Investigation and molecular typing of Brucella isolated from dairy cattle herd immunized with Brucella abortus RB51 vaccine in Egypt. Dr. Gamal wareth Friedrich-Loeffler-Institut, Federal Research Institute for Animal Health NRL for Brucellosis, The Institute for Bacterial Infections and Zoonoses (IBIZ). History of brucellosis Sir David Bruce

1855-1934 (microbiologist) In 1886 a cocco-bacillus isolated from from the spleen of a man who had died of Malta Fever. He named it Micrococcus melitensis Human disease was associated with people who consumed goat milk and had other close contact with goats. History of brucellosis In 1886 first isolation from human In 1897 a similar microbe was isolated from the udder of cows

In 1914 isolated from swine. In about 1920 the genus was renamed Brucella and its species became respectively B. melitensis, B. abortus, and B. suis. Brucellosis is a highly contagious major bacterial zoonosis spread worldwide and has different names: Infectious abortion, contagious abortion, enzootic abortion in animals; and Crimean fever, Mediterranean fever, rock fever, undulant fever or Malta fever in humans. Brucellosis According to WHO brucellosis is classified among the top seven world neglected zoonotic diseases. The true incidence of human brucellosis is not easy to estimate globally but an estimated number of more than 500,000 persons get infected newly every year.

Genus Brucella includes 11 nomo-species: The six classical spp. B. melitensis, B. abortus, B. suis, B. canis, B. ovis, and B. neotomae. Two of marine origin: B. pinnipedialis and B. ceti. B. microti isolated from the common vole Microtus arvalis. B. inopinata isolated from a breast implant wound of a female patient Finally, B. papionis was described from baboons (Papio spp.) Brucellosis in Egypt 1886 In Malta After 53 years

1939 In Egypt Animal population and seroprevalence in Egypt The total number of animals was steadily increasing during the last years in Egypt. 1999-2011 (FAO, 2012). Number of seropositive animals according to GOVS, Egypt, 2012 Brucellosis in Egypt It is likely that brucellosis has been an endemic disease in Egypt for thousands of years In 1939, brucellosis was reported in a scientific report from Egypt for the first time Brucellosis is prevalent nationwide in all farm animal species, in the environment, and in carrier hosts.

Wareth et al., 2014: Animal brucellosis in Egypt_review_ J Infect Dev Ctries 2014; 8(11):1365-1373. doi:10.3855/jidc.4872 Brucellosis in Egypt Cross species transmission of brucellosis was reported http://webhosting.web.com/imagelib/sitebuilder/misc/ show_image.html?linkedwidth=actual&linkpath=http:// www.alsorat.com/sitebuildercontent/sitebuilderpictures/ GoogleOverQamar.jpg&target=tlx_pichqfc&title=. What is the Solution? Men bathing their water buffalo in the Nile Delta Region http://muirgilsdream.tumblr.com/post/27296121225/menbathing-their-water-buffalo-in-the-nile-delta

Control and Surveillance program Surveillance and control programs in endemic developed or developing countries based on Test and slaughter strategy with assistance of vaccination. Vaccination Live attenuated vaccine B. abortus S19 hampered the serodiagnosis immunisation of pregnant cows may lead to abortion B. abortus RB51 Thanks

Google The study Status vaccinated animals Animals pregnant cows and heifers 3 months of pregnancy non-pregnant cows and

heifers 8 months No. A negative in the past 3years 197 20 cows, 1 heifer

5-6 months post fertilization 20 cows, 1 heifer positive B negative in the past 3years 93

- - negative Stable a vaccinated total nonvaccinated animals Serology post

vaccination Serology ante vaccination Abortion Time of abortion Other b 290

Bulls C negative in the past 3years 50 negative kept for meat production

young calves D negative in the past 3years 185 negative 8 months E

negative in the past 3years 75 negative 3 months of gestation pregnant cows and heifers nonvaccinated total

310 cattle total 600 - - Results Bacteriology Two B. abortus rough strains Two B. abortus smooth strains

No RB51 vaccine strain was detected Genotyping Two different genotypes among the four isolated B. abortus strains with low genetic diversity Histopathology Extensive necroses accompanied by mild to moderate suppurative inflammation Brucella antigen was detectable.

Genotyping using MLVA-16 Histopathology Fetal placenta. Severe necrosis (asterisks) of placental tissue, accompanied by mild to moderate infiltration of leukocytes (mainly neutrophils, arrowheads) and dystrophic calcification (tilde). HE. Liver of aborted foetus. Immunohistochemical detection of scattered Brucellae (arrows) within the cytoplasm of hepatocytes

Summary of the study Aborted cows were tested seronegative for brucellosis in the past 3 years. 10 % of vaccinated cows aborted after 3 months. All aborting cows had previously delivered at least twice healthy calves without difficulties . All aborted cows are seropositive for standard agglutination test. All isolated B. abortus are filed strains with low (or no) genetic diversity. No RB51 strain was isolated. The vaccine offers no complete protection from infection by field strains but it is known to reduce spread of infection What is the possible explanation for that result?

The source of infection in the present study could not be clarified but several scenarios are plausible Scenario 1: Artificial insemination Use of contaminated sperm for artificial insemination Brucella spp. has been detected in semen of serologically negative bulls Scenario 2: Introduction of seropositive animals without previous testing of its serological status A cattle market hold before the Eid al-Adha festival, 10th

Oktober 2011,Nile delta province of Menufiya, Egypt http://news.xinhuanet.com/english2010/photo/2011-10/30/c_131220532_4.htm Scenario 3: Latent infection a latent carrier suckler cow without antibody production against Brucella but could shed of the agent and infecting other animals Scenario 4: Vector introduction of the agent via vectors such as rats and stray cats and dogs Conclusion B. abortus bv1 was the source of this outbreak but

not the RB51 vaccine applied. RB51 vaccine might reduce the number of abortions caused by field strains but offers no complete protection from infection. The most likely source of infection appears to be the uncontrolled introduction of the agent via vectors or latent carrier. Eradication of brucellosis is a multidirectional process required combination between ordinary control program and high biosecurity level in the farms. Acknowledgments We would like to acknowledge Dr. Mohamed Mosbah (Animal health research institute; Mansoura branch) for his help in sample collection.

Special thanks for AGr 130 members Thank you for attention, Gracias por su atencin, Vielen Dank fr die Aufmerksamkeit, Merci de votre attention,

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