, 2001).
4. Genitourinary Brucellosis:
Brucella are also excreted in urine but routine cultures are usually negative. Orchitis and epididymitis occur in up to 20% of men. In women, salpingitis, cervicitis and pelvic abscesses have been reported (Queipo-Ortuno et al., 2006).
5. Cardiovascular Brucellosis:
Endocarditis occurs in less than 3% of cases. This complication is mostly associated with the death of the patient (Reguera et al., 2003).
Invasion of CNS occurs in less than 5% of cases. It causes neuroasthenia, encephalitis
and meningitis. In endemic areas, flaccid paralysis of upper and lower limbs has also been reported (Madkour, 2001).
7. Cutaneous Brucellosis:
In brucellosis, cutaneous lesions include dermatitis, rashes, soft tissue abscesses, ulcers and vasculitis.
8. Endocrinal Brucellosis:
Most commonly involved endocrine glands in brucellosis are testicles and epididymis. The other less frequently localization sites of Brucella are pituitary gland, pancreas, breast, ovary, placenta, adrenals and prostate (Vermigilio et al., 1995).
9. Ocular Brucellosis:
Ocular manifestations of brucellosis is very rare but can lead to marked decreased in visual acuity, blindness, panophthalmitis and subsequent enucleation of eye (Tabbara, 1990).
10. Chronic Brucellosis:
The chronic brucellosis is defined as symptoms persisting for more than one year. Patients with chronic brucellosis fall into 3 categories.
Those with bacteriologic relapse.
Those with deep focus of infection.
Those with nonspecific symptoms that are apparently related to active infection.
Of these, the latter appears to be the most common.
11. Pregnancy and Brucellosis:
Brucellosis can occur at any period of gestation in humans but is most commonly reported in the first trimester (Madkour et al., 1996). The most commonly involved species is B. melitensis .The onset of symptoms in pregnant women is usually abrupt with fever, chills, sweating and generalized aches and pain with abortion in 41% of cases.
12. HIV and Brucellosis:
The HIV-patients are vulnerable to many opportunistic infections. The association between HIV and brucellosis has recently been reported (Galle et al., 1997). Brucellosis
in HIV-patients is oftenly misdiagnosed as an opportunistic infection caused by other organisms.
Fig. 7: Arthritis due to brucellosis. (http://epivet.blogspot.com/2007_02_01_archive.html)
Fig. 8: Aborted Human Fetus
Veterinarian Mostly husbandry practices are
done by ladies in the villages.
Fig. 9: Occupationally exposed workers
The patient's history is very important in diagnosis. The diagnosis of human brucellosis is based on epidemiological evidence, clinical presentation and results of laboratory investigations (Abduljabbar, 1994). The clinical findings are often non specific and systemic brucellosis can be misdiagnosed and confused with other diseases such as typhoid, rheumatic fever, spinal tuberculosis, pyelitis cholecystitis, tumor of testis and thrombophlebitis (Young, 1995). In addition overlap in the clinical features makes diagnosis of various stages of brucellosis difficult, particularly when the time of onset is unknown. Diagnosis of chronic and complicated cases becomes difficult when the symptoms are mild or a typical or when there are reasons to suspect intercurrent disease (Young et al., 1985). Following techniques are used for diagnosis of brucellosis.
Successful culture of Brucella is confirmatory, but the yield of positive culture from clinical specimens especially in chronic cases and neurobrucellosis, is very low, i.e less than 20% (Lulu et al., 1988). Moreover, culture poses a great risk for laboratory workers. For Brucella, biosafety level 3 labs are required.
Serological tests for brucellosis are more useful in diagnosis than culture (Lulu, et al., 1988). Conventional serological procedures e.g. Serum agglutination test (SAT), Rose Bengal test (RBT), Compliment fixation test (CFT) and Enzyme Linked Immunosorbent Assay (ELISA) are all based on detection of anti-LPS antibodies, which remain very high even after recovery of disease (Goldbaum et al., 1994).
(a) Serum Agglutination Test:
SAT developed by Wright and colleagues (Wright and Smith, 1897) remains the most popular and yet used worldwide diagnostic tool for the diagnosis of brucellosis because it is easy to perform, does not need expensive equipments and training. SAT measures the total quantity of agglutinating antibodies (IgM and IgG) (Young, 1991). SAT titres above 1:160 are considered diagnostic in conjunction with a compatible clinical presentation. However, in areas of endemic disease, using a titre of 1:320 as cutoff may make the test more specific.
(b) Rose Bengal Test and Compliment Fixation Test:
The most widely used buffered antigen method is RBT. The RBT shows a high degree of correlation with SAT and is found to be useful screening method for large number of sera (Diaz et al., 1976). CFT was first used in 1912 for the diagnosis of brucellosis (Larson, 1912). It is relatively insensitive to antibodies arising from immunization of Brucella vaccines. However, CFT is laborious and reagent intensive, and some antisera show anti-complimentary activity.
(c) Enzyme Linked Immunosorbent Assay:
ELISAs are more sensitive than conventional tests used in the diagnosis of human brucellosis by detecting more positive sera, higher titers and different classes of immunoglobulins (Ariza et al., 1992). A close correlation between the persistence of IgG and IgA in the sera of some individuals and the chronicity of disease has been pointed out (Ariza, et al., 1992). Hence, IgG, IgA and IgM estimations are used to differentiate between acute and chronic human brucellosis patients. Acute cases have elevated titers of IgM alone or IgG , IgA and IgM while chronic cases have high titers of IgG and IgA.
4. Skin Test:
Brucellins are protein antigenic preparations, which are used to detect brucellosis by intradermal injection. Recent studies with ribosomal protein L7/L12 from B. melitensis was shown to induce strong delayed type hypersensitivity reaction in Brucella sensitized guinea pigs (Bacharach et al.,1994), suggesting that this test might be used for diagnostic purpose in future.
5. Polymerase Chain Reaction:
Polymerase chain reaction (PCR) is fast and can be performed on any clinical specimen (Queipo-Ortuno et al., 2006). A number of nucleic acid sequences have been targeted for the development of Brucella genus-specific PCR assays, including 16S rRNA, the 16S-23S intergenic spacer region, omp2 and bcsp31 ( Navarro et al., 2002). Recently, Redkar et al. (Redkar et al., 2001) described real-time PCR assays for the detection of
B. abortus, B. melitensis and B. suis biovar1. These PCR assays target the specific integration of IS711 elements within the genome of the respective Brucella species or biovar. Currently, a real-time multiplex PCR assay has been developed for rapid confirmatory identification of Brucella with speciation. The genus, B. abortus and
B. melitensis specific primers confirm the organism from isolates (Probert et al., 2004). One case of neurobrucellosis was confirmed in laboratory with the CSF being positive by PCR but undetectable from the blood. The agglutinins were positive in CSF and blood. However, culture of blood and CSF was negative showing the utility of molecular methods in tertiary care centers. Molecular characterization techniques described in the literature are very useful tools for differentiating Brucella spp. especially follow-up testing of unusual phenotypic results of Brucella isolates. Although PCR is very promising, standardization of extraction methods, infrastructure, equipment and expertise are lacking and a better understanding of the clinical significance of the results is still needed (Navarro et al., 2004). The use of molecular methods in Brucella endemic areas needs to be explored before they can be used in these areas to diagnose brucellosis.
The prerequisites for an effective therapy are: treatment should start on time, should consist of combination of drugs along with at least one drug having a good penetration into cells and should be prolonged. The treatment of human brucellosis is a controversial area because of the spectrum of disease, the possibility of chronic infection and the development of complications (Radolf, 1994). In all cases it is important that the patient completes the full course of therapy because the risk of incomplete recovery and relapse is otherwise increased considerably. In 1986, the World Health Organization issued guidelines for the treatment of human brucellosis. The guidelines discuss two regimens, both using doxycycline for a period of six weeks, in combination with either streptomycin for two to three weeks or rifampin for six weeks. Both combinations are the most popular treatments worldwide, although they are not used universally. The streptomycin-containing regimen is slightly more efficacious in preventing relapse (Bingol and Togay-Isikay, 2006). This may be related to the fact that rifampin down-regulates serum doxycycline levels (Wise, 1980). However, parenteral administration of streptomycin mandates either hospital admission or the existence of an adequate health care network.
Dose and duration of therapy
100mg twice daily for 6 weeks
Doxycycline combined with streptomycin, with rifampin,with gentamicin or with ciprofloxacin; doxycycline and streptomycin combined with rifampin or trimethoprim-sulfamethoxazole; doxycycline combined with rifampin and trimethoprim-sulfamethoxazole.
15mg/kg b. wt I/M for 2-3 weeks
Streptomycin and doxycycline; Streptomycin and doxycycline combined with rifampin or trimethoprim-sulfamethoxazole.
600-1200 mg/day for 6 weeks
Rifampin and doxycycline; rifampin and doxycycline combined with streptomycin or trimethoprim-sulfamethoxazole; rifampin and ofloxcin; rifampin and ciprofloxacin.
290 mg twice daily for 6 weeks
Trimethoprim-sulfamethoxazole combined with doxycycline, with rifampin, or with streptomycin; Trimethoprim-sulfamethoxazole and doxycycline combined with rifampin, or with streptomycin.
5 mg/kg/day in 3 divided I/V doses for 5-7 days.
Gentamicin and doxycycline.
400 mg twice daily for 6 weeks
Ofloxacin and rifampin.
500 mg twice daily for 6 weeks
Ciprofloxacin with doxycycline or rifampin.Table 3: Antibiotics used in the treatment of Human Brucellosis (Pappas et al., 2005).
Alternative drug combinations have been used, including other aminoglycosides (e.g., gentamicin and netilmicin) (Corbel, 1997). Trimethoprim-sulfamethoxazole is a popular compound in many areas, usually used in triple regimens. Relapses occur at a rate of about 10% and are often milder in severity than the initial disease and can be treated with a repeated course of the usual antibiotic regimen.
The prognosis of brucellosis is generally very good. With appropriate treatment, most patients with brucellosis recover within weeks or months. However, treatment of patients with cardiac or central nervous system involvement is little difficult (Reguera et al., 2003; Madkour, 2001)
Human Brucella Vaccines:
A vaccine has not been developed for human brucellosis. Although there are adequate scientific and financial tools for such development in some quarters, knowledge is still incomplete about the molecular pathogenesis of brucellosis. Numerous vaccines have been tested in the past, but none have gained wide acceptance (Caksen et al., 2002).
A derivative of strain 19, strain 19-BA, given intradermally by scarification, was formerly used in the Asian republics of the former USSR. It gave limited protection for a relatively short duration and re-immunization was necessary, but was accompanied by an increasing frequency of hypersensitivity reactions (Kolar, 1989). Other attenuated strains, such as B. abortus 84-C and 104-M given intradermally or as aerosols were also used in China and the USSR (Lu and Zhang, 1989). Although apparently effective, these vaccines could provoke severe reactions if not administered correctly or if given to sensitized individuals, and they appear to be no longer in routine use.
Attempts have been made to develop sub-unit vaccines for use in humans. An SDS-insoluble peptidoglycan fraction of Brucella melitensis M15 was used in France (Roux and Serre, 1971), but conclusive evidence of efficacy based on double blind controlled clinical trials was not obtained (Hadjichristodolou et al, 1994). Similarly, an acetic acid extracted polysaccharide-protein fraction developed in the USSR was reported to have low reactogenicity even in previously vaccinated individuals, but evidence of protective efficacy derived from controlled clinical trials is still awaited. It is unclear if more recent approaches such as lipopolysaccharide-protein conjugates ( Jacques et al., 1991) or purified protein antigens such as L7/L12 ( Oliveira and Splitter, 1996) or Cu-Zn SOD (Tabatabai and Pugh, 1992) or glyceraldehyde dehydrogenase (Rosinha et al., 2002) would be effective in humans but they seem worth considering. Recent efforts have focused on developing live attenuated strains. Strain RB51 has minimal human pathogenicity and would seem to have potential but its rifampicin resistance makes it an unsatisfactory candidate for human vaccination. Other efforts have focused on purE mutants (Hoover et al., 1999), but these may retain too much residual virulence. Other candidates such as rfbK mutants of B. melitensis could offer a useful starting point. The World Health Organization has indicated the need for further study in this field (Anon, 1997).
A major difficulty affecting the development of a vaccine against human brucellosis, is the absence of well-established correlates of protection. Although not usually a lethal infection, performance of challenge experiments in human subjects is likely to encounter ethical objections. This means that reliance will have to be placed on animal models. Both the mouse model and guinea pig model have a role to play in human vaccine development. The former is the most useful for monitoring vaccine consistency, whereas the latter may give a better indication of performance in an out-bred population. Any candidate vaccine will probably also need to be assessed for protective efficacy in non-human primates. It would seem advisable to use a vaccine with a known track record in humans, such as B. abortus strain 19-BA, as a baseline reference for efficacy. The cost implications and limited commercial possibilities for vaccines against human brucellosis mean that development is likely to be restricted to national defense agencies.
In animals, both live, attenuated and killed vaccines against brucellosis are used. Two main live, attenuated vaccines used to control B. abortus infection in cattle are B. abortus strain S19 and B. abortus strain RB51.The only approved live attenuated vaccine to control B. melitensis infection is B. melitensis Rev.1 (Rev 1). Killed vaccines against brucellosis are B. abortus strain 45/20 and B. melitensis H38.
Prevention and control of brucellosis:
Prevention of human brucellosis is dependent on control of the disease in domestic livestock mainly by mass vaccination (Nicoletti, 2001). In many countries, the use of B. abortus strain vaccine in cattle and B. melitensis strain Rev-1 vaccine in goats and sheep has resulted in the elimination or near-elimination of brucellosis in these animals. Since the treatment of animal brucellosis is very expensive, one should encourage the mass vaccination of livestock. Animal owners should be taught about the importance of vaccination of their animals. In spite of the clinical efficacy and cost effectiveness of vaccination, the limited availability of vaccines and lack of awareness has led to the persistence of brucellosis in most countries including Pakistan. The lack of human vaccines and effective control measures make it necessary for the doctors and other health care workers to take protective measures. Protective clothing/barriers while handling still births/products of conception and cultures can reduce occupation-related brucellosis (Madkour, 2001). The avoidance of unpasteurised dairy products will prevent infection in the general population (Busch and Parker, 1972).
Brucellosis in Pakistan and South-East Asian countries:
In the South-East Asian region, most of the countries are agricultural and majority of population is involved in land cultivation and livestock farming. The large number of human population is exposed daily to a huge number of animal population and their excreta. This is particularly the case with dairy production units which are increasing in rapidly growing cities of the SAARC (South Asian Association for Regional Cooperation) countries including Pakistan, India, Bangladesh, Sri Lanka and Nepal. However, there are not enough studies on this disease at national level in most of SAARC countries. For example, there is only one published report regarding the presence of Brucella antibodies in the sera of domestic livestock in Bangladesh (Mustafa, 1984). Unfortunately, there is no veterinary public health unit of the World Health Organization (WHO) in most of SAARC countries (Joshi, 1991). Consequently, farmers and their families are helpless to work in the poor hygienic conditions. The occurrence of acute, often most incapacitating infection in humans caused by B. melitensis, usually coincides with the outbreak of brucellosis in sheep and goats (Abdussalam and Fein, 1976).
The first case of brucellosis dates back to 1942 in the Indian part of Indo-Pak subcontinent, and the disease was reported in cattle , buffaloes , sheep, goats, dogs and humans (Renukaradhaya et al., 2002). Except a few published reports, there is no detailed data of brucellosis in human and animal population in Pakistan. Human cases of brucellosis have reported in mid 1980's in Multan region of Pakistan (Noor et al., 1986). A sero-diagnostic survey of human brucellosis among T.B patients in Islamabad, Pakistan revealed 19.2% seropositivity for brucellosis with slide and semi-quantitative agglutination tests (Qazilbash and Bari, 1997). According to the annual report released in the year 2000 by Ministry of Food, Agriculture and Livestock of Pakistan, prevalence of brucellosis in cattle and buffaloes ranged from 8.8 to 16.8% and 7.6 to 10.5%, respectively.