The use of different Brucella vaccines for protection against Brucella melitensis infection in cattle.

Transcription

1 Cairo University Faculty of Veterinary Medicine Department of Internal Medicine and Infectious Diseases The use of different Brucella vaccines for protection against Brucella melitensis infection in cattle. A thesis presented by Mahmoud Hassan Abd ELHaleem Awwaad (B.V.SC, Cairo University, 1987M.V.SC, Cairo University, 1998) For Ph.D. Degree (Infectious Diseases) Under The supervision of Prof. Dr. M.E.A. Shawkat Professor of Infectious Diseases Faculty of Veterinary Medicine, Cairo University Prof. Dr. S.A Barsoum Dr. S.I. Ibraheem Professor of Infectious Diseases Emeritus Chief Researcher Faculty of Veterinary Medicine Animal Health Research Cairo University Institute

2 Acknowledgement Thank God for granting me the ability to complete this work till it comes to an end. I cannot find enough words to express my deep feeling towards Prof. Dr. Sabry Aziz. Barsoum prof. of Infectious Diseases, faculty of veterinary medicine, Cairo University for his kind supervision and continuous help during work, Many Thanks and gratitude to Dr. Samy Ismail. Chief researcher of Animal Health Research Institute. Many thanks and gratitude are due to Dr. Abdel Khalek. Montaser, head of Brucella Department and other members and colleagues in the department 2

3 Contents Item Page Introduction 8 Literature 10 Material and methods 34 Experiments and results 51 Discussion 79 Summary 89 References 91 3

4 LIST OF ABBERVIATIONS ABTS: 2.2Azinobis (3ethylbenz6Sulphonic acid). AHRI: Animal Health Research Institute. AST: Allergic Skin Test BAPA: Buffer acidified plate antigen. BCIP/NBP: 5bromo4chloro3indoly1 phosphate/ Nitro blue tetrazolium. C o : Degree centigrade. i ELISA: indirect Enzymelinked immunosorbent assay. i ELISAr: Enzymelinked immunosorbent assay using rough antigen. E.U. ELISA unit. FAO: Food and Agriculture Organization of the United Nation. GOVS General Organization for Vet. Services. IgG: Immunoglobulin gamma (γ). IgM: Immunoglobulin mu (μ). ISAbS: International standard antibrucella abortus serum. Iu/ml: International unit per ml. LPS: Lipopolysaccharide. MET: Mercapto ethanol test. METr: Mercapto ethanol test using rough antigen. MRT: Milk Ring Test. O I E Office International de Epizotiec (World Organization for Animal Health). PAT: Plate Agglutination Test. PBS: Phosphatebuffered saline. RBPT: Rose Bengal plate test. R: Rough. RPM: revolution per minute. 4

5 RTD: Routine test dilution. S 19: strain 19. SLPS: Smooth lipopolysaccharide. TAT: Tube agglutination test. TATr: Tube agglutination test using rough antigen. Tb: Tbilisi phage. USDA: United States Department of Agriculture. VSVRI: Veterinary Serum and Vaccine Research Institute. WHO: World Health Organization. 5

6 List of tables No Title Page 1 Distribution of cows in the three herds used in this study. 2 Distribution of heifers in the three herds used in this study. 3 Degree of agglutination and sedimentation in TAT. 4 Conversion of brucella TAT titers to international units. 5 Results of Milk Ring Test. 6 The incidence of brucellosis in the three investigated herds. 7 Results of bacteriological culture of different samples collected from different herds before vaccination. 8 Distribution of investigated animals according to their status of pregnancy. 9 Gestation stage and lactation season of animals under investigation. 10 Frequency of abortion in different herds before vaccination (July 2000 November 2001). 11 Breeding troubles in the investigated animals before vaccination. 12 Mean international units of serological tests of heifers vaccinated with Rev Results of allergic skin test in cattle vaccinated with Rev1 after 2 years from vaccination. 14 Mean international units of serological tests of heifers vaccinated with S Results of allergic skin test in cattle vaccinated with S 19 after 2 years from vaccination. 16 Mean international units of heifers vaccinated with RB51 17 Results of allergic skin test (AST) in cattle vaccinate with RB51 after 2 years from vaccination. 6

7 List of figures No Title Page 1 Antibody production profiles (expressed as international units) following Rev.1 vaccination of heifers (dose of 1x10 9 CFU). 2 Antibody production profiles (expressed as international units) following S 19 vaccination of heifers (dose of 310x10 9 CFU). 3 Antibody production profiles (expressed as international units) following RB51 vaccination of heifers. 7

8 Introduction Brucellosis is contagious disease that infects different animal species causing severe economic losses due to abortion, infertility and sharp decrease in milk yield. Among the different species of genus brucella, Brucella abortus is the common strain infecting cattle all over the world while Brucella melitensis which is affecting mainly sheep and goats and also other animal species (Alton 1990, Hamdy 1992 and ElBauomy 1993 ). Unfortunately, Brucella melitensis infection occures in many parts of the world including the Mediterranean countries, the middle East, Southwest Asia and Latin America (Sayour et al 1970 and OIE, 2004a). In Egypt, serological incidence of the disease showed that it was 0.81% and 0.97% among cattle as reported by the General Organization of Vet. Services in 2003 and 2005 respectively. These incidences were lower, to a certain extent, than other incidences detected earlier in the late 80s and early 90s which was (1.4%) and 1% respectively. This can be attributed to the control program against brucellosis applied by the General Org. of Vet Services through American project USDA by the use of reduced dose of S x10 9 CFU together with test and slaughter policy. However another problem arose after the use of the strain 19 vaccination program in which Brucella melitensis was reported to be the main strain of brucella isolated from cattle and animal species. Brucella strains isolated from cattle were originally reported to be Brucella abortus and occasionally Brucella melitensis as reported by Samira EL Gibaly (1969), Corbel and Morgan (1984) and Alton (1990). Starting from 1998 Brucella melitensis was reported to be the common strain isolated from cattle as 8

9 reported by Abdel Haleem (1998), Shalaby et al., (2003), Sayour (2004) and Shehata (2004). RB51 strain of brucella as a vaccine was introduced to Egypt since (2000) in a trial to overcome the main disadvantages of strain 19 in which interference with serological diagnosis of brucellosis was present using the conventional serological tests. Yet, many data were reported that its efficacy in protection susceptible animals against brucellosis was less than expected (Fosgate et al., 2000 and Hamdy., 2002). The prevalence of Brucella melitensis among the isolated brucella species from different animal species especially cattle in Egypt nowadays necessitates the use of specific Brucella melitensis vaccine which appeared as a logic step towards disease control in our country. So, the aim of this work is to study the use of Rev1 brucella vaccine along with other brucella vaccines namely RB51 and strain 19 to compare between their protective efficacy against brucellosis in different groups of cattle. This investigation was planned to study the humoral and cellular immune response of the 3 vaccines in 3 different farms of cattle and their protective immunity as reflected by the epidemiological data of disease collected before and after vaccination for 2 years. 9

10 Literature i. Disease situation in Egypt. Zaki (1943), in Egypt, examined 200 sheep by agglutination test and recorded 9.5% incidence of brucellosis. goats. El Nahas (1951), in Egypt, isolated Brucella melitensis from blood of two Kamel (1951) examined 1600 blood samples from cows obtained from Cairo abattoir.he found that 1.1% were positive reactors to the infection with brucellosis by TAT. Kamel et al. (1960), in Egypt, recorded 5.8% incidence of brucellosis among goats out of 4618 examined goats using tube agglutination test. Hamada et al. (1963) examined 510 cattle sera by tube agglutination test and mentioned that there were no positive reactors among cattle. EL Dawi (1966) carried out a serological survey in Wadi Halfa using tube agglutination test and he found that 1.7% of sheep and 1.5% of goat were positive. Mathur (1967) found that out of 856 goat milk samples positive to milk ring test, 28 yielded brucella and out of 1129 pooled milk ring test negative samples from 4661 goats, 11 strains of Brucella organisms were isolated. For sheep13 strains 10

11 were isolated from 629 MRT positive samples and 8 strains from 1351 pooled MRT negative samples from 5224 sheep. Samira El Gibaly (1969), in Egypt, reported that the incidence of brucellosis was 4.72% in cows and 1.49% in buffaloes using TAT. The author succeeded to isolate 17 strains of Brucella abortus from cattle and 12 strains of Brucella melitensis, 8 from cattle and 4 from buffaloes. Sayour et al. (1970), in Egypt, recovered 10 strains of brucella organisms from sheep at different localities. All the isolated strains proved to be Brucella melitensis biovar 3. Shawkat (1973) examined 530 sheep and 480 goats for brucellosis using TAT, CFT and RBPT. He found that 2.86% of sheep and 7.08% of goats were positive by using TAT, 2.44% of sheep and 5.62% of goats were positive by using CFT while 2.26% of sheep and 5.2% of goats were positivly reacted to RBPT. Samira El Gibaly el al. (1975) recorded brucellosis abortion storm among a Friesian dairy farm (551 animals) in Menofia Governorate. They isolated 32 strains of brucella organisms from milk samples which were identified as Brucella abortus biovar 1. Samira El Gibaly et al. (1977) stated that the incidence of brucellosis among goats was 5.85% using TAT. Bacteriological investigation of 13 goats positive to TAT revealed the isolation of one strain of Brucella melitensis biovar 3 from lymph nodes of one goat, while the samples of other goats were negative on culture. 11

12 EL Olemy et al. (1984) found that 17 (5.2%) out of 326 goats serum samples had brucella agglutinins. Examination of 93 milk samples by MRT revealed 4.3% positive samples. Three strains of Brucella melitensis were isolated from three milk samples. AbdelAal (1985), in Egypt, investigated 362 cows and 250 buffaloes for brucellosis using TAT, BAPAT, RIVT and CFT. The results in cows revealed that 16.85%, 15.75%, 13.81% and 16.26% were positive reactors respectively. 75 milk samples, 4 aborted faeti and 11 retained placenta of the infected cows were examined bacteriologically and 11 isolates were obtained and typed as 4 Brucella abortus biovar 3 and 7 Brucella melitensis biovar 3. El Sheery (1987) examined 577 cow using PAT, TAT, RBPT, RivT and CFT in Egypt. He found that 14.7%, 16.1%, 12.99%, 11.8% and 12.3% were positive to these serological tests respectively. 13 isolated strains were recovered from milk of 83 cows which reacted positively to serological tests. 7 out of them were typed as Brucella abortus biovar 3 and the other 6 strains were typed as Brucella melitensis biovar 3. Salem et al. (1987) recovered 11 brucella isolates from 75 milk samples, 4 aborted foeti and 11 retained placentas. These 11 isolates were typed as 9 strains of Brucella melitensis biovar 3 and 2 strains of Brucella abortus biovar 3. Sayour (1988) selected 160 bovine sera according to TAT titers 30 positive, 50 suspicious and 80 negative i ELISA detected 95 as positive, 20 suspicious and 45 negatives the author found that the i ELISA detected 35 from 80 cows that were negative by TAT, BAPAT, RBPT and CFT. He indicated that i ELISA is a valuable and a reliable test to add to brucellosis serum test. Alton (1990) stated that, in regions where Brucella melitensis is prevalent in sheep and goats, cattle are liable to catch the infection from them. Brucella melitensis 12

13 infection in cattle sometimes causes abortion, but less frequently than Brucella abortus. Unfortunately colonization of the udder is frequent and the excretion of the organism in the milk may be has frequently led to epidemics of brucellosis in people working with cattle or drinking their milk. Samira El Gibaly et al. (1990) revealed that out of 142 cows examined by using TAT, RBPT, BAPAT, RivT and MRT. 28 brucella isolates were isolated from milk samples from cows positive to milk ring test and serological tests. All the isolated strains were typed as Brucella melitensis biovar 3. Abdel Rahman (1991) reported that a total of 77 serum samples collected from a herd of cattle suffering from abortion and breeding troubles were examined by TAT, RBPT and BAPA tests. The number of positive reactors were 25 (32.5%), 32 (41.5%) and 32 (41.5%) for TAT, RBPT and BAPAT respectively. One isolate of Brucella melitensis biotype 3 was recovered from a lymph node of serologically positive cow. Hosein et al. (1991) isolated Brucella melitensis biotype 3 from 94 cows milk positive to milk ring and complement fixation tests and stated that the rate of isolation increases with the intensity of milk ring test reaction. Montasser (1991) in Egypt examined 167 different lymph nodes from 38 slaughtered cattle, which had been proved to be positive to serological tests. 27 brucella strains were recovered and all the isolated strains were typed as Brucella melitensis biovar 3.The author attributed this phenomenon to the fact that sheep and goats are usually kept in close contact with cattle in Egypt. Mahmoud (1991) found that the incidence of brucellosis in Kafr ELSheikh Governorate was 2.27% and 3.09% in cattle and buffaloes respectively using Rose Bengal plate test. While in ELMenia villages the incidence was 3.03% and 5.5% in 13

14 cattle and buffaloes respectively. The author added that the infection rate of brucellosis among farm animals in Egypt is not static; however the evolution changes in the animal husbandry as well as the extent of population movement are considered the main factors which increase the exposure potential of individual animals or herds to brucellosis. Aldomy et al (1992) revealed that out of 206 samples from sheep, goat and cattle. 34 isolates (16.5%) of Brucella melitensis were recovered. 26 out of 31 isolates from sheep were typed as Brucella melitensis biotype 3, 2 isolates were typed as Brucella melitensis biovar 1, 3 isolates from vaginal swabs of aborted animals were not biotyped. The remaining 3 isolates were one isolate from a goat was typed as Brucella melitensis biovar 1 and the 2 isolates from cattle were typed as Brucella melitensis biovar 3. Hamdy (1992) examined 1683 cattle and 1280 buffaloes for brucellosis at different Governorates in Egypt by TAT, BAPAT, RBPT and Rivanol test. The author isolated 91 strains of brucella from cattle and 5 strains from buffaloes. Out of these strains, 86 were belonged to Brucella melitensis biovar 3 and 5 strains proved to be Brucella abortus biovar 1.The 5 strains isolated from buffaloes were typed as Brucella melitensis biovar 3. Ali et al. (1993) reported that the cause of abortion occurred in 17 cases of buffaloes during August to November 1992 at different localities in Assuit Governorate were due to brucella infection. Bacteriological examinations showed 10 (58.82%) of brucella isolates were recovered from buffaloes milk. All isolates were typed as Brucella melitensis biovar 3. Samira ElGibaly et al. (1993) stated that, in a serological survey on 2930 dairy cattle using TAT, BAPAT, RBPT and Riv.T, 38 (1.3%) cows were reactors. Out of 14

15 the positively reacted cows, 27 brucella strains were recovered and all the isolated strains were Brucella melitensis biovar 3. EL Sheery (1993) examined sera and milk of 600 cows and 300 buffaloes from different localities in Egypt by serological and bacteriological methods. The author isolated 21 brucella isolates which was typed as Brucella melitensis biotype 2 and 3. Hosein and Gabal (1994) isolated Brucella melitensis biovar 3 from cows indicating the prevalence of this biovar among cattle in Egypt. AbdelHafeez et al (1995) stated that 15% of brucella infected cows could not be detected serologically until after abortion or calving. They added that, in regions where Brucella melitensis is prevalent among sheep and goats, cattle are liable to catch the infection of Brucella melitensis. Ammar (1995) stated that the prevalence of brucellosis among animals showing reproductive troubles was 8.06% and among apparently healthy animals which in close contact with the previous animals was 0.69% by using BAPAT. He isolated 8 brucella strains from cattle, 6 from lymph nodes, one from milk and one from stomach content of aborted foeti. All the isolated strains were Brucella melitensis biovar 3. Salem et al. (1995) examined a total number of 894 sheep, 25 goats, 140 cows and 110 buffaloes using allergic skin test (AST), tube agglutination test (TAT), mercaptoethanol test (MET), buffered acidified plate antigen test (BAPAT), Rose Bengal plate test (RBPT) and Rivanol test (RivT). He found that the agreement between AST and these tests were 88.5%, 89.2%, 88.6%, 88% and 88.9% respectively in sheep, 92% to all test in goats, 77.9%, 76.5%, 77.9%, 77.1% and 80.7% in cattle and 65.5%, 78.2%, 78.2 and 62.7% in buffaloes. The agreement between results of bacteriological examination of milk from cattle and buffaloes and results of AST, 15

16 TAT, MET, BAPAT, RBPT and Riv T test were 72.8%, 82.2%, 84.9%, 81.7%, 86.4%, 86.4%, 82.2% and 93.8% respectively in cattle and 61.5%, 77.1%, 77.0%, 80.8%, 80.8%, 84.6% and 88.4% in buffaloes respectively. Sayour (1995) examined 119 Friesian non vaccinated cattle in a herd by Tube agglutination test (TAT),Buffered acidified plate antigen test (BAPAT) and Rose Bengal plate test (RBPT). 33 strains of Brucella melitensis biovar 3 as well as 39 field strains of Brucella abortus biovar 1 were isolated from cattle. AbdelGawad (1996) examined 1816 cows in Ismailia and PortSaid provinces and found that the ratio of positive reactors were 9.3%, 10.4%, 6.2% and 6.2% using tube agglutination test (TAT), Buffered acidified plate antigen test (BAPAT), Rose Bengal plate test and Rivanol test respectively. The author succeeded to isolate 8 brucella strains and all were typed as Brucella melitensis biotype 3. Kadry (1996) examined 2330 cattle and 1850 buffaloes from different localities at Sharkia Governorate for detection of brucellosis. The bacteriological examination of lymph nodes, milk samples and aborted foeti yielded 76 brucella isolates. Brucella abortus biotype 3 was the only biotype isolated from cattle herds while Brucella melitensis biovar 3 was isolated from other animal groups. AbdelHaleem (1998) reported that the isolated strains of brucella from a herd of cattle suffering from brucellosis were typed as Brucella melitensis biotype 3. ii. Use of Brucella vaccines: Jones et al. (1964) noted that Rev1 vaccine was better than S 19 in protection of sheep and goats against natural exposure to Brucella melitensis infection. 16

17 Horwell and van Drimmelen (1971) stated that the use of S 19 vaccine for protection of cattle against infection often causes marked local lesions and strong persistent serological reactions in older animals while the use of Brucella melitensis Rev1 showed to be superior to S 19 as immunizing agent for cattle even when used in much smaller doses. The authors reported that the use of Rev1 in adult cattle revealed a number of good features. The strains proved safe, was not excreted, stimulated titer which were not persistent and caused less sever local reaction. GarciaCarrillo (1980) reported that Brucella melitensis Rev1 vaccine had a superior efficacy than S 19 in protecting pregnant cows against challenge with 2 doses of Brucella abortus strain 2308, (8x10 6 ) & (1x10 9 ) viable cells. All Rev1 vaccinated heifers resist the lower dose of Brucella abortus strains2308 but one out of 10 heifers vaccinated with S 19 excreted virulent brucella as tested by vaginal swab culture. Out of 10 cows vaccinated with Rev1 and challenged with a dose of Brucella abortus strain 2308 (1x10 9 ) viable cells, 4 aborted and 4 calved normally. The other 2 cows were found to be infected. All ten cows vaccinated with S 19 and exposed to the same challenged dose aborted and were found infected. Corner and Alton (1981) recovered Brucella abortus S 19 at necropsy from cows vaccinated with reduced doses of S 19 three to six months previously. Strain 19 was isolated from 4 of 9 cows vaccinated with 5.8x10 8 viable cells during pregnancy. In two of these nine cows vaccination had led to colonization of the uterus with S 19 and consequent abortion of premature calving. In one of another group of nine pregnant cows the vaccine strain was recovered from the colostrum. There was no interference with the course of pregnancy in over 700 cows Vaccinated with 3x10 8 viable cells. Thirteen of these cows were serologically positive three months after vaccination and S 19 was isolated from six cows at necropsy. This lower dose has already been showed to produce immunity equal to that of higher dose. 17

18 Alton et al. (1983) vaccinated three groups each of 14 heifers using strain 19 vaccine and strain 45\20. About12 months later, those who became pregnant were challenged at about 6.5 months of pregnancy by the conjunctival route with virulent Brucella abortus. Group 1 of heifers received 2 doses of Brucella abortus 45/20 vaccine 2 months apart. Only 5 of the 14 heifers become pregnant and of these 5, only one resisted challenge. Group 2 heifers received one dose of 45/20 vaccine. 5 of the 10 become pregnant and challenged,and resist the infection. Group 3 of heifers received 38x10 9 CFU of S 19 six of the 10 challenged heifers resisted infection. All of 5 nonvaccinated control cows become infected. It appeared advantageous to give only one dose of 45/20 rather than 2 as recommended. A single dose of 45/20 vaccine stimulated transient serological positive reaction in 2out of 28 heifers where as the reduced dose of S 19 gave rise to persistent titers in 2 of 14 vaccinated heifers. Hitos et al. (1983) isolated 61 strains of Brucella abortus from milk of all 100 cows, formerly negative to the card test, that become positive 4 months after revaccination with a reduced dose of strain 19. Of the 61 strains in which the biotype was identified, 24 were classified as Brucella abortus biotype 1 (the field strain), 34 biotype 4 and one for each biotype 2, 7 and 9. No vaccinal strain was recovered. Results of complement fixation test are presented; 57 of the Brucella abortus isolates were from cows with CF titers of 1:160, but 4 were from cows with a negative titer of 1:20. It is concluded that revaccination with a reduced dose of strain 19 dose not give sufficient protection against natural infection. Hitos (1983) isolated Brucella abortus from 40 aborted fetuses from were 2 groups of cows (revaccinated and non revaccinated) 28 aborted fetuses from cows of the 1 st group which had been revaccinated with reduced dose of strain 19 (18 months previously) and 12 aborted fetuses from cows of the second group which had not revaccinated. In the first group 16 strains were typed as Brucella abortus biotype 1 (field strain), 11 strains were biotype 4 and 1 was vaccinal strain. In the second group, 18

19 7 strains were biotype 1 (field strain), 3 were biotype 4, 1 was biotype 2 and 1 was biotype 9. Alton et al (1984) reported that vaccination of mature or pregnant heifers with 3x10 8 CFU of S 19 produced immunity at least as good as that produced by calf hood vaccination with serological response greatly reduced in the majority of cattle. Tserendash (1984) stated that the use of Rev1 Brucella melitensis vaccine in cattle and yaks provided better resistance to experimental infection with Brucella abortus strain 544 or Brucella melitensis strain 678 than Brucella abortus strain 19 vaccine. He concluded that it produced titre which lasted for 46 months in calves and year in adult cattle. Chukwu (1985) found that the serum agglutinin titers of 6 heifers inoculated with live Brucella abortus strain 45/20 were similar to those produced in 6 bullocks inoculated with virulent Brucella abortus strain 544.However, the agglutinin response appeared slowly and disappeared earlier in the heifers than in the bullocks. This evidence seemed to confirm the instability of strain 45/20 which could revert to the smooth form becoming highly agglutinogenic in non pregnant heifers. Confer et al. (1985) studied the effect of two challenge doses (9.4x10 6 and 5.2x10 7 ) of Brucella abortus strain 2308 on unvaccinated heifers and vaccinated heifers with 2 doses (1x10 9 and 1x10 10 ) CFU of Brucella abortus strain 19 vaccine. He found that in unvaccinated heifersout of 9 heifers 8 were aborted with 9.4x10 6 CFU challenge dose and all were aborted with 5.2x10 7 CFU challenge dose. In vaccinated heifers with a dose of 1x10 9 CFU out of 11 heifers one aborted when challenged with 9.4x10 6 and 8 of 10 aborted when the challenge dose was 5.2x10 7. In vaccinated heifers with a dose of 1x10 10 CFU, 3 from 11 heifers were aborted when 19

20 the challenge dose was 9.4x10 6 CFU and all of the 8 challenged with a does of 5.2x10 7 CFU were aborted. Herr et al. (1985) measured the serological reaction following adult cow inoculation with 412x10 10 CFU (standard dose) of Brucella abortus strain 19 by using complement fixation test (CFT) and found that the antibody titers recorded could not be distinguished from those arising from infection for a period of 611 months. This was in the absence of the booster effect of brucella antigen from either field or vaccine strains. Chukwu and Cunninghm (1986) inoculated 12 cattle with Brucella abortus killed strain 45/20 adjuvant vaccine with 2 doses six weeks apart.they found that agglutinin titers did not exceed 60 I.U during 26 weeks after primary immunization and the Rose Bengal test was negative throughout. The second dose of vaccine produced rises in antibody titers measured by complement fixation test, while the reaction to a delayed hypersensitivity skin test and the lymphocyte transformation test reached a peak 6 weeks after the second dose. 12 months after primary immunization, 10 out of 14 cattle were still positive to the skin and the lymphocyte tests, while after 18 months only 9 out of 14 cattle were still positive. GarciaCarrillo (1986) inoculated guinea pigs with 1.08x10 9 or 6.2x10 5 of Brucella melitensis Rev1 vaccine (group 1) and with 1.5x10 10 or 1.5x10 7 of Brucella abortus strain 19 (group 2) and challenged both groups with virulent Brucella abortus 2308 and found that the protection occurred in Rev1 vaccinated group was better than that produced by strain 19 vaccinated group. He decided that all vaccinated groups were better protected a month after vaccination than after a year. Chukwu (1987 a) revealed that Brucella abortus 45/20 adjuvant vaccine elicited higher cell mediated immune responses than the attenuated strain 19 vaccine. The 20

21 inability of strain 19 to stimulate adequate cellmediated immune response might be due to lack of sensitised lymphocytes in the circulation or to insensitivity of the assays used (the lymphocytes transformation test and delayed hypersensitivity skin test) to detect the subsets of T lymphocytes stimulated by the vaccine. The degree of increased cellmediated immune responses in cattle immunized with 45/20 vaccine was probably due to stimulation of the reticuloendothelial system by the oil adjuvant. Chukwu (1987 b) immunized 38 months old heifers with strain 19 vaccine and killed 45/20 vaccine. He challenged heifers with virulent Brucella abortus strain 544 at the sixth month of gestation. He determined the cell mediated immune response by the wholeblood lymphocyte transformation test both before and after challenge and found that heifers from which Brucella abortus was recovered after calving or abortion had very high lymphocyte transformation responses, where as animals from which no brucella isolation had low blastogenic reaction. He concluded that there was no correlation between the lymphocyte transformation responses and protective immunity.he conclulded also that the assay is probably ineffective in measuring effector cells, and that the duration of high lymphocyte blastognic reaction in bovine brucellosis is probably related to a persisting infection which continues to provide antigenic stimulation. Hassanin (1987) reported that no antibody could be detected for 7 days post vaccination of guinea pigs with S 19 but after 7 days only TAT and indirect ELISA tests were positive. At 14 to 63 days post vaccination, TAT, ELISA, Riv and CFT tests were positive. After 70 days only TAT and ELISA were still positive. Crawford et al. (1991) administrated 3 different doses of strain 19 in 3 different groups of yearling heifers as follow 10 8 (n= 40), 10 9 (n= 44), or (n= 44) colony forming units. He found that, the proportion of heifers with positive serological test results at 1 month following vaccination increased as the dose of S 19 increased. This 21

22 proportion decreased with time and all heifers had negative results with card, rivanol and complement fixation tests within 4 months yet all heifers become ELISA negative within 9 months after vaccination. Lymphocyte transformation activity was stimulated by S 19 dose of 10 9 or and half of the heifers in both groups had a positive stimulation index at 9 months. Immunity of the pregnant heifers was challenged 9 months after vaccination with 10 7 Brucella abortus strain 2308 and the relative risk of brucellosis was reduced to 0.38, 0.15 and 0.06 for Brucella abortus S 19 doses of 10 8, 10 9 and respectively. Schurig et al. (1991) discovered Brucella abortus strain RB51 by repeated passage of Brucella abortus strain 2308 on media containing varying concentrations of rifampin and penicillin. After 51 passages, a rough colony was selected and tested as a possible Candidate for brucellosis vaccine. Early work demonstrated that the organism designated RB51 protected mice when challenged with virulent Brucella abortus strain 2308.This vaccine candidate was administered to rabbits, goats and cattle without any side effects and demonstrated clearance within a reasonable period of time. Repeated passage of the organism in vitro and in vivo failed to demonstrate reversion to smooth characteristic. The rough character of Brucella abortus strain RB51 based on clinical morphology, staining with crystal violet, spontaneous auto agglutination in saline, agglutination in acriflavine solution and the lack of oside chain of LPS. Gramstained preparation show rough Gramnegative rods and coccobacillary forms that are smaller than those of strain 19 and 45/20 strain. RB51 is urease positive grows in the presence of erythritol and does not require Co 2 or blood for growth. Strain RB51 grows in media containing thionin blue (1:500000) or penicillin (5 IU/ml). RB51 is sensitive to the antibiotics used in the treatment of human brucellosis (oxytertracycline, streptomycin and doxycycline) but resistant to rifampin, penicillin, cloxacillin and oxacillin. 22

23 Cheville et al. (1992) reported that 7 groups of 3 calves each were inoculated S/C with one of the following strains of Brucella abortus, virulent strain 2308 (2 groups), vaccinal strain 19 (2) groups, mutant strain RB51, 19 delta 31k and S 19 delta SOD (3) groups. Sera, lymph node and tissues were examined at 2 week intervals post inoculation for evidence of infection. At the 12 th week post inoculation 2 calves in each group were given dexamethasone for 5 days. Calves were then euthanatized and lymphoid tissue; spleen, liver and bone marrow were examined for evidence of Brucella abortus. Calves given strain 2308 had large numbers of bacteria in their lymph nodes, marked glaucomatous lymphadenitis in the deep cortex, and loss of lymphoid cells in superficial cortical areas. In addition, the nonslaughtered calves had high serum antibody titers at 16 th week post Inoculation.Calves given strain 19 or genetic mutants derived from strain 19, cleared bacteria from lymph nodes more rapidly, had less lymphoid destruction and developed antibody titers that did not persist for 16 week. The RB51 strain was cleared most rapidly from lymphoid tissues and induced serum antibody responses only to the core of the lipopoly saccharied molecule. Treatment of calves with dexamethasone did not cause Brucella abortus to reappear in tissues of any calves, nor did serum antibody titers increase. Cheville et al. (1993) inoculated 24, 10 months Polled Hereford old heifers S/C with live cells of one the following strains of Brucella abortus, S 19 delta 31k (4 heifers), S 19 delta SOD (4heifers), RB51 (4heifers), strain 19 (6heifers). Controls (6 heifers) were given saline solution. Heifers given the deletion mutants S 19 delta 31k and S 19 delta SOD and those given S 19 developed antibody responses to Brucella abortus and cutaneous reaction to brucellin. Heifers given strain RB51 did not develop antibodies that reacted in the tube agglutination test, but sera reacted in test using an antibody dotblotassay containing RB51 antigen. All heifers were bred naturally at the 1617 month of age. They were challenged by intraconjunctivally with a virulent Brucella abortus strain 2308 during their fifth month of pregnancy. All vaccinated heifers were protected i.e., none aborted and none had Brucella abortus 23

24 isolated from their tissues after parturition. Antibody response in heifers after challenge exposure was an indicator of immunity. All the 6control non vaccinated heifers developed serum antibodies after challenge exposure, then 3 were aborted and 1 delivered a small weak calf at 8.5 month of gestation. They concluded that live mutant strains of Brucella abortus strain RB51 can induce protective immunity when given at 10 month of age. Samira ElGibally et al (1993) inoculated 1413 calves, with a dose of 38x10 9 of Brucella abortus S 19. One month after vaccination, 385 calves were picked up and tested at different intervals by using tube agglutination, buffered acidified plate antigen, RoseBengal plate and the rivanol tests up until parturition and then periodically every 6 months. They reported that, 266 calves (69.1%) developed humoral antibodies to the vaccine (Group A), 119 calves (30.9%) did not react to this tests (Group B). One year after vaccination 6 of calves (0.42%) showed persistent titers (Group C). The 2 groups A and B had negative serological results until parturition. Three months post parturition, 9 cows (all from group B) become reactors and 7 brucella isolates were recovered from their milk. The isolates were found to be Brucella melitensis biovar 3, which is the local filed strain. It was suggested that the upper limit of the reduced dose (10x10 10 CFU) of strain 19 should be tried in order to produce effective protection in this area where Brucella melitensis is the prevalent strain. Madsen (1993) reported that the serological response to vaccination with Brucella abortus strain 19 in heifers with standard dose (1x10 9 ) CFU and Brucella melitensis Rev.1 in sheep and goats with standard dose (4x10 9 ) CFU as measured by RoseBengal, tube agglutination and complement fixation tests were recorded over a period of 5 months following dosing in case of strain 19 vaccination. The titer had disappeared 3 months post vaccination in case of Rev1 vaccine. 24

25 Stevens et al. (1994 a) assessed the serological responses of cattle after vaccination with either Brucella abortus S 19 or the rough mutant strain RB51 using fluorescence immunoassay (FIA) and tube agglutination tests. S 19 vaccinated cattle were positive two weeks after vaccination while strain RB51 vaccinated cattle were negative to the 2 tests. These results indicated that the cattle vaccinated with RB51 failed to produce antibodies that can be detected by convention serological tests used to diagnose bovine broucellosis. Stevens et al. (1994 b) measured lymphocyte proliferation in response to protein from the Brucella abortus strain 2308 and strain RB51 in 2308 infected cattle following abortion. Lymphocytes from Supramammary and cervical lymph node of infected cattle proliferated mostly when incubated with 2718 KDa proteins of strain 2308 or proteins of strain RB51 which contain no O LPS antigen. Induced lymphocyte proliferation arose from rough RB51 strain was similar to that induced by strain 2308 which contained O LPS antigen. These results indicate that 2718 KDa proteins, but not O LPS antigen were immunodominant in strain 2308 infected cattle as assessed by lymphocyte proliferation assays. Stevens et al. (1995) inoculcated mice with two doses of strain RB51 (5x10 6 ) and (5x10 8 ) CFU and one dose of S 19 (5x10 6 ) CFU and found that the titre persisted for 12 weeks in mice vaccinated with S.19 in contrast mice vaccinated with strain RB51 did not produce antibodies against smooth LPS. The mice were challenged with Brucella abortus strain 2308 and found that mice vaccinated with 5x10 6 or 5x10 8 CFU of strain RB51 had increased resistance to infection with Brucella abortus strain 2308 at 12, 16 and 20 weeks after vaccination but the resistance was lower than that induced by vaccinating mice with 5x10 6 CFU of strain 19. Spleen cells obtained from mice vaccinated with S 19 or strain RB51 generally exhibited similar proliferative response to strain These results indicated that strain RB51 vaccinated mice have 25

26 similar cell mediated immune responses to strain 2308 but lower resistance to infection with strain 2308 compared with S 19 vaccinated mice. They concluded that the lower the resistance in strain RB51 vaccinated mice probably resulted from a combination of clearance of strain RB51 and an absence of antibodies to strain 2308 smooth LPS. Chivlle et al. (1996) inoculated calves S/C at 3, 5 and 7 months of age with strain RB51 (26 calves), S 19 (19 calves) and sterile saline solution (15calves). Calves were bred 16 to 17 months of age and challenged during the first Pregnancy with virulent Brucella abortus strain After vaccination none of the heifers given strain RB51 had developed serum antibodies that reacted in the standard agglutination test, but reacted in dotblotassay using RB51 antigen. Brucella abortus was cultured from biopsy specimens of superficial cervical Lymph nodes in the strain RB51 and S 19 vaccinates at 10 weeks, but not at 12 weeks after vaccination. All 4 heifers that had been vaccinated with RB51 at 3 months of age were protected against infection and abortion when challenged exposed and vaccination at 5 and 7 months of age gave equivalent protection. Heifers given S 19 were 95% protected and controls given saline solution had a high percentage of infection and abortion. Thus RB51 vaccine is protective at doses comparable to those of S 19 in calves at 3 to 7 months of age. Immunogenicity and failure to induce antibodies that interfere with the serologic diagnosis of filed infection of Brucella abortus made RB51 an effective vaccine. Dennis (1997) recorded the serological findings and persistant titers obtained in cattle herds immunized with the Brucella melitensis Rev1 (the new vaccination program) and the Brucella abortus S 19 vaccine (the existing program) in Mongolia. To reduce the economic losses caused by Brucella abortus infection under the conditions of nomadic cattle breeding. In the first year more than half a million cows over 3 months of age were immunized with the Brucella melitensis Rev1vaccine in 7 provinces of Mongolia. In the second year, only heifer calves over 3 months of age, 26

27 yearling animals not vaccinated in the first year, as well as heifers over 2 years of age and cows were vaccinated. The vaccine was administered S/C at a dose of 5x10 9 viable organisms. In cooperatives that had the Brucella abortus S 19 vaccine, the existing vaccination program was maintained and all cattle over 3 months of age were vaccinated with a dose of 5x10 10 viable organisms, using the same vaccination program as in herds immunized with the Rev1 vaccine. Before vaccination, 3 serological tests were carried out at an interval of 2025 days to measure the incidence of brucella infection. Serum samples were tested by the RoseBengal test (RBT), tube agglutination test and complement fixation test (CFT). An infection rate of 3.835% was found in the herds tested and the animals that were serologically positive for brucellosis were removed from the herds 15 to 21 days after vaccination, blood samples were taken from 10% of the animals in all immunized herds. The serum samples were tested by TAT and CFT to check the seroconversion. In herds immunized with the Rev1 vaccine, 90% of the animals were the seropositive, while the seropositive rate of S 19 vaccinated cows and heifers was 68.8% and 89.7% respectively. The serological status of the vaccinated animals was checked lately 12 months post vaccination. After Rev1 vaccination antibodies were present in 7% of cows, 4.6% of 3yearold heifers, 3.1% of the 2yearold heifers and 1.6% 0f the yearold heifers by comparison 13.2% of cows vaccinated with the Brucella abortus S 19 vaccine were serologically positive and 7.9% of 3year old heifers, 5% of 2 year old heifers and 2.7% of 1 year old heifers. Ribeiro et al. (1997) studied the serological profiles of 33 heifers vaccinated at 38 months of age with Brucella abortus S 19 over a period of 728 days using the plate agglutination, tube agglutination, Rose Bengal and mercaptoethanol, tests. Maximum antibody titers were detected to the plate agglutination and tube agglutination tests between the 14 th and 42 nd days post vaccination. While Animals become negative to the tube agglutination and plate agglutination tests at 245 and

28 days post vaccination respectively. All animals were negative to the mercaptoethanol and RoseBengal tests 308 days after vaccination. Elzer et al. (1998 a) vaccinated 20 beef cattle orally by 1034x10 9 CFU of strain RB51 and 10 unvaccinated heifers control group. Six weeks after vaccination, heifers were pastured bred to brucellosis free bull. Approximately 6 months after gestation, heifers were challenged conjunctively with 10 7 CFU of virulent Brucella abortus strain Vaccination with the rough RB51 did not stimulate antibodies against o lipopoly saccharied of Brucella abortus.after challenge exposure and parturition, strain 2308 was recovered from 80% of controls and only 20% of vaccinates. Only 30% of the vaccinates delivered dead, premature, or weak calves where as, 70% of controls had dead or weak calves. They concluded that cattle vaccinated orally with strain RB51 developed significant (P < 0.05) protection against abortion and colonization and do not produce Olipopoly saccharied specific antibodies. Edmonds et al. (1999) inoculated 6 bulls and 7 pregnant heifers in mid gestation with the standard calf hood dose of 3x10 10 CFU of RB51 intramuscularly. After vaccination, none of the vaccinated bulls or heifers shed RB51 in their secretion. Also there were no reproductive problems appeared in sexually mature cattle vaccinated with RB51. Olsen et al. (1999) vaccinated 6 heifer calves S/C with 16x10 10 CFU of strain RB51 while another 6 heifer calves were vaccinated with 32x10 10 CFU. The vaccine strain was recovered from the superficial cervical lymph node, 14 week after vaccination in 2 of six animals that vaccinated with 16x10 10 CFU, dose but not from any cattle vaccinated with 32x10 10 CFU. The higher RB51 dosages stimulated greater antibody titers using dot blot assay. Protection against abortion or infection following Brucella abortus strain 2308, challenge was similar for both strain RB51 dosages and greater than the resistance of the non vaccinated animals. 28

29 Rosanna Adone and Franco Ciuchini (1999) vaccinated 67 months old Frisian heifers (from brucellosis free herds) with 5x10 9 CFU of Brucella abortus strain RB51 and collected sera at 7, 15, 30, 60 and 110 days post vaccination. They found that none of heifers vaccinated with strain RB51 developed antibodies detected by brucella abortus strain 99based CF and Rose Bengal plate tests but all of the heifers developed antibodies to Brucella abortus strain RB51 antigen that reacted in CF test. Antibodies were produced 7 days post vaccination and the highest titers occurred 15 to 30 days and decreased at 110 days when 2 cows were still weakly seropositive. None of the samples from unvaccinated cattle reacted in the CF test. Villegas et al. (1999) revealed that out of 55 cows vaccinated with Brucella abortus strain 19, 6 cows (11%) were positive to card test and the authors succeeded to isolate Brucella abortus biotype 1 from milk samples of these cows. BengnatEjmanetti (2000) inoculated seventyfive, 9month old Aberdeen Angus calves in three groups with anti brucellosis vaccine strain 19 (group1) and anti brucellosis vaccine RB51 (group2) while group3 served an non vaccinated controls. The animals were challenged with Brucella abortus strain 2308 at 18 months of age. The authors found that the strain 19 vaccine was almost twice as efficient as RB51 vaccine in preventing infection. Fosgate et al. (2000) vaccinated 32 young domestic water Buffalo with 1 3.4x10 10 CFU of Brucella abortus strain RB51.Vaccination did not result in positive serological results as measured by tube agglutination test (TAT), buffered acidified plate antigen test (BAPAT) and card test (CT).Study animals were maintained in a brucellosispositive herd with an estimated 56% prevalence to allow for natural exposure to Brucella abortus, which was evaluated using TAT, BAPAT and CT. Animals were sampled seven times over 2 years and 6 of vaccinated animals 29

30 classified as positive and Brucella abortus biovar 1 were isolated from 4 of serologically positive vaccinated animals. Olsen (2000) vaccinated heifers with 3 RB51 vaccine dosing regimens, 3x10 9 CFU once, 1x10 9 CFU once or 1x10 9 CFU twice. Cattle vaccinated with one dose of 3x10 9 CFU had greater antibody response than the other two groups of vaccinates. The authors suggested that vaccination with a dose of (3x10 9 once) of strain RB51 protects adult cattle against abortion or infection caused by exposure to virulent Brucella abortus during the subsequent pregnancy. Uzal et al. (2000) vaccinated 13 cows with 1x10 9 CFU of Brucella abortus Strain RB51 which they had been vaccinated as calves with reduced dose of strain19 their serological responses following adult vaccination remained negative to conventional brucellosis surveillance tests. Vaccination with strain RB51 during the eighth month of pregnancy did not induce abortion, although strain RB51 was recovered from milk for up to 69 days after vaccination. The authors suggested that 10 9 CFU of strain RB51 was safe for use in pregnant cattle. HerMoon, et al. (2001) reported that out of 28 serum sample of cattle vaccinated with Brucella abortus strain RB51, 16 sera were negative to tube agglutination test 45 months after vaccination While 12 samples were positive, 12 of the 16 sera from cattle vaccinated with RB51 were positive by dotblot assay. Darwish and Benkirane (2001) presented the epidemiological status of brucellosis in cattle and sheep in Syria from 1990 to 1996 using Rose Bengal plate, complement fixation and tube agglutination tests and found that the serological response in cattle decreased steadily from 17.48% in 1992 to 2.59% in 1996 in the Governmentowned farms, while seroprevalence increased in the private sector during the same period. The difference may be explained by the restriction of brucellosis 30

31 vaccination to public farms, combined with partial application of a test and slaughter policy. Mathias et al. (2001) vaccinated 108 heifers, 18 months of age with a standard dose of 60120x10 9 CFU of Brucella abortus strain 19 vaccine. The presence of antibody titers was measured using plate agglutination, Rose Bengal and complement fixation tests. Serum samples were collected just before vaccination and 45 days, 6, 12 and 18 months post vaccination. Before vaccination all the heifers showed negative antibody titers in all tests. After 45 days, all the heifers had titers higher than or equal to 1:100 in plate agglutination test, while 2 animals had titers of 1:2 and 106 animals had titers higher than or equal to 1:4 in complement fixation test. One year after vaccination, almost all heifers did not show significant titers. It was suggested that vaccination of heifers in endemic areas at 18 months of age would reduce the risk of brucella infection. Aguirre et al. (2002) inoculated 524, 8 monthold Holstein heifers with 3x10 10 CFU of Brucella abortus strain 19 and determined the antibody response by buffered plate agglutination test (BAPAT), Rose Bengal plate test (RBPT), tube agglutination test (TAT), mercaptoethanol test (MET), (i ELISA) and complement fixation test (CFT). They found that antibodies measured with the BAPAT and RBPT were similar and the proportion of positive reaction in these tests reaches 100% one week after vaccination and remained at this level for seven week after which the proportion of positive samples slowly decline to 8% (BAPAT) and 2% (RBPT) at the week 50. The response in the i ELISA was similar, but shorter than that observed with the BAPAT and RBPT. Hamdy et al. (2002) challenged immunized Balb/C mice with rough Brucella abortus RB51 and smooth Brucella melitensis Rev1 vaccines with a Brucella 31

32 melitensis filed strain. Humoral Immune response was assessed by tube agglutination, Rose Bengal plate, buffered acidified plate antigen and mercaptoethanol tests. The authors found that Rev1 vaccine was superior to RB51 vaccine in protection of mice against Brucella melitensis infection, yet they recommended the use of RB51 vaccine for control of Brucella melitensis infection in adults as it did not elicit antibodies to standard serological test. Soliman (2002) evaluated Brucella abortus strain RB51 vaccine in 150 guinea pigs inoculated S/C with (5x10 8 ) CFU of strain RB51. Serological examination of guinea pigs using TAT employing rough antigen showed that antibody production started one week post vaccination and reached the peak at the 4 th week post vaccination then declined and disappeared 8 weeks post vaccination. Thirty vaccinated and 20 non vaccinated guinea pigs were challenged with Brucella melitensis biovar 3, 10 weeks post vaccination. The author detected strong positive serological reactions in vaccinated and non vaccinated guinea pigs, using TAT, BAPA, RBPT and Riv T and succeeded to isolate Brucella melitensis biovar 3 from both vaccinated and non vaccinated animals. Jamal et al. (2003) evaluated strain 19 in 15 guinea pigs and 5, month old female buffalo and found that the vaccine induced a good immune response in guinea pigs at a dose of 5x10 9 as evidenced by a serological titer of 328 international units 10 days post vaccination. Vaccinated guinea pigs also withstood an experimental challenge of 5,000 CFU of a locally isolated virulent Brucella abortus strain. In buffalo, the dose of 7x10 10 CFU of strain 19 induced a significant immune response in 8 to 10.5 month old female buffalo calves. The titer was followed up by using tube agglutination test (TAT) and 2mercaptoethanol test (MET) and they found that TAT titers were seen on day 7 postvaccination and the rate of decrease was slow from day 14 to day 49 postvaccination; however, a rapid decrease in titres was seen from day 49 to day 91 post vaccination. 32