Chapter 2.4.2. – Caprine and ovine brucellosis (excluding Brucella ovis)
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 cases of natural infection are rare in sheep and goats. Brucellamelitensisis 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 and Central 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 (PCR) 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, spleen, uterus, testes and epididymes. Species and biovars should be identified by phage lysis, and by cultural, biochemical and serological criteria. Polymerase chain reaction (PCR)has recently been introduced as a complementary identification method based on specific genomic sequences.
Serological and allergic skin tests: The rose bengal plate agglutination test(RBT) and the complement fixation test(CFT) tests are usually recommended for screening flocks and individual animals. The standard serum agglutination test is not considered to be reliable for use in small ruminants. The indirect enzyme-linked immunosorbent assay(ELISA)can also be used for screening purposes and the native hapten gel precipitation test can be used to increase diagnostic specificity in vaccinated populations. 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 RBT and CFT 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. Antigens for indirect ELISA and gel precipitation tests are prepared from the B.melitensis biovar 1 reference strain 16M.
A. introduction
Brucellosis in sheep and goats(excluding Brucella ovis infection) is primarily caused by one of the three biovars of B.melitensis. Sporadic infections caused by B.abortus or B.suis have been observed in sheep and goats, but such cases 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[BGB1]). 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, spleen and organs associated with reproduction (uterus, epididymides and testes), and from arthritic lesions (23).
Brucella melitensis infection in cattle and in other domestic and wild sensitive species (see Chapter 2.3.1) is not rare when these species are reared in close contact with sheep and goats in enzootic areas. The manifestations of brucellosis in these animals are similar to those in cattle or sheep and goats.
The World Health Organization (WHO) laboratorybiosafety 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 placentas. 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 productsof abortion. Specific recommendations have been made for the safety precautions to be observed with Brucella-infected materials(for further details see Chapter 1.1.6 and refs 12, 2826, 67 83 and 68 85 of Chapter 2.3.1). 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 1.1.6, 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[BGB2])42. Nevertheless, in 2005, based on relevant differences in host preference and epidemiology displayed by the major variants, as well as molecular evidence of genomic variation, the International Committee on Systematics of Prokaryotes, Subcommittee on the Taxonomy of Brucellatook a clear position to return to pre-1986 Brucella taxonomy; the consequences of this imply the re-approval of the six Brucella nomenspecies with recognised biovars. The classical names related to the six Brucella nomenspecies are published in the Approved Lists of Bacterial Names, 1980, and the designated type strains are attached to these published names:Brucella abortus, B.melitensis, B.suis, B.neotomae, B.ovis and B.canis[BGB3]( 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[BGB4]). 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 refs10 13 and 1825). 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).[BGB5]
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, 183, 21). 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 RBT and the CFT are the most widely used methods (1720) and are the only tests prescribed for international trade. The RBT is not completely specific, but is 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 (67). 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, 62, 5, 7). 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 (67). Because antibodies induced after Rev.1 vaccination cannot be differentiated in both tests from those induced by B.melitensis infection, RBT and CFT results should be carefully interpreted according to the vaccination status in the flock. In addition, both 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 the ELISAs that use antigens with a high content of smooth lipopolysaccharide (sLPS) are the most adequate. These tests provide similar (competitive ELISA – C-ELISA) or better (indirect ELISA – I-ELISA) sensitivity than both RBT and CFT, but like the classical tests, both ELISAs areunable to differentiate B.melitensis infected animals from those recently vaccinated with the Rev.1 vaccine (1922) or infected with cross-reacting bacteria. I- and C-ELISAs with a highly immunogenic periplasmic protein from B.abortus (2024) and B.melitensis (1112) have been applied in sheep and reported to be promising in differentiating Rev.1 vaccinated from B.melitensis infected animals (10, 1311, 14). Some of these ELISAs have potential advantages in sensitivity and/or specificity with respect to both RBT and CFT, but the standardisation of reagents is still required (1518).
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[BGB6]).
- Production of cells
Please refer to Chapter 2.3.1 Bovine brucellosis[BGB7]. 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 (Rose rose bengal plate agglutination test) (a prescribed test for international trade)
Please refer to Chapter 2.3.1 Bovine brucellosis[BGB8].
Antigen production
Please refer to Chapter 2.3.1 Bovine brucellosis[BGB9]. Note that RB antigen made with B.abortus is usually used to test for B.melitensis. The standardisation of RB antigen, as it is prescribed in Chapter 2.3.1, provides a sufficient sensitivity to the RBT for international trade purpose. Moreover, it guarantees an adequate specificity in free areas where a lot of FPSR occurbecause of cross-reacting bacteria such as Yersinia enterocolitica O:9. However this standardisation is probably the main cause of the reduced sensitivity of some RB antigen batches and of the discrepancies with the CFT (67). Therefore, when RBT is used in eradication programmes in endemic areas, it could be advisable to adjust the RB antigen titre in order to detect the highest OIEISS dilution without affecting the specificity of the test. The discrepancies with the CFT can also be minimised by using 75µl of serum and 25µl of antigen in place of an equal volume of each as mentioned in the standard test procedure.
Test procedure
Please refer to Chapter 2.3.1 Bovine brucellosis[BGB10].
b)Complement fixation test (a prescribed test for international trade)
•Antigen production
Please refer to Chapter 2.3.1 Bovine brucellosis[BGB11]. 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[BGB12].
c)Enzyme-linked immunosorbent assays
Several variations of the I-ELISA have been described employing different antigen preparations, antiglobulin-enzyme conjugates, and substrate/chromogens. Several commercial I-ELISAs are available but to date there is no internationally recognised procedure for standardising those tests for use for international trade purpose in small ruminant brucellosis.
The test method described below is an example of a test whose sensitivity and specificity are appropriate for use in eradication programmes (Alonso, 191, 22).
The antigen-coating buffer is phosphate buffered saline (PBS),10mM, pH7.2 (50ml of PBS stock solution composed of 28.0ml of 0.2M sodium dihydrogen phosphate [NaH2PO4.2H2O; 31.8g/1000ml]and 72.0ml of 0.2M disodium hydrogen phosphate [Na2HPO4.7H2O; 53.6g/1000ml]in 950ml of distilled or deionised water added with 8.0g of sodium chloride [NaCl]). Keep in the cold, and do not add sodium azide or other anti-microbial agents.
The wash buffer (PBS,10mM, pH7.2, 0.05% Tween20 [PBST]) is prepared by adding 0.5ml of Tween20 to 1000ml of the above 10mM PBS (pH7.2). Rinse the pipette several times using the PBS (Tween is viscous and tends to remain in the pipette). Mix thoroughly and keep in the cold.
Several conjugates of Fc specificity or anti-IgG monoclonal antibody-based conjugates can be used. However, a recombinant protein G/HRPO (horseradish peroxidase) conjugate (Pierce, Ref 31499ZZ; other commercial products are available) is recommended.The commercial recombinant protein G/HRPO conjugate is available freeze-dried in 0.5mg vials and should be kept frozen at –20°C until reconstitution. Astock solution containing 100µg of protein G/ml has to be made in 5ml of a PBS solution containing 50% glycerol and 0.1% bovine serum albumin (BSA). This stock conjugate solution should be maintained also at –20°Cuntil use. For use, this stock has to be properly diluted in PBST. The optimal concentration of the immunoconjugate should be established against a panel of control sera (see below). Under normal conditions, the titre of the conjugate should be in the range of 0.2–0.3µg/ml (i.e. a total of 20–30µl of stock solution should be diluted in 10ml of PBST and then the adequate volume placed in the wells).
Substrate buffer is citrate buffer, 0.05M, pH4:
-330ml of a solution of 11.48g citric acid.1H2O in 500ml of distilled water
-235ml of a solution of 14.7g of trisodium citrate (Na3C6H5O7).2H2O in 500 ml of distilled water.
-Makeup to 1000ml with distilled water
Check that the pH is 4. This solution can be stored in the cool and keeps for at least 10days.
The substrate working solution is obtained by dissolving 5.5mg ABTS (2,2’-azino-di-[3-ethyl-benzthiazoline]-6-sulphonic acid) in 50ml of the above citrate buffer. In a separate container mix 5µl of 30% commercial hydrogen peroxide solution (good grade and fresh stocks) and 120µl of H2O. Just prior to use, add 20µl of this last hydrogen peroxide dilution to the 50ml of the ABTS solution and mix thoroughly.
The enzymatic reaction-stopping solution is 4% sodium dodecyl sulphate (SDS), but the stopping step can be avoided if optical density (OD)readings are made immediately after the substrate incubation (see below).
At least a negative and a positive control serum of known ELISA titre should be used. Ideally a panel of several sera from B.melitensis culture positive and Brucella free animals should be available. The optimal concentrations of the sera should be determined to result in the best discriminating ability (i.e. the maximum differences in OD readings between positive and negative animals).
•Antigen production
A smooth lipopolysaccharide (S-sLPS) rich extract, obtained from B.melitensis 16M as described elsewhere (Díaz15 R., Toyos J., Salvo M.D., Pardo M.L., 1981. A simple method for the extraction of polysaccharide B from Brucella cells for use in the radial immunodiffusion test diagnosis of bovine brucellosis. Ann. Rech. Vet., 12, 35-39.) is recommended. These sorts of extracts are most often used in the ELISA at concentrations between 1.0 and 2.5µg per /ml, and this titre should be determined against an adequate panel of sera from B.melitensis infected sheep and Brucella free-sheep. It is convenient to keep a stock solution of 1mg/ml of distilled water at -–20º°C and make up the coating solution before use by mixing 10-–25µl of stock (according the antigen titre) with 10ml of 10mM PBS,pH pH7.2 (ELISA coating buffer).
Test procedure (example)
The indirect ELISA includes many variables (antigen, antigen coating, conjugate, time of incubations with serum, conjugate and substrate, temperature, etc.) and every laboratory should determine the optimal conditions that are more appropriate in each case. The optimal concentrations of the reagents should be determined to result in the best discriminating ability (i.e. the maximum differences in OD readings between positive and negative controls).
I)The frozen antigen is thawed and is diluted according to the previously established titre in the antigen-coating buffer, (for example, 2.5 µg of smooth lipopolysaccharide per ml -[see above]-). To coat the microplates, 100µl volumes of the diluted antigen solution are added to all wells, and the plates are covered and incubated for 16-–18 hours (overnight) at 4°C. Unbound antigen is removed by washing all microplate wells with wash buffer four times. Let the plates air dry at room temperature. The plates may be used immediately, or sealed and stored at 4°C for up to several months.
ii)Test sera are added to specified wells (100µl of optimum dilutions in PBST previously determined with control sera. ; uUsing the protein G/HRPO conjugate the optimal serum dilutions are around of 1/50). They may be tested singly or, better, in duplicate to minimise plate variability. The controls are set up in duplicate wells and include a positive and a negative control serum, as well as a buffer control. Plates are sealed or covered and incubated at 37°C for 60minutes.