Brucellosis

Brucellosis is also known as Bang’s disease, Mediterranean Fever, Undulant Fever and Malta fever.

Brucellosis is an important Zoonotic disease, causes a significant reproductive losses through abortions, placentitis, epididymitis and orchitis in infected animals.

Brucellosis is caused more often by B. abortus, less frequently by B. melitensis and occasionally by B. suis.

Etiology

Brucella is a Gram negative, non-motile, facultative intracellular, coccobacillary / short, rod shaped organism. They are strict aerobes and grow very slowly (fastidious) on blood agar. Chlamidophila and Coxiella brunetti are indistinguishable from Brucella. Some species of this genus and their host specificity has been listed below:

• Brucella abortus – Cattle, bison and buffalo, human
• Brucella melitensis, B.ovis – Sheep and goats
• B. suis – Pigs
• B. canis – Dogs
• B. maris – Marine mammals
• B. neotomae – Rodents (found in this species but not causing the clinical disease)
• B. pinnipdedialis – Pinnipeds (seals, sea lions and walruses)
• B. cetaceae – Cetaceans (whales, porpoises and dolphin)

There are five biovars in B. suis and three for B. melitensis and nine for B. abortus present. B. suis contains more diverse isolates and broader host range as compared to any other species of Brucella.

B. abotus, B. melitensis, B. suis and B. canis causes brucellosis in human. In addition, some species of Brucella has been used in biological warfare.

Epidemiology

Prevalence of Infection

• It is worldwide in distribution.
• Brucellosis is endemic in India.

Source of infection

• Brucella species have also been found in secretions and excretions which include urine, feces, hygroma fluids, saliva, nasal and ocular discharge.
• After the first full term abortion, cattle become a asymptomatic chronic carriers, and continue to shed the organism in milk and uterine discharges.
• Dogs may or may not show clinical signs but shed the specific organism in later stage of pregnancies. In ruminants, udder colonizes the organisms after parturition and the in the uterus during pregnancy.

Transmission

Brucellosis occurs by ingestion, inhalation while having direct contact with food and other materials contaminated with brucella in addition the organism spread to skin injury and conjunctival route. 

Mostly, Brucella abortus, B. melitensis are shed through bull semen for a long period but venereal spread is seems to be uncommon.

The venereal route is the primary route of transmission for B. ovis, B. suis and B. canis and occurs frequently.

General spread of infection occurs through ingestion, mucous membranes, broken skin. 

The infection spread also from milkers and farmers hands but long-term shedding not occurs normally in ruminants milk.

Host Affected

Cattle, bison, buffaloes, sheep, goats, pigs, dogs, marine mammals, rodents, pinnipeds such as seals, sea lions and walruses, cetaceans such as whales, porpoises, European otter and dolphins are affected. Sheep and goats acts as reservoir for B. melitensis and deer as a maintenance host for B. ovis.

Wildlife reservoirs which includes feral pigs, bison, elk and European hares maintain the organisms complicate the eradication processes of B. abortus and B. suis. B. abortus, B. melitensis and B. suis affects horses, camels, moose, chamois, alpine ibex, raccoons, opossums, dogs, coyotes, foxes and wolves (accidental hosts).

Dogs are the only species known to be naturally infected with B. canis , although antibodies to this organism has been found in other carnivores.

Pathogenesis

Following entry, the bacteria are swallowed by neutrophils, monocytes and macrophages. Within the phagocytic cells they proliferate intracellularly and remain in lymph nodes if not killed by the host immune cells and get localised in regional lymph nodes.

Brucella primarily localised in the organs rich in sugar, erythritol ie; breast, uterus, epididymis and testes etc. Within the phago-lysosome B. abortus releases 5′ -guanosine and adenine which inhibits the de-granulation of peroxidase-containing granules and thus inhibit the myeloperoxidase-peroxide-halide system of bacterial killing.

The intracellular persistence of the brucella leads to granuloma formation in organs and tissues are due to hypersensitivity reactions, mostly type-IV. The organism is distributed from lymph nodes to lympho-reticular organs such as spleen, bone marrow, liver and testes where they develops granulomas or micro abscesses which resulted in abortion in the third term pregnancy.

Clinical Signs

Cattle (B. abortus)

• Abortion in second half to full term pregnancy, still births, weak calves, retained fetal membrane, reduction in lactation noticed.
• Once aborted animals become symptomatically normal for the further pregnancy but actively shed the organism in uterine discharge and in milk in the subsequent parturition.
• In bulls, epididymitis, seminal vesiculitis, orchitis, testicular abscess, infertility, in both sexes, unilateral or bilateral hygroma and arthritis following long term infection.
• Rarely causing death of fetus.

Sheep and Goats (B. melitensis)

• It causes abortions in second weeks to fifth weeks of pregnancy, still birth, weak offspring, retained fetal membrane, re-invasion during subsequent pregnancies, reduction in milk yield, absence of mastitis.
• In acute cases, orchitis, epididymitis in males which results in infertility.
• Arthritis develops in both males and females.

Sheep (B. ovis)

• It affects only sheep not goats.
• Epididymitis (unilateral occasionally bilateral), orchitis, impaired infertility and in rams, poor quality semen, atrophied testes etc. Ram shed B.ovis for long periods while it is asyptomatic.
• Abortion and placentitis but mortality in ewes is not common.

Pigs (B. suis) (Porcine and rangiferine Brucellosis)

• It is causes abortion at any time in pigs, weak and still birth piglets, vaginal discharge, infertility, metritis is occasional.
• Temporary or permanent orchitis and sterility in boars common.
• Infected boars are persistent carriers.
• Biovar-2 in hares produces nodules in internal organs, reproductive organs, subcutaneous tissues, muscles, and that may be purulent in nature.
• Biovar-4 causes abortion, retained placenta, metritis, mastitis and in male orchitis, lameness, arthritis, bursitis, tenosynovitis, and/or hygromas and subcutaneous abscess in caribou and reindeer.

Dogs (B. canis)

• It produces abortion in 7-9 weeks of gestation followed by a mucoid, serosanguineous / gray green vaginal discharge that persists for up to 6 weeks.
•  Early embryonic death, resorption in a few weeks after mating, and may be mistaken for a failure to conceive.
• Pups born alive, weak, later develop disease.
• Epididymitis, scrotal edema, orchitis, poor sperm quality, unilateral or bilateral testicular atrophy in chronic infection and lymphadenitis is common.
• Lethargy, decreased appetites, poor performance, loss of alertness, discospondylitis of the thoracic and lumbar vertebrae cause stiffness, lameness, back pain, uveitis, endophtholmitis, polygranulomatous dermatitis, endocarditis, and meningoencephalitis without fever noticed.

Horses (B. abortus and B. suis)

• It causes inflammation of supra spinous or supra atlantal bursa and leads to “fistulous withers” or “poll evil”.
• The bursal sacs becomes distended or thick wall, rupture leading to secondary inflammation. In chronic cases, nearby ligaments and dorsal vertebral spines may become necrotic.
• Rarely it cause abortion in horses.

Zoonosis

Brucellosis is an occupational hazard. Veterinarian, butchers, farmers and some animal handlers dealing with shandy and exhibition animals, exposed to infection while contact with infected animals.

Ingestion of unpasteurized milk and other dairy products, aerosol laden particles contact with laboratory cultures, tissue samples, accidental injection of live brucellosis vaccines spread the disease.

It is a serious, debilitating and chronic disease develops a systemic infection in human. Although, human to human transmission is rare, the disease occurrence has been reported following blood transfusion, bone marrow transplantation or sexual contact.

Transmission due to crossing of placental barrier and ingestion of breast milk leads to young ones. In clinical cases, acute febrile illness with non-specific flu like signs such as fever, head ache, malaise, back pain and muscular pain noticed.

Drenching sweats particularly at night, spleenomegaly, hepatomegaly, cough and pleuritic chest pain are sometimes seen.

Necropsy Findings

B. abortus, B. melitensis and B. suis develops autolysis of fetus, subcutaneous edema, blood stained fluid in body cavity in ruminants, enlargement of spleen, liver, lung, pneumonia and fibrinous pleuritis, placentitis. Cotyledeons becomes red, yellow, normal or necrotic.

In cattle, small ruminants inter-cotyledonary parts typically leathery, with focal thickening and exudate.

In adult granulomatous to purulent lesion in mammary gland, superficial lymphnodes, lymphoid tissues, bones, joints and other tissues and organs lesions found.

Mild to severe endometritis after an abortion, in male unilateral or bilateral epididymitis or orchitis.

Hygroma of knee joint, fetlock, hock, stifles, haunch, nuchal ligaments and thoracic spines etc.

• B. suis: Purulent nodules in hares present.
• B. ovis: Fibrous atrophy of the testes noticed
• B.canis: Atrophy of testes, Subcutaneous edema, congestion, hemorrhage in abdominal region, duodenum, serosanguineous fluid, degenerative lesion in liver, spleen, kidneys, and in intestines. Enlargement of retropharyngeal and inguinal lymphnodes, scrotal edema, dermatitis, epididymitis, orchitis, prostatitis, testicular atrophy, fibrosis, metritis and vaginal discharge seen in adult animals.

Diagnosis

• Based on clinical signs and necrospy findiings Identification of organism by Stamp’s modification Ziehl Neelsen method.
•  Stained smears reveals coccobacilli or short rods, arranged in a single chain, but sometimes in pairs or in small groups. .
• Isolation of organism by culture.
• Immuno-staining demonstrate the presence of Brucella spp. in a clinical specimen.      
• PCR techniques can also be used for diagnosis.
• Rose Bengal Plate agglutination testIt is a very sensitive, qualitative, antibody screening test for Brucellosis. But sometimes the test result may become positive in response to animal vaccinated with S19 vaccine, and false positive serological reactions. Hence, the test result should be confirmed with some other confirmatory test. False negative reactions occur due to prozoning. Antigen for this test is prepared from killed B. abortus S99 or S1119-3 cells
• Complement fixation test: It is an international trade test. It is a very specific and widely used confirmatory test for testing animals meant for export and import. It gives positive result in S19 vaccinated animals.
• Cows vaccinated with B. abortus S19 between 3 and 6 months are usually considered to be positive if the sera gives positive fixation at a titre of 30 or great ICFTU/ml when the animals are tested at an age of 18 months or older.
• Indirect Enzyme Linked Immuno Sorbent Assay: It is a test of international trade.
• Fluorescent polarization assay: It is a simple and international trade test. Its sensitivity and specificity (99%) is similar to the C-ELISA.
• Intradermal Brucellin Skin test: It is a screening test of un-vaccinated herds in which purified standarfdized Brucellin INRA antigen is used.
• This inoculation may stimulate temporary anergy in the cellular immune response. An interval of 6 weeks is required to re-conduct this test in the same animal.
• Serum Agglutination test: It is an antibody detection test. Its specificity is improved with addition of EDTA to the antigen. The antibody titre of 1:40 is considered as positive in animals infected for the first time in uninfected area and 1:80 is considered positive for animals maintained in an endemic areas.
• Milk Ring test: It is a simple, qualitative, antibody detection test in which 1 or 2 ml of milk from either bulk milk take for herd screening or individual animal diseas.amma Interferon test : It is a test of choice for detection of cell mediated immune response.
•  Most human infections are diagnosed by serology. Tests used include serum agglutination, a modified Coombs’ (antiglobulin) technique, ELISAs and Western blot. Blood, uterine discharge, foetal stomach content, hygroma fluid and milk.

Differential Diagnosis

• Trichomoniasis
• Leptospirosis
• Campylobacteriosis
• IBRT

Treatment

• No treatment is advised in animals.
• Long term antibiotics treatment is advised in human with combination of antibiotics.

Prevention

Brucella abortus strain 19 (cotton strain) vaccine

This vaccine is most widely used in the prevention of brucellosis. It is a reference live vaccine normally given to female calves of 3-6 months in subcutaneous route with dose rate of 5-8×10¹⁰ viable organisms where as in adult cattle 3×10⁸ organism is given s/c route but some animals develop persistent antibody titers and may abort and excrete the vaccine strain in milk.

Brucella abortus strain RB51 vaccine

It is an official vaccine  being used since 1996. This strain was produced from a laboratory derived rough mutant of smooth B.abortus strain 2308. Calves of 4-12 months old are vaccinated with 1-3.4×10¹⁰ viable strain RB51 organisms.

Brucella Melitensis strain Rev.1 vaccine

This vaccine may be more effective in countries with a high prevalence of brucellosis in small ruminant and this vaccine should not be used in cattle because the safety of the vaccine in cattle is not studied yet.

Control

• All positive animals must be separated and disposed.
• Vaccination of all susceptible in contact animals with either B. abortus
• S19 vaccine or B.abortus RB51 vaccine.
• B.abortus 45/20 vaccine is also used.
• In endemic area pasteurization of dairy products is an important safety measure.
• Good hygiene and protective clothing/equipment are very important in preventing occupational exposure.
• Measures should be taken to exclude the entry of organism through skin, inhalation, ingestion while performing a necropsy, butchering and parturition or while removing fetal membranes and fluids.
• The Strain 19 B. abortus vaccine and B. melitensis Rev-1 vaccine must be handled with precaution to avoid accidental injection or exposure.
• Brucella organism should be handled under bio-safety containment level 3 or higher.
• 2.5% sodium hypochlorite, 2-3% caustic soda, 20% fresh slaked lime suspension, 2% formaldehyde solution (all tested for one hour) can be sprayed for inactivating the organism in contaminated surfaces.
• Moist heat of 121°C for at least 15 minutes (autoclave) is used to destroy contaminated equipments.
• Dry heat for 160-170°C for at least 1 hour (Hot air oven) for inactivation of glasswares is preferable.
• Boiling of liquids for 10 minutes is usually effective.
• Xylene (1ml/liter) and calcium cyanamide (20 kg/m3) are used to decontaminate liquid manure in 2 to 4 weeks if its use Gamma irradiation (e.g. in colostrum) and pasteurization can inactivate the organism.
• Screening, culling or isolation or depopulation of infected animals
• Proper disposal of infected carcass or aborted fetus and placental membrane.
• Stamping out policy by quarantine, vaccination, test and slaughter, depopulation, cleaning, disinfection, surveillance, trace back of animals, eradication and neutering.