Although the brucellae munitions never were used against human targets, the ... Brucella is coccobacilli or short rods 0.6-1.5 μm long by 0.5 to 0.7 μm in width. ... with a typical doubling time of 2 hours (Hoover and Friedlander, 1997). Most ...... are between 20 and 30 nucleotides in length which allows a reasonably high.
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2.0 Literature review 2.1 History of brucellosis Brucellosis in humans has a strong association with military medicine. In 1751, Cleghorn, a British army surgeon stationed on the Mediterranean island of Minorca, described cases of chronic, relapsing febrile illness and cited Hippocrates description of a similar disease more than 2,000 years earlier (Hoover and Friedlander, 1997). Three additional British army surgeons working on the island of Malta during the 1800s were responsible for important observations of the disease. J.A Marston described clinical characteristics of his own infection in 1861 (Hoover and Friedlander, 1997). In 1887, British army doctor David Bruce isolated the organism that bears his name from the spleens of 5 patients with fatal cases on Malta and placed it within the genus Micrococcus (Coghlan, 1997; Maloney and Fraser, 2001). At that time the disease known as Malta or Mediterranean fever, had a high incidence among army and navy personnel and among the island’s civilian population. Ten years later, M.L.Hughes, who had coined the name undulant fever, published a monograph that detailed clinical and pathological findings in 844 patients (Hoover and Friedlander, 1997). In that same year, B. Bang, a Danish investigator, identified an organism, which he called the Bacillus of abortion. In 1917, A.C. Evans recognized that Bang’s organism was identical to that described by Bruce as the causative agent of human brucellosis. The organism infects mainly cattle, sheep, goats, and other ruminants in which it causes abortion, fetal death, and genital infection (Hoover and Friedlander, 1997). The name of Brucella was subsequently given in honour of Bruce, who established it as the cause of the disease by transmitting the infection to monkeys (Coghlan, 1997). Twenty years later, Zammit, a Maltese bacteriologist, showed that the organism was transmitted to man in goats milk.
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Given the ease of aerosol transmission of Brucella species, researchers attempted to develop it into a biological weapon beginning in 1942, and in 1954 it became the first agent weaponized by the old US offensive biological weapons program. Field testing on animals soon followed. By 1955, the US was producing B suis-filled cluster bombs for the US Air Force at the Pine Bluff Arsenal in Arkansas. Of note, B melitensis actually produces more severe disease in humans. Development of brucellae as a weapon was halted in 1967, and President Nixon later banned development of all biological weapons on November 25, 1969 (Maloney and Fraser, 2001). Although the brucellae munitions never were used against human targets, the research performed resulted in concern that Brucella species someday may be used as a weapon against either military or civilian objectives (Maloney and Fraser, 2001).
2.2 Description of the causative organism 2.2.1 Morphology and general characteristics Brucella is coccobacilli or short rods 0.6-1.5 μm long by 0.5 to 0.7 μm in width. They are arranged singly and less frequently in pairs or small groups. The morphology of Brucella is fairly constant except in old cultures, where pleomorphic forms may be evident. Brucella is non-motile. They do not form spores, flagella or pili. True capsules are not produced. Brucella is Gramnegative and usually does not show bipolar staining. They are not truly acid fast but resist decolouration by weak acids, thus stain red by the Stamp’s modification of Ziehl-Neelsen method, which is sometimes used for the microscopic diagnosis of brucellosis from smears of solid or liquid specimens (Scientific Committee on Animal Health and Animal Welfare, 2001; Baron, et al., 1994).
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The guanine-plus-cytosine content of the deoxyribonucleic acid (DNA) is 55-58 moles/cm. No Brucella species has been found to harbor plasmids naturally although they readily accept broad-host range plasmids (Alton and Forsyth, 1997). The metabolism of the Brucella is mainly oxidative and they show little action on carbohydrates in conventional media. Multiplication is slow at the optimum temperature of 37oC and enriched medium is needed to support adequate growth (Alton and Forsyth, 1997).
2.2.2 Cultural and growth characteristics Brucella members are aerobic, but some strains require an atmosphere containing 5-10% carbon dioxide (CO2) added for growth, especially on primary isolation. The optimum pH for growth varies from 6.6 to 7.4 and culture media should be adequately buffered near pH 6.8 for optimum growth. The optimum growth temperature is 36-38 oC, but most strains can grow between 20 oC and 40 oC (Scientific Committee on Animal Health and Animal Welfare, 2001). Brucella grows best on trypticase, soybased, or other enriched media with a typical doubling time of 2 hours (Hoover and Friedlander, 1997). Most biovars of Brucella abortus require incubation in an atmosphere of 5% to 10% carbon dioxide for growth. Brucella may produce Urease, oxidize nitrite to nitrate, and are oxidase and catalase positive. Species and biovars are differentiated by their carbon dioxide requirements; ability to use glutamic acid, ornithin, lysin, and ribose; hydrogen sulfide production; growth in the presence of thionine or basic fuchsin dyes; agglutination by antisera directed against certain lipopolysaccharide epitopes; and susceptibility to lysis by bacteriophages. Recently, analysis of fragment lengths of DNA cut by various restriction enzymes has also been used to differentiate brucella groupings (Hoover and Friedlander, 1997). 7
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Brucella utilizes carbohydrates but produce neither acid nor gas in amounts sufficient for classification. It is moderately sensitive to heat and acidity and killed in milk by pasteurization. Brucella colonies become visible on suitable enriched media in 2-5 days. They appear as small, round convex but dissociation, with loss of the O chains of the LPS, occurs readily to form rough or mucoid variants (Brooks, et al., 2004). These latter forms are natural in B canis and B ovis as the LPS of these lack O chain (Alton and Forsyth, 1997). In other words, the typical virulent organism forms smooth, transparent colonies; upon culture, it tends to change to the rough form which is avirulent. The serum of susceptible animals contains a globulin and lipoprotein that suppress growth of non-smooth, avirulent types and favor the growth of virulent types. Resistant animal species lack these factors, so that rapid mutation to avirulence can occur. D-Alanine has a similar affect in vitro (Brooks, et al., 2004). Brucella requires biotin, thiamin and nicotinamide. The growth is improved by serum or blood, but heamin (V-factor) and nicotinamide-adenine dinucleotide (X-factor) are not required. The growth of most Brucella strains is inhibited on media containing bile salts, tellurite or selenite (Scientific Committee on Animal Health and Animal Welfare, 2001). Growth is usually poor in liquid media unless culture is vigorously agitated. Growth in static liquid media favours dissociation of smooth-phase cultures to non-smooth forms. Continuous and vigorous aeration will prevent this, provided a neutral pH is maintained. In semisolid media, CO2-independent Brucella strains produce a uniform turbidity from the surface down to a depth of a few millimeters, while cultures of CO2-requiring strains produce a disk of growth a few millimeters below the surface of the medium (Scientific Committee on Animal Health and Animal Welfare, 2001).
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2.2.3 Biochemistry The metabolism of Brucella is oxidative and Brucella cultures show no ability to acidify carbohydrate media in conventional tests. The Brucella species are catalase positive and usually oxidase positive, and they reduce nitrate to nitrite (except B ovis and some B canis strains). The production of H2S from sulphur containing amino acids also varies. B. melitensis does not produce H2S. Urease activity varies fast to very slow. Indole is not produced from tryptophane and acetylmethylcarbinol is not produced from glucose (Scientific Committee on Animal Health and Animal Welfare, 2001).
2.2.4 Antigenic structure Different species of Brucella cannot be differentiated by agglutination test but can be distinguished by agglutination absorption reactions. It is probable that two lipopolysaccharide (LPS) antigens, A and M, are present in different proportions in the four species. In addition, a superficial L antigen has been demonstrated that resembles the Vi antigen of Salmonellae (Brooks, et al., 2004). The mechanism of pathogenesis of these organisms is not well defined, except that endotoxin is involved; i.e., When the O antigen polysaccharides are lost from the external portion of the endotoxin, the organism loses its virulence. No exotoxins are produced (Levinson and Jawetz, 2000). The lipopolysaccharide of Gram-negative bacteria is composed of a hydrophobic lipid A linked to a charged, densely compact oligosaccharides core associated (smooth-LPS) or not (rough-LPS) with a hydrophilic polysaccharide chain (Ochain). Brucella possesses a peculiar LPS that call non-classical LPS as compared with the so-called classical LPS from enterobacteria such as Escherichia coli. B abortus lipid A possesses a diaminoglucose backbone (rather than glucosamine), and acyl groups are longer (C18-C19, C28 rather than C12 and C14) and are only linked to the core by amide bounds (rather 01
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than ester and amide bonds) (Moriyon, 2003; Ko and Splitter, 2003; Lapaque, et al., 2005). This unique structural feature may underlie the remarkably reduced pyrogenicity (less than 1/100 th) of Brucella LPS, compared with the pyrogenicity of Escherichia coli (Goldstein, et al., 1992). In addition, the Opolysaccharide portion of LPS from smooth organisms contains an unusual sugar,
4,
6-dideoxy-4-formamido-alpha-D-mannopyranoside,
which
is
expressed either as a homopolymer of alpha-1,2-linked sugar (A type), or as 3 alpha-1,2 and 2 alpha-1,3-linked sugar (M type). These variations in Opolysaccharide linkages lead to specific, taxonomically useful differences in immunoreactivity between A and M sugar types (Hoover, and Friedlander, 1997). This non-classical structure confers to B abortus LPS peculiar characteristics that make it a virulence factor because it alters the LPS pathogen-associated molecular pattern (PAMP) and reduces the endotoxinrelated properties typical of LPSs. In contrast to enterobacterial LPSs, Brucella LPS is several-hundred-times less active and toxic than E. coli LPS (Rasool, et al., 1992; Moriyon, 2003; Lapaque, et al., 2005). All smooth Brucella strains show complete cross reaction with each other agglutination tests with unabsorbed polyclonal antisera, a cross-reaction which does not extend to non-smooth variants. Cross reaction between nonsmooth strains can be demonstrated by agglutination tests with unabsorbed anti-R sera. LPS comprises the major surface antigens of the corresponding colonial phase involved in agglutination (Scientific Committee on Animal Health and Animal Welfare, 2001). Serological cross reactions have been reported between smooth Brucella and various other Gram-negative bacteria, e.g., Escherichia coli O: 116 and O: 157, Salmonella group N (O: 30) of Kaufmann-White, Pseudomonas multophilia, Vibrio cholera and especially Yersinia enterocloitica O: 9. These organisms can include significant levels of antibodies which cross-react with
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S-LPS Brucella antigens in diagnostic tests (Scientific Committee on Animal Health and Animal Welfare, 2001). While serological cross-reactivities between common pathogens can cause difficulties in the differential diagnosis of infections by such pathogens, cross reactivity can have diagnostic usefulness if one of the cross-reacting species is not pathogenic and is unlikely to enter the host. Under these conditions, cross reacting antigens could be obtained from the nonpathogenic species and used for diagnosis of infections caused by the pathogenic agent. Few studies have investigated the serologic cross-reactivity between proteins from Brucella and those from related alpha-proteobacteria (Delpino, et al., 2004).
2.2.5 Molecular genetics of Brucella Characterization of the molecular genetics of Brucella has taken place almost entirely within the past 10 years. The average molecular complexity of the genome is 2.37 x 109 daltons and the molar G+C 58-59% (De Ley, 1987; Corbel, 1997). The genus itself is highly homogenous with all members showing > 95% homology in DNA-DNA pairing studies, thus classifying Brucella as a monospecific genus (Verger, et al., 1985). However, the nomenclature proposed by Veger and Colleagues, 1985 in which all types would be regarded as biovars of B melitensis, has not been generally adopted on practical grounds. For this reason, although its shortcomings are well known, the old nomenclature has been retained with the former specie’s names B. abortus, B. melitensis, B. suis, B. neotoma, B. ovis and B. canis being used for the corresponding nomen species. Within these, seven biovars are recognized for B. abortus, three for B. melitensis, and five for B. suis (Corbel, 1997). Restriction fragments patterns produced by infrequently cutting endonuclease provide support for the current differentiation of the nomen
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species (Allardet-Servent, et al., 1988). Restriction endonuclease analysis has generally been unsuccessful for typing when applied to the whole genome but polymerase chain amplification of selected sequences followed by restriction analysis has provide evidence of polymorphism in a number of genes including omp 2, dna K, htr, and ery (the erythrulose-1-phosphate dehydrogenase gene) (Cellier, et al., 1992). The outer membrane protein type 2 (omp2) gene is taxonomically important because it determines dye sensitivity, one of the traditional typing methods for biovar differentiation. Its polymorphism and capacity for posttranslational modification of its product may explain the tendency for variation in dye sensitivity patterns and have been used as the basis for a genetic classification of Brucella. The dnaK gene of B. melitensis is cleaved into two fragments by Eco RV endonuclease, whereas the genes of the other nomen species all produce a single fragment. The ery gene is reported to have undergone a 7.2 kbp deletion in B. abortus strain 19. This could explain this strain’s erythretol sensitivity, a major factor in its attenuation (Corbel, 1997). The genome of Brucella contains two chromosomes of 2.2 and 1.5 mbp, respectively. Both replicons encode essential metabolic and replicative functions and hence are chromosomes and not plasmids. Natural plasmids have not been detected in Brucella, although transformation has been effected by wide host range plasmids after conjugative transfer or electroporation (Rigby and Fraser, 1989; Corbel, 1997).
2.2.6 Susceptibility to phages Over 40 Brucella phages have been reported to be lytic for Brucella members. All phages are specific for the genus Brucella, and are not known to be active against any other bacteria that have been tested. Thus, lysis by Brucella phages is a useful test to confirm the identity of Brucella spp. and for speciation within the genus. The Brucella phages currently used for Brucella
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typing are: Tbilisi (Tb), Weybridge (Wb), Izatnagar 1 (Iz1) and R/C. The three former phages are used for differentiation of smooth Brucella species. R/C is lytic for B. ovis and B. canis (Scientific Committee on Animal Health and Animal Welfare, 2001).
2.2.7 Susceptibility to dyes and antibiotics Susceptibility to the dyes, thionine and basic fuchsin (20 μg/ml), which varies between biovars, is one of the routine typing tests of Brucella. Brucella melitensis grows in the presence of both dyes. On primary isolation, Brucella is usually susceptible in vitro to gentamicin, tetracycline and rifampicin. Most strains are also susceptible to the following antibiotics: ampicillin, chloramphenicol, cotrimoxazole, erythromycin, kanamycin, novobiocin, spectinomycin and streptomycin, but variation in susceptibility may occur between species, biovars and strains. Most strains are resistant to β-lactams, Cephalosporins, polymyxin, nalidixic acid, amphotericin B, bacitracin, cycloheximide, clindamycin, lincomycin, nystatin, and vancomycin at therapeutic concentrations (Baron, et al., 1994; Scientific Committee on Animal Health and Animal Welfare, 2001). Penicillin is used for the routine differentiation of the vaccinal strain B. abortus species biovars 1 strain 19, and streptomycin for B. melitensis biovars 1 strain Rev. 1, the vaccines widely used for immunization of cattle and small ruminants, respectively, from the virulent field strains of their respective biovars by virtue of their different sensitivity to these antibiotics (Alton, et al., 1988; Scientific Committee on Animal Health and Animal Welfare, 2001).
2.2.8 Taxonomy of Brucella species and biovars Brucella belongs to the alpha-2 subgroup of proteobacteria, along with Ochrobactrum, Rhizobium, Rhodobacter, Agrobacterium, Bartonella, and Rickettsia (Delpino, et al., 2004; Pappas, et al., 2005). There are several lines
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of evidence about the close genetic and antigenic relationship between Brucella and other alpha-proteobacteria (Delpino, et al., 2004). Brucella is a monospecific genus that should be termed Brucella melitensis, and all other species are subtypes, with an interspecies homology above 87%. The phenotypic difference and host preference can be attributed to various proteomes as exemplified by specific outer-membrane protein markers (Verger, et al., 1985; Cloeckaert, et al., 2002; Pappas, et al., 2005). The traditional classification of brucella species is largely based on its preferred hosts. There are six classic pathogens (B. melitensis, B. abortus, B. suis, B. neotoma, B. ovis and B. canis (Corbel and Brinley, 1984; Brooks, et al., 2004). The first 4 species are normally observed in the smooth form, whereas B.ovis and B. canis have only been encountered in the rough form. Three biovars are recognized for B. melitensis (1-3), seven for B.abortus (1-6 and 9), and five for B. suis (1-5). Two new brucella species, provisionally called Brucella pinnipediae and Brucella cetaceae, have been isolated from marine hosts within the past few years (Ross, et al., 1996; Pappas, et al., 2005). Species identification is routinely based on lysis by phages and on some simple biochemical tests (oxidase, urease, and etc). For B. melitensis, B. abortus and B.suis, the identification at the biovars level is currently performed by four main tests, i.e. carbon dioxide (CO 2) requirement, production of hydrogen sulphide (H2S),dye (thionine and basic fuchsin) sensitivity, and agglutination with monospecific A and M antisera. Moreover, a recently developed co-agglutination test, using latex beads coated with a pair of monoclonal antibodies directed against the rough lipopolysaccharide (RLPS) and the 25 kDa outer membrane protein (omp 25), respectively (Bowden, et al., 1997), makes it possible to accurately differentiate B. ovis from B. canis and the occasional rough isolates of the smooth Brucella species. B. melitensis biovars 3 appears to be the most frequently biovars
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isolated in Mediterranean countries (Scientific Committee on Animal Health and Animal Welfare, 2001). Several methods, mainly polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and southern blot analysis of various genes or loci, have been employed to find DNA polymorphism which would enable the molecular identification and typing of the Brucella species and their biovars (Cloeckaert, et al., 1995; Ficht, et al., 1996; Mercier, et al., 1996; Ouahrani-Bettache, et al., 1996; Vizcanio, et al., 1997). Taxonomic knowledge of Brucella has progressed a great deal since the techniques of molecular biology have been applied to these bacteria. A number of molecular tools (nucleic acid probes, primers...) are now available which make the elaboration of a more objective and reliable classification of the genus possible (Scientific Committee on Animal Health and Animal Welfare, 2001).
2.3 The disease (brucellosis) 2.3.1 Pathogenesis and Pathophysiology Brucella can enter mammalian hosts through skin abrasions or cuts, the conjunctiva, the respiratory tract, and the gastrointestinal tract (Hoover and Friedlander, 1997). Because the infection is systemic, it is often not possible to determine which portal of entry was involved in a particular case. Oral entry, by ingestion of contaminated animal products (often raw milk or its derivatives) or by contact with contaminated fingers probably represents the most common route of infection even though this portal may not be the most vulnerable one. Inhalation of aerosol containing the bacteria, or aerosol contamination of the conjunctiva is another route. Inhalation probably underlies some industrial outbreaks. Precutaneous infection though skin
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abrasions or by accidental inoculation has frequently been demonstrated (Alton and Forsyth, 1997, Madkour and Kasper, 2001). Brucella species differ markedly in their capacity to cause invasive human disease. Brucella melitensis is the most pathogenic; Brucella abortus is associated with less frequent infection and a greater proportion of subclinical cases. The virulence of Brucella suis strains for human varies but is generally intermediate (Alton and Forsyth, 1997). The successful co-existence for each brucella spp. with its preferred host is the outcome of the battle between the host genome and brucella genome (Adams, 2002). The pathogen has evolved to survive within the biological systems of the host, and the host has evolved innate and acquired immune system which allows controlled survival of infection by the pathogen ultimately supporting survival of the host pathogen system (Gavora and Spencer, 1983; Adams, 2002). Serum
opsonized
Brucella
organisms
for
ingestion
by
polymorphonuclear leucocytes and activated macrophages. Brucella resist intracellular phagocytic killing by mechanisms such as the suppression of the meyloperoxide-hydrogen halide system by releasing of 5’-guanosine and adenine (Alton and Forsyth, 1997; Madkour and Kasper, 2001). It is well documented that brucellosis is caused by facultative intracellular pathogen that invades both professional and non-professional phagocytic cells. Resistance to killing in professional phagocytic cells controls survival and chronic infection. Resistance of the organism to killing appears to derive from altered intracellular trafficking of Brucella containing vacuoles to the endoplasmic reticulum via autophagic pathway (Ficht, 2003). For intracellular bacteria, survival and replication within phagocytic cells is the key to pathogenesis. Successful strategies for intracellular survival include the ability to survive in acidified-bound vesicles (Rathman, et al., 1996; Eskra, et al., 2001), inhibition of macrophage apoptosis (Monack et al., 1996), prevention of phagosome-lysosome fusion (Cerda-Pizarro, et al., 1998; 06
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Rittig, et al., 2001), and detoxification and repair mechanisms (Eskra, et al., 2001). Brucella abortus likely utilizes a number of strategies to counter the hostile macrophage environment. Within the context of the macrophage, bacterial genes are likely activated in an orchestrated fashion as the macrophage displays an array of potentially bactericidal pathways (Eskra, et al., 2001). Further, Arenas and associates, (2000) concluded that Brucella abortus bacteria do not prevent phagosome acidification and alter phagosome maturation in macrophages. However, acidification does occur in these phagososmes, and some of them can eventually mature to phagolysosomes. Rittig, and associates (2001) concluded that (i) human monocytes readily internalize Brucella in a conventional way using various phagocytosispromoting receptors, (ii) the maturation of some Brucella phagosomes is passively arrested between the steps of acidification and phagosome-lysosome fusion, (iii) Brucella are killed in maturing but not in arrested phagosomes, and (iv) survival of internalized Brucella depends on an acidic intraphagosomal pH and/or close contact with phagosomal wall. The majority of studies addressing the intracellular survival of internalized Brucella focused on a pathogen-induced reduced killing capacity of the macrophages, such as blockade of the oxidative burst or inhibition of cytokines release (Liautard, et al., 1996). Canning and associates, (1986); Caron and associates, (1994); Corbel (1997) documented that an important determinant of virulence is the production of adenine and guanine monophosphate, which inhibit phagolysosome fusion; degranulation and activation of the meylo-peroxidasehalide system; and production of tumor necrosis factor. Bricker, and coworkers, (1990) demonstrated that the production of these inhibitors is prevented in pur E mutants, which are substantially attenuated in consequence. Cu-Zn superoxide dismutase is believed to play a significant role in the early phase of intracellular infection. 07
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In macrophage, Brucella may inhibit fusion of phagososme and lysosomes, and replicate in the phagosome. If unchecked by macrophage microbicidal mechanisms, the bacteria destroy their host cells and infect additional cells. Brucella can also replicate extracellularly in host tissues. Histopathologically, the host cellular response may range from abscess formation to lymphocytic infiltration to granuloma formation with caseous necrosis (Hoover and Friedlander, 1997). On the other hand, it is known that one of the conditions needed for intracellular bacteria to survive is their capability to obtain iron inside host cells (Guerinot, 1994; Ratledge and Dover, 2000). Most of the iron in mammals is intracellular, mainly as part of the heme molecule. Many pathogenic bacteria express receptors for heme or hemoproteins in their membranes under iron-restricted conditions (Craig, 1995). Almiron and coworkers (2001) concluded that ferrochelatase (the last enzyme in heme metabolic pathway and it introduces on iron molecule into the porphyrin ring) play an important role in the intracellular survival and virulence of Brucella abortus. In the gastrointestinal tract, the organisms are phagocytosed by lymphoepithelial cells of gut-associated lymphoid tissue, from which they gain access
to
the
submucosa.
Organisms
are
rapidly
ingested
by
polymorphonuclear leucocytes, which generally fail to kill them and are also phagocytosed by macrophages. Bacteria transported in macrophages which traffic to lymphoid tissue draining the infection site, may eventually localize in lymph nodes, liver, spleen, mammary gland, joints, kidneys, and bone marrow (Hoover and Friedlander, 1997). The brucellae that infect humans have apparent differences in pathogenicity. B. abortus usually causes mild disease without suppurative complications; non-caseating granulomas of the reticuloendothelial system are found. B. canis also causes mild disease, B Suis infection tends to be chronic 08
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with suppurative lesions; caseating granulomas may be present. B melitensis infection is more acute and severe (Brooks, et al., 2004). Persons with active brucellosis react more markedly (fever, myalgia) than normal persons to injected brucella endotoxin. Sensitivity to endotoxin thus may play a role in pathogenesis. Placentas and fetal membranes of cattle, swine, sheep, and goats contain erythretol, a growth factor for brucellae (Brooks, et al., 2004). Further, in ruminants, the presence of erythretol in the placenta may further enhance growth of brucellae and the proliferation of organisms in pregnant animals lead to placentitis and abortion in these species. The products of conception at the time of abortion may contain up to 10 10 bacteria per gram of tissue (Anderson, 1986, Hoover and Friedlander, 1997). It must be stressed that there is no erythretol in human placentas, and abortion is not part of brucella infection of humans (Brooks, et al., 2004).
2.3.2 Immune response to disease The host response in humans reflects unique features of brucella. Smooth lipopolysaccharide does not activate the alternative complement pathway. Brucella is resistant to damage from polymorphonuclear cells owing to suppression of the myeloperoxidase-hydrogen peroxide-halide system and copper-zinc superoxide dismutase and the production of inhibitors of adenylate monophosphate and guanyl monophosphate. Impaired activity of natural killer cells and impaired macrophage generation of reactive oxygen intermediates and interferon regulatory factors have been documented (KO, et al., 2002; Pappas, et al., 2005). The primary method of control is cell-mediated immunity rather than humoral immunity (Malony and Fraser, 2001). Macrophages have been shown to process brucellar antigen and present this to T lymphocytes which produce lymphokines. Interferon is the most active one of these agents in this context. Interferon-γ has a central role in the pathogenesis of brucellosis by activating 11
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macrophages, producing reactive oxygen species and nitrogen intermediates; by inducing apoptosis, enhancing cell differentiation and cytokine production; by converting immunoglobulin G to immunoglobulin G2a; and by increasing the expression of antigen-presenting molecules (Yingst and Hoover, 2003). This inflammatory response is enhanced by cytokines, such as the colony stimulating factors, tumor necrosis factor and interleukin-1, produced by a number of cell types. Cytokines, including interleukin (IL) 1, IL-12, and tumor necrosis factor, appear to be important in host defense against Brucella infection (Madkour and Kasper, 2001). Brucella, like other facultative or obligate intramacrophage pathogens, is primarily controlled by macrophages activated to enhanced microbicidal activity by IFN-γ and other cytokines produced by immune T lymphocytes. It is likely that antibody, complement, and macrophage activating cytokines produced by natural killer (NK) cells play supportive roles in early infection or in controlling growth of extracellular bacteria (Hoover and Friedlander, 1997). Corbel, 1997; Lapaque and associates, 2005 demonstrated that the smooth-LPS is the main antigen responsible for containing protection against infection in passive transfer experiments with monoclonal and polyclonal antibodies. The protection is usually short-term and incomplete. The elimination of virulent Brucella depends on activated macrophages and hence requires development of Th1 type cell-mediated responses to protein antigen. On the other hand, an antibody response occurs with infection, and it is probable that some resistance to subsequent attacks is produced. Immunogenic fractions from brucella cells wall have a high phospholipid content, lysine predominates among eight amino acids, and there is no heptose (thus distinguishing the fractions from endotoxin) (Brooks, et al., 2004). The specific host defenses against Brucella resemble those against other intracellular bacteria. Passively administered monoclonal antibody directed against LPS has been shown to reduce the numbers of brucellae surviving in 10
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the spleens and livers of experimental mice, indicating the role of antibodies in protection (Alton and Forsyth, 1997). The immunity to reinfection is provided by serum immunoglobulins. Initially, immunoglobulins M (IgM) level rise, followed by immunoglobulins G (IgG) titers. IgM antibody level rise during the first week of acute illness, peak at 3 months, and may persist during chronic disease. Even with appropriate antibiotic therapy, high IgM levels may persist for up to 2 years in a small percentage of patients. IgG antibody levels rise about 3 weeks after onset of acute disease, peak at 6-8 weeks, and remain high during chronic disease. The usual serologic tests may fail to detect infection with B. canis (Brooks, et al., 2004). The appearance of calls A immunoglobulins in conjunction with class G immunoglobulins for longer than six months was consistent with the presence of chronic disease (Pappas, et al., 2005). Antibody response in brucellosis, although extremely useful diagnostically, play a limited part in the overall host response.
2.3.3 Clinical aspects of brucellosis There is various clinical classification of brucellosis in different references and there is no standard classification adopted by WHO or others. Some authors classified the disease according to duration of illness into subclinical, acute, subacute, and chronic stage and define the chronic cases if the duration of symptoms more than one year (Gotuzzo and Carrillo, 1998; Salsta, 2000; Doganay and Aygen, et al., 2003). Timothy, 2002 classified the disease into acute, chronic and localized forms while Madkour and Kasper, (2001) classified the disease into active and inactive form. Others considered that the classic categorization of brucellosis as acute, subacute, or chronic is subjective and of limited clinical interest (Pappas, et al., 2005). The incubation period of brucellosis varied from 1-3weeks and may extend to months, depending on the virulence of microorganism, route of entry,
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the infecting dose, and the host preexisting status and immunity (Madkour and Kasper, 2001; Timothy, et al., 2002). B.melitensis and B. Suis are known for increased virulence compared with brucella abortus or canis ( Madkour and Kasper, 2001). Human brucellosis can occur in any age group, but the majority of cases are found in young men between the ages of 20-40 years of age (Salsta, 2000; Doganay and Aygen, 2003). Human brucellosis is notably a disease of protean manifestations (Madkour and Kasper, 2001; Pappas, et al., 2005). Acute brucellosis is the classical form of the disease and can occur acutely or insidiously. Most patients complain of fever, it is seen in 98% of cases (Timothy, et al., 2002), and it is over 38.5 C in 85% initially (Doganay and Ayagen, 2003). The undulant pattern described by Bruce description of Malta fever is uncommonly seen now. The fever of brucellosis has no distinctive pattern but may exhibit diurnal variation, with normal temperature at morning and high temperature in the afternoon and evening. The febrile illness is accompanied by chills, marked aches, malaise, lassitude, arthralagia, backache, headache, anorexia, and drenching sweating (Madkour and Kasper, 2001). Gastrointestinal symptoms are seen in 50 % of cases. Examination of the patients is usually normal with the exception of fever, but occasionally splenomegaly and hepatomegaly are found (Salsta, 2000; Madkour and Kasper, 2001). With time fever lessens and the most prominent symptoms by the patient is low grade fever, aches, arthralgia and excessive sweating. As the symptoms continue over six months the disease enters the chronic form which in general is similar to chronic fatigue syndrome (Doganay and Aygen, 2003). Chronic brucellosis can develop from acute infection or it develops directly by it self. It is extremely rare in children but is more frequent in older patients (Irmak, et al., 2003). Fever is rare, but patients have non-specific complaints of aches, muscular pain, weakness, lassitude, tiredness, fatigue, 12
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arthralgia, excessive sweating, irritability, insomnia, psychoneurosis and depressive symptoms, and weight loss. Examination is usually normal and the patient is usually afebrile (Madkour and Kasper, 2001). Brucellosis is a systemic disease and any organs might be affected (4). Localized brucellosis, called also complications of brucellosis by some authors, can occur as the presenting feature of the disease or occurs as a complication of acute or chronic brucellosis. Osteoarticular disease universally is the most common form of complication of brucellosis seen in 17-37%, and includes arthralgia, reactive or septic arthritis, sacroiliitis, spondylitis, tenosynovitis and vertebral osteomyelitis (Madkour and Kasper, 2001; Pappas, 2005). Skeletal brucellosis in Iraqi patients was studied by Al-Rawi and associates, (1989) who reported the clinical features of 21 episodes of skeletal brucellosis among these patients. The 2nd common local manifestation of brucellosis is genitourinary manifestation, seen in 1-20% of cases, and the commonest form is unilateral epidedymo-orchitis. Pyelonephritis, interstitial nephritis, renal abscess, cystitis, amenorrhea, cervicitis, salpingitis, glomerulonephritis, tubo-ovarian abscess and prostatitis are rare complications. Brucellosis during human pregnancy can cause abortion or intrauterine fetal death (Madkour and Kasper, 2001; Doganay and Aygen, 2003). Cardiovascular complications occur in 2% of cases, and endocarditis is the most serious clinical form, although myocarditis, pericarditis, aortic root abscess,
thrombophlebitis
and
pulmonary
aneurysm
can
occur .
Neurobrucelosis is uncommon and seen in 2-6.5% of cases, and includes meningitis, encephalitis, brain abscess, myelitis, Guillian Barre syndrome, cranial nerve lesions, hemiplegia, myositis, and papillitis could be seen (Madkour and Kasper, 2001; Doganay and Aygen, 2003). Respiratory tract could be involved, in the forms of flu like illness and sore throat, dry cough, pneumonia, lung abscess, and 13
empyema.
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Gastrointestinal tract and hepatobiliary systems is involved in brucellosis but usually mild and unimportant clinically, in the forms of nausea, vomiting, constipation, abdominal pain, diarrhea, liver abscess, ileitis, colitis, liver transaminasemia,
ascitis,
pancreatitis,
cholecystitis,
and
jaundice.
Hepatomegaly and splenomegaly are seen in 15-20% of cases (Madkour and Kasper, 2001; Doganay and Aygen, 2003). Eye is also involved, also uncommonly, in the form of conjunctivitis, keratitis, corneal ulcer, uveitis, choroiditis, scleritis, optic neuritis, and endophthalmitis. Skin manifestations are uncommon in brucellosis, and usually include maculopapular rash, purpura and petechiae, chronic ulcerations, skin abscesses, superficial thrombophlebitis, erythema nodosum and pemphigus (Madkour and Kasper, 2001; Doganay and Aygen, 2003). Endocrine system is involved rarely in the form of thyroiditis with abscess formation, adrenal insufficiency, and syndrome of inappropriate antidiuretic hormone secretion. Hematopoietic system manifestations includes, anemia, leucopenia with relative lymphocytosis, and thrombocytopenia is classical hematological manifestations (Madkour and Kasper, 2001; Doganay and Aygen, 2003).
2.4 Epidemiology Although typical brucellosis is easily recognized in areas where it is endemic, there are other, more difficult cases which may go unnoticed, especially in those places where incidence of this infection is generally very low (Ruiz, et al., 1997). Spain, where the predominant species by far (98%) is Brucella melitensis, is one of those areas of endemicity for this disease together with all the Mediterranean area, the Arabian Peninsula, Mexico, Central America, and South America. In the United States, where the disease is far less frequent, changes in the predominant species have been observed throughout the last three decades. Thus, in the 1970s the highest number of 14
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isolates corresponded to B. suis, in the 1980s it was B. abortus, and now the most frequently isolated species is B. melitensis (Rodriguez, et al., 1992; Chomel, et al., 1994). The global incidence of human brucellosis is not known because of the variable quality of disease reporting and notification systems in many countries. The only countries believed to be free of brucellosis are Norway, Sweden, Finland, Denmark, Iceland, Switzerland, the Czech and Slovak republics, Romania, the united Kingdom (including the Channel Islands), the Netherlands, Japan, Luxembourg, Cyprus, and Bulgaria; the U.S. Virgin Island are also free of the disease. Reports indicated that, even in developed nations, the tru incidence of brucellosis may be up to 26 times higher than official figure suggest. In the United States, about 200 new cases are reported every year; however, it is estimated that only 4 to 10% of cases are recognized and reported (Madkour, 2001). Dajani et al., 1989; Mousa, and co-workers, 1998 documented that the incidence of human brucellosis is much higher in other regions such as the middle east; countries bordering the Mediterranean sea; and china, India, Mexico, and peru; for example, 33 cases per 100,000 population in Jordan (1987) and 88 cases per 100, 000 population in Kuwait (1985) respectively.
2.4.1 Modes of transmission The Brucella organism is transmitted most commonly through the ingestion of untreated milk or milk product; raw meat (i.e., blood) and bone marrow have also been implicated. However, the organism can be contracted via inhalation during contact with animals, especially by children and by slaughterhouse, farm, and laboratory workers. Other routes of infection for atrisk workers include skin abrasion, autoinoculation, and conjunctival splashing. The organism has occasionally been transmitted from person to
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person through the placenta, during breast-feeding, and during sexual activity. Aerosolized B melitensis is a classic agent of biological welfare (Madkour, 2001). Animals may transmit Brucella organisms during septic abortion, at the time of slaughter, and in their milk. The incidence of human disease is thus closely tied to the prevalence of infection in sheep, goats, and cattle, and to practices that allow exposure of humans to potentially infected animals or their products. Brucellosis is an occupational disease in shepherds, abattoir workers, veterinarians, dairy-industry professionals, and personnel in microbiologic laboratories. One important epidemiologic step in containing brucellosis in the community is the screening of household members of infected persons (Almuneef, et al., 2004; Pappas, et al., 2005). With regard to the laboratory workers, Brucella is also highly infectious in laboratory settings; numerous laboratory workers who culture the organism become infected. Fewer than 200 total cases per year (0.04 cases per 100,000 populations) are reported in the United States (Dajani et al., 1989; Mousa, et al., 1998).
2.4.2 Survival of Brucella in the environment The ability of Brucella to persist outside mammalian hosts is relatively high compared with most other non-sporing pathogenic bacteria, under suitable conditions. Numerous studies have assessed the persistence of Brucella under various environmental conditions. Thus, when pH, temperature and light conditions are favorable, i.e., pH > 4, high humidity, low temperature and absence of direct sunlight, Brucella may retain infectivity for several months in water, aborted fetuses and fetal membranes, faeces and liquid manure, wool, hay, on buildings, equipment and clothes. Brucella is able to withstand drying particularly in the presence of extraneous organic material
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and will remain viable in dust and soil. Survival is prolonged at low temperature, especially below 0 0C (Alton, 1985). Contaminated equipment can be sterilized by autoclaving (121 0C). Chemical treatment is recommended to destroy Brucella in contaminated premises. The survival of Brucella in milk and dairy products is related to a variety of factors including the type and age of product, humidity level, temperature, change in pH, moisture content, biological action of other bacteria present and conditions of storage. At low concentration in liquid media, Brucella is fairly heat-sensitive. Thus, dilute suspensions in milk are readily inactivated by pasteurisation (high-temperature short-time) or by prolonged boiling (10 min). Brucella does not persist for a long time in ripened fermented cheese. The optimal fermentation time to ensure safety is not known, but is estimated at 3 months (Nicoletti, 1989). However, in normally acidified soft cheese, the strictly lactic and short-time fermentation and drying increase the survival time of Brucella. Previous pasteurisation of milk or cream is the only means to ensure safety of these products. Brucella are fairly sensitive to ionizing radiation and are readily killed by normal sterilizing doses of gamma-rays under conditions which ensure complete exposure, especially in colostrums (Garin-Bastuji, et al., 1990). In contrast to dairy products, the survival time of Brucella in meat seems extremely short except in frozen carcasses where the organism can survive for years.
2.5 Diagnostic tests 2.5.1 Isolation of bacteria by culture It is well known that unequivocal diagnosis of brucellosis requires isolation of the causal agent (Serra and Vinas, 2004). Although a presumptive diagnosis of brucellosis can be made by demonstrating high or rising antibody
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titers to Brucella antigens, isolation of the organism from blood, bone marrow, or tissues cultures is the only irrefutable proof of the disease (Yagupsky, 1999). Overall, blood cultures are positive in 53.4 to 90% of patients with brucellosis but the chances of successful isolation of the organism decrease over time (Yagupsky, 1999). Because of the suboptimal recovery rate of brucellae from blood, it has been suggested that cultures of bone marrow (Young, 1995), liver tissue or lymph nodes (Yagupsky, 1999) may improve the recovery rate of the organism. The rationale of these alternative approaches is that Brucella organisms survive the intracellular killing by phagocytes and polymorphonuclear leucocytes and localize in the reticuloendothelial system (Young, 1995; Solera, et al., 1997). There are several methods of blood culture for Brucella. The following techniques are the most commonly used: 1-Broth culture monophasic media: - It is based on the experience obtained with traditional methods (manual monophasic blood culture methods), 30 day incubation of blood cultures has been advocated to maximize the recovery of brucellae (Yagupsky, 1999). 2-Broth biphasic method:- To avoid the need to make repeat subcultures, a biphasic medium made of a solid and liquid phase in the same blood culture bottle was develop by Castaneda. After inoculation, the air in the bottle is replaced by a mixture of air with added 10% Co2 and tilted so that liquid flows over the solid medium- the bottle is then incubated in the upright position and examined every 3 days. Any colonies that appear in the solid media should be subculture. If none are seen, the bottles are re-incubated and re-examined after other 3 days. Cultures processed by this method usually became positive within 1 week. No ones detected after 15 days (Hurtado, 2001). 3-Lysis centrifugation blood culture technique: - A method whereby osmotic lysis of blood cells followed by concentration of organisms by a centrifugation 18
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step and dispersion of the concentrate on the surface of agar media has also been used in the detection of Brucella (Hurtado, 2001; Mantur and Mangalgi, 2004). 4-Automated blood culture system: - Initially studied with the BACTEC 460 system. An outbreak of B. melitensis among travelers to Spain 78.9% blood cultures was positive between 4th to 8th days of incubation. This system recovered Brucella in 35% Vs 27.8% yet the time to detection was shorter with the lysis centrifugation method (3.5 days Vs 14 days) (Hurtado, 2001). Some advocate isolation of Brucella form bone marrow, liver tissue and lymph nodes as means to improve recovery the reason is that Brucella survives the intracellular killing by phagocytes and polys and localizes in the reticuloendothelial system (Hurtado, 2001).
2.5.2 Serological tests Human brucellosis is an infectious disease of world wide importance. Due to the extraordinary variety of manifestations of this disease, its diagnosis cannot be made solely on clinical grounds and it is always essential to perform bacteriological and serological tests. There are two broad categories of serologic methods for diagnosis brucellosis: those based on antibody production against lipopolysaccharide and those based on antibody production against other bacterial antigens (Pappas, et al., 2005).
A-Slide agglutination test (RoseBengal test) The Rose Bengal test was developed more than 20 years ago for the diagnosis of bovine brucellosis. Despite the scanty and sometimes conflicting information available (MacMillan, 1990; Alton, 1990, Blasco, et al., 1994a, 1994b), this test is internationally recommended for the screening of brucellosis in small ruminants (Garin-Bastuji and Blasco, 1997). An important problem affecting the sensitivity of the RB test concerns the standardisation of 21
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the antigen. However, if the antigen is standardised differently to give a higher analytical sensitivity, the diagnostic sensitivity is much improved (MacMillan, 1997). It has been suggested that RB test is useful in surveying population groups for the presence of antibody titer, where both IgG and IgM are involved in this reaction (Baraton and Finegold, 1990). However, the test can persist positive for 6 months or more but with a decreasing titer (Corbel, 1989). This will add to the difficulty in the diagnosis of brucellosis especially in endemic areas and among workers with domestic animals. It has been concluded that false positive reaction might be due to cross reaction with other bacteria like Vibrio cholera, Yersinia enterocloitica, and Salmonella spp. (Corbel, 1989; Brooks, et al., 2004).
B-Standard tube agglutination test (STA) Serological diagnosis of brucellosis began more than 100 years ago with simple agglutination test (Nielsen, 2002). It was realized that this type of test was suspected false positive reaction result from, for instance, exposure to cross reacting microorganisms caused other illnesses such as salmonellosis, tularemia, cholera, in addition to others like lupus erythematosus and meyloma (Al-Attas and co-workers, 2000; Nielsen, 2002). Further, while brucellosis agglutinins are cross-reactive with tularemia agglutinins, thus, tests for both diseases should be done on positive sera; usually the titer of one disease will be much higher than that of the other (Brooks, et al., 2004). On the other hand, false negative reaction may occur early in the course of disease or in case of focal infection (Al-Attas, et al., 2000). Further, sporadic studies shed the light on false negative results caused by blocking antibodies represented by IgG and IgA classes, resulting in prozone phenomenon which forms antigen-antibody complexes in high antibody
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concentration (Madkour and Kasper, 2001). This phenomenon can be avoided by testing of sera at both low and high dilutions. In endemic areas, a brucella antibody titer of 1:320 or 1:640 is significant while in non endemic areas an antibody titer 1:160 is considered significant (Madkour and Kasper, 2001; Pappas, et al., 2005).
C-2-Mercaptoethanol test The addition of 2-mercaptoethanol (2-ME) destroys IgM and leave IgG for agglutination reactions. The test is not as sensitive as the standard tube agglutination test, but the results correlate better with the activity of the disease (Hentges, 1986; Brooks, et al., 2004). It is regarded superior to other tests in the determining of the efficacy of antimicrobial therapy (Madkour and Kasper, 2001). Marrodan, and associates (2001) documented that mercaptans (2mercaptoethanol) or dithiothreitol cause the cleavage of disulphide bonds of IgM and loss of agglutination activity. This comparision of results obtained in the absence or presence of these agents is often used to distinguish between early and persistent infection in human brucellosis. A titer of 1/20-1/40 is indicative of active Brucella infection (Al-Abbasi, 2001; Taha, 2001).
D-Coomb’s (antiglobulin) test The antiglobulin test or Coomb’s test was developing to detect antibodies which, although they combine with cellular antigens of Brucella, do not give rise to agglutination. The presence of blocking antibodies, or the so called "prozone" phenomenon (i.e., the inhibition of agglutination at low dilutions due to an excess of an antibodies or to non specific serum factors (Pappas, et al., 2005). Some of these shortcomings can be overcome by modification such as the addition of antihuman globulin. 21
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Blocking antibodies are IgA antibodies that interfere with agglutination by IgG and IgM and cause a serologic test to be negative in low serum dilutions although positive in higher dilutions. These antibodies appear during the subacute stage of infection, tend to persist for many years in dependently of activity of infection (Brooks, et al., 2004).
E-Complement fixation (CF) test The CF test is the most widely used test for the serological confirmation of brucellosis. It is of particular value in the diagnosis of disease especially for distinguishing between serological reaction resulting from vaccination with living vaccine and that produced by active infection (Alton, et al., 1988). Both IgG and IgM antibodies fix complement, the test remains positive for a long time (Madkour, and kasper, 2001). The complement fixation test mostly identifies IgG antibodies predominant in the later stage of disease or in chronic disease but has several disadvantages, such as the occurrence of anticomplement activity, the need to use a highly labile reagent (such as complement), a failure to detect a CFT response in the early stage of the disease, and technical demands (Lucero, et al., 1999). On the other hand, the CF test has many drawbacks such as complexity, variability of reagents, prozones, anticomplementary activity of sera, difficulty to perform with hemolysed sera, and subjectivity of the interpretation of low titers. Therefore, while the sensitivity of RB is sufficient for the surveillance of free areas at the flock level, RB and CF should be used together in infected flocks to obtain accurate individual sensitivity in test (Scientific Committee on Animal Health and Animal Welfare, 2001).
F-Passive haemoagglutination test (PHAT) It is sensitive test with high specificity but not routinely used (Madkour and Kasper, 2001), because it is closely and time consuming. 22
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G-Enzyme immunoassay (EIA) method In endemic areas, the detection of elevated levels of antibody to Brucella organisms in the absence of symptoms during the screening of potential blood donors is common in these areas. To establish a diagnosis in these regions, clinical and serological evaluation should be repeated after 2-4 weeks and a further rise in titer sought. A high titer of specific IgM suggests recent exposure, while a high titer of specific IgG suggests active disease. Lower titers of IgG may indicate past exposure or treated infection (Madkour and Kasper, 2001). IgM antibodies to the smooth lipopolysaccharide (S-LPS) predominate in the first days of infection, after which there is a switch to IgG isotype synthesis in individuals who have not received treatment (Marrodan, and associates, 2001). Consequently, the evaluation of Brucella-specific IgM and IgG antibodies allows descrimination between patients with acute or recent brucellosis and those who have undergone a long infectious process before diagnosis. Descrimination of both classes of antibodies can not be achieved in the conventional tests for brucellosis, i.e., the RoseBengal, serum agglutination and complement fixation tests, because although IgM antibodies specific to the smooth lipopolysaccharides are efficient agglutinins, IgG antibodies can behave as either agglutinating or non-agglutinating (incomplete) antibodies and both classes are active in the complement fixation tests (Wilkinson, 1996). Thus, complementary tests such as the indirectenzyme
linked
immunosorbent
assay
(iELIZA)
with
smooth
lipopolysaccharides and anti-IgM and anti-IgG conjugates are used (marrodan, et al., 2001). The large majority of EIAs in use in the diagnosis of brucellosis are iELISA. ELISAs are methods that involve the immobilization of one of the active components on a solid phase, and iELISAs are those in which the antigen is bound to a solid phase, usually a polystyrene microtiter plate so that 23
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antibody, if present in a sample binds to the immobilized antigen and may be detected by an appropriate antigen-enzyme conjugate which in combination with a chromogenic substrate gives a coloured reaction indicative of the presence of antibody in the sample. It is the method that is now familiar to most diagnostician (Scientific Committee on Animal Health and Animal Welfare, 2001). Another method which is gaining prominence in the publications on brucellosis diagnosis is the competitive ELISA (cELISA) (Nielsen, et al., 1991; Marin, et al., 1999). In this test, Brucella antigen is immobilized on the plate as with the indirect ELISA. Following that, the serum under test and a monoclonal antibody directed against an epitope on the antigen are coincubated. This anti-Brucella monoclonal antibody is conjugated to an enzyme, the presence of which is detected if it binds to the antigen. This will only occur if there is no antibody in the serum sample which is bound preferentially (Scientific Committee on Animal Health and Animal Welfare, 2001). Among the newer serological tests, primary binding assays were developed to improve sensitivity and specificity. The indirect enzyme immunoassay (iELISA) appears to be the most sensitive; however, interpretation may be difficult, as false-positive reactions may occur due to exposure to, for instance, Yersinia enterocolitica O: 9. Another problem with the iELISA is the standardization of reagents, which should be improved in order to make interlaboratory results easy to interpret. The competitive enzyme immunoassay (cELISA) for the detection of serum antibody to Brucella is a multispecies assay which appears to be capable of differentiating vaccinal antibodies from antibodies elicited by field infection in cattle. The monoclonal antibody used in this assay is specific for a common epitope of smooth lipopolysaccharide (S-LPS). The test is fairly rapid but does require some equipment or manipulation. 24
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Hurtado, (2001) documented that ELISA appears to be more specific than classic tests for monitoring the clinical course of brucellosis. While an increase in ELISA titers of IgG and IgA were a good marker of relapse, the persistence of a high ELISA titer of IgG long after therapy, was often detected in patients with a satisfactory clinical outcome and was strongly linked to a high titer of this antibody at admission. It is well recognized that the most important factor affecting initial immunoglobulins titers was the duration of disease at admission. Patients with longer duration of illness before hospitalization had relatively low ELISA IgM titers whereas IgG titers were relatively high in patients ill for a long period before admission (Hurtado, 2001). ELISA is the most sensitive of the Brucella serological tests and is useful to monitor antibodies in patients undergoing treatment. False positive ELISA’s occur because of nonspecific binding of antibody by smooth lipopolysaccharides from Brucella abortus when the latter is used as the solidphase antigen.
H-Counter immunoelectrophoresis It is one of the immunoprecepitation tests especially when used with protein antigen. It is important for testing for the brucella antibodies in the serum and cerebrospinal fluid (Madkour and Kasper, 2001).
I-Immunocapture test The Immunocapture test is a one step technique, very easy to perform, able to detect antibodies of medium to high affinity against Brucella, and suitable for simple standardisation and automation. The test is based on a blue coloured cellular antigen of Brucella melitensis (strain 16 M) and on anti total species immunoglobulins coated polystyrene microtiter plates of 96 U-wells.
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The test was initially evaluated for the serodiagnosis of human brucellosis and has shown high sensitivity and specificity both in the first stages of the disease and, in particular, in chronic cases as well as in relapses and reinfections (Gomez et al., 1999; Orduna, et al., 2000). Moreover, the Immunocapture test and Coomb’s test have a similar performance in the diagnosis of human brucellosis but the Immunocapture test is more sensitive than the Coomb’s test and usually shows high titers (Orduna, et al., 2000).
J-Fluorescence Polarization Assay (FPA) Fluorescence polarization immunoassay (FPA) makes use of molecular rotational properties, measuring antibody binding to antigen directly, eliminating the need for separation procedures. The principle of the method relies on a fluorescent dye attached to a small antigen (or antibody fragment) that is excited by plane-polarized light at the appropriate wavelength. The rate of rotation of the antigen molecule is reduced when its molecular size is increased by its binding to antibody (or antigen) (Lucero, et al., 2003). This change in rate can be measured. The method has been applied to the detection of bovine antibody to Brucella abortus, resulting in a sensitive and specific test (Nielsen et al., 2000). The assay, which can be completed in a few minutes, requires a one-step serum dilution, assessment of background fluorescence, addition of labelled antigen and, finally, measurement of antigen–antibody interaction. In this study, we compare results obtained with the FPA with the CELISA and conventional tests for the diagnosis of human brucellosis (Lucero, et al., 2003). The technique was firstly described by Perrin in 1926. It is a simple technique for measuring antigen/antibody interaction and may be performed in a laboratory setting or in the field. It is a homogenous assay in which analytes neither are nor separated and it is therefore very rapid (Corbel and MacMillan, 2000). 26
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The premise for the fluorescence polarization assay (FPA) is that a small molecule in solution randomly rotates at a rate inversely proportional to its size. Plane polarized light allows measurement, using an attached fluorochrome, of the rate of rotation through a given angle. Thus, a small molecule rotating at a high rate will revolve through the angle rapidly, resulting in a low polarization value. If an antibody is attached to the small molecule, the increased size will cause a decrease in the rate of rotation resulting in a higher polarization value. The FPA is a homogenous assay, requiring no steps to remove unreacted reagents, and can therefore be performed in minutes, even outside the laboratory, and is very cost effective (Scientific Committee on Animal Health and Animal Welfare, 2001). The sensitivity and specificity values of the FPA for bovine brucellosis are almost identical to those of the cELISA (Corbel and MacMillan, 2000). The diagnostic sensitivity has been determined to be over 99%, while the diagnostic specificity approaches 100%. Despite the fact that FPA considered as more sensitive test for diagnosis of human brucellosis than RB and STA, it needs standardisation and purity of antigen used (Mohammed Ali and Bagdasaian, 1990; Al-Abbasi, 2001).
2.5.3 Tests based on cell mediated immunity A-Brucellin test Brucella cytoplasmic antigens, known also as Brucellin have been used with variable success for the allergic diagnosis of brucellosis (Blasco, et al., 1994). Cold saline protein extracts of the rough Brucella melitensis 115 a strain devoid of the O-chain polysaccharides were used as allergen (Jones, et al., 1973). When a protein brucella extract is injected intradermally, erythema, edema, and induration develop within 24 hours in some infected individuals.
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The skin test is unreliable and is rarely used. Application of skin test may stimulate the agglutinin titer (Brooks, et al., 2004).
B-Interferon-gamma test Tests for the in vitro detection of cell mediated immunity (lymphocyte transformation and proliferation assays) showed a lack of acceptable efficacy in order to be applied for the large scale routine diagnosis of Brucella infection (Nicoletti and Winter, 1990). However, in the last decade, the recognition the role of some cytokines such as interferon (IFN) gamma, in immunity against intracellular agents has enabled the development of an in vitro test with useful diagnostic applications. The test was first designed for the diagnosis of bovine tuberculosis (Rothel et al., 1990; Wood et al., 1990, 1991). IFN-gamma is one of the most important T-cell stimulated cytokines in the course of an infection. It is a potent activator of macrophages and monocytes and up-regulates their metabolic activities to produce oxidative metabolites and other microbicidal molecules (Scientific Committee on Animal Health and Animal Welfare, 2001).
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2.5.4 Molecular methods 2.5.4.1 Polymerase chain reaction (PCR) A-History In 1983, the Cetus scientist Kary Mullis developed an ingenious “in vitro” nucleic acid amplification technique termed the polymerase chain reaction (PCR). This technique involves the use of a pair of short (usually 20 bp long) pieces of synthesized DNA called primers and a thermostable DNA polymerase to achieve near-exponential enzymatic amplification of target DNA (Jones, 2002). The 1993 Nobel Prize for chemistry was awarded to Dr. Mullis for having invented PCR. The original protocols for PCR amplification (Newton and Graham, 2000) used the Klenow fragment of E coli DNA polymerase I to catalyze the oligonucleotide extension. However, this enzyme is thermally inactivated during the denaturation step of a PCR cycle and so the researchers had to add a fresh aliquot of enzyme at each cycle of the amplification process. The use of the Klenow enzyme worked well for the amplification of short fragments of DNA (
