Updated: 23.07.2004
Manual of Diagnostic Tests
and Vaccines for Terrestrial Animals
PART 2
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SECTION 2.4.
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Chapter 2.4.2.
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Summary
? - Index


CHAPTER 2.4.2.

CAPRINE AND OVINE BRUCELLOSIS
(excluding Brucella ovis)


 

SUMMARY

Brucella melitensis (biovars 1, 2 or 3) is the main causative agent of caprine and ovine brucellosis. Sporadic cases caused by B. abortus have been observed, but clinical disease is rare. Brucella melitensis appears to be endemic in the Mediterranean region, but infection is widespread world-wide. North America (except Mexico) is believed to be free from the agent, as are Northern Europe, South-East Asia, Australia and New Zealand.
 
Clinically, the disease is characterised by one or more of the following signs: abortion, retained placenta, orchitis, epididymitis and, rarely, arthritis, with excretion of the organisms in uterine discharges and in milk. Diagnosis depends on the isolation of Brucella from abortion material, udder secretions or from tissues removed at post-mortem. Presumptive diagnosis can be made by assessing specific cell-mediated or serological responses to Brucella antigens.
 
Brucella melitensis is highly pathogenic for humans, causing one of the most serious zoonoses in the world, and all infected tissues, cultures and potentially contaminated materials should be handled at a high biohazard containment level.
 
Identification of the agent: Presumptive evidence of Brucella is provided by the demonstration, by modified acid-fast staining of organisms, of Brucella morphology in abortion material or vaginal discharge, especially if supported by serological tests. The recently developed polymerase chain reaction methods provide additional means of detection. Whenever possible, Brucella spp. should be isolated using plain or selective media by culture from uterine discharges, aborted fetuses, udder secretions or selected tissues, such as lymph nodes, testes or epididymes. Species and biovars should be identified by phage lysis, and by cultural, biochemical and serological criteria.
 
Serological and allergic skin tests: The rose bengal plate agglutination and complement fixation tests are usually recommended for screening flocks and individual animals. The serum agglutination test is not considered to be reliable for use in small ruminants. For pooled samples, there are no useful tests such as the milk ring test in cattle. The brucellin allergic skin test can be used as a screening or complementary test in unvaccinated flocks, provided that a purified, lipopolysaccharide (LPS)-free, standardised antigen preparation is used. Results must then be interpreted in relation to the clinical signs, history, and results of serological or cultural examination.
 
Requirements for vaccines and diagnostic biologicals: Brucella melitensis strain Rev.1 remains the reference vaccine to immunise sheep and goats at risk of infection from B. melitensis and to which any other vaccines should be compared. Production of Brucella antigens or Rev.1 vaccine is based on a seed-lot system. Seed cultures to be used for antigens for serological and allergic skin tests and for vaccines should originate from reference centres. They must conform to minimal standards for viability, smoothness, residual infectivity and immunogenicity, if applicable. Brucellin preparations for the intradermal test must be free of smooth lipopolysaccharide and must not produce nonspecific inflammatory reactions or interfere with serological tests. Antigens for serological diagnosis must be prepared from smooth strains of B. abortus, strain 1119-3 or strain 99 and comply with minimum standards for purity, sensitivity and specificity.
 

A. INTRODUCTION

Brucellosis in sheep and goats (excluding Brucella ovis infection) is primarily caused by one of the three biovars of B. melitensis. Sporadic cases caused by B. abortus or B. suis have been observed in sheep and goats, but clinical signs are rare. Pathologically and epidemiologically, B. melitensis infection in sheep and goats is very similar to B. abortus infection in cattle (see Chapter 2.3.1. Bovine brucellosis). In most circumstances, the primary route of transmission of Brucella is the placenta, fetal fluids and vaginal discharges expelled by infected ewes and goats when they abort or have a full-term parturition. Shedding of Brucella is also common in udder secretions and semen, and Brucella may be isolated from various tissues, such as lymph nodes from the head and those associated with reproduction, and from arthritic lesions (2).
 
Brucellosis has also been reported in the one-humped camel (Camelus dromedarius) and in the two-humped camel (C. bactrianus), related to contact with large and small ruminants infected with B. abortus or B. melitensis. In addition, brucellosis due to B. abortus has been observed in the domestic buffalo (Bubalus bubalus), American and European bison (Bison bison; Bison bonasus), yak (Bos grunniens), elk/wapiti (Cervus elaphus) and also occurs in the African buffalo (Syncerus caffer) and various African antelope species. The manifestations of brucellosis in these animals are similar to those in cattle or sheep and goats.
 
The World Health Organization (WHO) laboratory biosafety manual classifies Brucella (and particularly B. melitensis) in Risk group III. Brucellosis is readily transmissible to humans, causing acute febrile illness – undulant fever – which may progress to a more chronic form and can also produce serious complications affecting the musculo–skeletal, cardiovascular, and central nervous systems. Infection is often due to occupational exposure and is essentially acquired by the oral, respiratory, or conjunctival routes, but ingestion of dairy products constitutes the main risk to the general public. There is an occupational risk to veterinarians and farmers who handle infected animals and aborted fetuses or placentae. Brucellosis is one of the most easily acquired laboratory infections, and strict safety precautions should be observed when handling cultures and heavily infected samples, such as products of abortion. Specific recommendations have been made for the safety precautions to be observed with Brucella-infected materials (for further details see Chapter I.1.6. Human safety in the veterinary microbiology laboratory and refs 1, 28, 67 and 68 of Chapter 2.3.1. Bovine brucellosis,). Laboratory manipulation of live cultures or contaminated material from infected animals is hazardous, as is handling large volumes of Brucella, and must be done under containment level 3 or higher conditions, as outlined in Chapter I.1.6., to minimise occupational exposure.
 
Genetic and immunological evidence indicates that all members of the Brucella genus are closely related and it has been proposed (but not yet accepted by the Taxonomy Subcommittee) that the genus contains a single species of which the classical species (B. abortus, B. melitensis, etc.) would be mere biovars (for a review see Chapter 2.3.1., ref. 36). Nevertheless, there are real differences in host preference and epidemiology displayed by the major variants, as well as molecular evidence of genomic variation. From a practical point of view, it is convenient to maintain the classification into the six classical nomenspecies: Brucella abortus, B. melitensis, B. suis, B. neotomae, B. ovis and B. canis. The first three of these are subdivided into biovars based on cultural and serological properties (see Tables 1 and 2 in Chapter 2.3.1.). Strains of Brucella have been isolated in the last decade from marine mammals that cannot be ascribed to any of the above-recognised species. Investigations are continuing to establish their correct position in the taxonomy of the genus and it is proposed that they could be classified into two new species, B. cetaceae and B. pinnipediae (see Chapter 2.3.1. refs 10 and 18). Finally, Brucella shows close genetic relatedness to some plant pathogens and symbionts of the genera Agrobacterium and Rhizobium, as well as animal pathogens (Bartonella) and opportunistic or soil bacteria (Ochrobactrum).
 

B. DIAGNOSTIC TECHNIQUES

1.   Identification of the agent
 
     Please refer to Chapter 2.3.1. Bovine brucellosis.
 
2.   Serological tests
 
     In situations where bacteriological examination is not practicable, diagnosis of Brucella infection must often be based on serological methods (2, 18). In routine tests, anti-Brucella antibodies are detected in serum. The most widely used serum-testing procedures for the diagnosis of smooth Brucella infections in sheep and goats are the buffered Brucella antigen tests (BBAT), i.e. the card and rose bengal (RB) plate agglutination tests, which are essentially the same, and the complement fixation test (CFT). The bulk milk ring test, which has been very useful in cattle, is ineffective in small ruminants.
 
     In small ruminants, the RB test (RBT) and the CFT are the most widely used methods (17) and are the only prescribed tests. The RBT is not completely specific, but appears to be adequate as a screening test for detecting infected flocks or for guaranteeing the absence of infection in brucellosis-free flocks. However, due to the relative lack of sensitivity of both tests, discrepancies between results obtained using the RBT and the CFT are not rare in infected sheep and goats (6). The results of the two tests should therefore be considered simultaneously to increase the likelihood of detecting infected individuals and to improve control of the disease in areas where it has not been completely eradicated (1, 4, 6). When, for practical or economic reasons, the CFT cannot be used simultaneously with the RBT in eradication programmes, it is recommended to improve the sensitivity of the RBT by using 75 µl of serum and 25 µl of antigen in place of an equal volume of each. This simple modification increases RBT sensitivity and minimises the discrepancies between RBT and CFT results (6). Because Rev.1-induced antibodies cannot be discriminated from wild type, brucellosis serological tests should be interpreted in the context of the vaccination status in the flock. In addition these tests are not specific enough to discriminate serological reactions due to B. melitensis from the false-positive reactions (FPSR) due to cross-reacting bacteria such as Yersinia enterocolitica O:9.
 
     Good diagnostic results have been obtained in sheep and goats with indirect or competitive enzyme-linked immunosorbent assays (ELISAs) using various antigens, but generally those with a high content of smooth lipopolysaccharide (LPS) are the most reliable. These ELISAs provide similar or better sensitivity than both RBT and CFT, but like the classical tests, ELISAs are unable to differentiate infected animals from animals recently vaccinated with the Rev.1 vaccine (19) or animals infected with cross-reacting bacteria. A highly immunogenic periplasmic protein from B. abortus (20) and B. melitensis (11) has been applied to brucellosis diagnosis in different host species. Indirect and competitive ELISAs with this antigen could be sensitive and specific tests for diagnosing B. melitensis infection in sheep and have been reported to be useful in differentiating Rev.1 vaccinated from infected animals (10, 13). All these ELISAs have potential advantages in sensitivity and specificity with respect to both RBT and CFT, but a great deal of work is still required on standardisation of reagents to be used for diagnosing B. melitensis infection in sheep and goats (15).
 
   Reference sera
 
     The primary reference serum for standardising RBT and CFT in sheep and goats is the OIE International Standard Serum (OIEISS; see Chapter 2.3.1. Bovine brucellosis).
 
   Production of cells
 
     Please refer to Chapter 2.3.1. Bovine brucellosis. Brucella abortus biovar 1 strains 99 or 1119 are the only strains recommended for the preparation of RBT and CFT in sheep and goats.
 
      a)    Buffered Brucella antigen test (a prescribed test for international trade)
 
          Rose bengal plate agglutination test
 
            Please refer to Chapter 2.3.1. Bovine brucellosis.
 
            •    Antigen production
 
            Please refer to Chapter 2.3.1. Bovine brucellosis. Note that RB antigen made with B. abortus is usually used to test for B. melitensis.
 
            •    Test procedure
 
            Please refer to Chapter 2.3.1. Bovine brucellosis.
 
      b)    Complement fixation test (a prescribed test for international trade)
 
            •    Antigen production
 
            Please refer to Chapter 2.3.1. Bovine brucellosis. Note that CF antigen made with B. abortus is usually used to test for B. melitensis.
 
            •    Test procedure
 
            Please refer to Chapter 2.3.1. Bovine brucellosis.
 
3.   Other tests
 
     a)   Brucellin skin test
 
          An alternative diagnostic test is the brucellin skin test, which can be used for screening unvaccinated flocks, provided that a purified (free of sLPS) and standardised antigen preparation (e.g. brucellin INRA) is used.
 
          The brucellin skin test has a high sensitivity for the diagnosis of B. melitensis infection in small ruminants and, in absence of vaccination, is considered one of the most specific diagnostic tests (2, 4, 15, 17). This test is of particular value for the interpretation of FPSR due to infection with cross-reacting bacteria (FPSR affected animals are always negative in the skin test), especially in brucellosis-free areas.
 
          Despite its high sensitivity not all infected animals show positive skin test responses to brucellin and, moreover, Rev.1 vaccinated animals can react in this test for years (15). Therefore this test cannot be recommended either as the sole diagnostic test or for the purposes of international trade.
 
          To obtain suitable results it is essential to use standardised brucellin preparations that do not contain sLPS, as this antigen may provoke antibody-mediated inflammatory reactions or induce antibodies that interfere with subsequent serological screening. One such preparation is brucellin INRA, which is prepared from a rough strain of B. melitensis that is commercially available (Brucellergčne OCB®, Synbiotics Europe, 2 rue Alexander Fleming, 69007 Lyon, France).
 
             Test procedure
 
          i)   A volume of 0.1 ml of brucellin is injected intradermally into the lower eyelid.
 
          ii)   The test is read after 48 hours.
 
          iii)   Any visible or palpable reaction of hypersensitivity, such as an oedematous reaction leading to an elevation of the skin or thickening of the eyelid (> or = 2 mm), should be interpreted as a positive reaction.
 
          Although in the absence of vaccination the brucellin intradermal test is one of the most specific tests in brucellosis, diagnosis should not be made exclusively on the basis of positive intradermal reactions and should be supported by adequate serological tests. The intradermal inoculation of brucellin might induce a temporary anergy in the cellular immune response. Therefore an interval of 6 weeks is generally recommended between two tests repeated on the same animal.
 
     b)   Native hapten tests
 
          The native hapten-based gel precipitation tests (see Chapter 2.3.1.) are also of interest in sheep and goats as they are very specific for discriminating the serological responses of infected animals (positive) from those induced in Rev.1 vaccinated animals (usually negative). (The detailed procedure could be obtained from the Departamento de Sanidad Animal, Servicio de Investigacion Agraria/DGA, Apartado 727, 50080 Zaragoza, Spain.)
 
          The optimal diagnostic sensitivity (around 90%) is obtained in the double gel diffusion (DGD) or reverse radial immunodiffusion tests for sheep and goats, respectively (14, 19).
 

C. REQUIREMENTS FOR VACCINES AND DIAGNOSTIC BIOLOGICALS

C1.   Brucellin
 
     Please refer to Chapter 2.3.1. Bovine brucellosis.
 
C2.   Vaccines
 
Brucella melitensis strain Rev.1 vaccine
 
     The most widely used vaccine for the prevention of brucellosis in sheep and goats is the Brucella melitensis Rev.1 vaccine, which remains the reference vaccine with which any other vaccines should be compared. The Rev.1 vaccine is used as a freeze-dried suspension of live B. melitensis biovar 1 Rev.1 strain for the immunisation of sheep and goats. It is normally given to lambs and kids aged between 3 and 6 months as a single subcutaneous injection. The standard dose is between 0.5 x 109 and 2.0 x 109 viable organisms. The subcutaneous vaccination induces strong interferences in serological tests and should not be recommended in combined eradication programmes (14, 19). However, when this vaccine is administered conjunctivally to lambs and kids aged between 3 and 6 months it produces a similar protection without inducing a persistent antibody response, thus facilitating the application of eradication programmes combined with vaccination (14, 19). Care must be taken when using Rev.1 vaccine to avoid the risk of contaminating the environment or causing human infection. In many developing countries and endemic areas, vaccination of the whole population has to be considered as the best option for the control of the disease (5). However, Rev.1 vaccine is known to often cause abortion and excretion in milk when animals are vaccinated during pregnancy, either with a full or reduced dose (5). These side-effects are considerably reduced when adult animals are vaccinated conjunctivally (full dose) before mating or during the last month of pregnancy. Therefore, when mass vaccination is the only means of controlling the disease, a vaccination campaign should be recommended using the standard dose of Rev.1 administered by the conjunctival route when the animals are not pregnant or during the lambing season (5).
 
     The subcutaneous vaccination of young animals and the vaccination of adult animals, even at reduced doses, may lead to long-term persistence of vaccinal antibodies in a significant proportion of vaccinated animals that creates serious interferences in the serological diagnosis of brucellosis. As indicated above, conjunctival vaccination minimises these problems. Therefore, the serological diagnosis of brucellosis should take into account the vaccinal state of the herd and the overall frequency distribution of antibody titres detected in the group of animals tested.
 
1.   Seed management
 
     a)   Characteristics of the seed
 
          Brucella melitensis biovar 1 strain Rev.1 original seed for vaccine production can be obtained commercially (obtainable from Institut National de la Recherche Agronomique (INRA), Laboratoire de Pathologie Infectieuse et Immunologie, 37380 Nouzilly, France). A European reference Rev.1 strain that possesses the characteristics of the Rev.1 original seed has been recently produced by the European Pharmacopoeia (obtainable from the European Pharmacopoeia, BP 907, 67029 Strasbourg Cedex 1, France).
 
          Production of Brucella live vaccines is based on the seed-lot system described above (Section B.2.) for RBT and CFT antigens. Strains should be cultured in a suitable medium. Strain Rev.1 must conform to the characteristics of B. melitensis biovar 1, except that it should grow more slowly. Additionally, when incubated in air (atmospheres containing CO2 alter the results) at 37°C, it should grow on agar containing streptomycin (2.5 µg/ml), and it should be inhibited by the addition to a suitable culture medium of sodium benzylpenicillin (3 µg [5 International Units (IU)]/ml), thionin (20 µg/ml) or basic fuchsin (20 µg/ml). Recently, polymerase chain reaction amplification and molecular techniques have been used to further characterise the vaccine (3, 12). It must also conform to the characteristics of residual virulence and immunogenicity in mice of the original seed.
 
     b)   Method of culture
 
          Serum–dextrose agar, and trypticase–soy agar, to which 5% serum or 0.1% yeast extract may be added, are among the solid media that have been found to be satisfactory for propagating the Rev.1 strain (2, 21). Rev.1 strain does not grow well on potato agar.
 
          For vaccine production, Rev.1 may be grown under conditions similar to those described for S99 and S1119-3 (see Chapter 2.3.1.), except that Rev.1 generally needs 3–5 days to grow, the phenol saline is replaced by a freeze-drying stabiliser, and the organisms are not killed but are stored at 4°C while quality control examinations are carried out as described below. Moreover, the specific requirements for Rev.1 vaccine production recommend that: each seed lot (i.e. the culture used to inoculate medium for vaccine production) should be no more than three passages removed from an original seed culture and that the harvest of a vaccine lot should be no more than three passages from a seed lot or an original seed. The original seed culture should always be checked for the absence of dissociation before use. The recommended method for preparing seed material is given in ref. 2. The following freeze-drying stabiliser (sterilised by filtration) is of proven value: enzymatic digest of casein (2.5 g); sucrose (5 g); sodium glutamate (1 g); distilled water (100 ml).
 
     c)   Validation as a vaccine
 
          Numerous independent studies have confirmed the value of B. melitensis strain Rev.1 as a vaccine for protecting sheep and goats from brucellosis. Its virulence is unchanged after passage through pregnant sheep and goats. Abortions may result when the Rev.1 vaccine is inoculated into pregnant ewes or goats. The vaccine-induced abortions are not avoided using reduced doses, and doses as low as 106 used either subcutaneously or conjunctivally, have been demonstrated to induce abortions and milk excretion of the vaccine virus (5).
 
          A Rev.1 vaccine is efficient if it possesses the characteristics of the Rev.1 original strain, i.e. those of B. melitensis biovar 1 reference strain 16M (ATCC No. 23456), except those specific for the strain Rev.1 (2, 17), and if it proves to be satisfactory with respect to immunogenicity and residual virulence (8) (see below).
 
2.   Method of manufacture (2, 21)
 
     For production of B. melitensis strain Rev.1 vaccine, the procedures described above for antigens (2) can be used except that the cells are collected in a freeze-drying stabiliser and deposited by centrifugation. The yield from one fermenter run or the pooled cells from a batch of Roux flask cultures inoculated on the same occasion from the same seed lot constitutes a single harvest. More than one single harvest may be pooled to form the final bulk that is used to fill the final containers of a batch of vaccine. Before pooling, each single harvest must be checked for purity, cell concentration, dissociation and identity. The volume of the final bulk is adjusted by adding sufficient stabiliser so that a dose contains an appropriate number of viable organisms. After adjusting the cell concentration of the final bulk, tests for identity, dissociation and absence of contaminating organisms are conducted (see below).
 
3.   In-process control
 
     In-process checks should be made on the growth of Rev.1 vaccine from either solid or liquid medium to verify identity and to ensure purity and freedom from dissociation to rough forms during preparation of seed lots, single harvests, final bulks and the final (filling) lots. At least 99% of cells in seed lots and 95% of cells in final lots should be in the smooth phase.
 
     Cell concentration should be estimated on the bulks and precisely determined on final lots. Immunogenicity and the residual virulence (50% persistence time or 50% recovery time) should also be determined on seed lots and final lots.
 
4.   Batch control
 
     With freeze-dried vaccine, the control tests should be conducted on the vaccine reconstituted in the form in which it will be used.
 
     a)   Sterility (or absence of extraneous microorganisms)
 
          The Rev.1 vaccine should be checked for bacterial and fungal contamination as prescribed in Chapter I.1.5.
 
     b)   Safety
 
          The Rev.1 vaccine is a virulent product per se, and it should keep a minimal virulence to be efficient (see Section C2.4.c. in Chapter 2.3.1.). A safety test may be performed on sheep and goats when a new manufacturing process is started and when a modification in the innocuousness of the vaccine preparation is expected. This control should be done as follows: the test uses 12 ewe lambs and 12 goat kids, aged 4–6 months. Six young females of each species are injected with one or three recommended doses. Each lot of six young females are kept separately. All animals are observed for 21 days. No significant local or systemic reaction should occur. If, for a given dose and route of administration, this test gives good results on a representative batch of the vaccine, it does not have to be repeated routinely on seed lots or vaccine lots prepared with the same original seed and with the same manufacturing process.
 
     c)   Potency
 
          A Rev.1 vaccine is efficient if it possesses the characteristics of the Rev.1 original strain, i.e. if it is satisfactory with respect to identity, smoothness, immunogenicity, and residual virulence (9). Batches should also be checked for the number of viable organisms.
 
             Identity
 
               The reconstituted Rev.1 vaccine should not contain extraneous microorganisms. Brucella melitensis present in the vaccine is identified by suitable morphological, serological and biochemical tests and by culture: when incubated in air at 37°C, Rev.1 strain is inhibited by addition to the suitable culture medium of 3 µg (5 IU) per ml of sodium benzyl-penicillin, thionin (20 µg/ml) or basic fuchsin (20 µg/ml); the strain grows on agar containing 2.5 µg per ml of streptomycin.
 
             Smoothness (determination of dissociation phase)
 
               Please refer to Chapter 2.3.1. Bovine brucellosis.
 
               Sometimes slight and difficult to observe differences, can be seen in the size of Rev 1 colonies. The small colonies (1–1.2 mm in diameter) are typical for Rev.1, but larger Rev. 1 colonies can appear depending on the medium used, the amount of residual moisture in the incubator atmosphere, and the presence or absence of CO2. The frequency of variation in colony size occurs normally at a ratio of 1 large to 103 small colonies. Both Rev.1 variants are of the S (smooth) type. To avoid an increase in this colony size variation along successive passages, it is important to always select small colonies for preparation of seed lots.
 
             Enumeration of live bacteria
 
               Please refer to Chapter 2.3.1. Bovine brucellosis.
 
             Residual virulence (50% persistence time or 50% recovery time) (7, 16)
 
               The same technical procedures indicated for 50% recovery time (RT50) calculation of S19 vaccine (see Chapter 2.3.1.) have to be applied for Rev.1, except that B. abortus S19 seed lot or batch to be tested (test vaccine) and the S19 original seed culture (used as a reference strain), respectively, are replaced by the corresponding B. melitensis Rev.1 seed lot or batch to be tested (test vaccine) and the B. melitensis Rev.1 original seed culture as the reference strain. For the reference original Rev.1 strain, RT50 and confidence limits are around 7.9 ± 1.2 weeks. A given Rev.1 vaccine seed lot or batch should keep similar residual virulence to be efficient.
 
          If this test has been done with good results on a representative batch of the test vaccine, it does not have to be repeated routinely on other vaccine lots prepared from the same seed lot and with the same manufacturing process.
 
             Immunogenicity in mice
 
               The same technical procedures indicated for immunogenicity calculation of S19 vaccine (see Chapter 2.3.1.) have to be applied for Rev.1, except that B. abortus S19 seed lot or batch to be tested (test vaccine) and the B. abortus S19 original seed culture (used as a reference strain), respectively, are replaced by the corresponding B. melitensis Rev.1 seed lot or batch to be tested (test vaccine) and the B. melitensis Rev.1 original seed culture as the reference strain.
 
               Conditions of the control experiment are satisfactory when: i) the response in unvaccinated mice (mean of Y) is at least of 4.5; ii) the response in mice vaccinated with the reference Rev.1 vaccine is lower than 2.5; and iii) the standard deviation calculated on each lot of six mice is lower than 0.8.
 
          If this test has been done with good results on a representative batch of the test vaccine, it does not have to be repeated routinely on other vaccine lots prepared from the same seed lot and with the same manufacturing process.
 
     d)   Duration of immunity
 
          It is accepted that subcutaneous or conjunctival vaccination with standard doses of Rev.1 confers a solid and durable immunity in sheep and goats. However, growing field evidence shows that the immunity conferred declines with time, and revaccination could be advisable in endemic areas.
 
          The use of reduced doses of Rev.1 produces a less efficient immunity, while side-effects, such as antibody responses or induction of abortion, are not fully avoided.
 
     e)   Stability
 
          Strain Rev.1 vaccine prepared from seed stock from appropriate sources is stable in characteristics provided that the in-process and batch control requirements described above are fulfilled, and shows no tendency to reversion to virulence. The lyophilised vaccine shows a gradual loss of viable count, but should retain its potency for the recommended shelf life. Allowance for this phenomenon is normally made by ensuring that the viable count immediately following lyophilisation is well in excess of the minimum requirement. Maintenance of a cold chain during distribution of the vaccine will ensure its viability.
 
     f)   Preservatives
 
          Antimicrobial preservatives must not be used in live Rev.1 vaccine. For preparation of the freeze-dried vaccine, a stabiliser as described in Section C2.4.f. of Chapter 2.3.1. is recommended.
 
     g)   Precautions (hazards)
 
          Brucella melitensis Rev.1, although an attenuated strain, is still capable of causing disease in humans. The cell cultures and suspensions must be handled under appropriate conditions of biohazard containment (see Chapter I.1.6.). Reconstitution and subsequent handling of the reconstituted vaccine should be done with care to avoid accidental injection or eye or skin contamination. Vaccine residues and injection equipment should be decontaminated with a suitable disinfectant (phenolic, iodophor or aldehyde formulation) at a recommended concentration. Medical advice should be sought in the event of accidental exposure. The efficacy of the antibiotic treatment of infections caused by Rev.1 in humans has not been adequately established. If Rev.1 contamination occurs, a combined treatment with doxicycline plus rifampicin could be recommended.
 
5.   Tests on the final product
 
     a)   Safety
 
          See Section C2.4.b. of Chapter 2.3.1.
 
     b)   Potency
 
          For the freeze-dried vaccine, the potency must be determined on the final product. The tests are as described in Section C2.4.c. of Chapter 2.3.1.
 

REFERENCES

1.   Alton G.G. (1990). Brucella melitensis. In: Animal Brucellosis, Nielsen K.H. & Duncan J.R., eds. CRC Press, Boca Raton, Florida, USA, 383–409.
 
2.   Alton G.G., Jones L.M., Angus, R.D. & Verger J.M. (1988). Techniques for the Brucellosis Laboratory. INRA, Paris, France.
 
3.   Bardenstein S., Mandelboim M., Ficht T.A., Baum M. & Banai M. (2002). Identification of the Brucella melitensis vaccine strain Rev.1 in animals and humans in Israel by PCR analysis of the PstI site polymorphism of its omp2 gene. J. Clin. Microbiol., 40, 1475–1480.
 
4.   Blasco J.M. (1992). Diagnosis of Brucella melitensis infection in small ruminants. In: Prevention of Brucellosis in the Mediterranean Countries, Plommet M., ed. Pudoc Scientific, Wageningen, The Netherlands, 272–277.
 
5.   Blasco J.M. (1997). A review on the use of B. melitensis Rev 1 vaccine in adult sheep and goats. Prev. Vet. Med., 31, 275–283.
 
6.   Blasco J.M., Garin-Bastuji B., Marin C.M., Gerbier G., Fanlo J., Jimenez De Bagues M.P. & Cau C. (1994). Efficacy of different rose bengal and complement fixation antigens for the diagnosis of Brucella melitensis infection in sheep and goats. Vet. Rec., 134, 415–420.
 
7.   Bosseray N. (1991). Brucella melitensis Rev.1 attenuated vaccine: Stability of markers, residual virulence and immunogenicity in mice. Biologicals, 19, 355–363.
 
8.   Bosseray N. (1992). Le vaccin Rev.1: dérive des caractčres d’immunogénicité et de virulence indépendante des marqueurs classiques. In: Prevention of Brucellosis in the Mediterranean Countries, Plommet M., ed. Pudoc Scientific, Wageningen, The Netherlands, 182–186.
 
9.   Bosseray N. (1993). Control methods and thresholds of acceptability for anti-Brucella vaccines. Dev. Biol. Stand., 79, 121–128.
 
10.   Cloeckaert A., Baucheron S., Vizcaino N. & Zygmunt M.S. (2001). Use of recombinant BP26 protein in serological diagnosis of Brucella melitensis infection in sheep. Clin. Diag Lab. Immunol., 8, 772–775.
 
11.   Cloeckaert A., Debbarh H.S., Vizcaino N., Saman E., Dubray G. & Zygmunt M.S. (1996). Cloning, nucleotide sequence and expression of the Brucella melitensis bp26 gene coding for a protein immunogenic in infected sheep. FEMS Microbiol. Lett., 140, 139–144.
 
12.   Cloeckaert A., Grayon M. & Grépient O. (2002). Identification of Brucella melitensis vaccine strain Rev.1 by PCR-RFLP based on a mutation in the rpsL gene. Vaccine, 20, 2546–2550.
 
13.   Debbarh H.S., Zygmunt M.S., Dubray G. & Cloeckaert A. (1996). Competitive ELISA using monoclonal antibodies to the Brucella melitensis bp26 protein to evaluate antibody response in infected and B. melitensis Rev1 vaccinated sheep. Vet. Microbiol., 53, 325–337.
 
14.   Díaz-Aparicio E., Marín C., Alonso B., Aragón V., Pérez S., Pardo M., Blasco J.M., Díaz R. & Moriyón I. (1994). Evaluation of serological tests for diagnosis of Brucella melitensis infection of goats. J. Clin. Microbiol., 32, 1159–1165.
 
15.   Garin-Bastuji B., Blasco J.M., Grayon M. & Verger J.M. (1998). Brucella melitensis infection in sheep: present and future. Vet. Res., 29, 255–274.
 
16.   Grillo M.J., Bosseray N. & Blasco J.M. (2000). In vitro markers and biological activity in mice of seed lot strains and commercial Brucella melitensis Rev.1 and Brucella abortus B19 vaccines. Biologicals, 28, 119–127.
 
17.   Joint Food and Agriculture Organization of the United Nations/World Health Organization Expert Committee on Brucellosis (1986). Technical Report Series 740, Sixth Report. WHO, Geneva, Switzerland.
 
18.   MacMillan A. (1990). Conventional serological tests. In: Animal Brucellosis, Nielsen K.H. & Duncan J.R., eds. CRC Press, Boca Raton, Florida, USA, 153–197.
 
19.   Marin C.M., Moreno E., Moriyon I., Diaz R. & Blasco J.M. (1999) Performance of competitive and indirect enzyme-linked immunosorbent assays, gel immunoprecipitation with native hapten polysaccharide, and standard serological tests in diagnosis sheep brucellosis. Clin. Diagn. Lab. Immunol., 6, 269–272.
 
20.   Rossetti O., Arese A., Boschiroli L. & Cravero S. (1996). Cloning of Brucella abortus gene and characterisation of expressed 26 kilodalton periplasmic protein: potential use for diagnosis. J. Clin. Microbiol., 34, 165–169.
 
21.   World Health Organization (1977). Requirements for Brucella melitensis strain Rev.1 vaccine (Live – for veterinary use). World Health Organization (WHO) Technical Report Series No. 610, 28th Report, Annex 4. WHO, Geneva, Switzerland, 85–97.
 

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NB: There are OIE Reference Laboratories for Caprine and ovine brucellosis (excluding Brucella ovis) (please consult the OIE Web site at: http://www.oie.int/eng/OIE/organisation/en_LR.htm).

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