Animal Health Research Journal Vol. 5, No. 4(A), November 2017 pp

Animal Health Research Journal Vol. 5, No. 4(A), November 2017 pp. 74-83 Efficacy of Brucella abortus RB 51 as calf hood vaccine for protection of imported cattle under field conditions in Egypt Soliman, S. Hazem; Rania, I. Ismail and Mahmoud, H. Abdel-Halim Animal Health Research Institute, Brucella Department Dokki- Giza Received in 4/10/2017 Accepted in 14/11/2017 ISSN: 2356-7767 Abstract A total of 100 heifer calves of 6-8 months age born to an imported Holstein herd at El- Ismailia Governorate, Egypt, were used in this study. These calves were subcutaneously vaccinated with 1.0 3.4 10 10 CFU Brucella abortus RB51 rough mutant vaccine. Blood samples were collected from 100 vaccinated heifer calves for separation of blood sera before and after vaccination. Serological examination of Brucella abortus SRB51vaccinated heifer calves using smooth antigens, revealed 6 (6%) positive calves 90 days post vaccination and another 6 (6,38%) positive calves 120 days post vaccination using SAT, BAPA, RBT, RIV, and CFT. Bacteriological examination of the slaughtered 6 seropositive heifer calves 90 days post vaccination revealed isolation of brucella from retropharyngeal lymph nodes, internal iliac lymph node, liver, and spleen of the six calves. Brucella cultures showed typical characteristics for the genus Brucella. Typing of Brucella isolates recovered from vaccinated animals revealed Brucella melitensis biovar 3 which indicates brucella field infection and non-vaccinal strain. Molecular examination using duplex PCR was performed on DNA isolated from blood sera of vaccinated calves. Results of PCR of ten heifer calves vaccinated with RB51 vaccine 30 days post vaccination, revealed amplification of 1682 bp in all heifer calves. On the other hand, the results of PCR of heifer calves vaccinated with RB51 vaccine 90 days post vaccination, revealed amplification of 1682 bp in all calves and amplification of the 839 bp, in 6 heifer calves. Examination at 120 days post vaccination, revealed amplification of 1682 bp in vaccinated heifer calves and amplification of the 839 bp, in other 6 heifer calves. The results indicate that the Brucella abortus SRB51 did not produce sufficient protection in vaccinated heifer calves. Failure of RB51 vaccine to protect such calves might be due to vaccination of calves prior to the development of an efficient level of protection against virulent infection. Also, we concluded that the duplex PCR performed on DNA isolated from sera of vaccinated heifer calves is very convenient as it significantly shortens the time of detection of the brucella and has a low cost using two primer pairs in one step. Keywords: Brucella abortus, RB51; Vaccination, PCR; Cattle, serological tests. Introduction Bovine brucellosis is one of the most important zoonotic diseases worldwide, especially in developing countries affecting cattle, buffaloes and numerous wildlife species. The causative agent of brucellosis in cattle is mainly Brucella abortus followed by infections with Brucella melitensis and Brucella suis (Aznar et al., 2014). Brucellosis is economically important as it causes financial losses from abortions, sterility, decreased milk production, veterinary fees and costs of replacement animals. However, most data and evidence on the economic burden of brucellosis and benefits of its control are from the developed world even though the losses are believed to be higher in the developing countries (McDermott et al., 2013). Surveillance and control programs in endemic developed or developing countries aim at the control and eradication of the disease fre- 74

First International Conference of Animal Health Research Institute Soliman et al. quently involving vaccine campaigns (Moriyon et al., 2004). The aim of vaccination is the reduction of susceptible individuals in the population, and the success of any vaccination program depends mainly on the effectiveness of the vaccine used and its coverage in the target population. One of the major Gabs in Brucellosis control programs is a disorganized application of vaccination programs and selection of proper vaccine. Brucella abortus S19 is the most commonly used vaccine for protecting cattle against brucellosis, although this vaccine has several disadvantages. One major disadvantage is that S19 induces antibodies against the O-chain of the B. abortus lipopolysaccharide (LPS), which may persist for several months or years and interfere with serological tests (Nielsen 2002). In Egypt, brucellosis control programs for bovines are based on a test and slaughter policy in combination with vaccination, Refai, (2002). However, the use of this vaccine hampers serodiagnosis because the vaccinated and naturally infected animals are difficult to distinguish (Yang et al., 2013), and immunization of pregnant cows may lead to abortion in up to3% of the vaccinated cows (Beckett and MacDiarmid, 1985; Smith and Ficht, 1990). To reduce these drawbacks, current vaccination programs for cattle frequently use the rough mutant Brucella abortus RB51 vaccine derived from wild-type Brucella abortus 2308, (Yang et al., 2013). Prevention of infection, and hence disease, can be achieved with vaccines. As a general rule, induction of an effective, long-lasting protective immune response to facultative intracellular parasites requires the use of live vaccines, WHO (1997) Since Brucella abortus RB51 lacks an O-chain, it does not induce anti-o-chain antibodies measurable by conventional serological tests regardless of age, dose or frequency of injections. Therefore, the antibody response induced by vaccination does not interfere with serological tests. In Egypt, brucellosis control programs for bovines are based on a test and slaughter policy in combination with vaccination, Refai, (2002). Recently, vaccination with attenuated Brucella abortus RB51 has been approved, although it may also lead to abortion in some cases (Fluegel Dougherty et al., 2013). Most studies about the protection induced by RB51 were performed under strictly controlled conditions in mice and/or heifers, and have concluded that animals are protected against moderate challenge, but contradictory results in field experiments under high or moderate challenge appears to indicate that more research is necessary to evaluate the level and duration of immunity under field conditions (Moriyon et al., 2004). In Egypt, Brucella melitensis biovar3 remains the prevalent type of brucella affecting cattle, Salem and Hosein (1990); Afifi et al., (2013); Menshawy et al., (2014); Hosein et al..,(2016). Several efforts have been made to control the disease through the use of vaccines including Brucella abortus SRB51. Recently, with the gradual use of RB51 vaccine, it became necessary to develop a method capable of identifying animals vaccinated with RB51 vaccine (Robles et al., 2003). This include a dot-blot assay using killed irradiated RB51 bacteria as an antigen (Olsen et al., 1997), ielisas using a 5% optical density heat-killed whole RB51 organisms as an antigen (Edmonds et al., 1999), and a crude rough LPS preparation from RB51 (Uzal et al., 2000), Dot-Blot ELISA (Fosgate et al., 2003; Diptee et al., 2007), an immunoblot analysis using sonicated cell lysates from RB51 (Edmonds et al., 1999), a complement fixation test, using RB51 cultured cells in calcium magnesium-veronal buffer (Diptee et al., 2007; Galiero, 2009; Caporale et al., 2010) and an agar gel immunodifusion test using hot saline extract antigen from Brucella ovis (Robles et al., 2009). Unfortunately, all these methods were unpractical and time-consuming. 75

Animal Health Research Journal Vol. 5, No. 4(A), November 2017 pp. 74-83 Therefore, the aim of the current study was to evaluate the efficacy of Brucella abortus RB51 as calf hood vaccine in protection of heifers born from imported cattle against brucellosis under field conditions in Egypt on serological, bacteriological and molecular basis as well as to develop a rapid duplex PCR that can differentiate the DNA of Brucella melitensis field strains prevalent in Egypt from that of the Brucella abortus RB51vaccine strain. Materials and Methods 1-Animals A total of 100 heifer calves of 6-8 months age born to an imported Holstein herd at El- Ismailia Governorate, Egypt, were used in this study. These calves were subcutaneously vaccinated with the recommend dose 1.0 3.4 10 10 CFU Brucella abortus RB51 vaccine. 2 Samples: a. Blood samples were collected by jugular vein puncture from 100 heifer calves under study for separation of blood sera before vaccination and periodically after vaccination. Sera were subjected to serological examination and PCR. b. Retropharyngeal, internal iliac lymph nodes, liver, and spleen were collected from 6 seropositive slaughtered calves after 90 days post vaccination for bacteriological examination. 3 Vaccine The commercial vaccine strain Brucella abortus SRB51,U.S.Vet. Licence No. 188 Professional Biological Company USA was used in this study. 4- Serological tests: a. Slow Agglutination Test (SAT), was carried out according to Alton et al., (1988) a white Brucella. abortus strain 99 at a concentration of 4% packed cells in phenol saline. It is prepared by the Veterinary Serum & Vaccine Research Institute, Abbasia. b. Buffered Acidified plate antigen test (BPAT) was carried out according to Alton et al., (1988) using Buffered acidified plate test antigen (killed Brucella abortus strain 99 antigen, at concentration of 11% in lactate buffer, ph 3.7±0.03) obtained from the Veterinary Sera and Vaccine Research Institute, (VSVRI) Abbassia, Cairo, Egypt. c. Rose Bengal test (RBT) was carried out according to OIE (2012) using Rose Bengal test antigen (Rose Bengal stained, 8% cells killed Brucella abortus strain 99 antigens in lactate buffer, ph 3.65±0.05) obtained from VSVRI, Abbassia, Cairo, Egypt. The rivanol test was carried out according to Alton et al. (1988) using rivanol test antigen and solution obtained from the Veterinary Sera and Vaccine Research Institute, (VSVRI) Abbassia, Cairo, Egypt. e. Complement fixation test (CFT) was carried out according to Alton et al., (1988) using Complement fixation test antigen (Brucella abortus biovar 1 strain 1119-3 cells in phenol saline, at a concentration of 4.5%, ph 6.8) obtained from the National Veterinary Services Laboratories (NVSL) Ames, Iowa state, USA.Warm microtechnique was used. 5- Bacteriological examination: Tissue homogenates were cultured on tryptose agar medium with antibiotics selective antibiotic supplement (Ewalt et al., 1983), (Oxoid) according to Alton et al., (1988). Plates were incubated at 37 C in an atmosphere of 5-10% CO 2 and examined daily for 12 days for growth. Isolates were identified as Brucella by assessing colony morphology, biochemical reactions (oxidase, catalase, and urease), CO 2 requirement, production of H 2 S, growth in the presence of the dyes thionine and basic fuchsine, reaction with monospecific antisera (A, M and R), phage lysis (Tb, Wb) as well as the use of acriflavine and staining of colonies with crystal violet to assess the colonial morphology of the isolated strains and to differentiate between rough and smooth type colonies prior to identification according to the methods described by Alton et al., (1988); OIE (2012). 6-Molecular examination: Bricker and Halling,( 1994) and OIE, (2009). 76

First International Conference of Animal Health Research Institute Soliman et al. DNA extraction: DNA extraction from serum samples was performed using the QIAamp DNA Mini kit (Qiagen, Germany, GmbH) with modifications from the manufacturer s recommendations. Briefly, 200 µl of the sample suspension were incubated with 10 µl of proteinase K and 200 µl of lysis buffer at 56 O C for 10 min. After incubation, 200 µl of 100% ethanol was added to the lysate. The sample was then washed and centrifuged following the manufacturer s recommendations. Nucleic acid was eluted with 100 µl of elution buffer provided in the kit. Oligonucleotide Primers. Primers used were supplied from biobasic (Canada) and are listed in table (1) Table (1). Primers sequences, target genes, amplicon sizes for conventional PCR according to Bricker and Halling,( 1994) and OIE, (2009). Target gene Target agent Primers sequences Amplified segment (bp) 1S711 Brucella melitensis 1S711-specificPrimer Fw TGC-CGA-TCA-CTT -AAG-GGC-CTT-CAT Rv AAA-TCG-CGT-CCT -TGC-TGG-TCT-GA 839 wboa RB51 vaccinal strain Fw ATC-CTA-TTG-CCC -CGA-TAA-GG Rv GCT-TCG-CAT-TTT- CAC-TGT-AGC 1682 PCR amplification. Oligonucleotide Primers supplied from biobasic (Canada), Table (1) were used in this study. Primers were utilized in a 50- µl reaction containing 25 µl of Emerald Amp Max PCR Master Mix (Takara, Japan), 1 µl of each primer of 20 pmol concentration, 13 µl of water, and 8 µl of DNA template. The reaction was performed in an applied biosystem 2720 thermal cycler. Analysis of the PCR Products. The products of PCR were separated by electrophoresis on 1% agarose gel (Applichem, Germany, GmbH) in 1x TBE buffer at room temperature using gradients of 5V/cm. For gel analysis, 30 µl of the products were loaded in each gel slot. A 100 bp DNA ladder H3 RTU (Genedirex, Taiwan) was used to determine the fragment sizes. The gel was photographed by a gel documentation system (Alpha Innotech, Biometra) and the data was analyzed by computer software Results Table (2). Serological examination of RB51 vaccinated calves using smooth antigens. Days post-vaccination 0 30 90 120 150 240 No. of positive * 0 0 6 6 0 0 % 0% 0% 6% 6.38% SAT: serum agglutination test, BAPA: Buffered acidified plate antigen test, RB: Rose Bengal, RivT: Rivanol test, CFT: Complement fixation test. 0% 0% 77

Animal Health Research Journal Vol. 5, No. 4(A), November 2017 pp. 74-83 Table (3). Detailed serological results of reactors of RB51 vaccinated calves using smooth antigens Serial No. DPV SAT BAPA RBT Riv.T CFT 1 90 1\160 4+ 3+ 1\100 1\32++++ 2 90 1\160 4+ 3+ 1\100 1\32++++ 3 90 1\160 3+ 3+ 1\100 1\16+++ 4 90 1\640 4+ 4+ 1\100 1\128 5 90 1\640 4+ 4+ 1\100 1\128 6 90 1\320 4+ 4+ 1\100 1\64++ 7 120 1\160 4+ 4+ 1\100 1\64++ 8 120 1\80++++ 4+ 3+ 1\100 1\32++ 9 120 1\320 4+ 4+ 1\100 1\128 10 120 1\160++ 4+ 4+ 1\100 1\64+ 11 120 1\160++ 3+ 3+ 1\100 1\32+ 12 120 1\80+++ 3+ 2+ 1\100 1\8++ DPV: Days post vaccination, SAT: serum agglutination test, BAPA: Buffered acidified plate antigen test, RB: Rose Bengal, RivT: Rivanol test, CFT: Complement fixation test. Serological examination of Brucella abortus RB51 vaccinated heifer calves using smooth antigens, Tables (2 and 3) revealed that 6 (6%) calves 90 days post vaccination and another 6 (6,38%) calves 120 days post vaccination, where positive using SAT, BAPA, RBT, Riv.T and CFT table 2. Detailed serological results of the 12 seroconverted positive cases are illustrated in table 3 Table (4). Isolation of Brucella organisms from 6 slaughtered seropositive calves 90 days post vaccination No of examined calves Specimens NO. of positive calves Type of Brucella Isolates Retropharyngeal LN 2 6 Internal iliac LN Liver 2 1 Br.melitensis biovar 3 Spleen 1 Bacteriological examination of the 6 seropositive heifer calves 90 days post vaccination revealed isolation of brucella from retropharyngeal lymph nodes, internal iliac lymph node, liver, and spleen of the six calves. Brucella cultures showed typical characteristics for the genus Brucella. Colonies were smooth elevated, transparent, and convex, with intact borders, brilliant surface and had a honey color under transmitted light. Typing of Brucella isolates recovered in this study revealed Brucella melitensis biovar 3 (Table 4) which indicates brucella field infection and non-vaccinal strain. 78

First International Conference of Animal Health Research Institute Soliman et al. Figure (1). Results of Duplex PCR of calves vaccinated with RB51 vaccine 30 days post vaccination. control positive, RB51 and control positive Br. melitensis lane 1-10 positive RB51 vicinal strain. Figure (2). Results of Duplex PCR of calves vaccinated with RB51 vaccine 90 days post vaccination. control positive, RB51 and control positive Br. melitensis lane 1-10 positive RB51 vicinal strain, lane 1, 2, 3, 4, 6 and 8 positive Br. melitensis field strain. Figure (3). Results of Duplex PCR of calves vaccinated with RB51 vaccine 120 days post vaccination control positive, RB51 and control positive Br. melitensis lane 1-10 positive RB51 vicinal strain, lane 2, 3, 4, 5, 6 and 7 positive Br. melitensis field strain. 79

Animal Health Research Journal Vol. 5, No. 4(A), November 2017 pp. 74-83 Molecular examination using duplex PCR was performed on DNA isolated from blood sera of vaccinated calves using specific primers Brucella meltensis IS711, which amplify 839 bp, and RB51wboA which amplify 1682 bp. Results of PCR of heifer calves vaccinated with RB51 vaccine 30 days post vaccination, Figure (1) revealed amplification of 1682 bp in all heifer calves. On the other hand, the results of PCR of heifer calves vaccinated with RB51 vaccine 90 days post vaccination, Figure (2) revealed amplification of 1682 bp in all calves and amplification of the 839 bp, in 6 heifer calves. Examination at 120 days post vaccination, Figure (3) revealed amplification of 1682 bp in vaccinated heifer calves and amplification of the 839 bp, in other 6 heifer calves. Discussion Brucellosis is still a worldwide disease caused by gram-negative bacteria of genus brucella. The disease causes sever economic losses in many countries including Egypt. Serological examination of 100 heifer calves vaccinated with Brucella abortus SRB51, using smooth antigens, Table 2 revealed that 6 (6%) of heifer calves were serologically positive, 90 days post vaccination and another 6 (6,38%) were positive 120 days post vaccination, (Table 2) using SAT, BAPA, RBT, Riv T and CFT. Development of brucella antibodies in these calves indicates their exposure to smooth brucella organisms. RB51 confers immunity through induction of the host s cellmediated immunity using Th1 cells rather than a humoral immunity (Vemulapalli et al., 2000). Therefore, vaccinated animals do not produce O-chain antibodies, and thus the vaccination does not result in interference with serological testing (Shurig et al., 2002). The results also indicate that the Brucella abortus SRB51 did not produce sufficient protection in vaccinated heifer calves. Failure of RB51 vaccine to confer complete protection such calves might be due to vaccination of calves prior to the development of an efficient level of protection against virulent infection. This may be attributed to that the cell-mediated immunity which has the essential role in protection takes a longer time in vaccinated animal. This means that investigated calves were still susceptible to Brucella infection as they were kept within an infected herd in which chronically infected cows exist and shedding huge numbers of Brucella organisms following abortion or parturition. Moreover, close contact with the surrounding grazing area and management procedures, the persistence of Brucella in the environment pose a risk for reintroduction of Brucella to livestock that facilitates the transmission of the disease and allows multiple sources of infection that are difficult to control. Delay in the removal of infected animals after serological identification and keeping these animals inside the farm even for a short period also permit the spreading of infection to susceptible animals. Development of brucella seropositive cases among RB51 vaccinated calves may be the results of previous latent infection either in-utero infection or calves consuming infected milk as reported by Nielsen and Duncan, (1990) that as high as 20% of calves born by infected heifers could be found persistently infected with brucella. Heifer calves that develop latent disease remain asymptomatic and serologically negative until the first parturition at which time abortion and seroconversion are frequentl y observed (Nicoletti, 1980). Also, the obtained results can be explained by Poester et al. (2006) who reported that RB51 prevent 59% of abortions, 59% of cow infections, and 61% of fetal infections. Bacteriological examination of the slaughtered 6 seropositive calves detected 90 days post vaccination confirmed the results of the serological examination. The results revealed isolation of Brucella from retropharyngeal lymph nodes, internal iliac lymph node, liver, and spleen of the six calves. Brucella cultures showed typical characteristics for the genus Brucella. Colonies were smooth elevated, transparent, and convex, with intact borders, 80

First International Conference of Animal Health Research Institute Soliman et al. brilliant surface and had a honey color under transmitted light. Typing of Brucella isolates recovered in this study revealed Brucella melitensis biovar 3, (Table 4) which indicates Brucella field infection and nonvaccinal strain. Brucella melitensis biovar 3 was previously identified and considered as the prevalent biovar in Egypt as recorded by Salem and Hosein (1990); Afifi et al., (2013); Menshawy, et al., (2014); Hosein et al.,(2016). This also indicates the higher susceptibility of imported cattle herds to field Brucella infection. Molecular examination using duplex PCR was performed on DNA extracted from sera of vaccinated calves using specific primers for Brucella meltensis and Brucella abortus RB51 Results of PCR of calves vaccinated with RB51 vaccine 30 days post vaccination, Figure (1), revealed amplification of 1682 bp in all calves indicating Brucella abortus RB51. On the other hand, the results of PCR of calves vaccinated with RB51 vaccine 90 days post vaccination, Figure (2), revealed amplification of 1682 bp, in all calves indicating Brucella abortus RB51 and amplification of the 839 bp, in 6 calves suggesting field Brucella melitensis infection. Examination at 120 days post vaccination, Figure (3), revealed amplification of 1682 bp in vaccinated calves indicating Brucella abortus RB51 and amplification of the 839 bp, in other 6 heifer calves suggesting further field Brucella melitensis infection. Such results indicate that both culture and molecular techniques can definitively establish the presence of infection as well as determine the Brucella species responsible. Several authors have attempted to differentiate Brucella melitensis field strains from Brucella vaccine strains, Banai, (2002); Benkirane et al., (2014). Differentiation of Brucella melitensis field strains from the Brucella abortus RB51 vaccine strain has been achieved in this study by using duplex PCR technique. The results showed that the duplex PCR performed on DNA isolated from blood of vaccinated heifer calves is very convenient as it significantly shortens the time of detection of the Brucella and has a low cost using two primer pairs in one step. Similar results were reported by Alvarez, et al., (2017) who could differentiate Brucella melitensis from the vaccine strain REV-1. Recommendation It is essential to employ more research using various techniques such as genetic, proteomics, metabolic engineering to develop an ideal vaccine against Brucellosis under field conditions in Egypt, where Brucella melitensis is prevailing in most domesticated animal's species. References Afify, M.; Al-Zahrani, S.H. and El-Koumi, M.A. (2013). Brucellosis-Induced Pancytopenia in Children: A Prospective Study. Life Sci. J. 10: 1.1364-8. Alton, G.G.; Jones, L.M.; Angus, R.D. and Verger, J.M. (1988). Techniques for the brucellosis laboratory. 1st. Edn., Institut National de la Recherche Agronomique (INRA), 147, Paris, France. PP: 13-61. Alvarez, L.P.; Marcellino, R.B. and Martinez, A. Robles C.A. (2017). Duplex PCR for the diagnosis of Brucella melitensis and its differentiation from the REV-1 vaccine strain. Science Direct Small Ruminant Research 146 (2017) 1 4. Aznar, M.N.; Samartino, L.E.; Humblet, M.F. and Saegerman, C. (2014). Bovine brucellosis in Argentina and bordering countries: update. Transbound. Emerg. Dis. 61,121 133. Banai, M. (2002). Control of small ruminant brucellosis by use of Brucella melitensis Rev.1 vaccine: laboratory aspects and field observations Vet Microbiol. 2002 Dec 20;90 (1-4): 497-519. Beckett, F.W. and MacDiarmid, S.C. (1985). The effect of reduced-dose Brucella abortus strain 19 vaccination in accredited dairy herds. Br. Vet. J. 141, 507 514. Benkirane, A.; El Idrissi, A.H.; Doumbia, A. and de Balogh, K. (2014). Innocuity and 81

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