Brucellosis in pigs is caused by Brucella suis, a bacterial infection that, after an initial bacteraemia, causes chronic inflammatory lesions in the reproductive organs of both sexes, with occasional localisation and lesions in other tissues. The species Brucella suis consists of five biovars, but the infection in pigs is caused by B. suis biovars 1, 2 or 3. The disease caused by biovars 1 and 3 is similar, while that caused by biovar 2 differs from 1 and 3 in its host range, its limited geographical distribution and its pathology. Biovar 2 is rarely pathogenic for humans, whereas biovars 1 and 3 are highly pathogenic, causing severe disease. Porcine brucellosis is of widespread occurrence; generally, however, the prevalence is low, with the exception of South America and South-East Asia where the prevalence is higher. In some areas, B. suis infection has become established in wild or feral pigs – diagnostic methods recommended for wild and feral pigs are the same as for domestic pigs. Various biovars of B. suis cause infections in animals other than pigs, such as reindeer, caribou, hares and various murine species, and occasionally in cattle and dogs. Brucella suis infections in animals other than pigs are reviewed in an Appendix at the end of this chapter.

Signs of disease in sows include abortion at any stage of gestation, and birth of dead or weak piglets. In boars, the most prominent sign is orchitis, and the secondary sex organs may be affected. Brucella suis may be present in the semen, sometimes in the absence of clinical signs. Transmission during copulation is more common than is the case with brucellosis in ruminants. In both sexes, bones and especially joints and tendon sheaths may be affected, causing lameness and sometimes paralysis. Pigs are susceptible to artificial infection with B. abortus and B. melitensis, but reports of natural disease in pigs being caused by either of these organisms are rare. In humans, the infection is usually confined to those who are occupationally exposed to pigs, and to laboratory workers. The capability of B. suis to colonise the bovine udder with subsequent shedding in milk means that it has the potential to be a serious human health risk.

Identification of the agent: Brucella suis is readily isolated from live pigs by culture of birth products, and from carcasses by culture of lymph nodes and organs. Selective media are available for culture of contaminated samples. In nature, B. suis occurs invariably in the smooth phase – the appearance on solid medium is typical of smooth brucellae. Biovars of porcine origin agglutinate with monospecific A antiserum, and not with M antiserum. Definite identification of species and biovars may be effected by phage typing and biochemical tests, preferably carried out in specialised laboratories.

Serological tests: To date, none of the conventional serological tests has been shown to be entirely reliable in routine diagnosis in individual pigs. Their preferred use is for the identification of infected herds. The indirect enzyme-linked immunosorbent assay (ELISA) and competitive ELISA are the prescribed tests for international trade purposes. The buffered Brucella antigen tests (BBATs), i.e. the buffered plate agglutination test (BPAT) and the rose bengal test (RBT), are suggested as alternative tests for screening purposes or complete herd tests. A fluorescence polarisation assay has also been developed. The allergic skin test is also useful for identifying infected herds.

Requirements for vaccines and diagnostic biologicals: Brucella suis strain 2 vaccine has been used for immunising pigs in China (People’s Rep. of). Confirmation of the results obtained in China is required before strain 2 vaccine can be recommended for general use. In other countries, experimental work has shown that B. melitensis Rev.1 vaccine is superior to B. suis strain 2 in protecting sheep against B. melitensis. Sufficient data are not available to conclude if B. abortus strain RB51 vaccine is efficacious in protecting swine against exposure to B. suis. In practice, no product has yet found general acceptance. Preparation, testing and use of an established allergen, brucellysate (or brucellin fraction F) are described.

A. INTRODUCTION

Porcine brucellosis is an infection caused by biovar 1, 2 or 3 of Brucella suis. It occurs in many countries where pigs are raised. Generally, the prevalence is low, but in some areas, such as South America and South-East Asia, the prevalence is much higher. Porcine brucellosis may be a serious, but presently unrecognised, problem in some countries. Brucella suis biovar 1 infections have been reported from feral pigs in some of the southern States of the United States of America (USA), and in Queensland, Australia. In Queensland, a number of human infections have been reported from people who hunt and handle material taken from feral pigs (16).

The disease is generally transmitted by consumption of feed contaminated by birth and/or abortion products and uterine discharges. Pigs will readily eat aborted fetuses and membranes. Transmission during copulation also occurs frequently, and this has implications for those practising artificial insemination.

In pigs, as in ruminants, after the initial bacteraemia, B. suis colonises cells of the reproductive tract of either sex. In females, placentas and fetuses are invaded, while in males, invasion occurs in one or more of the following: testis, epididymis, seminal vesicles, and/or bulbo-urethral glands. In males the lesions, which are most often unilateral, start with a hyperplasia that may progress to abscess formation; the final stage is characterised by sclerosis and atrophy. Arthritis may occur in various joints, and sometimes spondylitis occurs.

The most common manifestation of brucellosis in female pigs is abortion, occurring very early or at any time during gestation. Vaginal discharge is not often evident, and the problem may appear to be infertility rather than abortion. In males, brucellosis is more likely to be persistent, with lesions in the genital tract often leading to interference with sexual activity, which can be temporary or permanent. The boar may excrete brucellae in the semen without any apparent abnormality in the sex organs or interference with sexual activity.

In both sexes, there may be swollen joints and tendon sheaths, lameness and, occasionally, posterior paralysis. A significant proportion of both male and female pigs will recover from the infection, often within 6 months, but many will remain permanently infected.

Brucellosis caused by B. suis biovar 2 differs from infection caused by biovars 1 and 3 in its host range, its distribution, and in its pathology. In recent outbreaks in Europe, wild pigs have been implicated as the source of transmission of biovar 2 to pigs that are reared outdoors (7). In addition to wild swine, the European hare (Lepus capensis) is also a reservoir of B. suis biovar 2 and has been implicated as a possible source of transmission to domestic livestock (1, 7). The geographical distribution of biovar 2 has historically been in a broad range between Scandinavia and the Balkans (1). Brucella suis biovar 2 causes miliary lesions in tissues, particularly reproductive tissues, that often become purulent. To date, biovar 2 has only once been reported as the cause of human brucellosis.

The common biovars of B. suis (1 and 3) are serious human pathogens and precautions are needed when handling and disposing of potentially infective material. This is especially so in the laboratory after culture has greatly increased the number of organisms present. Laboratory manipulation of the cultures or contaminated material from infected animals must be done under strict biosecurity conditions to safely handle this dangerous zoonotic agent. Biosecurity containment level 3 or higher is recommended, as described in Chapter I.1.6. Human safety in the veterinary microbiology laboratory.

B. DIAGNOSTIC TECHNIQUES

In porcine brucellosis, culture methods are at least as sensitive as serology (4). As the produce of almost all pig-raising enterprises passes through abattoirs, surveillance methods (serology and culture) can be applied effectively at this point. In many areas, traditional village pig breeding is being replaced by or accompanied by the development of larger commercial units, thereby increasing the use of artificial insemination. Therefore artificial insemination using brucellosis-free boars can be a valuable aid in the control of porcine brucellosis, the inadvertent use of infected semen could, obviously, cause incalculable damage.

1.

Identification of the agent

Optimal samples for bacteriological culture and methods for processing samples are similar to those described for in Chapter 2.3.1. Bovine brucellosis. Standard and selective media used for other species of brucellae are suitable for B. suis (see Chapter 2.3.l. Bovine brucellosis). The addition of serum is not essential, but basal medium containing 5% serum is a satisfactory medium, both for isolation, maintenance of cultures and typing. The addition of CO2 to the atmosphere is not required.

In nature, B. suis invariably occurs in the smooth form and colonies are indistinguishable from other smooth brucellae, described in Chapter 2.3.1. Bovine brucellosis.

Biovars 1, 2 and 3 of B. suis are all A surface antigen dominant, and growth may be presumptively identified by slide agglutination with monospecific A antiserum. Confirmatory identification of species and biovar should be performed in a specialised reference laboratory. The OIE Reference Laboratories for brucellosis are listed in the Table given in Part 3 of this Terrestrial Manual.

Confirmation of species and biovar depends on phage tests, production of H2S (only biovar 1 produces H2S), and growth in the presence of dyes. Some strains of B. suis biovar 1 are atypical in that they grow on media containing 20 µg/ml basic fuchsin. Most strains of B. suis are inhibited by safranin O at a concentration of 1/10,000, whereas B. suis reacts more rapidly in the urease test than either B. abortus or B. melitensis. Oxidative metabolic tests are supplemental tests that can be used for distinguishing B. suis from other smooth Brucella species.

Molecular genetic techniques using the polymerase chain reaction (PCR) and specific primers are available that can distinguish B. suis from other smooth species of Brucella (3, 18). However, these PCR techniques cannot distinguish biovars within B. suis, neither have they been fully evaluated and standardised. The 3.3 Mb complete genomic sequence of B. suis strain 1330 has been determined, and has a similar chromosome structure and gene content to that of B. melitensis strain 16M (14). The B. suis sequence will be beneficial for basic research on taxonomy, virulence, and protective antigens of B. suis, and may prove beneficial in developing new diagnostic tests.

2.

Serological tests

None of the conventional serological tests used for the diagnosis of porcine brucellosis are reliable for diagnosis in individual pigs. A significant problem is the fact that weaners up to 2–3 months of age are susceptible to infection with B. suis, but their agglutinating antibody response to the infection is very limited.

These conventional tests use antigens that are dependent on smooth lipopolysaccharide (LPS) for their activity. Due to the sharing of an ‘O’ chain polysaccharide, such antigens react equally with the LPS of Yersinia enterocolitica serotype 0.9 and are not, therefore, able to distinguish between antibodies to these two infections. Yersinia enterocolitica infection in pigs is not uncommon in some areas (21). Studies have suggested that the sensitivities and specificities of the buffered acidified plate antigen assay, the 2-mercaptoethanol test, the indirect enzyme-linked immunosorbent assay (I-ELISA), a competitive ELISA (C-ELISA), and the fluorescent polarisation assay (FPA) are similar (13). Swine serum may sometimes also contain nonspecific antibody, thought to be IgM, further reducing the specificity of conventional serology, especially the serum agglutination test (SAT). Also, swine complement interacts with guinea-pig complement to produce a pro-complementary activity that reduces the sensitivity of the complement fixation test (CFT). Sensitivity levels as low as 38% (15) and 49% (17) have been reported for the CFT; therefore this test cannot be recommended for the diagnosis of brucellosis in individual pigs. For international and other trade purposes, such as purchasing boars, the disease status of the herd and of the area in which the herd is situated are of more importance than tests on individual animals. In spite of this, the European Union and some other countries still insist that only pigs whose serum shows an agglutination titre less than 30 International Units (IU) per ml and a CFT of less than 20 ICFTU (international CFT units) should be allowed to cross international borders.

•

Reference sera

Primary reference standards are those against which all other standards are compared and calibrated. These reference standards are currently being developed and will be available to national reference laboratories when completed.Biological reagents for the C-ELISA and I-ELISA for the diagnosis of porcine brucellosis are available in small quantities for research or standardisation purposes (obtainable from the OIE Reference Laboratory for Brucellosis at the Animal Diseases Research Institute, 3851 Fallowfield Road, Nepean, Ontario K2H 8P9, Canada).

a)

Enzyme-linked immunosorbent assay (the prescribed test for international trade)

•

Indirect ELISA

Indirect and competitive ELISAs have been developed for the diagnosis of brucellosis in individual pigs and for screening large numbers of sera. These techniques promise to be more efficient than any of the tests mentioned above, and the C-ELISA appears to be better at distinguishing antibody reactions that are due to infection with Y. enterocolitica serotype 0.9 from those that are due to infection with Brucella sp. The method for the I-ELISA is described in detail in Chapter 2.3.1. Bovine brucellosis, however, monoclonal antibody specific for porcine IgG conjugated with horseradish peroxidase should be used.

•

Competitive ELISA

C-ELISA procedures for detection of porcine antibody to Brucella sp. (11) are identical to the procedures used for bovine antibody to B. abortus described in Chapter 2.3.1. This assay is capable of eliminating some reactions due to Y. enterocolitica serotype 0.9 and other cross-reacting antibody, such as IgM, which have lower affinity for Brucella epitopes as compared with the monoclonal antibody used in the assay. The C-ELISA is recommended as a confirmatory test as its sensitivity and specificity exceeds those of the agglutination tests.

The FPA for detection of porcine antibody to Brucella sp. is essentially the same as that described for cattle (for more details see Chapter 2.3.1); the serum dilution can vary depending upon validation with local samples but is often used at 1/25 for the tube test and 1/10 for the plate test (11). It is a simple technique for measuring antigen/antibody interaction and may be performed in the laboratory or in the field. This assay may assist in eliminating some of the reactivity resulting from exposure to Y. enterocolitica serotype 0.9 and other cross-reacting antibody. Lyophilised porcine sera tend to increase background activity in this assay. The FPA may be used as a screening and/or confirmatory test.

For screening purposes or complete herd tests, the buffered Brucella antigen tests (BBAT), i.e. the card test, the rose bengal plate agglutination test (RBT) or the buffered plate agglutination test (BPAT), are recommended as alternative tests. The preparation and standardisation of BBAT antigens and the methods of performing the tests are described in Chapter 2.3.1. Bovine brucellosis. All biovars of B. suis affecting pigs have the same immunodominant A antigen as do most of the B. abortus biovars, which makes the B. abortus antigens appropriate for testing swine sera.

3.

Other tests

a)

Allergic (hypersensitivity) tests

Allergic tests have been widely used for the diagnosis of brucellosis in herds of pigs in Eastern Europe, in the countries that were formerly in the Union of Soviet Socialist Republics (USSR), and in the People’s Republic of China. The specificity is very high, however, the sensitivity of the allergic test is similar to that of the serological tests (BBATs) and is not reliable for diagnosis in individual pigs. Some infected animals that are negative serologically, react positively to the skin test, and vice versa. Thus, where practicable, it is useful to perform both tests. As the active principle of the allergic test is protein, there should be no cross-reaction with Y. enterocolitica serotype 0.9, at least in theory, and the authors are not aware of any demonstration of this cross-reactivity.

Many different allergens have been used for diagnosis, but one that is simple to prepare, that has been used effectively as a herd test in pigs, and that is still in use in some countries is an acid hydrolysate, sometimes called brucellysate or brucellin fraction F. Despite containing some polysaccharide, this preparation does not stimulate agglutinins or complement-fixing antibodies, nor does it sensitise animals (9). The method for preparing brucellin fraction F (Section C2), acid hydrolysis, will give rise to a substantial quantity of O-polysaccharide in the supernatant fluid, thus not helping in the differentiation of Yersinia from Brucella antibody. A method of preparation is given in Section C2. As a diagnostic agent in pigs, 0.2 ml of the allergen is injected intradermally into the skin at the base of the ear. The reaction is read after 48 hours. A positive reaction shows erythema on nonpigmented skin and an oedematous swelling. In severe reactions there may also be some necrosis.

More recently, brucellin INRA has been developed for use in ruminants; the authors are not aware of it having been used in pigs. A rough strain is used in its preparation, thereby avoiding the presence of smooth LPS. The preparation, testing, and use of brucellin INRA is described in detail in Chapter 2.3.1. Bovine brucellosis.

C. REQUIREMENTS FOR VACCINES AND DIAGNOSTIC BIOLOGICALS

Numerous attempts have been made to develop a vaccine to immunise pigs against B. suis. Only one product has found any acceptance for field use – B. suis strain 2 (S2) vaccine has been used extensively in south China (People’s Rep. of). To date, it does not appear to have been used elsewhere in pigs, probably because it has been shown to confer less protection in sheep against B. melitensis than the Rev.1 vaccine (20). Sufficient data are not available to conclude if B. abortus strain RB51 vaccine is efficacious in protecting swine against exposure to B. suis.

C1.

Brucella suis strain 2 vaccine

S2 is a smooth, stable, naturally attenuated strain of B. suis biovar 1. Extensive studies have failed to detect any characteristic to distinguish S2 from field strains of biovar 1, except the level of virulence, which, as judged by its persistence in mice and guinea-pigs, is much lower than that of the average field strain. The low level of virulence and its stability has been demonstrated by passage in pregnant sows and boars (22).

The S2 vaccine may be given orally, for example in the People’s Republic of China it is often mixed in food, although it is warned that fermented food rapidly inactivates the vaccine. Oral vaccination could be an attractive possibility for the control of brucellosis in feral pigs. Challenge experiments in both sexes have shown appreciably lower levels of infection in the vaccinated compared with unvaccinated control animals, even in sows challenged with infected semen. For oral vaccination, doses of around 2 (1010 are required, and usually two doses are given at an interval of approximately 2 months. Antibody produced in response to oral vaccination is said to disappear by 6 months after vaccination. Liang Xingxian (10) reports that in Guangdong Province in south China, porcine brucellosis has been controlled by a combination of annual serological testing of breeding stock with elimination of serologically positive animals and vaccination of serologically negative animals. In 1985, the reactor rate was 10%; in 1987 it had fallen to 1.2%, and no reactive boars had been found in the 4 years to 1991. Before the S2 vaccine is accepted for general use, its safety and immunogenicity should be thoroughly investigated under the conditions pertaining in each country.

1.

Seed management

Freeze-dried seed cultures for the experimental production and use of S2 vaccines are available from recognised institutions (Obtainable from the National Institute for the Control of Veterinary Products and Pharmaceuticals, Ministry of Agriculture, 30 Baishiqiao Road, Beijing 100081, People’s Rep. of China, or from VLA Weybridge, New Haw, Addlestone, Surrey KT15 3NB, United Kingdom. Supply from the Weybridge laboratory needs the prior permission of the World Health Organization). This freeze-dried material may be propagated by culture on nutrient agar or trypticase–soy agar for 2–3 days to produce seed lots that should not be more than three subcultures away from the starting material.

Any country interested in introducing this vaccine should organise adequate challenge experiments and field trials in pigs.

2.

Method of manufacture

The S2 vaccine is produced in fermenters using the same procedures and media as described for B. abortus strain 19 (S19) (see Chapter 2.3.1. Bovine brucellosis). However, for the propagation of S2 vaccine on solid medium, either nutrient agar or trypticase–soy agar is preferred to potato agar.

3.

In-process control and further testing

In-process control, batch control and tests on the final product are all the same as for B. abortus S19. With regards the length of immunity, annual revaccination is recommended.

C2.

Brucellysate (brucellin fraction F)

a)

Preparation

i)

Grow a smooth culture of Brucella (e.g. B. suis) on agar medium or in a fermenter.

ii)

Harvest cells grown on solid medium in normal saline (0.15 M NaCI).

iii)

Deposit by centrifugation and resuspend to 50–100 ( 109 cells/ml in normal saline.

Check for sterility. Check for lack of toxicity by injecting 0.5 ml into each of three to five mice.

Potency may be tested in sensitised guinea-pigs in comparison with a product of known potency. Guinea-pigs may be sensitised by repeated injections of killed B. suis in Freund’s incomplete adjuvant (2).

b)

Field use

As a diagnostic agent in pigs, 0.2 ml of the allergen is injected intradermally into the skin at the base of the ear. The reaction is read after 48 hours. A positive reaction shows erythema on nonpigmented skin and an oedematous swelling. In severe reactions there may also be some necrosis.

APPENDIX: BRUCELLA SUIS INFECTIONS IN ANIMALS OTHER THAN PIGS

1.

Rangiferine brucellosis

Brucella suis biovar 4 causes serious disease in reindeer or caribou (Rangifer tarandus and its various subspecies) throughout the Arctic region, Siberia, Canada and Alaska (12). Some of these animals are domesticated, others are wild and migratory. Rangifer tarandus is very susceptible to B. suis infection, which causes fever, depression and various local signs, such as abortion, retained placentas, metritis, sometimes with blood-stained discharge, mastitis, bursitis and orchitits. In the Arctic region, B. suis biovar 4 constitutes a serious zoonosis (5). Transmission to humans may be by direct contact or through consumption of milk and other inadequately heated products from reindeer. Bone marrow, which is considered to be a special delicacy in this region, is also a source of human infection.

The methods already described for isolating and identifying B. suis in samples taken from pigs are equally applicable to B. suis biovar 4 in samples taken from reindeer. Biovar 4 grows well on all the usual media used for the culture of Brucella. It reacts positively with both A and M monospecific sera. For serology, the tube agglutination test has been reported to be satisfactory, with titres from 1/20 being considered diagnostic. The CFT has also been used, but the clinical interpretation of these tests in reindeer has not been established.

Vaccination of reindeer with B. abortus S19 vaccine, or alternatively with B. abortus 45/20 adjuvant vaccine, has been tried experimentally without any clear-cut result. In the case of S19, the reaction to vaccination was rather severe and immunity in the vaccinated animals could only be demonstrated against challenge with very small doses of B. suis biovar 4. Gall et al. (6) compared several serological tests and found that the specificity values for the BPAT and CFT using reindeer/caribou sera was lower than the I-ELISA, C-ELISA and the FPA, while sensitivity values were similar for all tests.

2.

Brucella suis infection in other nonporcine species

There are two different types of epidemiological situation with regard to B. suis infection in other nonporcine species. In the first case, B. suis infection occurs in animals that are not the natural host of the particular infection through the ingestion of contaminated materials or by co-habitation with infected natural hosts. For example, Arctic foxes and wolves may contract B. suis biovar 4 from reindeer; dogs and rodents, such as rats and mice, may acquire other B. suis biovars by cohabitation with infected hosts. Cattle may become infected by cohabitation with infected feral swine. The infecting bacteria are invariably the well defined biovars of the natural host species.

In the second case, wildlife species that are natural hosts for B. suis or B.-suis-like infections become infected. One example is the so-called murine brucellosis of the former USSR, where small rodents are infected with B. suis biovar 5. Other similar situations have been reported from Queensland, Australia and from Kenya. In all three cases, B. suis strains with different characteristics were involved, and at least one of them was difficult to classify.

Brucellosis caused by B. suis biovar 2 is perhaps a special case. The reservoir of infection is in wild pigs (Sus scrofa) living in the same area (7, 8), or in the European hare (Lepus capensis) (19), or in both. Biovar 2 infection historically has been confined to an area between Scandinavia and the Balkans. Domestic swine reared outdoors in this area are at highest risk for transmission of biovar 2 from wildlife vectors. After invading domestic pig herds, biovar 2 is likely to spread as rapidly as biovars 1 and 3. The disease in hares is characterised by the formation of nodules, varying in size from that of a millet seed to a cherry or even larger; these often become purulent. Such nodules may occur in almost any location, sometimes subcutaneosly or intramuscularly, in the spleen, liver or lung and in the reproductive organs of either sex. The bodily condition of the hare may be surprisingly unaffected.

Serological investigations in nonporcine species are usually carried out for screening purposes. In these particular circumstances, specificity is more important than sensitivity. Here the CFT is recommended, although the buffered Brucella plate agglutination test may be useful because of its simplicity. In many previous investigations, the tube agglutination test was used, apparently with satisfaction. However, in nonporcine species the interpretation of serological results may be problematic. Where supposedly positive samples are encountered, serological screening should be followed by bacteriological investigation.

For bacteriological investigations in situations such as these, where the infecting organisms may have unusual characteristics, it is advisable to duplicate the culture on selective media by culture on plain medium supplemented with 5% serum, and to broaden the investigation by incubating the cultures in an atmosphere containing 10% CO2. Colonies resembling Brucella can be tentatively identified by Gram staining, by slide agglutination tests with monospecific A and M sera, and by anti-rough Brucella serum (Chapter 2.3.1 Bovine brucellosis) Brucella suis biovar 5 is unusual in that it reacts with monospecific M serum, and not with monospecific A serum. Further identification is best carried out in a specialised laboratory.

Liang Xingxian (1991). The prevalence, prevention and control of swine brucellosis in Guangdong Province, China. Document AGU/BRU/92/21 Food and Agriculture Organization of the United Nations (FAO), Rome, Italy.