Brucella

Genus Brucella has 6 species namely: B. abortus, B. canis, B. melitensis, B. ovis, B. suis except B. neotome (important pathogens of domestic animals and man) Brucellosis = disease cause of this bacterial pathogen Infections = reproductive organs and reticuloendothelial tissues Abortion = in cattle, sheep, goat because of the lesions of the female reproductive tract and of the placenta and fetus. Transmission = directly or indirectly from infected animls to humans (there is a zoonotic importance because of this) Brucella melitensis = first member of the genus to be isolated by David Bruce in 1887 Source = from the spleens of patients who died in Mediterranean or Malta fever Brucella abortus = the second member was isolated by Fredrick Bang in 1897 Source = aborted bovine fetus

Brucella suis = isolated by Jacob Traum in 1914 Source = aborted piglet B. Ovis and B. canis = more highly host adapted and they were isolated in 1956 and 1969 respectively Morphological and cultural features of the 6 members = not sufficiently characteristic to differentiate. Nor the host from which the organism is isolated . But each species have a predilection site for certain hosts. *Isolation of a Gram-negative rod from a suspected case of brucellosis requires reliance on the results of the laboratory tests.

The species of Brucella and other principal hosts B. suis B. melitensis B. canis B. abortus humans B. neotome wood rats B. suis B melitensis B. abortus B. canis dogs B. abortus B. melitensis B. suis pigs B. suis B. abortus horses B. abortus B. melitensis goats B. ovis (epididymitis) B. abortus B. melitensis sheep B. suis B. melitensis B. obortus cattle Other pathogenic Brucella sp . Isolated Principal pathogenic Brucella sp . Isolated Host

Brucella abortus (Bacillus abortus, Bang’s bacillus) Affected animal = cattle population (absent in countries where eradication programs have been actively pursued) Morphology and Staining reaction = short rod / a coccobacillus (0.5 to 0.7 µ m by 0.6-1.5 µ m.) =rods are short that are mistaken to be a cocci =arranged singly, although in cultures appear to be in short chains = a facultative intracellular bacterium = found in clumps and smears made from exudates =Gram negative and stains with ordinary stains although with some difficulty =not acid fast =Can resist decolorization with some mild acids.

Brucella abortus (Bacillus abortus, Bang’s bacillus) Koster stain = which the organism stains deep red Other characteristics = nonmotile, doesn’t form spore, doesn’t have a well-developed capsule (poorly developed capsules have been demonstrated on freshly isolated strains using special stains) Cultural and biological features =aerobic growth (many strains require increased tension of CO 2 for growth especially on primary isolations = is catalase and oxidase positive and usually produce H 2 S from protein or peptides rich in amino acids containing sulfur =produces urease =8 biotypes are currently recognized by dye sensitivity test, CO 2 requirement, H 2 S production, and presence of A or M Antigen. = not hemolytic, doesn’t liquefy gelatin, doesn’t produce acid from glucose = It has a characteristic substrate oxidation pattern

Brucella abortus (Bacillus abortus, Bang’s bacillus) Growth = enhanced by serum or blood Tool for primary isolation = complex media (serum dextrose agar or Albimi Brucella agar or broth) Antibiotics = they are sometimes added to these basal media to inhibit contaminants in specimens such as milk and vaginal discharge. Primary isolation = they are slow-growing and barely visible at 48 hours = reach maximum size after 5- 7 days at the optimum growth temperature (37 o C) = may be on a smooth type characterized by a round convex colonies with an entire edge or they may be associated by rough type colony in which they have large flat colonies with a dull granular appearance. =growth is sparse in fluid media and appear as a faint clouding.

Brucella abortus (Bacillus abortus, Bang’s bacillus) Bacteriophage Typing Bacteriophage = often used for identifying and typing cultures of Brucella sp. Tb, Wb, Fi, Bk2, and R strains = the phages currently in routine Rotuine Test Dilution (RTD ) = dilution of the phage; the minimum number of phages that will produce confluent lysis on a lawn of the propagating strain Phage typing = is rapid and very useful tool of identifying B. abortus Antigen cell wall of B. abortus = consists of an outer layer of lipopolysaccharide protein on which polysaccharide chains are exposed Polysaccharide chains = carries the major antigen (A and M) involved in the agglutination reaction

Brucella abortus (Bacillus abortus, Bang’s bacillus) cell wall proteins have been grouped into three = 88,000-94,000. 95,000-40,000 (porins); and 25,000-30,000 Epizootiology and Pathogenesis B. abortus = an obligate facultative intracellular parasite of cattle and some wild ruminants; they are not very resistant to sunlight and drying and therefore survives better in winter than summer; can survive and grow in host macrophages and epithelial cells Transmission = ingestion of contaminated discharges or feed; venereal transmission is possible but unusual; intramammary and congenital transmission also occur Other infected animals = horses, sheeps, chickens, and dogs

Brucella abortus (Bacillus abortus, Bang’s bacillus ) survives = in milk destroys = pasteurization Large dairy herds = more likely to be infected with a high prevalence of infection within the herd. the greater level of intensivism and opportunity for transmission within large herds together with the greater likelihood of introduction of a latently infected animals are some of the factors that contributes to the higher risk of infection in large herds factor of transmission = common pastures, contaminations of streams, activities of carrion feeders Brucella infection in free-living animals = bison, moose and elk (comingling with this animals in cattle enhances the possibility of cross-infection) Prepubescent animals = resistant to infection

Brucella abortus (Bacillus abortus, Bang’s bacillus) Heifer calves = can be infected early in life either in utero from infected dams or by ingestion of contaminated milk (susceptibility increases with sexual development and pregnancy) Latent infection = cannot be detected by serological test in calves exposed in this way Male cattle = resistant to infection but infection of the epididymis and testicle has been recorded (Bulls can transmit the infection in their semen) Main portals of entry = oral mucosa (milk), nasopharynx, and conjunctiva of cattle as well as the genital tract in both bulls and cows experimental conditions = organisms has been shown to penetrate the unbroken skin of guniea pig and cattle. after penetrationof the host, B. abortus initially localizes in the regional lymph node and then enters to the blood stream.

Brucella abortus (Bacillus abortus, Bang’s bacillus) bacteremic phase = results in dissemination of the organisms to the udder, uterus, and associated nodes. B. abortus = has an unusal resistance to intraleukocytic killing; proliferates massively in cells with high levels of erythritol (found in genital tract in male and pregnant female) *it penetrates the epithelial cells of the chorion and proliferates, producing placentitis. * Endometritis is produced with ulceration of the epithelial lining of the uterus. *Lesions of the fetus include edema and congestion of the lungs along with hemorrhages of the epicardium and splenic capsule. *fetal death occurs (it is unclear whether endotoxin of the organism or to interference with placental function)

Brucella abortus (Bacillus abortus, Bang’s bacillus) * inflammation of the membranes (this interference with the circulation of the fetus may explain why abortion occurs) *organisms found in the stomach and lungs of aborted fetuses * abortion occur late in pregnancy *organism is present in large numbers in uterine discharges for a few days but is then gradually cleared from the reproductive tract (after calving or abortion) *infection is maintained in the reticuloendothelial tissue and the udder Milk = source of infection for calves and also humans (shed in large numbers)

Brucella abortus (Bacillus abortus, Bang’s bacillus) *abortion are usually seen only during the first pregnancy after infection (placenta is infected during each pregnancy) *organisms are seen in the lymph nodes of the digestive tract and in the spleen in the infected cattle. *it has been isolated from the blood and from hygromas of the knee (shows high correlation with brucella abortion in African cattle) B. abortus in horses = less frequency than in cattle; frequently localizes in bursae, joints, or tendon sheath and has been found in poll evil and fistulous withers, supra-atlantal bursitis, and supraspinous bursitis as well as in lesions of the fetlock and sternum.

Brucella abortus (Bacillus abortus, Bang’s bacillus) Immunity *the organisms are so readily escapes the bactericidal effects of antibody and complement in plasma protective immunity = depends mainly on cell-mediated responses in which the microbicidal activity of macrophages is enhanced following activation by lymphokines from sensitive T- lymphocytes. *the organism has been shown to multiply more slowly in the macrophages of vaccinated calves than in control animals. humoral antibodies = correlate poorly with protective immunity Strain 19 vaccines = cattle vaccinated by this have been shown to be resistant to challenge after antibody titers fall below detectable levels IgM agglutinins = first antibodies to appear in the plasma and their levels reach a peak at about 2 weeks; IgG antibodies appear later.

Brucella abortus (Bacillus abortus, Bang’s bacillus) IgG1 subclass = infected cattle have high titers of nonagglutinating antibody pH 3.6 = treatment with acid at this level activates their agglutinating ability. strain 19 = IgM antibody levels increase rapidly in calves vaccinated with this strain; IgG1 antibody levels rise more slowly, do not attain high high levels and do not persist. also, levels of nonagglutinating IgG1 antibodies are much lower than in naturally infected animals. protective immune responses = appear to be most effective when infection occurs before puberty. the majority of infections in sexually mature animals persist for life although the immune response does reduce the severity of disease in the uterus and placenta so that such animals are more likely to carry to term later in pregnancies.

Brucella abortus (Bacillus abortus, Bang’s bacillus) Vaccines strain 19 and the McEwen killed 45/20 = two vaccines that are important in the control of B. abortus infection Strain 19 = consists of viable culture of a strain that was discovered to have very little virulence for guinea pigs and cattle but to possess excellent immunizing properties. = it has a great stability = it is a smooth strain that is mildly pathogenic for guinea pigs = pregnant cattle can be made to abort by inoculating them with large doses of this

Brucella abortus (Bacillus abortus, Bang’s bacillus) = vaccine organisms can usually be demonstrated without difficulty in the fetal membranes and the fetus itself = susceptible cattle, associating with those have aborted as a result of inoculation with the vaccine strain, do not become infected = it is unlikely transmitted from one animal to another; if any damage is done by the use of this vaccine, it is limited to the animal injected. = it is rarely shed in milk of vaccinated animals = it can cause infection to humans, although it is usually mild with recovery in a much shorter time than with virulent strains = it is dangerous to humans and should be handled with caution = calves are vaccinated 2-10 months of age (vaccination at this age is advocated to avoid persistent agglutinins that could create diagnostic problems later in the animal’s life)

Brucella abortus (Bacillus abortus, Bang’s bacillus) = it protects about 65-70 percent of animals for 4 or 5 pregnancies and is most effective for protection of young breeding animals when applied in a herd basis. = animals vaccinated as adults are also protected but develop persisting agglutinins. = Latently infected animals or animals in the early stages of infection do not benefit from vaccination. Colostral antibodies may interfere with response to strain 19 during the first 5 months of the calf's life. = vaccination of cows in early pregnancy with large doses produces probability of uterine infection. this risk is greatly diminished when the dose is reduced. = horses with fistulous withers sometimes treated by this vaccination

Brucella abortus (Bacillus abortus, Bang’s bacillus) McEwen killed 45/20 vaccine = a rough strain that is inactivated and adjuvanated for use as a vaccine or antigen in the anamnestic diagnostic test = it is administered in two doses (6- 12 weeks apart) = it stimulates complement fixing antibodies and agglutinins for rough B . abortus antigens of the IgG1 subclass = it has been used widely for the control of bovine brucellosis

Brucella abortus (Bacillus abortus, Bang’s bacillus) Diagnosis bacteriologically and serologically = B. abortus can be diagnosed by these. Bacteriological diagnosis = isolation and recognition of B. abortus are often accomplished by direct culture on a basal medium such as tryptose agar or Albimi agar to which serum and selected antibiotics are added. = cultures are incubated at 37 o C in an atmosphere of 10 percent co2 tension and are examined 2-3 days =serologic typing of Brucella strains and phage typing with the Tbilis (Tb) strain of bacteriophage are useful aids to definitive identification, but their use in restricted to specifically equipped reference laboratories

Brucella abortus (Bacillus abortus, Bang’s bacillus) Specimens should be examine by the following: 1. The aborted fetus = direct cultures of specimens of the stomach contents, intestinal contents, or the lung tissue 2. the placenta = direct stained smears from the outer surface of the chorion, especially from the margins of the characteristic thickenings, will usually suffice to make positive diagnosis without the need for cultural method (the organisms occur free or enclosed in the epithelial cells). these are visualized using the modified Koster stain by which appear red against a blue background.

Brucella abortus (Bacillus abortus, Bang’s bacillus) 3. The uterine exudate = after abortion or calving, when the placenta has been infected in the lochia and can be recognized by guniea pig inoculation. (organisms seems to disappear and cannot be found at the uterus until the animal is again pregnant and reinfection of the organ occurs) 4. milk = udder is infected and detected by the intraperitoneal injection in milk into guinea pigs or by direct culture 5. abscesses = direct culture of specimens from abscesses of the testicle and epididymis usually produced pure cultures, and isolations have been made from hygromas in cattle and from infected bursae in horses.

Brucella abortus (Bacillus abortus, Bang’s bacillus) Inoculation of guinea pigs = most reliable method of detecting B. abortus in infected materials. their serums are examined for the presence of antibodies and specimens from the spleen, liver, regional lymph nodes, testicles are cultured. Direct examination of tissues and fluorescent antibody = aid to diagnosis and practically useful for examining contaminated materials (placental membranes, cotyledons, fetal or vaginal discharges).

Brucella abortus (Bacillus abortus, Bang’s bacillus) serological diagnosis =the test are applied to serum, whole blood, vaginal mucus, seminal plasma, whey, or milk. Serum tube agglutination test (SAT) = used in eradication programs; The test is performed in small test tubes on dilutions of serum. Complete agglutination at a serum dilution of 1:100 and higher is considered positive. In Great Britain, a titer at least 50 percent agglutination at 1:40 is considered positive Flase-positive reaction = can be caused by reactions of antibodies to Yersinia enterocolitica 09 and other Gram-negative organisms. It occur in a significant number of serum sampls from latently or chronically infected animals. Animals in the early stage of infection similarly may fail to react.

Brucella abortus (Bacillus abortus, Bang’s bacillus) Buffered antigen plate agglutination tests = replaced standard SAT because the use of buffered antigen reduces the frequency of false-positive reactions Rose bengal plate tests = B. abortus cells stained with rose bengal and suspended in acidic buffer to inhibit nonspecific agglutinins. The test can be used as a screen or definitive test. The frequency of false-negative reactions is less than for the SAT Card test = the antigen is a buffered, stained, whole cell suspension of B. abortus strain. the antibody source is plasma separated from blood following clumping or erythrocytes by lectins. the test is useful for the detection of early infection where IgM is the dominant antibody.

Brucella abortus (Bacillus abortus, Bang’s bacillus) Plate agglutination = test is a heavy suspension of strains of B. abortus stained with gentian violet and brilliant green to make the test easier to read. Serum, whole blood, or whey can may be used in this test. (using glass slide or plate) whole-blood method = used for testing range cattle when it is desirable to hold the animals in chutes until the results are known Milk ring test = the antigen is a hematoxylin-stained suspension of killed B . abortus . It is mixed with fresh milk in a tube in the proportion of one drop per milliliters of milk. the mixture is then incubated in a water bath at 37oC for 1 hour.

Brucella abortus (Bacillus abortus, Bang’s bacillus) clumps of agglutinated organisms = a positive test is indicated by a bluish violet cream layer and a decolorized milk column. The test is widely used for the examination of bulk milk samples and so allows herds in an area to be quickly and economically screened. positive herds are then subjected to blood tests on individual animals . complement-fixation test = one of the most accurate tests in widespread use for the diagnosis of B. abortus infection. It detects IgM and IgG1 antibodies only. The test does not differentiate infected animals from animals recently vaccinated with strain 19 or 45/20. This test designed to test antibody to the 45/20 vaccine is also available. And for that, the test has been automated in many laboratories thus resulting in greater precision.

Brucella abortus (Bacillus abortus, Bang’s bacillus) Antiglobulin (coombs) test = measures binding of nonagglutinating antibodies to B. abortus antigen. The ravinol and mercapteothenol tests = used to help distinguish between agglutinin titers caused by chronic infection and those caused by vaccination with strain 19. IgM antibodies are sensitive to mercaptoethenol. Enzyme-linked immunosorbent assay (ELISA) = offer promise of greater sensitivity and specificity than the test described above. Allergic skin test = based on purified antigens; is to be a valuable addition to serology for detecting animals.

Brucella abortus (Bacillus abortus, Bang’s bacillus) Control 1. affected animals are detected and eliminated from the herd (detection using serological test - milk ring test - agglutination test - slaughter) 2. resistance of the remaining animals or any replacements is increased by vaccination 3. General principles of hygiene are imposed to prevent spread or reintroduction of infection systematic calf vaccination = a protective measure in herds that are only lightly infected systematic calfhood vaccination with strain 19 or 45/20 = is used in heavily infected herds

Brucella abortus (Bacillus abortus, Bang’s bacillus) calfhood vaccination = it is maintained in such a herd as long as there is a real danger of the exposure to the disease from any source periodic milk-ring tests and random testing on serum = surveillance of herd is maintained on bulk milk samples when they are sent to slaughter back-tagging = allows infection to be traced back to the her of origin

Brucella abortus (Bacillus abortus, Bang’s bacillus ) Antimicrobial sensitivity Gentamicin, kanamycin, tetracycline, and rifampicin = B. abortus is sensitive to these substances tetracycline + streptomycin = treatment for human brucellosis *cattle are not usually given antimicrobials for prophylaxis or therapy of B. abortus infection

Brucella suis (porcine type of Brucella ) =caused by brucellosis in swine. =has wider range of host specificity (humans, dogs, rodents, horses, reindeer, musk, oxen, and wild carnivores) Five types are recognized = Biotype 1, 2, 3, 4, and 5 Biotype 1, 2 , 3 = have the pig as their natural host but can be transmitted to other host Biotype 2 = occurs naturally in horses and relatively avirulent for humans Biotype 4 and 5 = reindeer and rodents are the primary natural hosts (but others can be infected).

Brucella suis (porcine type of Brucella ) Morphology and Staining reaction B suis is closely resembles B. abortus Cultural and Biochemical Features =does not require increased levels of CO 2 for growth. = large amounts of H 2 S are produced by Biotype 1 but more produce by other biotypes = Urea is highly hydrolyzed

Brucella suis (porcine type of Brucella ) Antigen =surface antigen predominates a smooth cultures of all biotype except biotypes 4 and 5 Biotype 4 = has both A and M antigens in equal amounts Biotype 5 = has mostly M antigens on its surface Epizootiology and Pathogenesis = it occurs to most swine raising areas (not been reported in Great Britain, Iceland, or Canada) = Survival in the environment has not been well studied (but similar to B. abortus ) Feces and urine = the organisms survives at least 6 weeks

Brucella suis (porcine type of Brucella ) Feral swine = is most infected than domesticated swine; can be also a source of infection for cattle Biotype 1 = is isolated from tissues of wild swine wild boars = important reservoirs of B. suis biotype 2 source of infection = contaminated pastures from these hosts can infect domesticated swine Reindeer ( Rangifer tarandus ) = brucellosis is an important disease; B. suis biotype 4 has been isolated from them; it cause bursitis, spondylitis, arthritis, and orchitis Larvae of reindeer warble fly = seen the B. suis organism and there is a speculation that this insect may act as a vector of this disease

Brucella suis (porcine type of Brucella ) Wolves of Siberia and sled dogs in Alaska = seen B. suis because they fed on infected reindeer meat Soviet Union = brucellosis is of particular interest and significance because of the large number of their free-living reindeer Domestic guard dog = seen to be a carrier of B. suis biotype 2 (source of infection for a herd of domestic swine) *although feral animals can be a source of B. suis infection for pigs, the organism is transmitted almost exclusively from pig to pig (by coitus or by the ingestion of feed contaminated by urine or genital excretions from infected boars or sows)

Brucella suis (porcine type of Brucella ) * spread is rapid in a susceptible herd * outbreak of disease followed by intermittent cases as susceptible newcomers contact chronically infected pigs. initial infection = B. suis is localized in the regional lymph nodes. It proliferates and may cause extended bacteremia with generalized infection of the spleen, lymph nodes, joints, udder, and genitalia B. suis = a facultative intracellular parasite and owes a great deal of its pathogenic properties to its ability to survive in the host's phagocytic cells. Erythritol = has a growth-stimulating effect upoin B. suis and is found in placenta of sows and seminal vesicles of boars

Brucella suis (porcine type of Brucella ) stimulatory action of eythritol + its presence on tissues = lesions in male and female genital tracts clinical signs and lesions produce = depends on animal's age, previous exposure, organ or organs involved Suckling and weanling piglets = they can be affected adults = can have abortion, metritis, spondylitis (vertebra in the lumbar and sacral region are not uncommon), lameness, and paralysis posterior paralysis = caused by pressure from the necrotic tissue on the spinal cord nodular splenitis = also been associated with brucellosis

Brucella suis (porcine type of Brucella ) Immunity field and experimental evidence = indicates the swine immunity to brucellosis is very slight and that after a period of herd resistance, animals are again susceptible to the disease Abortion = don't occur after the first exposure but when reexposed cell-mediated immunity = important in protecting the host Diagnosis cultural methods and agglutination test = positively diagnose brucellosis in swine

Brucella suis (porcine type of Brucella ) Isolated B. suis = from blood, spleen, uterus, lymph nodes, and mammary glands of sows and testes and semen of boars *methods are the same as B. abortus except that an increase of the CO 2 tension of the culture jar (unnecessary) Agglutination test = used as an aid in diagnosis but not as reliable in swine as it is in cattle; it is valuable in determining whether the infection exists in a herd Card test and rose bengal test = superior to most other serological test for detection of antibody in swine serum ELISA = it replaced the tube agglutination test

Brucella suis (porcine type of Brucella ) Control sell all stock to slaughter = simplest way to eradicate the disease *the premises should be thoroughly cleaned and disinfected For breeding herd in which blood lines must be preserved = pigs are raised from the infected unit. They are weaned (8 weeks old) and tested individually by agglutination test If negative = they are removed from the infected herd, placed on a clean ground and raised in isolation *all pigs are tested periodically (reactors are immediately removed) when of Breeding age = serological negative gilts are bred to noninfected boars. the original herd is disposed of as soon as the replacement unit has grown to sufficient size

Brucella suis (porcine type of Brucella ) Antimicrobial Susceptibility B. suis is sensitive to = vitro to aminoglycosides, rifampicin, and tetracyclines clinical efficacy of antimicrobials = very low

Brucella melitensis (Bacterium melitensis, caprine type of Brucella, Micrococcus melitensis) =first isolated by Bruce in 1887 from the spleen of a resident of the Island of Malta who had died of a disease known as Malta fever or Mediterranean fever = it is a disease of goats and sheep Morphology and Staining Reactions = forms small rods that are so short that they are mistaken to be a cocci stains = same as B. abortus

Brucella melitensis (Bacterium melitensis, caprine type of Brucella, Micrococcus melitensis) Cultural and Biochemical Features = does not require supplementary CO 2 for growth and either does not produce H 2 S (does in trace amounts) solid and liquid media of B. abortus = suitable for B. melitensis 3 biotype = Biotype 1, 2, 3 Biotype 1 = the most common Antigens smooth cultures = have the A and M antigens or both antigens together.

Brucella melitensis (Bacterium melitensis, caprine type of Brucella, Micrococcus melitensis) Epizootiology and Pathogenesis natural hosts = goats and sheep other infected animals = pigs, cattle, camels, and humans, feral animals including hares and impala Goats = more susceptible than sheep Source = ingestion of feed or water contaminated by uterne and vaginal discharges from infected does Milk = contains large numbers of this organisms Pathogenecity = same as B. abortus in cows B. melitensis = facultative intracellular parasite that survives and multiplies within the phagocytic cells of the reticuloendothelial system

Brucella melitensis (Bacterium melitensis, caprine type of Brucella, Micrococcus melitensis) Erythritol = the growth is stimulated Extended bacteremia with fever = 5 to 10 weeks or longer is followed by abortion storm (highly susceptible goat herd or sheep flock) Infected does = shows lameness, hygromas, and mastitis Billy goats = develops orchitis Kid goats = can remain latently infected until sexually mature Immunity Goats and sheep = develop effective protective immune responses to this organism that eventually clear the infection Humoral antibody = appears about 2-3 weeks after infection

Brucella melitensis (Bacterium melitensis, caprine type of Brucella, Micrococcus melitensis) Infected sheep = develop delayed hypersensitivity to antigens of B. melitensis live avirulent Rev 1 and strain 53H38 = vaccines used to prevent and control B. melitensis Rev 1 = more effective than 53H38; prevents abortion and shedding of the organism; animals immunized by this become serologically negative 6 months later. disadvantage of this is that it may localize in the placenta of pregnant does and cause abortion. Localization in the mammary gland and shedding in the milk also occur 53H38 = stimulates more persistent antibody response then Rev 1

Brucella melitensis (Bacterium melitensis, caprine type of Brucella, Micrococcus melitensis ) Diagnosis bacteriological methods = the same as those used to detect brucellosis phage sensitivity, agglutination with monospecific antiserum, guniea pig inoculation = isolations may be identified complement fixation, agar-gel immunodiffusion, and rose bengal plate test = serological tests that are available and give comparable results with the classic agglutination test Whey test = can be performed also Milk-ring test = not reliable on sheep milk Intrapalpebral allergic test = more sensitive but slightly less specific than serological assays for detection of infection in sheep flocks

Brucella melitensis (Bacterium melitensis, caprine type of Brucella, Micrococcus melitensis) Antimicrobial Susceptibility Aminoglycosides, rifampicin, and tetracyclines = sesitive to B. melitensis Efficacy of antimicrobials in therapy = has not been reported

Brucella neotomae = first isolated by Stroenner and Lockman from a dessert wood rat ( Neotoma lepida ) trapped alive in the Great Salt Dessert of Utah = it has not been recovered from any other naturally infected host wood rat = well tolerated and upon experimental infection it persist for at least a year without producing significant lesions

Brucella ovis =causes epididymitis in rams = early isolation in New Zealand and Australia = produces late abortions in females and lowers flock fertility Morphology and Staining Characteristics =acid fast under certain conditions of staining korster staining method = it stains blue, not red cultural and biochemical features =requires supplementary CO 2 for growth = does not produce H 2 S =most strains are urease negative = does not metabolized erythritol or other carbohydrates

Brucella ovis colonies = appear to be smooth, they are always in the rough phase on primary isolation = there are no biotypes = the only species to show less than 100 percent DNA homology with other species in the genus Antigens = it has an R surface antigen that cross reacts with rough strains = does not react with A or M antigens = shares a large number of protein antigen with other Brucella sp.

Brucella ovis Epizootiology and Pathogenesis sheep = only natural hosts of B. ovis British breeds = more susceptible than Merinos rams = clinically affected much more frequently than ewes Transmission = venereal, and rams become infected by mating with ewes previously covered by other infected rams ewes = developed placental infections with subsequent abortion Pasture = B. ovis can survive for a month or two Route of natural infection = ingestion; rams can be experimentally infected by the oral route as can ewes in the early stages of pregnancy; rams as young as 4 months old can be come infected

Brucella ovis clinical signs = transient, inapparent bacteremia with subsequent localization of the organism in the epididymis of the male. (tail of the epididymis is affected often unilaterally). the ram may show no clinical signs at this time but shed the organism in its semen for a prolonged period . epididymis = a permatocele forms and ruptures, and finally spermatic granulomas form with later testicular atrophy Rams = have lowered fertility Type 3 hypersensitivity responses = plays a role in the pathogenesis of the epididymis because lesions in experimentally challenged rams are more severe in animals previously vaccinates than in non-vaccinated controls.

Brucella ovis Ewes = less susceptible to infection by B. ovis but lesions occur. They vary from a superficial purulent exudates on an intact chorioallantoic membrane to advanced fibrosis and necrosis of this membrane Lambs = organism is pathogenic to this in the utero, but the fetus may survive in the presence of infection Placentitis = interferes with fetal nutrition Immunity cell-mediated = immunological basis of the protective immune response *many rams that become infected do not develop epididymitis humoral antibodies = detectable in about 3-4 weeks after infection, and delayed cutaneous hypersensitivity to B. ovis antigens develops later Vaccination of ewes = does not influence spread of infection in a flock

Brucella ovis vaccines = consists of saline oil adjuvant bacterins of formalinized B. ovis or live B. melitensis Rev 1 B. ovis 7-26 = a live vaccine was shown to be effective in 6-10 weeks old lambs (it was not transmitted to unvaccinated sheep) Vaccination of ewes = does not influence spread of infection in a flock Diagnosis =palpation of the testicles, culture of the semen, and demonstration of antibodies in the serum Palpation = is of limited value since many advanced cases show no palpable lesions of the testes (enlarged epididymis with testicular atrophy is valuable in diagnosis)

Brucella ovis culture of the semen = important adjunct to diagnosis and has been facilitated by the use of selective media modified for the growth of the organism flourescent antibody test = use to detect the organism in a semen smear complement-fixation, ELISA, immunodiffusion, and hemagglutination test = used for the detection of the antibody in the serum ELISA = less accurate and sensitive as the complement-fixation test nad is easier to perform complement-fixation test = rams are five times more likely than ewes to be positive

Brucella ovis Control = depends on preventing spread of infection between rams Infected older rams = must kept separate from young rams and the latter are used only on ewes known to be free of infection eradication of the disease = reacts in the complement-fixation and ELISA

Brucella canis (canine type of Brucella) first isolation = in 1966 from outbreaks of abortion and whelping failures among dogs in the US and Great Britain 1968 = it was characterized and named Brucella canis = highly adopted to domestic dogs =not readily transmitted to other animals, although it will infect humans. Morphology and Staining reactions =a small rod-shaped coccobacillus similar to other Brucella sp. Stains = similar to other Brucella sp.

Brucella canis (canine type of Brucella) Cultural and Biochemical Features =produces only rough or mucoid colonies = smooth phase has not been observed = it is inhibited by 10 percent CO 2 Broth =growth becomes quite mucoid and a ropy, viscous sediment is formed =does not utilize erythritol as a preferred nutrient =produces large amounts of urease = does not form H 2 S.

Brucella canis (canine type of Brucella) Antigens = it carries R but not A or M surface antigens =species-specific antigen is associated with the mucoid antigen shed by the organism = cross-reactivity with Actinobacillus equuli , Pseudomonas aeruginosa , and P. multicoda also occur = it has been found in breeding kennels, pet and stray dogs, and in lkaboratory colonies of beagles = infection appears to be more prevalent in stray dogs than in pets = 2-3 years age group had a higher prevalence of seropositivity than did older dogs

Brucella canis (canine type of Brucella) Transmission = occurs principally at the time of abortion, when may bacteria are shed in the persistent vaginal discharge. it continues 4- 6 weeks after an abortion Male dogs = harbor the organism in their genital tracts, from it shed intermittently (transmitted by coitus to the female) =after initial infection, bacteremia follows for 2 years. = infected dogs do not show no clinical signs but some do exhibit clinical signs have generalized lymphadenitis, splenitis, and embryonic deaths and abortions at approximately 50 days of gestation. Infected males = have epididymitis, scrotal dermatitis, and testicular atrophy.

Brucella canis (canine type of Brucella) Sperm agglutination in the semen, phagocytosis of spermatozoa, delayed skin hypersensitivity to testicular antigens = isoimmune responses to sperm antigens plays a role in the pathogenesis of B. canis infections and male fertility *there is evidence that damage to epithelial cells of the testes, epididymis, and prostate caused by B. canis (triggers production of sperm antibody)

Brucella canis (canine type of Brucella) Immunity cell-mediated = protective immune response of B. canis Humoral antibodies = appear at between 2-7 weeks nad are present in highest concentration in bacteremic animals Titers = diminish with resolution of the bacteremia *there is no successful vaccines against B. Canis infection in dogs B. ovis vaccines = can stimulate some resistance to B. Canis , but this resistance is much inferior to that observed dogs following recovery from B . canis infection

Brucella canis (canine type of Brucella) Diagnosis *diagnosis is easy in animals that show clinical signs of abortion and infertility (females) or epididymis (males) Clinical signs, direct culture of blood, lymph nodes, or bone marrow; and serological test = are used in diagnosis mercaptoethanol, tube agglutination, complement-fixation, counter immunoelectrophoresis, agar-gel immunodiffusion, and a rapid slide agglutination test = are not completely standardized and are subject to occasional but important "false-positives" and hard to interpret "low titers" and to broad heterotypic reactivity shown by B. canis antigens

Brucella canis (canine type of Brucella) Agar-gel immunodiffusion test = based on internal antigens of B. canis is the most sensitive and accurate serologic means of detecting infection. Isolation of B. Canis in infected dogs = most certain diagnostic method Antimicrobial Susceptibility Vitro to aminoglycosides, rifampicin, and tetracycline = B. canis is sensitive * B. canis infection is unlikely to be successful aminocycline (25mg/lb twice daily) + streptomycin (25 mg/lb ) = given by intramuscular injection for 7 days is effective in curing only 3 out of 11 experimentally infected dogs.

Brucellosis (Undulant Fever) of Humans = can be caused by B. abortus, B. canis, B. melitensis , and B. suis (all biotypes) =species involved is in great part determined by opportunity for exposure B. suis infection = common in swine belt (north central of US) B. abortus = southeastern US is found B. melitensis = common in Mexico and in localized areas of the southwestern part of US =this species is also found with some frequency in the midwestern states (where it is contracted from swine) Caribou (North American reindeer Rangifer tarandus ) = brucellosis in humans have been derived in Alaska infected with B. suis biotype 4

Brucellosis (Undulant Fever) of Humans B. canis = many people were found to be infected with this species ten years after it was isolated. Half were laboratory personnel who were working with the organism, and most of the owners of infected dogs were infected Cases of B. abortus and B. suis = usually seen in veterinarians, farmers, slaughterhouse personnel, and others connected to cattle and swine *men are more susceptible than woman (2/3 of the cases were men that was infected partly B. abortus and partly B. suis infection) direct contact = is more hazardous than drinking infected milk (infections in men are more common even in areas where the predominant type is B. abortus )

Brucellosis (Undulant Fever) of Humans 20 and 45 years = age group that can have brucellosis infants = who drinks milk is seldom become infected although the disease has been diagnosed in children as young as 4 years old B. suis = more virulent for humans then B. abortus ; most often contracted by exposure to blood or tissue fluids from infected swine in the slaughterhouse

Brucellosis (Undulant Fever) of Humans Brucellosis as an Occupational Hazard for Veterinarians brucellosis = poses a hazard to veterinarians in rural practice because they often come in contact with infected secretions Blood test surveys = indicates that a comparatively large percentage of veterinarians reacted positively, although may had no clinical history of the disease infection in veterinary colleges = was not uncommon *the veterinarians who showed no evidence of antibodies to brucella infection were either recent graduates or involved in commerce, laboratory work, or teaching.

Brucellosis (Undulant Fever) of Humans practicing veterinarian = is exposed to more viable Brucella organisms than other people in rural communities. during the common procedure of removing a retained placenta from a cow, the vet. comes in intimate contact with the uterine discharges rich in organisms that can enter his or her body through the conjunctiva or the intact skin, or by inhalation. wounds on the hands = make ideal sites of entry Accidental inoculation with B. abortus strain 1 9 = major source of infection in vet in vaccinating cattle. Veterinarians may jab the needle into his thigh or scratch thumb. strain19 = it usually does not cause chronic infections in humans but can cause severe effects in those previously infected with B. abortus

Brucellosis (Undulant Fever) of Humans Diagnosis *the acute disease is frequently overlooked or misdiagnosed. *patients frequently decide they have influenza or chills and then recover acute stage = the disease may be misdiagnosed because of its sporadic in nature and its vague signs diagnosis = based on the patient's history, clinical signs, isolation of the organism, and serological tests. severe phase of the disease = individual is acutely ill, suffers from prostration and weakness, develops daily fever (afternoon and evening), suffers chills and night sweats during which fever disappears only to have the cycle recur on following days.

Brucellosis (Undulant Fever) of Humans the acute symptoms usually lessen after a few days, but , following an interval of varying length during which the patient feels better, another period of acute symptoms may appear. There may be several remissions intermittent fever = responsible for the name undulant fever Infections with B. melitensis and B. suis = are usually more severe than those with B. abortus , but this is not always the case. *the mortality is low, but recovery from infections often is very slow. *many persons never fully recover from the effects of this disease.

Brucellosis (Undulant Fever) of Humans chronic cases = this form is more difficult to diagnose. usually the patients suffers from great debility, weakness, a low-grade remittent fever, and joint pains; there may be sweating, lassitude and malaise, gastritis, abdominal pain, skin rashes, headache, irritability, depression, insomnia, arthritis, and backache. *patients may be labeled neurotic (complains are hard to substantiate by laboratory test)

Brucellosis (Undulant Fever) of Humans Melitococcic spondylitis = an adteoarticular complication of brucellosis of humans in Italy septic arthritis of the hip= came from B. suis Brucella organisms = implicated cases of osteomyelitis and diseases in the nervous system blood culture = when positive, are diagnostic isolation of the organism = from the blood is usually difficult and often possible, particularly when the offending organism is B. abortus *Greater success is achieved in acute rather than in chronic cases

Brucellosis (Undulant Fever) of Humans Standard agglutination test, antiglobulin test, and the complement-fixation test = three serological test used in aid to diagnosis Agglutination test = it has been used for years, but it must be interpreted with great caution because it may produce aberrant results, such as occasional prozones, and "nonagglutination" antibodies may be present, giving a negative test even in persons who have had the disease for a long time; useful particularly in recently acquired acute cases of brucellosis Antiglobulin test (Coomb's test) and complement fixation test = are supplementary and are value in the diagnosis of long-stranding brucellosis

Brucellosis (Undulant Fever) of Humans Treatment Tetracyclines = are recommended by FAO/WHO Expert Committee on Brucellosis be given for 21 days Tetracycline + streptomycin = must be given to severe infection and for all B. suis infections Chloramphenicol = is of value but urged caution in its prolonged use. *although these antibiotics often produce a good initial clinical response, a large percentage of patients suffer recurrence of the disease.

Brucella

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