Brucella
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This document provides information on Brucella, the causative agent of brucellosis. It discusses that Brucella is a facultative intracellular, Gram-negative coccobacillus that is a zoonotic pathogen. Six main species that can infect humans are described: B. melitensis, B. abortus, B. suis, B. ovis, B. canis, and B. neotomae. Transmission to humans typically occurs through consumption of unpasteurized dairy products. Brucellosis presents as an acute or subacute febrile illness that can become chronic and involve any organ system. The global epidemiology and scenarios in India and a local medical college
Brucella
- 1. Brucella Dr. Kanwal Deep Singh Lyall M.D. Microbiology
- 2. • Facultative intracellular, GNCB, partially acid fast, aerobic, non-sporing, non-capsulated, non-motile • Zoonotic infection • Oxidase + ve & urease + ve
- 3. • Bang – Br. abortus – cattle - Copenhagen • Tratum (1914) – Br. suis – pigs - USA • Br. neotomae, Br. ovis, Br. canis (does not infect humans) • Mediterranean fever , Malta fever , Gibraltar fever , Cyprus fever • Bruce (1887) – spleen of fatal case in Malta – B. melitensis (Melita – Roman name for Malta)
- 4. Organism Basic fuchsin (1:50.000) Thionin (1:25000) Lysis by Tblisi RTD 10’000 RTD B. Melitensis Grows Grows Not lysed Not lysed B. Abortus Grows Inhibited Lysed Lysed B. Suis Inhibited Grows Not lysed lysed Culture • Strict aerobes, Br. abortus – capnophilic • Growth ↑ by serum, glucose, blood, liver extract • Serum-dextrose agar, srum potato-infusion agar, trypticase soy agar • small., moist, translucent colonies
- 5. Obsolete names for brucellosis • Brucelliasis • Bruce's Septicemia • Continued Fever • Crimean Fever • Cyprus Fever • Febris Melitensis • Febris Undulans • Goat fever • Melitensis septicemia • Melitococcosis • Milk sickness • Mountain fever • Neapolitan fever • Slow fever
- 6. • Sir David Bruce - a Scottish physician - 1887 numerous small coccal organisms in stained sections of spleen - a fatally infected soldier • Isolated & identified organism in culture from spleen tissue of four other British soldiers stationed at Malta • Micrococcus melitensis, -remittent fever in inoculated monkeys • 1 animal died - organism recovered in pure culture from liver & spleen • Species name from Melita (honey), Roman name for Isle of Malta
- 7. • Hughes ML- undulant fever • Wright and Smith - Abs to M. melitensis • Zammit concluded -goat was reservoir of M. melitensis & consumption of raw milk & cheese infects man • Bang in Denmark - cattle which had aborted • Traum in 1914 -foetus of aborted swine • Buddle and Boyce (1956)-B. ovis - epididymitis in rams • Stoenner and Lackman (1957)-B. neotomae -desert wood rat in Utah in USA • Carmicheal and Bruner (1968)-B. canis -epidemic of abortions in beagles
- 8. • Alice Evans an American bacteriologist (1918)- similar morphology, clutural characteristcs & biochemicals, antigenically different • In 1920 showed that M. melitensis was also a bacillus • Meyer and Shaw confirmed Evan's observations & suggested generic name Brucella in honour of Sir David Bruce
- 9. • Two new brucella species, provisionally called B. Pinnipediae and B.Cetaceae - isolated from marine hosts in past few years • 3 reports in literature of humans infected with marine mammal strains of brucella -1 in a research laboratory worker after occupational exposure • Other two community- including recent report in a patient of new zealand with spinal osteomyelitis.
- 10. Taxonomy • Still unclear & unresolved • Classified according to differences in pathogenicity & host preference, into 6 species: B. Melitensis, B. Abortus, B. Suis, B. Ovis, B. Canis and B. Neotomae • Verger & colleagues - DNA-DNA hybridization studies - investigated 51 brucella strains of all species -found them to be identical • With these results- proposed that all species should be considered as biovars of B. Melitensis • Differences in animal reservoirs & clinical severity associated with different species- this proposal not widely accepted
- 11. • Transmission & prevalence in a region - several factors like food habits, methods of processing milk & milk products, social customs, husbandry practices, climatic conditions, socio-economic status & environment hygiene
- 12. • Animal products - milk & meat products • Dairy products prepared from unpasteurized milk - soft cheese, yoghurts & ice-creams • Camel milk also considered important source in middle east countries & mongolia
- 13. Other means of infection • Include skin abrasions or inhalation of airborne animal manure particles • Laboratory acquired brucella infection -accidental ingestion, inhalation & mucosal or skin contact -laboratory workers • Recognized as one of the common laboratory- transmitted infections
- 14. Antigenic Components • No.of antigenic components • Dominating Ag in Ab response -lipopolysaccharide (LPS) • Numerous outer & inner membrane, cytoplasmic & periplasmic protein Ags
- 15. • Omp25 -highly conserved in all Brucellae -associated with both LPS & peptidoglycan • Recently, ribosomal proteins have emerged as immunologically important components - confer protection against challenge with Brucella • One such example is L7/L12.-has been shown to stimulate protective response-potential as candidate vaccine component.
- 16. Genome • complete genomic sequence B. melitensis, B. abortus and B. suis has been achieved • average size of the genome is 2.37 x 10 9 dalton • All types show > 95% homology in DNA-DNA pairing studies- justify nomination of Brucella as a monospecific genus • The omp 2 gene is believed to determine dye sensitivity • Genome contains 2 circular chromosomes • Both replicons encode essential metabolic & replicative functions & therefore are chromosomes, not plasmids.
- 17. Virulence Factors, Pathogenic Mechanisms & Immune response • pathogenicity related to various factors • S-LPS - major determinant of virulence & dominates Ab response • Main component responsible for conferring incomplete & short-term protection against infection • Relatively poor inducer of γ-IFN & TNF-α • An effective inducer of IL 12, which stimulates Th1 type response • Survival within macrophages associated with synthesis of stress induced proteins of different molecular weight - induces acid environment of pH< 4-limts antibiotic action
- 18. • Urease protect Brucellae in passage through stomach when acquired by the oral route - major way of infection in human brucellosis • Entering body - taken up by local tissue lymphocytes- transferred through regional lymphnodes into circulation - subsequently seeded throughout the body- tropism for the reticuloendothelial system.
- 19. Epidemiology • complex & it changes from time to time • Wide host range & resistance to environment & host immune system facilitate its survival • Major source of disease in humans - domesticated animals • Worldwide, reported incidence in endemic disease areas - <0.01 to >200 / 100,000 population • true incidence -unknown for most countries including India • True incidence may be 25 times higher than reported incidence due to misdiagnosis & underreporting
- 20. Global scenario • endemic - countries of Mediterranean basin, Arabian gulf, the Indian subcontinent & parts of Mexico & Central & South America • significant presence in rural/nomadic communities - close association with animals • Sheep, goats & their products - 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 - particularly problematic because B. abortus vaccines do not protect effectively against B. melitensis infection
- 21. • Recent isolation of distinctive strains of brucella from marine mammalsas well as humans - extended the ecological range of human brucellosis • Well-characterized occupational disease in shepherds, abattoir workers, veterinarians, dairy industry professionals & personnel in microbiologic laboratories • Males are affected more commonly than females • Human brucellosis affects all age groups
- 22. Indian Scenario • Established early in previous century & reported from almost all states • Mathur reported 8.5% seroprevalence of brucellosis among dairy personnel with the isolation of brucella strains from 7 cases of human brucellosis • In gujarat-8.5% prevalence of brucella agglutinins was recorded in human cases • In haryana, 34% prevalence of human brucellosis was recorded among veterinarians, attendants & compounders in contact with animals • Thakur et al revealed a prevalence rate of 4.97% in samples obtained from persons exposed to animals with markedly higher prevalence of 17.39% among field veterinarians
- 23. • Hemashettar et al - 24(8.2%) veterinary workers showed Brucella specific Abs in significant titres • Handa & coworkers identified 4(3.3%) cases with acute brucellosis in a group of 121 patients with PUO • Sen & co-workers identified 28 (6.8%) seropositive cases in a group of 414 patients with PUO • Kadri& co-workers identified 28 (0.8%) seropositive cases in a group of 3,532 patients with PUO • Prevalence of 3% was observed among patients attending Karnataka Medical College, Hubli • Mantur & colleagues reported 93 children with brucellosis identified from 5726 children in Bijapur during a period of 13 years • Seroprevalence was 1.6% by standard tube agglutination test (≥ 1:160) - Dx confirmed in 43 of these paediatric patients by isolation of B. melitensis
- 24. DMCH Scenario • Total no. of samples from Jan 09-Oct09 = 68 2 ½ mnths data (Aug-10Sep 09) • Total samples = 24 • Positive samples = 6 (25%) • Last positive on Oct 09
- 25. Name Age Sex Ward Date Widal Blood culture Rajesh kumar 37 y M Pvt 12/08/09 - NG Joginder kaur 73 y F MU 4 21/08/09 - NG Hans raj 32y M Skin 07/09/09 - - Neha jain 24 y F Pvt 20/09/09 To-320 Th-640 - Srdesh kumar M 10/10/09 - - Ajmer singh M 12/10/09 Th-40 -
- 26. Spectrum of Disease • Systemic disease - involve any organ or system of the body • 4 species responsible for most human cases: B. melitensis (found in sheep and goats), B. abortus (found in cattle), B. suis (found in swine) & B. canis (found in dogs) • Disease from marine species has also emerged • B. melitensis principal cause of human brucellosis worldwide • Sufficient data on virulence & clinical presentation of biotypes of B. melitensis lacking • separate biotypes that predominate in various regions such as type 1 in India & Spain, type 2 in northwestern Greece & type 3 in Turkey
- 28. • usually manifests as an acute (< 2 months) or subacute (2-12 months) febrile illness- may persist & progress to a chronically (> 1 year) • B. melitensis assoc. with ac infection , infections with other species usually subacute and prolonged • Ac form characterised by an undulating fever • t° remains normal during early part of the day & rises during evening
- 30. Lab Diagnosis • High degree of clinical suspicion • Travel history , h/o exposure to animals & exotic foods • In all cases a blood sample should be collected • Definite diagnosis of brucellosis is impossible without laboratory confirmation • Proper & prompt diagnosis is important, as Rx requires specific & prolonged antibiotics.
- 31. • isolation & identification , detection of Ag, demonstration of genome & Brucella specific antibodies • Blood culture definite proof of brucellosis but may not provide a +ve result for all patients even under ideal conditions • Brucellae are relatively slow growing & culture result may not become available for several days or weeks. • In ch disease, sensitivity of culture can be low. • Recently, higher rates of positive blood cultures (91% in ac & 74% in ch. brucellosis) along with the rapid confirmation of clinical diagnosis have been reported by lysis centrifugation technique
- 32. • Automated blood culture systems improved the speed of detection but are still too slow to make a rapid diagnosis • Bone marrow culture - considered the gold standard - in some studies,- results not universally reproducible-suggesting that the bacteraemia is as unpredictable as clinical manifestations in human brucellosis • Identification - standard classification tests, including Gram stain, a modified ZN stain, growth characteristics, oxidase activity, urease activity, H 2 S production (four days), dye tolerance such as basic fuchsin (1: 50000 and 1: 100000) & thionin (1:25000, 1:50000 and 1:100000) & seroagglutination
- 33. • Mantur & colleagues - recommended Gram stain morphology & modified ZN staining, coupled with the urease test, for rapid identification genus level • Only one report suggesting Ag detection by ELISA as an acceptable alternative to blood culture -sensitivity & specificity 100% and 99.2% respectively • PCR - fast & can be performed on any clinical specimen • Nucleic acid sequences targeted for the development of Brucella genus- specific PCR assays, include 16S rRNA, 16S-23S intergenic spacer region, omp2 & bcsp31 • Recently, Redkar et al described RT-PCR assays
- 34. • Unequivocal diagnosis of brucellosis requires isolation of the causal agent • Blood obtained early prior to antibiotic administration & need prolonged periods of incubation-failure rate high • PCR cannot be considered a routine diagnostic method yet. • These limitations make serology for Ab detection most useful • Abs- begin to appear at end of 1st wk of the disease,IgM →IgG • Serological tests like Rose Bengal Plate Agglutination Test (RBPT), standard tube agglutination test (SAT), Coombs test, immunocapture agglutination test, latex agglutination, CFT, ELISA, dipstick assay, fluorescence polarization assay (FPA), have all been applied in the diagnosis of human brucellosis
- 35. • RBPT is - a rapid screening test , sensitivity very high (>99%), specificity disappointingly low • Value as a screening test in high risk rural areas-where not always possible to perform tube agglutination titration test • serial dilution (1:2 through 1:64) of the serum sample - increase specificity & positive predictive value • Specificity increases when higher dilutions agglutinate & titres of 1:8 or 1:16 & above regarded as +ve-lower sensitivity • +ve result confirmed by a more specific test • Has value in rapid confirmation of neurobrucellosis, arthritis, epididymoorchitis, hydrocele due to Brucella
- 36. SAT • Wright and colleagues • most popular, used worldwide, it is easy to perform, does not need expensive equipments and training • measures total quantity of agglutinating Abs (IgM and IgG) • quantity of specific IgG is determined by treatment of the serum with 0.05M 2- mercaptoethanol (2ME), which inactivates the agglutinability of IgM • SAT titres above 1:160 are considered diagnostic in conjunction with a compatible clinical presentation • In endemic aresa, using a titre of 1:320 as cutoff may make test more specific • IgG Abs considered a better indicator of active infection than IgM • Rapid fall in level of IgG Abs is said to be prognostic of successful therapy.
- 37. • Almuneef and coworkers (2002) in Saudi Arabia found various levels of SAT antibodies in many clinically cured patients • Mantur and colleagues followed-up 79 patients diagnosed as having active brucellosis for different lengths of time and monitored for Brucella antibodies by SAT and 2ME agglutination • In most cases, Brucella SAT titres remained measurable, in spite of falling to low levels ranging from 1:160 to 1:640 despite an effective therapy and clinical cure. • Sustained drop in 2ME titres in 97.5% of cases -importance of the 2ME test for diagnosis in conjunction with the SAT, as well as for follow up brucellosis in Brucella -endemic countries
- 38. • Gazapo et al claimed that ELISA was an excellent method for follow up of brucellosis; however, author's clearly indicate that 2ME agglutination test is a useful assay, as it is inexpensive and technologically simple with stable reagents • Coombs test & immunocapture-agglutination tests have shown similar performance with higher sensitivity and specificity • ELISA reported to be most sensitive test for diagnosis of CNS brucellosis • Among newer serologic tests, ELISA appears to be the most sensitive
- 39. Treatment • WHO recommends for acute brucellosis in adults - rifampicin 600 to 900 mg & doxycycline100 mg BD x 6 wks • I/M streptomycin (1 g/day for 2-3 wks) + an oral tetracycline (2 g/day for 6wks) • Trimethoprim-sulfamethoxazole -triple regimens • Quinolones are an alternative • combinations of ciprofloxacin and ofloxacin have been tried clinically
- 40. Prevention • Control of disease in domestic livestock mainly by mass vaccination • B. abortus strain vaccine in cattle & B. melitensis strain Rev-1 vaccine in goats and sheep has resulted in the elimination or near-elimination of brucellosis in these animals • Studies are ongoing to develop an effective vaccine against B. suis • 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
- 41. • good clinical efficacy & cost effectiveness of vaccination, limited availability of vaccines & lack of awareness has led to the persistence of brucellosis in most areas including India.