Introduction

Brucella belongs to the class Alphaproteobacteria. Also in this class is Bartonella species. Bartonella is dealt together with the Rickettsias in a lecture named “vector-borne bacterial infections”.

Brucella spp. are gram-negative, non-motile, non-spore-forming, small cocci or short rods. It resists decolouration by weak acids, and stains red with Stamp’s modification of the Ziehl-Neelsen stain = partially acid-fast. Most wild strains require complex media and CO₂ (5-10%) supplementation for growth and will take between 2 to 3 days to grow. The use of selective media, such as Farrell’s medium, may substantially enhance the chances of isolation by inhibiting the growth of contaminants, but the growth rate of brucellae may also be retarded by them, and for this reason, such cultures should be incubated for 5 days or longer. Smooth and rough colonies are recognised, the former being more pathogenic than the latter. Since all the brucellae are highly related, a combination of growth characteristics, colonial and cellular morphology, staining properties, agglutination antisera, phage typing, dye sensitivity and biochemical reactions will allow an accurate identification.

Brucellae can be present in the milk of infected cows where they can survive in the fat layer even when the milk is soured (fat protects from acid destruction of the bacteria). However, they will be destroyed by pasteurisation. They do not survive in well-hung carcasses due to the low pH of the carcass. They can survive for up to 8 months in liquid manure and in foetal membranes for 2-3 months on irrigated pastures.

Brucella species have been identified in all mammals. However, serious disease is only caused by Brucella melitensis (goats and sheep), Brucella abortus (contagious abortion in cattle), B. suis (infertility in pigs), B. canis (infertility in dogs) and B. ovis (contagious epididymitis in rams).

Brucella species are true parasites that are carried in intracellularly in the leucocytes of their host species.

diseases caused by Brucella species

Diseases caused by Brucella species are lymphohaemopoetic and multisystemic, but in animals they tend to cause a predominantly reproductive disease (abortions and orchitis) in animals. In people, infections results in multisystemic disease with a low-grade bacteraemia. Hence people often show joint disease with recurrent bouts of fever. Neurobrucellosis is a particularly debilitating disease in people. 29 causes of brucellosis were reported in Australia in 2023 mainly from Queensland and NSW.

The list of diseases caused by Brucella species in animals is shown below.

Table of diseases caused by Brucella species and their status in Australia

pathogenesis of brucellosis

In animals transmission of brucellae is mainly by ingestion of infected material i.e. foetal membranes or contaminated pastures. This is especially recognised for B. abortus and B. melitensis. Venereal transmission is also common, especially for B. suis and B. canis infections. Infection via wounds is less common.

Brucella organisms readily penetrate mucous membranes and are then phagocytosed by neutrophils and macrophages, which carry them to the regional lymph nodes. Here they multiply intracellularly and cause a lymphadenitis, which may persist for months. This may be followed by a bacteraemia, which may last for several months, resolve itself, or be recurrent for at least 2 years. Recurrence in the case of B. abortus and B. melitensis occurs particularly at the time of parturition.

In the bloodstream, organisms are carried intracellularly in neutrophils and macrophages or free in the plasma and localise in various organs, especially the gravid uterus, udder and supramammary lymph nodes, but also elsewhere, such as other lymph nodes, spleen, testes and male accessory sex glands. Occasionally localisation occurs in synovial structures causing e.g. tenovaginitis, bursitis or arthritis.

In cattle, localisation of B. abortus in the endometrium of the gravid uterus and in foetal membranes of animals appears to be the result of the special affinity of the organism for erythritol, elevated levels of which occur in the placenta and foetal fluids from about the 5^th months of gestation. The chorionic epithelium becomes parasitized and infection extends to the placenta stroma, blood vessels, and ultimately to the foetus. Depending on the severity of the placentitis, abortion, premature birth or the birth of a viable or non- viable calf may result. Because the non-pregnant uterus is not particularly susceptible to B. abortus, it is cleared of the organisms within a few weeks of abortion or parturition. The same is true for B. melitensis in goats.

How an intracellular lifestyle allows brucellae to evade the host immune response and persist in the host

It is the ability to survive in the cytoplasm of macrophages allows brucellae to persist in the body, often for years. Once ingested by cells they restrict lysosome-phagosome fusion. The phagosomes traffic to the endoplasmic reticulum (ER) where they are surrounded by the ER, which protects them against intracellular destruction.  They survive in the phagosome, where the acidic condition stimulates the upregulation of genes that code for proteins that modify the phagosome and protect it against oxidation. They are also able to scavenge iron and manganese and to enter a stationary phase which protects them from pathogen directed destruction as well as antibiotic destruction. Smooth strains are able to resist Complement-mediated lysis. The lipopolysaccharide of all Brucella species does not stimulate a strong inflammatory response.

Although humoral immunity affords some protection, it is the cellular mediated immunity (CMI) that is important. This immunity is, however, incomplete. Thus vaccine-induced immunity can be overcome, either by immunosuppression or by a high infectious dose. Passive immunity acquired by the ingestion of colostrum does not last long, although it may reduce infection of suckling animals via milk.

specific diseases

CAPRINE (OVINE) BRUCELLOSIS

Brucella melitensis (biovars 1, 2 or 3) is the main causative agent of caprine and ovine brucellosis. It is endemic in the Mediterranean region, but infection is widespread world-wide; including Mexico, Latin America, Russia and China. Notifiable disease, absent from Australia.

Clinically, the disease in animals (goats and sheep) 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.

B. melitensis is highly pathogenic for humans, causing Malta fever, one of the most serious zoonoses globally. All infected tissues, cultures and potentially contaminated materials should therefore be handled at biocontainment level 3.

CANINE BRUCELLOSIS

Notifiable disease, absent from Australia.

In dogs, the male serves as the primary reservoir of B. canis where females are infected venereally. Contact with aborted foetuses may also allow the agent to spread further. Unlike B. abortus in cattle, B. canis tends to cause a persistent bacteraemia (epidemiological evidence in an infected dog suggest that he was infected 8 years prior to testing positive) and in addition to the reproductive organs (orchitis with swollen testicles), it will cause osteomyelitis, discospondylitis, uveitis and renal infections. Since this is a rough strain of Brucella, serological tests aimed at detection of the smooth brucellae are not effective, thus a species-specific test must be used. In endemic countries, only those dogs that test negative on 2 consecutive tests, 4 – 6 weeks apart should be used for breeding.

BRUCELLOSIS OF MARINE MAMMALS

Pathogenic findings associated with brucellosis in cetaceans include placentitis, orchitis, abortion, mastitis, pneumonia, subcutaneous lesions, arthritis, non-suppurative meningoencephalitis, and encephalitis. Brucella-induced neurologic disorders may lead to strandings. To date there are 4 known cases of humans infected with Brucella spp. from marine mammals.

epididymitis in rams

Contagious ovine epididymitis caused by Brucella ovis was first recognised in Australia in 1953 and is endemic in the Australian sheep industry. It does not infect humans nor is it a notifiable disease in Australia. This venereal and orally transmitted disease affects predominantly the tail of the epididymis in rams causing persistent infections. Although some ewes may abort, they are usually transiently infected and don’t tend to pass on the infection to their lambs. The disease is more common in older rams that are used in multi-sire flocks where a susceptible ram will “join” the same ewe that was just mated by an infected ram. During the non-breeding season rams kept together may mount each other or lick each others prepuce. Rams may develop and fever and scrotal oedema soon after infection and will over a period of about 9 weeks develop palpable lesions in one or more testes. Note that not all infected rams will have visible or palpable lesions in the scrotal sac.

Similar disease can occur from opportunistic infections by gram-negative bacteria belonging to the Pasteurellaceae, namely Actinobacillus seminis and Histophilis somni as well as the gram-positive pyogenic bacteria such as Trueperella pyogenes and Corynebacterium pseudotuberculosis.

The table and flow chart below shows the differences in transmission and pathogenesis of a contagious (Brucella ovis) versus an opportunistic bacterial cause of ovine epididymitis.

Diagnosis of ovine epididymitis

Epididymitis in rams is diagnosed by palpation of the scrotum (see pictures below) for any swelling, nodules, ulcerations and changes in consistency. Ejaculated semen is examined for the presence of neutrophils and bacteria and in the case of ovine brucellosis serum testing for antibodies to Brucella ovis using the Complement fixation test.

Control of ovine epididymitis

The disease is controlled in flocks by maintaining closed flocks, keeping virgin rams in a separate flock, by 6-monthly palpation of testes, cytological examination of semen and serum screening for antibodies using the complement fixation test (CFT). Flocks free of ram epididymitis should only purchase new rams from stud flocks that are accredited under the voluntary Ovine Brucellosis Accreditation Scheme. The list of the accredited flocks are available in the provided website for most the the Australian States and Territories. Treatment with antibiotics such as doxycycline is generally not done as the lesions within the epididymis and testes often remain in spite of bacteriological cure. (Other countries may use the live Rev-1 vaccine in 2-3 month old rams. This vaccine is not registered in Australia).

Rams suffering from opportunistic infections may self-cure if the lesions of mild. Antibiotics such as oxytetracycline can be used, but rams must be monitored for fertility as the more severe lesions can result in permanent infertility.

Porcine brucellosis

Brucella suis biovars 1, 2 and 3 cause reproductive failure in domestic, feral and wild pigs. Thus swine brucellosis in other animal species and people is associated geographically with areas that have an abundance of feral pigs and where they are hunted. In Queensland and NSW B. suis biotype 3 is present in feral pigs, which are hunted. The distribution and prevalence of B. suis mirrors the distribution of feral pigs in these States.  The highest populations of feral pigs in Australia are in far North Queensland  and parts of the outback in NSW.   People, horses, cattle and dogs can also be infected. Pigs exhibit a persistent bacteraemia and humans can contract the disease through the skin, conjunctiva and by ingestion. Therefore, the slaughtering of infected pigs can result in human infection. Pig-hunting dogs are at particular risk of being infected. The Hunter region of NSW had confirmed cases in dogs in 2022.

Serological blood testing in Australia is not that reliable as it depends on cross-reactivity with B. abortus which is the test antigen.

About 15 to 30 humans cases are recorded annually, usually in pig hunters. There is an escalation of reports of infected pig hunting dogs especially in the outback of New South Wales (Moree area). Dogs develop systemic disease including osteomyelitis and reproductive disease – in males an orchitis and females a vaginitis and abortion. There is also evidence that some dogs become persistently infected and in utero transmission to the unborn pups occurs.

A fact sheet on porcine brucellosis is provided by Wildlife Australia

A fact sheet on Brucella suis infections in dogs is also provided by the NSW DPI.

BOVINE BRUCELLOSIS (contagious abortion in cattle)

Bovine abortion or contagious abortion (CA) is a highly contagious disease of cattle caused by Brucella abortus, which is characterized by mid- to late term abortion and retained placentae in cows and, more rarely, inflammation of the testes and accessory sex glands in bulls. Notifiable disease, absent from Australia. It was eradicated from Australia in 1989.

Cattle usually get infected after licking an infected placenta, new-born calf, foetus or the genitalia of an infected cow soon after it has aborted or calved, when very large numbers of B. abortus are present, particularly in the lochia. They can also be infected by ingesting contaminated feed or water, by inhaling organisms or though the conjunctiva. Calves may acquire the infection in utero or after the ingestion of colostrum. Most of these calves will rid themselves of the infection within a few months after infection; however, a few may retain the infection and spread the disease at their first and subsequent parturitions. Approximately 2.5 – 9 % of heifers born to seropositive cows may be latently infected, seroconverting during or after their first gestation.

In bulls, the testes (one or both) and accessory sex glands may be affected. Infected bulls may shed B. abortus organisms in their semen, seminal fluid and urine, and therefore bulls in infected herds should always be viewed with suspicion. Venereal transmission by natural service is, however, rare. In cows, which lactate following abortion, milk (including colostrum) is an important source of infection, and bacteria are excreted intermittently in milk throughout the lactation period. Urine and faeces of infected cattle are less important sources of the bacteria. There is a rapid reduction in the numbers of organisms shed after calving or abortion, and cows are usually non-infective until the next pregnancy, when there is again a rapid increase of Brucella organisms in the reproductive tract. During subsequent pregnancies there is invasion of the gravid uterus and allantochorion, but abortion rarely occurs. Subsequent calves are carried to full-term. Ninety per cent of infected cows remain chronically infected, sometimes life-long, with the infection confined to the udder and lymph nodes.

Joint inflammation with the accumulation of large volumes of fluid known as “hygromas” can occur in the weight-bearing joints i.e. the carpal joints. When newly introduced into a herd the abortion rate can be as high as 30%. In endemic herds the abortion rate is much lower with abortions occurring predominantly heifers. Other livestock and wild animal species are sometimes infected. Horses become infected particularly by ingesting B. abortus-contaminated feed. In this species, the bacteria localise in bursae, tendon sheaths and joints, and are therefore an unlikely source of infection to cattle.

diagnosis of brucellosis

Diagnosis of brucellosis is critical in any regulatory disease as it ensures that the correct animals are targeted for control and allows one to trace-back to the source of infection. It relies on:

1. Demonstration of the agent. The modified acid-fast stain (Stamp’s stain) can be used to demonstrate intracytoplasmic bacteria in affected tissues i.e. foetal lung impression smears. However, it must be confirmed by culture or Brucella DNA-specific PCR as abortigenic agents Coxiella burnettii and Chlamydia spp. will also have the same staining reaction.

2. Culture and identification by qPCR. In humans, dogs and pigs, blood and bone marrow cultures can be cultured.  In the case of abortions brucellae can be cultured from the maternal uterine fluids, spleen and placenta and also from the foetal stomach contents and lung. In affected males, brucellae are usually cultured from semen samples. To screen for the bacteria on slaughtered healthy animals, the lymph nodes draining the head and supramammary area should be used. Milk can also be used.

In the case of ovine epididymitis, semen can be used.

The growth of brucellae is slow and the culture poses a risk to laboratory personnel due to high infectivity of brucellae. It is a PC3 bacterium.

3. Demonstration of antibodies against the agent. Screening with the Bengal Rose reactions and then confirming positive reactors with the Complement-fixation Test or ELISA. Note that all the smooth strains of Brucella cross-react in tests that detect the O-antigen. Being a rough strain a Brucella ovis specific test is used to detect antibodies to this species.

control of brucellosis (Exception ovine brucellosis)

As brucellosis is notifiable in animals, animals can become persistent carriers even if treated and humans are very susceptible to infection, it is recommended that all animals that test positive should be euthanised.

To access information on brucellosis and its control in Australia refer to the AUSVETPLAN on Brucellosis.

1.Management of B. suis in Australia.

Control of the disease in commercial pigs may involve test and slaughter or whole herd destocking and restocking after decontamination. Biosecurity Queensland administers a voluntary Swine Brucellosis Accreditation Scheme.

Keeping the numbers of feral pigs down by hunting reduces the likelihood that domesticated livestock will be infected. About 70% of a feral pig population in an area has to be removed. However, it is important that pig hunters take measures to protect themselves and their dogs from infection.

The disease is controlled in dogs using antibiotic therapy and desexing.

2.   Brucella abortus/B.melitensis-free countries (Australia)

Australia was finally eradicated bovine brucellosis by 1989. By the OIE definition, it means that it has a prevalence of <0.2%, does not vaccinate against brucellosis and has a surveillance programme in place for monitoring for the disease. (Testing 17899 sera from January 2014 to July 2018 has revealed no positives). Generally live cattle are not imported into Australia, therefore, it is necessary that any semen or embryos be sourced from farms and areas accredited free from brucellosis.

3.  Brucella abortus/B. melitensis control in endemic countries

In countries with a >2% prevalence vaccination heifer calves between 3 – 10 months of age with a live attenuated Strain 19 or RB41 vaccine is considered the only practical way of controlling the disease. As the vaccine only provides partial protection, these countries remain endemic. Test-and-slaughter of positive animals is considered too expensive.

Countries with <2% prevalence and excellent laboratory facilities, the test-and-slaughter of positive animals becomes practical. Malaysia with a small national herd reduced the prevalence of Brucella from 3.3% in 1979 to 0.28% in 1988. However, it now has a prevalence of 5%. Reasons for this are many, but the following are of greater significance:

1. Inability to detect the remaining positive animals. Either by not being able to access them or due to delays in seroconversion. NB are heifers infected in utero or as a neonate, but evidence disease when they abort with their first pregnancy.
2. Movements of cattle are not fully controlled.
3. Purchasing of breeders from non-Brucella-free countries

END OF CHAPTER