(Maltese Fountain, Mediterranean Fountain, Undulant Fire, Crimean Fire, Cyprus Fire) BRUCELLOSIS

BRUCELLOSIS

(Maltese Fountain, Mediterranean Fountain, Undulant Fire, Crimean

Fire, Cyprus Fire)

Brucellosis

• Bacteria (Domain), Proteobacteria (phylum), Alphaproteobacteria (class), Rhizobiales (order), Brucellaceae (family), Brucella (genus)

• David Bruce isolated the bacteria from a human kidney in 1887 for the first time

• Brucellosis is considered to be one of the most important diseases of domestic animals when considering losses such as loss of offspring, loss of milk yield, loss of breeder value, infertility caused by animals

• It is zoonotic.

• Brucellosis is considered the most common zoonosis in the world by the Food and Agriculture Organization of the United Nations (FAO), the World Health Organization (WHO) and the International Agency for Epidemiological Diseases (OIE)

• Brucellosis has been largely eradicated over the years in Northern Europe, North American countries, Japan, Malta, Australia and New Zealand

• However, it continues to be prevalent in some Southern European countries,

particularly in the Mediterranean region, the Middle East, the developing

countries of West Asia, the Indian subcontinent, Africa, some parts of Central

and South America,

• It causes abortion and infertility by settling in testis, breast, uterus of cattle, sheep, goats and pigs. It is a cause of chronic, infectious and necrotic infection.

• Asymptomatic in non-pregnant !! In the pregnancy, abortion is observed in 5-9th months as a result of placentitis.

• It is placed in breast tissue and related lymph node, spread with milk

• In bulls: Orchitis

• In humans: widespread fever (during septicemia), sweating (wet straw smell), articulation pain, muscular pain, leucopenia and anemia are typical and orchitis is seen in men

• Genetic similarity with Brucella, Agrobacterium, Bartonella and Rhizobium

• Classification of all Brucella species as subtypes of B. melitensis was proposed by NCBI

• Despite genetic and immunological similarities, there are host differences

• International Committee on Systematics of Procaryotes; They met in 2005 and approved 6

Brucella groups

ETIOLOGY

• Gram negative small cocobacillus, without spore, without capsules, immotile,

• 5-10 % CO2 requirement (B. abortus), microaerophilic, facultative intracellular

• 2.1 Mb and 1.5 Mb have 2 chromosomes

• Blood/serum (serum dextrose) should be added in medium

• B. ovis and B. canis R typed colony, others S type

• Tibilisi, Weybridge, Berkeley, Izatnagar, R, R / O, R / C phage types

• Izatnagar, R, R / O, R / C phage lyses R types biovar CO2 need

H2S production

Reproduction of thionine, basic fuksin stain

L-alanine, L-asparagine, D-ribose, D-glucose etc. sugar tests Agglutination with monospecific serum (A, M, R)

Species

• B. abortus : Abortion, orchitis in sheep, goat, pig, human / 9 biotype

• B. melitensis : Abortion, orchitis, in sheep and goat, cattle (sporadic abort), human / 3 biotype

• B. suis : Abortion in swine, orchitis, epididymitis, human / 5 biotype

• B. ovis : Coach epididymitis, abdominal abortion

• B. canis : Dog abortion, epididymitis, human (less)

• B. neotomae : Wooden rats

• B. cetaceae : Sea mammals

• B. pinnipedialis : Sea mammals

• B. microt : Field mouse

• B. inopinata : The mace is not clear, the woman is isolated from the breast implant

Distinctive features of Brucella species and their biotypes Reproduction in

painted medium(x) Agglutination Genus Biotypes CO2

needs H2S B.füchsin

(b) Thionin Mono-

specific Serum

R-colony antiserum

Tb, RTD (xx) Phage sensitivity B.

melitensis a b A. M.

1 2 3

- - + - - + -

- - + - + + - -

- - + - + + + +

B. abortus

1 2 3 4 5

d + + - - + - -

d + - - - + - -

d + + + + + - -

d + + - - - + -

- - + - + - + -

6 7 8 9

- - + - + + + -

- d + - + + + -

+ - + - + - + -

- + + - + - + -

B. suis

1 2 3 4

- + + + + + - -

- - - - + + - -

- - + + + - -

- - - + + + + -

B.neotomea

- + - - + + - +

B. ovis

+ - + + + - - +

B.canis

- - - + + - - +

a=1:25.000 ; b=1:50.000

XX RDT: Routine Test Dilution

Bovine Brucellosis

• Agent B. abortus, very small Gram negative rod, brownian movement

• For the first isolation, the agent requires 10% CO2 (microaerophilic) and grows in 2 days

• There is no hemolysis on media, S-type small colonies, slightly bluish

• After 6-7 days, the colonies dissociate, the avirulant R form (I and M)

• Catalase, oxidase, urease and nitrate are positive

• Agglutination with A antiserum (A / M, 20/1)

• Does not have exotoxins, O-antigen is important in LPS construction

• Cross-reaction from aglutinins with F. tularensis, V. cholerae, C. fetus, Y. enterocolitca (O: 9), Leptospira, Bacillus, Salmonella urbana

• 10-15 minutes in pasteurization heat

• Transmission: digestion, respiration, intact and portentine skin, conjunctiva, mating, milking, snakes, frozen semen, bulls

• 2nd abortion is very rare

• Reticulohistiocytic system disease, located in certain organs

• Microorganisms enter the body from the digestive, skin, or mucosal surfaces multiply here and (via macrophages) reach the nearest lymph gland through the lymphatic channels

• Then they spread through the blood (2-3 weeks) to the whole body,

• After this transient period called bacteriemia, the factors are particularly less common with the pregnant uterus (erythritol), mammals, testes, lymphoid tissues and organs (such as lymph glands, liver and spleen), tendon sheaths and bursa

• It is located between the cornea and the uterine mucosa in the coricular villi epithelium of the uterine fetus. Villages are dispersed with oil degeneration and autolysis. Accumulating

fibrinoprulent exudate causes fetal ligament breakdown and fever

• In later stages of the disease, Brucella species either completely disappear or remain hidden in the nipple and genital lymph nodules, as in most cases

INFECTION

ABORTION

1,000-10,000 billion bacteria are sprinkled Can infect 60.000-600.000 pregnants

WITH THE DIGESTION SYSTEM

B. melitensis: 5000 jerm B. abortus: 106-107 jerm B. suis

RESPIRATORY SYSTEM (INHALATION) B. melitensis: 1300 jerm

B. abortus: 100 jerm

B. suis

SYMPTOMS

• Abortion, usually in the 6-8th month

• Placenta retention (second inf.)

• Dead or weak childbirth

• Infertility

• Epididymitis of the testis, previously acute then post-fibrosis stiff orchitis, spread with semen

• Artuculation inflammation

• In mastitis, the breasts appear normal, but the structure of the milk is distorted, localized in the breast and may cause abortion again by blood.

• Wavy fever in humans, articulation pain, fatigue

• The incubation time of brucellosis in animals varies considerably from 2 weeks to 1 year, even longer in some cases

• When the first symptom is abortion, the minimum incubation time is about 30 days

• Some infected animals do not abort at all

• Sensitivity to infection and the symptoms of the disease variable according to age, sex, race, gestational status, animal immunity status, amount of bacteria taken, type and virulence

• Infection while pregnant often results in abortion. It is suggested that non-

pregnant sheep do not make abort in their next pregnancy and develop an

immunity when they are infected for the first time.

DIAGNOSIS

A-CLINICAL DIAGNOSIS

Campylobacteriosis, Trichomonas fetus, Mikotik abort, Salmonella, Listeria abort, viral abort, food poisoning

B- LABORATORY DIAGNOSIS

1- BACTERIOSOCOPY Gram staining, Modification Ziehl-Neelsen, Stamp

(Differentiate from chlamydial abortions)

2- CULTURE

Blood, abort, cotyledons, stomach contents, liver, vaginal discharge, milk,

spleen, liver, lymph nerves of dead or cut animals etc.

• Suitable feed, adequate material, experienced staff

• The pathogenic material should be fresh and sufficient, antibiotics not applied,

• Inoculations should be cultured in more than one medium

• The surfaces of the medium should be dry,

• 5-10% CO2, incubation at least 10 days,

• Serum dextrose agar, Columbia agar, Brucella agar 3- ANIMAL EXPERIMENT

(cavy test) milk, organ extract, suspected culture, S.C. or I.P.

4 - SEROLOGICAL TESTS

A- TESTS WITH BLOOD SERUM

1- Slow Tube Agglutination Test (Sero-Agglutination Test-SAT)

• The test reveals antibodies that result in both vaccination and infection

• The sensitivity of the test is low during the incubation period.

• Antibody can not be detected immediately after the abortion

• In chronic form the reaction result is suspicious or negative

• Agglutination antigen (strain, production technique, antigen density, standardization)

• Non-specific reactions

Conditions affecting specificity in seroagglutination

CO- AGLUTININES

Y. enterocolitca O:9

E. coli O:116 ve O:157 F. tularensis

N Group Salmonella (S. Urbana) C. fetus

NON SPECIFIC AGLUTININES

Isı (56 C

), 2-Met, Rivanol, EDTA, DTT

Sat criterias

CATTLE SHEEP

Unvaccinated 1/40 ++ 1/20 ++

Vaccinated 1/80 ++ 1/40 ++

2- Rapid Agglutination (PLATE TEST)

-Serum and antigen should be in room temperature, -Antigen should be vigorously shaken before use, -The material to be used in the test should be clean, -The antigen should not spontaneously agglutinate,

-Serum and antigen should be placed in equal amounts,

-The result should be read in 1-2 minutes

Other Tests Made with Blood Serum

3- Rose Bengal Plate Test (RBPT) IgM İnaktive, IgG, pH 3.6 4- Complement Fixation Test(CFT) Chronic cases

5- Antiglobulin Test (Coombs)

6- Merkaptoetanol (IgM’s inactive , acute cases) 7- Rivanol Test (IgM inactive, acute cases)

8- Indirect Hemolysis Test

9- Indirect Hemaglutination Test

10- Fluorescence Test (Direct and Indirect FAT-IFAT) 11- Radioimmunoassay (RIA)

12- Enzyme Immunosorbent Assay (ELISA)

B. TESTS WITH MILK AND MILK SERUM

1- Milk Ring Test (blending milk, ring test antigen)

• Colostrum

• Pasteurized, sterilized and boiled milk,

• Non-fatt milks,

• Animal milk with mastitis,

• Distorted milk,

• Milk from the vaccinated animals is not used

• Cow's milk is kept in hematoxylin, sheep-goat's milk with tetrazolium- stained antigen for 1 hour at 37, the red ring is positive.

• 2-Aglutination with Milk Serum

C. OTHER SEROLOGICAL TESTS

1- VAGINAL MUKUS AGLUTINATION TEST 2- AGLUTINATION WITH SPERM

3- AGLUTINATION WITH MUSCLE LIQUID (MYOAGLUTINATION) 5- BACTERIOPHAGE TEST

Tibilisi : B. abortus Weybridge : B. abortus ve suis

Berkeley : B. abortus, suis ve melitensisi lize der

6- MOLECULAR TESTS

7- ALLERGIC TESTS (Brucellin ve Melitin)

• Negative result does not indicate no infection

• Some animals are negative when they are infected

• Sensitivity may continue after clinical symptoms have disappeared

• No acute infection

• Vaccinated and infected are indistinguishable

• The antigens used in the diagnosis of B. canis are not used in the diagnosis of B. abortus, B. suis and B. melitensis

Current status of diagnosis

• In order to avoid false positives, serological tests should not be used, except for the control of the quarantine and breeding animals.

• Only cultures made from aborted material will be evaluated

PROTECTION

•No treatment, compensation !!!

•9/10 of the value of the serological and bacteriologically determined animals are paid(2012)

•Do not infect herds

•Extraction of reactors

•Hygienic measures (disinfection of barns, waste material, infected milk, breastfeeding of calves, Vet. Hek., Etc.)

•Elimination of the source of the disease (waste, fetus, placenta)

•Periodic disinfection

•Vaccination

VACCINES

AGGLUTINOGEN VACCINES A- LIVE VACCINES

B. abortus S19 (5 x 10¹⁰ / ml)

B. abortus Adult (half dose 1-3 x 10⁹ / ml) B. melitensis Rev I (2 x 10⁹ / ml)

B. melitensis Rev I Adult(half dose 5-10x10⁴/ml) B- INACTIVE VACCINES

B. melitensis H38 NON-AGGLUTINGEN VACCINES B. abortus 45/20

B. suis S-2

P.B. Vaccine (B. abortus Buck 19) RB 51 Vaccine

(B. abortus 2308; R-form, rifampisin-resistant, immunosuppression can not be cured in humans)

BRUCELLOSIS LEGAL STATUS

• VETERINARY SERVICES, PLANT HEALTH, FOOD AND FOOD LAW No. 5996 must strictly adhere to the regulation of combating brucellosis. Brucellosis animal meat is processed according to the Meat Inspection Instruction.

According to this;

• In the case of suspicion or presence of brucellosis, it is obligatory to immediately notify the competent authority.

• The treatment of brucellosis is prohibited in cattle, sheep and goat animals.

• Animal entry and exit is prohibited in the cattle herd, except when authorized

by the competent unit for dispatch to the slaughter.

• The milk of infected cows can be used in the feed of the same farm animals after they have been subjected to appropriate heat treatment.

• The milk obtained from cows in an infected herd cannot be transferred to milk

processing facilities except for proper heat treatment and cannot be sold as raw milk.

• Straw, garbage or other substances that have been in contact with infected cows and calves or placentas are destroyed immediately after being burned or disinfected and buried.

• In order to remove the quarantine from the infected herd, the first test is carried out at least thirty days after the infected animals are separated from the herd and the second test is carried out thirty m sixty days after the first test. In the last two tests, when all the flock has negative results, the third test is performed six months after the third test. In this final test, quarantine is removed when the entire flock has a negative result.

• The test period is restarted when a positive animal is detected in any test.

Brucellosis Animal Meats

• Especially during periods of fever, the animals are killed and destroyed

• In the absence of fever, only the animals that have been diagnosed as having the disease by serological examination are cut off,

• Consumption is allowed by roasting. However, internal organs and breast and

tenaculum organs are completely destroyed

Brucella Eradication Program

• First national Brucella eradication program in 1984

• Goal; be able to withdraw the prevalence below 1%

• 26 years, 27.469.561.000 TL- 325.853.833.800 TL (contribution to the national income)

• Turkey is divided into zones 5

• MRT and serum from 10% of cows RBPT, SAT, CF

• Sheep-goat puppies were vaccinated with 4-8 months of consultation

• Microbiological and serological screening of bulls every 6 months

• Hygienic measures

• In 2011 the herd prevalence was found to be 22%

Current status

• In our country, it has been decided that mass vaccination is the most effective method in combating Brucella and conjugative vaccination can be applied to the animals of all ages reliably.

• Within this context, it is approved by Ministry's Authority to initiate the Control and Eradication Project of Brucella with Conjugtival

Vaccine for 10 years in cattle and 6 years in sheep and goats as of

01/01/2012

Why Konjuktival vaccine?

• After conjunctival vaccine, the agent is localized only to the head lymph nodes.

• There is no risk of generalized infection.

• Possible abortion and spreading risk is minimal.

• It can be applied to cattle and sheep-goats of all ages.

• Because it is not parenteral, it is safe for the personnel making the

vaccine.

• All female cattle, sheep and goats and male sheep and goats to be separated as breeders will be vaccinated with conjunctival vaccine according to the project to be implemented

• The project will be applied to all female cattle, except those vaccinated with young S-19 subcutaneous vaccine, in 2012. The B.abortus S-19 conjugate vaccine will be applied in 2012 as a strategy for mass vaccination in cattle. In 2013, the first grafting of adult female cattle that could not be vaccinated in 2012, the second grafting of the first grafted animals and the first grafting of the female cattle aged 3-6 months will be done. In 2014, the first

vaccination of animals in 2013, the second vaccination of animals and the first vaccination of 3 - 6 month old calves will be carried out and a 10 - year vaccination program will be implemented. Sufficient immunization is sufficient for each animal to be vaccinated twice throughout life. Two vaccinations in cattle will be done in 12 months.

• All female cattle, sheep and goats and male sheep and goats to be separated as breeders will be vaccinated with conjunctival vaccine according to the project to be implemented

• The project will be applied to all female cattle, except those vaccinated with young S-19 subcutane vaccine, in 2012. The B.abortus S-19 conjugate vaccine will be applied in 2012 as a strategy for mass vaccination in cattle. In 2013, the first grafting of adult female cattle that could not be vaccinated in 2012, the second grafting of the first grafted animals and the first grafting of the female cattle aged 3-6 months will be done. In 2014, the first

vaccination of animals in 2013, the second vaccination of animals and the first vaccination of 3 - 6 month old calves will be carried out and a 10 - year vaccination program will be implemented. Sufficient immunization is sufficient for each animal to be vaccinated twice throughout life. Two vaccinations in cattle will be done in 12 months.

• The project will be applied to all female sheep and goats and

breeding male sheep and goats, except those vaccinated with young Rev-1 subcutane vaccine, in 2012 as a strategy for mass vaccination on sheep and goats. For adequate immunity to occur, it is sufficient for each animal to be vaccinated once in a lifetime. In the second year of vaccination, only female lambs and kids aged 3-6 months old and adult female animals that remain unvaccinated from the

previous year and stud male animals will be vaccinated. In this way a

6 year vaccination program will be implemented.