Identification of the agent
P O R C I N E B R U C E L L O S I S
SUMMARY
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 dealt with in an Appendix at the end of this chapter.
Signs of disease in sows include infertility and 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, 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, molecular and biochemical tests, preferably carried out in specialised laboratories.
Serological tests: To date, none of the serological tests has been shown to be reliable in routine diagnosis in individual pigs. The indirect and competitive enzyme-linked immunosorbent assays (ELISAs), as well as the Rose Bengal test (RBT), complement fixation test (CFT) and fluorescence polarisation assay (FPA) are the prescribed tests for international trade purposes. The allergic skin test and the buffered plate agglutination test (BPAT) are also useful for identifying infected herds. The procedures for all the tests are the same as those described in Chapter 2.4.3 Bovine brucellosis.
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 is 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, brucellin is 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 both countries, a number of human infections have been reported from people who hunt and handle material taken from feral pigs (21, 22)
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 placental 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, prostate, 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, in chronically infected herds, infertility rather than abortion is the most relevant clinical sign of the disease. 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 general, the geographical distribution of biovar 2 has historically been in a broad range between Scandinavia and the Balkans (2). The prevalence in wild boars appears to be high throughout continental Europe (1, 3, 12, 14, 15). In recent outbreaks in Europe, wild boars have been implicated as the source of transmission of biovar 2 to outdoor reared pigs (12). In addition to wild boars, the European hare (Lepus capensis) is also considered as a reservoir for B. suis biovar 2 and has been implicated as a possible source of transmission to domestic livestock (2, 14, 24). Brucella suis biovar 2 causes miliary lesions, particularly reproductive tissues, that often become purulent. To date, biovar 2 has rarely been reported as the cause of
human brucellosis. However, biovar 2 infection has been reported in two immuno-compromised hunters, who had been extensively exposed through gutting or skinning boars or hares (13).
The most common B. suis biovars (1 and 3) are serious human pathogens and adequate 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 biosafety conditions to safely handle this dangerous zoonotic agent. Biosafety containment level 3 is recommended (see Chapter 1.1.2 Biosafety and biosecurity in the veterinary microbiology laboratory and animal facilities).
The classification, microbiological and serological properties of the genus Brucella and related species and biovars are given in the Chapter 2.4.3 Bovine brucellosis.
B. DIAGNOSTIC TECHNIQUES
As far as biovars 1 and 3 are concerned, culture methods are at least as sensitive as serology (6). Biovar 2 appears to be highly sensitive to selective media and could be more difficult to isolate (Garin-Bastuji & Blasco, unpublished data). As the product 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 now accompanied by the development of larger commercial units, thereby increasing the use of artificial insemination. Whereas 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.
Identification of the agent
Optimal samples for bacteriologic culture and methods for processing of samples are similar to those described for bovine brucellosis in Chapter 2.4.3 Bovine brucellosis. Standard and selective media used for other species of brucellae are suitable for B. suis (see Chapter 2.4.3 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 morphologically indistinguishable from other smooth brucellae, described in Chapter 2.4.3 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 the 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. However, some strains of B. suis biovar 1 are atypical in that they grow on media containing 20 µg/ml of basic fuchsin. Most strains of B. suis are inhibited by safranin O at a concentration of 1/10,000, whereas B. suis usually 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 identify B. suis and other species of Brucella (see chapter 2.4.3 Bovine brucellosis). Some of these techniques can distinguish biovars of B. suis (5, 7).
Serological tests
None of the serological tests used for the diagnosis of porcine brucellosis are reliable for diagnosis in individual pigs.
The major antigen involved in the serological tests currently available is the smooth lipopolysaccharide (LPS). The OPS moiety of this molecule contains epitopes that cross-react with those existing in the corresponding LPS from
Yersinia enterocolitica serotype O:9 (16). Therefore, available serological tests are unable to distinguish between antibodies raised to these two infections. Y. enterocolitica O:9 infection in pigs is not uncommon in some areas (1, 26). Studies have suggested that the sensitivities and specificities of the Rose Bengal test (RBT), the indirect and competitive enzyme-linked immunosorbent assay (I- and C-ELISAs), and the fluorescent polarisation assay (FPA) are similar (20). With the exception of acute stages of infection (<9 weeks) (16), the use of the FPA (18) or CELISA has been reported to eliminate cross-reactivity with Y. enterocolitica but this should be confirmed in additional field studies performed in various epidemiological situations. Swine serum may sometimes also contain nonspecific antibody, thought to be of the IgM isotype, further reducing the specificity of conventional tests, 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 may be low for the CFT; therefore caution should be taken when interpreting test results from individual animals. For international and other trade, e.g. 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.
Other Tests
a) Allergic (hypersensitivity) tests Brucelin-INRA is an sLPS free cytosolic extract from rough B. melitensis B115. This preparation does not stimulate the formation of antibodies that would be reactive in BBAT, CFT or ELISAs. The product has been developed for use in ruminants, but is also effective for confirming the disease at the herd level in pigs. A rough strain is used in its preparation, thereby avoiding the presence of sLPS. The preparation, standardisation and testing, of Brucellin-INRA is described in detail in Chapter 2.4.3 Bovine brucellosis. As a diagnostic agent in pigs 0.1 ml of the allergen is injected intradermally into the skin at the base of the ear or preferably next to the base of the tail. The latter appears more practical and less hazardous. The reaction is read after 48 hours. A positive reaction shows erythema of non-pigmented skin and an oedematous swelling. In severe reactions, there may also be some necrosis.