Brucellosis

In cattle: Contagious abortion - Bang's disease"

"In humans: Undulant fever, Malta or Mediterranean fever

Nature of the disease

History
Etiology
Hosts
Transmission
Key signs
Lesions
Diagnosis
Immunity
Treatment
Control
References

 

Nature of the disease

  • Brucellosis is the contagious disease caused by the genus Brucella, in which it generally cause reproductive failure; that is, abortion or birth of unthrifty newborn in the female, and orchitis and epididymitis with frequent sterility in the male in a wide range of animal species and can affect man.

  • Animal brucellosis caused by various Brucella spp. is widespread, it affects cattle and buffalo (B. aborts), sheep (B. ovis), sheep and goats (B. melitensis), and pigs (B. suis). Brucellosis occasionally affects dogs (B. canis) and horses (B. aborts - B. suis).

  • In man, infection by B. melitensis and B. aborts causes Malta, and undulant fever respectively.

  • Persistent "lifelong" infection is common with this facultative intracellular organism that is able to survive and multiply within phagocytic cells and reticuloendothelial tissues with shedding in reproductive and mammary secretions.

  • Brucellosis is considered by FAO, WHO, and 0IE as the most widespread zoonosis in the world. B. melitensis, being very pathogenic for humane beings and the possibility of infection occurring by the drinking of infected sheep and goat milk necessitates the pasteurization of milk.

  • Animal brucellosis has a major economic impact in the major cattle-farming regions of the world primarily due to abortions and calf losses, decreased milk production and infertility that increases the period between lactations and prolongs the average of intercalving period. In sheep and goats, the economic losses stems from decreased breeding efficacy due to rams infertility and reduced milk production.

  • 0IE classification: list "B."

History and Occurrence   

  • Bruce in Malta identified B. melitensis for the first time in 1887 whereas B. abortus was identified by Bang in Denmark in 1897. The first record of brucellosis in Africa was that of Gray in 1906 in South Africa. In Egypt, Ahmed was the first to investigate the problem of brucellosis serologically among animals in 1939.

  • Brucellosis is widespread in most undeveloped countries especially of the Mediterranean region Middle East, Western Asia, and parts of Africa including Egypt and Latin America. Many countries have made considerable progress with their eradication programs and some eradicated the disease.

 

Etiology

  • Brucella spp. are small Gram-negative, non-motile, non-sporulated, non-encapsulated rods or coccobacilli. They are partially acid-fast in that they resist decolorization by weak acids in the modified Ziel-Neelson stain and the retained carbol fuchsin stains red the coccobacilli against the blue background.

  • Brucella are aerobes that require enriched media for primary isolation, they grow well on 5-10% blood agar but specimens other than fetal abomasal contents and colostrums requires selective serum agar media, such as Farrell's medium. B. abortus biotype-2, B. ovis, and B. canis are fastidious, slow growing, and require 5-10 % C02 for primary isolation to start and later ordinary air is sufficient. On specific media, such as tryptose agar, brucella allbimi agar, liver infusion agar, blood agar, and serum dextrose agar, at pH 6.6 - 6.8, after 2-4 days incubation brucella colonies are round entirely smooth, convex, glistening and translucent. Young colonies are pale honey color, 1-2 mm in diameter. Later, colonies become larger and slightly darker on continuous incubations.

  • Brucella spp. and biovars can differentiated on the bases of C02 requirements, production of hydrogen sulfide (HS), sensitivity to azo dyes, phage susceptibility, agglutination in monospecific sera, and other in vitro laboratory procedures.

  • There are six species presently known in the genus Brucella. They are nearly similar morphologically, culturally, biochemically and serologically, B. abortus (9 biotypes-cattle), B. melitensis (3 biotypes-sheep and goats), B. suis (4 biotypes-swine), B. ovis (sheep), B. canis (dogs and other carnivores), and B. neotama (desert wood rat).

  • B. abortus will survive for up to 8 months in an aborted fetus in shade, 1-2 months in dry soil, 2-3 months in wet soil, 3-4 months in feces and for 8 months in manure storage tanks at 15C. The pathogen will survive for few hours only when exposed to direct sunlight.

  • Brucellae are sensitive to pasteurization temperatures, sour milk or other products with a low pH., and susceptible to chemical disinfectants such as 2- 5 % carbolic acid solution

       

Hosts

  • Cattle and buffalo are the natural hosts for B. abortus, and infection occurs in all ages but is most common in sexually mature animals, particularly dairy cattle. Cattle and buffalo are liable to catch B. melitensis and B. suis (rarely) in regions where these spp. are prevalent.

  • B. melitensis is the important spp. that ordinarily infects goat flocks, but goats are liable to catch B. abortus and B. suis. Sheep are naturally infected with B. ovis and B. melitensis, but are liable to catch B. abortus and B. suis.

  • Camels are liable to catch B. abortus and B. melitensis.

  • Horses may occasionally infected with B. abortus and B. suis.

  • Dogs and cats may occasionally infected with B. canis, but dogs may also catch B. abortus, B. melitensis and B. suis. Dogs are important mechanical vectors of brucellosis by transporting of infected placentas and fetuses.

  • In human brucellosis: B. melitensis is the most pathogenic and occurs more frequently, followed, in order by B. suis and B. abortus.

  • Immature animals often showing little or no clinical disease but sexually mature animals are the most susceptible especially in the breeding season.

  • Guinea pigs are susceptible to all brucellae except B. ovis.

 

Transmission

  • Brucellae are present in the placenta, fetal fluids, fetus, vaginal discharges, milk, semen and urine. Infectious bacteria are also found in the bursa of horses with poll evil or fistulous withers. The major sources of infection are the contents of the pregnant uterus, the fetus and the fetal membranes, which contain large number of organisms that contaminate the vulva, tail, legs of the animal, and the surrounding environment. Excretion of organisms may occur for several days before abortion and up to several weeks after. Some infected cows that aborted previously shed brucellae from the uterus at subsequent normal parturitions. Most cows and heifers excrete brucellae in colostrums and milk during the first month of lactation, but some animals, can shed long-term or lifelong. In addition, the organism is secreted in urine, semen, and feces of infected animals.

  • Infection occurs mainly by ingestion of contaminated feed and water or after licking an infected placenta, calf, fetus or the genitalia of infected cow soon after it has been aborted or calved. Infection can also occur by penetration of skin, mucous membranes, and conjunctiva, or via inhalation. Venereal transmission rarely occurs in bovine brucellosis. However, artificial insemination with infected semen often results in transmission of the disease.

  • Calves are infected in utero or by sucking infected dams.

  • Humans become infected by ingestion (raw milk - unpasteurized cheese), through the mucous membranes and breaks in the skin, and probably by aerosols (in abattoirs and laboratories).

  • Most wild animals, rodents, insects and blood sucking arthropods (such as ticks) are carriers and reservoirs for infection, so they play a role in transmission of the disease 

 

Key signs

Bovine brucellosis      

  • Cattle and buffalo are the natural hosts for B. abortus, and other animals that are liable to catch the infection are considered "dead-end hosts" as they rarely act as a source of infection to cattle.

  • The course and infection consequences are usually governed by the age of infected animal and the immune status of the herd. Systemic signs do not usually occur and abortion rates in full susceptible herds vary from 30-70%. Calves not yet at puberty are fairly resistant to infection.

  • In highly susceptible non-immune herds, there is a high incidence of abortion (storm of abortions) in pregnant cows last for a year or more during the second half of gestation especially in the last 3 months of pregnancy. In subsequent pregnancy, the fetus is usually carried to full term but she will continue, at subsequent normal calvings, to shed the bacteria from the uterus and in the milk, although second or even third abortions may occur in the same cows. In less acute infections, cattle may give stillbirths and weak calves, or merely show a retained placenta leading to increased incidence of retained placenta in a herd. After abortion, metritis will follow in the majority of cases, impairing the fertility of the animal. Other signs of brucellosis infection in a herd may be decreased milk yield. In infected bulls, an acute to chronic orchitis, epididymitis and seminal vesculitis occasionally occurs. Arthritis and hygromas, especially of the carpal joints may occur in chronic infections in both males and females. Septicemia and death may occur in rare cases.

  • Cattle and buffalo are readily infected with B. melitensis when in close contact with infected goats and sheep, which sometimes causes them to abort but often confined to the udder and supramammary lymph nodes and excretion of the organism in the milk may prolonged for months or years. B. suis infection in cattle is rare and self-limiting.

Ovine brucellosis

  • In sheep and goats, B. melitensis can cause abortion, retained placenta, orchitis and epididymitis. Abortions usually occur late in gestation in sheep and during the fourth month of gestation in goats. In goats, mastitis and lameness may be seen. Arthritis is rare in sheep.

  • B. ovis affects sheep only that can cause epididymitis, orchitis and impaired fertility in rams. Initially, only poor quality semen may be seen; later, lesions may be palpable in the epididymis and scrotum. The testes may atrophy permanently. Abortions, placentitis and prenatal mortality can be seen but are uncommon. Systemic signs are rare.

  • B. abortus and B. suis occasionally infect sheep and goats if kept in contact with cattle and pigs.

Brucellosis in pigs

  • In pigs infected with B.suis, the most common symptom is abortion at any time during gestation, weak or stillborn piglets, and temporary or permanent infertility. Temporary or permanent orchitis can be seen in boars that excrete B. suis asymptomatically in the semen and sterility may be the only sign of infection. Swollen joints or lameness can occur in both sexes.

Brucellosis in camels

  • B. abortus and B. melitensis occasionally can infect camels, which may leads to abortion or stillbirths, bilateral lacrimation, slight lameness, and reduced appetite.

Brucellosis in horses

  • In horses, B. abortus and occasionally B, suis can cause inflammation of the supraspinous or supra-atlantal bursa known as fistulous withers or poll evil respectively. The bursal sac becomes distended by a clear, viscous, straw-colored exudate and develops a thickened wall. It can rupture, leading to secondary inflammation. Arthritis, intermittent lameness, and abscesses in the region of the sternum and fetlock may be evident.

  • Brucella-associated abortions are rare in mares.

Brucellosis in dogs

  • Dogs occasionally become infected with B. canis and may catch B. abortus, B. melitensis and B. suis.

  • B. canis causes abortions, stillbirths, and infertility in dogs. Abortions usually occur during the last trimester and are followed by a prolonged vaginal discharge.

  • Infected dogs may have lymphadenitis, epididymitis, orchitis and prostatitis.

Human brucellosis

  • Asymptomatic infections are common in humans. In symptomatic cases, the incubation period varies from 1-3 weeks or longer. Some cases of brucellosis resemble influenza; the symptoms may include irregular fever, headache, generalized weakness, malaise, sweating, fatigue and severe limb or back pains. Gastrointestinal signs especially in adults. Coughing and pleuritic chest pain, irritability, insomnia, and mental depression are occasionally seen.

  • In many patients, the symptoms last for 2 to 4 weeks and are followed by spontaneous recovery. Others develop recurrent bouts at 2 - 14 day intervals. Most people with this undulant form recover completely in 3 to 12 months. A few patients become chronically ill, with symptoms of chronic fatigue, depressive episodes and arthritis. Relapses can be seen months after the initial symptoms, even in successfully treated cases. Hypersensitivity reactions, influenza, and malaria can mimic the symptoms of brucellosis.

  • Occasional complications include arthritis, endocarditis, granulomatous hepatitis, meningitis, uveitis, orchitis, cholecystitis, osteomyelitis, and rare cases of encephalitis.

lesions

  • In adult cattle and buffalo, there is considerable variation in the lesions that are not pathognomonic. There may be a mild to severe endometritis that becomes chronic. The chorion may appear normal or uniformly affected. The cotyledons may be normal or necrotic covered by a sticky, odorless brown exudate and are yellow-grey. Parts of the intercotelydonary region are typically leathery with a wet appearance and focal thickening. The fetus may be normal, autolyzed, or may have varying degrees of subcutaneous edema, blood-stained fluid in their thoracic and abdominal cavities, or have evidence of bronchopneumonia. Beside the reproductive tract, the granulomatous inflammatory lesions may be found in the mammary gland, supramammary lymph nodes and joints.

  • In bulls, there may be seminal vesiculitis, epididymitis, and orchitis.

  • In sheep infected with B. melitensis, the Placentitis, edema, necrosis of the cotyledons and the thickened and leathery intercotyledonary region, mimics that seen in cattle whereas the placenta is usually normal in goats.

  • Fetal and placental lesions are rare in pigs infected with B. suis.

        

 

Diagnosis

Brucellosis is generally of no unique set of symptoms and may even be asymptomatic for long periods, so its diagnosis has traditionally relied on the detection of circulating antibodies followed by the bacteriological isolation and other tests.

Samples and specimens:

  • For culture, aborted fetuses (abomasal contents, lung, spleen), fetal membranes, vaginal secretions (swabs), milk, semen, and arthritis or hygroma fluids. Post mortem, mammary tissues, supramammary or genital lymph nodes, spleen, and graved uterus. Tissue samples are removed aseptically with a small amount of sterile phosphate buffered saline.

  • For serology, serum, milk, whey, and semen samples are used.

  • Microscopic examination of direct smears made from vaginal swabs or cultivation of milk and vaginal swabs on specific media have the advantage of detecting the organism directly and thus limiting the possibility of false positive results but culture is an expensive and time-consuming procedure. Direct examination may not detect the small numbers of organisms present in milk and dairy products.

  • Serological tests: There is no single test available that completely satisfies an accurate diagnosis requirement. Some serologic tests are not useful in some hosts or for some species of Brucella.  Surveillance testing consists of conducting a series of tests, each of which has its own special applicability.

  • The brucella milk ring test "MRT" is a sensitive, valuable and inexpensive screening test used in bulk milk samples. The added stained antigen is agglutinated by the brucella antibodies/fat-globule complexes and the all rise to form a colored cream layer at the top.

  • Serum agglutination tests (rapid plate agglutination test and standard tube agglutination test) are one of the traditional standard diagnostic tests. Agglutination tests may also detect antibodies in milk, whey, plasma, and vaginal mucous. It detects specific and non-specific antibodies originating from brucella infection or vaccination and considered the last test to reach diagnostically significant levels during the incubation stage of the disease and after calving or abortion in cows or heifers. In addition, during the chronic stage of the disease, it often becomes negative due to the low serum agglutinins when the results of the other tests may be positive.

  • Rapid plate test has the advantage of speed and in not being affected by the prozone phenomenon but it is slightly less sensitive than the tube test.

  • The slow tube agglutination test "SAT" is sometimes used as supplementary test and has some value in detecting IgM, the persistent and predominant immunoglobulin resulting from strain 19 vaccination.

  • Buffered brucella antigen tests "BBAT" is the most commonly used tests and relies on the principles that the ability of nonspecific IgM antibodies to bind is markedly reduced in a low pH. They include card test, rose bengal test "RBPT", and buffered plate agglutination test "BPAT". Rose bengal test is simple, rapid, spot agglutination test used for large-scale screening of sera. The buffered antigen inhibits the IgM and detects IgG and IgA. RBPT may be false negative in cases of residual antibody from vaccination, colostral antibodies in calves, and cross-reaction with certain Gram-negative bacteria such as Yersina enterocolitica, Salmonella urbana, Escherichia coli or, Pseudomonas maltophilliia, and Vibro species.

  • Complement fixation test "CFT" is very sensitive and specific and is regarded as the definitive test of choice but it is difficult to carry out. The test was better indicator of infection than the SAT and reactions to the CFT receded sooner than those to the SAT after S19 calfhood vaccination.  It more accurately detects chronic infections and there is a more rapid decline of CFT titers following vaccination, than with infection.  The CFT is often the last test to become negative in calves with colostral antibodies

  • Recently, an ELISA test is used to detect brucella antibodies in milk and serum and brucella antigens in vaginal discharge, it is more sensitive but less specific.

  • Other, less commonly used, serologic tests include rivanol precipitation and indirect hemolysis test "IHLT" as a supplementary to CFT in the latter stages of eradication schemes. Fluorescence polarization tests are being developed.

  • Allergic tests: Intradermal brucellin allergic skin test is of little value due to its minor sensitivity.

Surveillance testing:

  • Screening tests: The brucella milk ring test " MRT " are commonly used to screen bulk milk samples for B. abortus for locating infected dairy herds, whereas rose bengal or tube agglutination tests are commonly used for testing sera collected from cattle at markets or abattoirs to screen nondairy herds in an area, and reactors are traced to the herd of the origin.

  • Negative animals should be retested 30-60 days later or 14 day after calving as the first test may be carried out during the incubation stage of the disease and some infected cows or heifers may remain serologically negative for up to 21 days or longer after calving or aborting.

  • A definitive test must be carried out on animals, which are positive to a screening test as the screening tests are very sensitive but not always specific. Non-specific reactions are caused by strain 19 vaccination and occasionally by infection with other Gram-negative bacteria as Salmonella and Yersinia. CFT is the most specific definitive test and commonly used to detect individual reactors. All sera that test positive to RBPT should subject to CFT.

  • CFT and ravinol precipitation are designed to detect primarily antibodies specifically associated with brucella infection, and are used to distinguish between residual vaccinal titers and those due to infection and improves the probability of detecting reservoirs.

  • All bulls from infected herds should be subjected to clinical examination including palpation of the seminal vesicles and ampullae, serological tests, and bacteriological and antibody examination in semen.

  • In sheep, B. ovis infection can be diagnosed by several methods, which include palpation of the external genitalia of rams, the presence of B. ovis and neutrophils in semen smears with repeated sampling especially of subclinical cases, and serological tests as rose bengal test, tube agglutination test, CFT, FAT, and gel-diffusion tests.

  • In sheep and goats, B. melitensis infection can be diagnosed by the presence of B. melitensis in fetal abomasal content, vaginal discharge, or epididymis smears, and serological tests as rose bengal test, CFT, and ELISA. Brucellin allergic skin test can be used as a screening test in unvaccinated flocks. The bulk milk ring test is not used in small ruminants.

  • In pigs, B. suis infection can be diagnosed bacteriologically and serologically with CFT and ELISA.

  • In camels, rose bengal test and CFT are the most practical tests. Differential diagnosis:

  •  A wide range of infectious and non-infectious causes may be implicated in an abortion problem. Abortion problem investigations should follow a standard pattern that may include:

  • History;

  • Ascertain the age and necropsy of the fetus and examination of the placenta for evidence of placentitis;

  • Clinical examination of the cow to exclude conditions such as acute salmonellosis, summer mastitis or tick borne fever;

  • In laboratory: Blood samples for serological tests for brucellosis and leptospirosis. Examination of vaginal discharge and the contents of the fetal abomasum at the earliest opportunity for trichomonads, and by culture methods for B. abortus, Campylobacter fetus, trichomonads, listeria, and fungi. Examination of urine for leptospirosis;

  • Interpretation of results.

  Immunity

  • Naturally, infected animals and those vaccinated are considered to have a relative immunity. Sexually immature and sexually mature but non-pregnant animals become immune to one degree or another following infection, in that they may never show clinical signs of disease. Aborted animals are also become immune and do not usually abort again.

  • Naturally infected animals and those vaccinated as adults with strain 19 remain positive to the serum, and other agglutination tests, for long periods. The infected cattle sera contain high levels of IgM, IgG1, IgG2, and IgA isotypes antibodies. After vaccination of cattle with strain 19, IgM, the persistent and predominant immunoglobulin begin to appear after 5 days, reaching peak values after 13 days whereas IgG1 antibodies may appear with IgM and reach the peak values at 28-42 days, after which they decline.

  • Most chronically infected animals remain positive to the CFT, but a significant proportion become negative to SAT as serum agglutinins tend to wane within a few months.

  • Calves nursing positive reactor cows are passively immunized via colostrums, the half-life of colostral antibodies is about 22 days.

Treatment     

  • Treatment of bovine brucellosis neither effective nor practical, because of the intracellular sequestration of the organisms in lymph nodes, the mammary gland and reproductive organs. The drugs are unable to penetrate the cell membrane barrier and some antibiotics may cause L-transformations to B. abortus that may prevent its serological detection leading to creation of carrier animals. Also there is no practical treatment for infected pigs.

  • Antibiotics can eliminate B. ovis infections in valuable rams but the fertility may remain poor. Long-term antibiotic treatment is sometimes successful for B. canis infections in dogs. In horses with fistulous withers or poll evil, the infected bursa may need to be surgically removed.

 

Prevention and control measure  

  • Control and eradication of bovine brucellosis strategies and policies will invariably differ according to the situation in each instance. The control and ultimately eradication of bovine brucellosis is broadly based on vaccination, test-and-slaughter policy, and the prohibition of the sale and movement of infected cattle.

  • In countries and areas with a high prevalence of brucellosis, the major goal of control is the reduction of infected cattle through vaccination and good hygiene and management. In this situation, large-scale vaccination is used as the first phase of control before test-and-slaughter procedures are adapted. Vaccination greatly reduces the risk of abortion and the excretion of organisms. It will reduce the number of infected animals in a herd by over 90% if carried out for about 5 years.

  • Strain 19 vaccine: Vaccination of heifer calves at 4-8 months of age with strain 19 B. abortus, a live strain of low pathogencity and high immunogenicity provides a relative immunity for about 16-18 months.

  • A small percentage of vaccinated animals develop antibodies especially SAT that may persist into adulthood, and thus may confuse diagnostic test results. To minimize this problem, calves are vaccinated with a greatly reduced dose.

  • Vaccination of the adult cattle is usually not permitted but it may be of value in the event of an outbreak for reducing the effects of an abortion storm.

  • Strain 19 vaccine is of low virulence in cows, but systemic reactions may occur rarely in both calves and adults. Strain 19 vaccine is of no value and may cause orchitis in bulls. In addition, it has a zoonotic risk for veterinarians and cattle handlers.

  • The cessation of immunization with strain 19 vaccine should be considered only when the prevalence of infection is reduced to 0.2% or less.

  • The inactivated strain 45/20 adjuvant vaccine "Abortex" is safe in all ages but is not as protective as strain 19 vaccine in animals less than 9 months old. For animals over six month's, two consecutive inoculations are usually recommended to with 6-12 weeks apart. It interferes greatly with the interpretation of the CFT but little with the SAT.

  • Strain RB-51 vaccine: Strain RB-51 is a live attenuated, stable rough mutant of B. abortus strain, capable of inducing protection against infection and abortions as strain 19 vaccine. Unlike strain 19 vaccine, it does not develop antibodies detectable by the traditional tests of brucellosis, hence enabling to identify vaccinated animals from those infected with field strains. It is indicated for active immunization of heifer calves over 4 months old but it may induces placentitis that can cause premature birth when given to pregnant heifers in the six month of gestation. Age limits for vaccination are the same as strain 19 vaccine, 4-7 months of age is preferable.

  • Complete eradication in a region or a country when the prevalence of infection is reduced to 0.2 % or less, needs detection and slaughter of infected animals, followed by proper disinfection of premises with 2.5 % formalin and continuous screening, possibly combined with vaccination of calves. Its economic implications should consider.

  • All calves of infected herds should be regarded as potential sources of infection as latently infected calves that remain serologically negative until mid-gestation or later during their first pregnancy "two-year breakdown syndrome".

  • Continuous surveillance should maintain through regular blood testing in infected herds at 2-3 months as well as bacteriological examination. In MRT, milk from negative animals should be retested regularly at short intervals. All animals diagnosed bacteriologically or serologically positive should be regarded as infected and must be permanently identified.

  • It is important to prevent spread of the disease especially in outbreak. Cows prior and after parturition should be isolated. Aborted cows should isolated immediately with incineration of aborted fetuses, placenta, and discharges and tested serologically. Positive animals should be culled and negative animals should be retested 2-3 weeks later.

  • The herd is regarded as negative after 2-3 successive tests are negative.

  • The negative herd must be maintained as such. Replacement animals should be purchased from herds certificated free and should be tested before introduction into the herd and/or they should be vaccinated animals only.

References

1.  Alton, G.G. ; Jones, L. J. and Pietz, D. E. (1975): "Laboratory techniques oin brucellosis " 2 nd edit. Geneva: WHO.

2.  Blowey, R.W. and Weaver, A.D. (1991): "A colour atlas of diseases and disorders of cattle" Wolfe Publishing England.

3.  Bishop, G.C.  Bosman, P. P. and Herr, S. (1994): "Bovine brucellosis: In Infectious Diseases of Livestock with special reference to Southern Africa. Volume II (Coetzer, J. H.W.; Thomson, G.R. and Tustin, R.C.), Chapter 1124 pp. 1052-1072.

4.  Carter, G.R. (1986): "Brucellosis In: Essentials of Veterinary Bacteriology and Mycology" 3 rd Edit. Lea and Febiger, Philadelphia.

5. El Sawalhy, A. A. (1999) : "Veterinary Infectious Diseases" 2 nd Edit. Ahram Distribution Agency , Egypt .

6.  FAO/OIE/WHO (1995): "Animal health yearbook" Rome, Roma, Italy.

7.  Genral Veterinary Services (1994): "Brucellosis (in Arabic) TTc, NARP, Ministry of Agriculture, Egypt.

8.  Hungerford, T.G. (1990): "Brucellosis In: Diseases of Livestock" 9 th Edit. Mcgraw - Hill Book Company Sydney, New York, London, Tokyo.

9.  Miodrag Ristic and Ian MCIntyre (1981): "Diseases of cattle in the tropics".  Martinus Nijhoff Publishers, The Hague, Boston / London.

10.  Merchant, I.A. and Barner, R.D. (1964): "Brucellosis In: An outline of the infectious diseases of domestic animals " Oxford, IBH Publishing Co, New Delhi, Bombay.

11.Nielsen, K., and Duncan, J.R. (1990): "Animal brucellosis". CRC Press, Boston, Massachusetts.

12.Quinn, P.J.; Carter, M.E.; Markey, B. K.; and Carter,G.R. (1994): "Clinical veterinary microbiology”. Mosby -Year Book Europe limited.

13.Radostits, O.M.; Gay, C.C.; Blood, D.C.; and Hinchcliff, K.W. (2000): "Brucellosis   In: Veterinary Medicine A textbook of the diseases of cattle, sheep, pigs, goats and horses".9th Edit.W. B. Saunders Company Ltd. London, New York, Philadelphia, San Francisco St Louis, Sydney.

14.Wernery, U. and Rueger Kaaden, O. (1995): "Infectious diseases of Camelids" Blackwell wissenschafts-Verlag Berlin 1995 Oxford, Edinburgh, Boston, London, Paris.