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Anaplasma phagocytophilum is a tick borne bacterium, causing disease in sheep and other mammals, including humans. The bacterium has great economic and animal welfare implications for sheep husbandry in Northern Europe. With the prospect of a warmer and more humid climate, the vector availability will likely increase, resulting in a higher prevalence of A. phagocytophilum. The current preventive measures, as pyrethroids acting on ticks or long acting antibiotics controlling bacterial infection, are suboptimal for prevention of the disease in sheep. Recently, the increased awareness on antibiotic- and pyrethorid resistance, is driving the search for a new prophylactic approach in sheep against A. phagocytophilum. Previous studies have used an attenuated vaccine, which gave insufficient protection from challenge with live bacteria. Other studies have focused on bacterial membrane surface proteins like Asp14 and OmpA. An animal study using homologous proteins to Asp14 and OmpA of A. marginale, showed no protective effect in heifers. In the current study, recombinant proteins of Asp14 (rAsp14) and OmpA (rOmpA) of A. phagocytophilum were produced and prepared as a vaccine for sheep. Ten lambs were vaccinated twice with an adjuvant emulsified with rAsp14 or rOmpA, three weeks apart and challenged with a live strain of A. phagocytophilum (GenBank acc.nr M73220) on day 42. The control group consisted of five lambs injected twice with PBS and adjuvant. Hematology, real time qPCR, immunodiagnostics and flow cytometric analyses of peripheral blood mononuclear cells were performed. Vaccinated lambs responded with clinical signs of A.phagocytophilum infection after challenge and bacterial load in the vaccinated group was not reduced compared to the control group. rAsp14 vaccinated lambs generated an antibody response against the vaccine, but a clear specificity for rAsp14 could not be established. rOmpA-vaccinated lambs developed a strong specific antibody response on days 28 after vaccination and 14 days post-challenge. Immunofluorescent staining and flow cytometric analysis of peripheral blood mononuclear monocytes revealed no difference between the three groups, but the percentage of CD4 + , CD8 + , γδ TcR + , λ-Light chain + , CD11b + , CD14 + and MHC II + cells, within the groups changed during the study, most likely due to the adjuvant or challenge with the bacterium. Although an antigen specific antibody response could be detected against rOmpA and possibly rAsp14, the vaccines seemed to be ineffective in reducing clinical signs and bacterial load caused by A. phagocytophilum. This is the first animal study with recombinant Asp14 and OmpA aimed at obtaining clinical protection against A. phagocytophilum in sheep., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Vibrio mimicus is the causative agent of ascites disease in fish. The heat-labile hemolytic toxin designated VMH is an immunoprotective antigen of V. mimicus. However, its epitopes have not been well characterized. Here, a commercially available phage displayed 12-mer peptide library was used to screen epitopes of VMH protein using polyclonal rabbit anti-rVMH protein antibodies, and then five positive phage clones were identified by sandwich and competitive ELISA. Sequences analysis showed that the motif of DPTLL displayed on phage clone 15 and the consensus motif of SLDDDST displayed on the clone 4/11 corresponded to the residues 134-138 and 238-244 of VMH protein, respectively, and the synthetic motif peptides could also be recognized by anti-rVMH-HD antibody in peptide-ELISA. Thus, both motifs DPTLL and SLDDDST were identified as minimal linear B-cell epitopes of VMH protein. Although no similarity was found between VMH protein and the consensus motif of ADGLVPR displayed on the clone 2/6, the synthetic peptide ADGLVPR could absorb anti-rVMH-HD antibody and inhibit the antibody binding to rVMH protein in enhanced chemoluminescence Western blotting, whereas irrelevant control peptide did not affect the antibody binding with rVMH. These results revealed that the peptide ADGLVPR was a mimotope of VMH protein. Taken together, three novel B-cell epitopes of VMH protein were identified, which provide a foundation for developing epitope-based vaccine against V. mimicus infection in fish., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Mannheimia haemolytica is a very important pathogen of pneumonia in ruminants. Bighorn sheep (BHS, Ovis canadensis) are highly susceptible to M. haemolytica-caused pneumonia which has significantly contributed to the drastic decline of bighorn sheep population in North America. Pneumonia outbreaks in wild BHS can cause mortality as high as 90%. Leukotoxin is the critical virulence factor of M. haemolytica. In a 'proof of concept' study, an experimental vaccine containing leukotoxin and surface antigens of M. haemolytica developed by us induced 100% protection of BHS, but required multiple booster injections. Vaccination of wild BHS is difficult. But they can be vaccinated at the time of transplantation into a new habitat. Administration of booster doses, however, is impossible. Therefore, a vaccine that does not require booster doses is necessary to immunize BHS against M. haemolytica pneumonia. Herpesviruses are ideal vectors for development of such a vaccine because of their ability to undergo latency with subsequent reactivation. As the first step towards developing a herpesvirus-vectored vaccine, we constructed a chimeric protein comprising the leukotoxin-neutralizing epitopes and the immuno-dominant epitopes of the outer membrane protein PlpE. The chimeric protein was efficiently expressed in primary BHS lung cells. The immunogenicity of the chimeric protein was evaluated in mice before inoculating BHS. Mice immunized with the chimeric protein developed antibodies against M. haemolytica leukotoxin and PlpE. More importantly, the anti-leukotoxin antibodies effectively neutralized leukotoxin-induced cytotoxicity. Taken together, these results represent the successful completion of the first step towards developing a herpesvirus-vectored vaccine for controlling M. haemolytica pneumonia in BHS, and possibly other ruminants., (Copyright © 2016. Published by Elsevier B.V.)

Current contagious bovine pleuropneumonia (CBPP) vaccines are based on live-attenuated strains of Mycoplasma mycoides subsp. mycoides (Mmm). These vaccines have shortcomings in terms of efficacy, duration of immunity and in some cases show severe side effects at the inoculation site; hence the need to develop new vaccines to combat the disease. Reverse vaccinology approaches were used and identified 66 candidate Mycoplasma proteins using available Mmm genome data. These proteins were ranked by their ability to be recognized by serum from CBPP-positive cattle and thereafter used to inoculate naïve cattle. We report here the inoculation of cattle with recombinant proteins and the subsequent humoral and T-cell-mediated immune responses to these proteins and conclude that a subset of these proteins are candidate molecules for recombinant protein-based subunit vaccines for CBPP control., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The efficiency of antigen capture, processing, and presentation by antigen-presenting cells is the key to induce an effective immune response. Cell-penetrating peptides (CPPs) are short peptides that facilitate cellular uptake of various molecular cargoes and have an attractive potential for vaccine delivery. In this study, the Drosophila Antennapedia homeoprotein (Antp) and the human immunodeficiency virus-1 transactivator of transcription (TAT) peptides were fused to the N- or C-terminus of Sia10, a protective antigen of Streptococcus iniae, resulting in four recombinant fusion proteins, i.e., rAntp-Sia10, rSia10-Antp, rTAT-Sia10, and rSia10-TAT. All fusion proteins were expressed and purified, and their ability to penetrate into cells was examined. The results showed that rTAT-Sia10 had the strongest ability to translocate through the cellular membrane into cells. Immunofluorescence microscopy and Western blot assay confirmed that rTAT-Sia10 could penetrate into the head kidney lymphocytes and gill cells of Japanese flounder (Paralichthys olivaceus). Immunological analysis showed that rTAT-Sia10 significantly enhanced macrophage activation and peripheral blood leukocyte proliferation, and induced production of specific serum antibodies at 2-8 weeks post-vaccination. Transcriptional analysis showed that vaccination with rTAT-Sia10 up-regulated the expression of the genes encoding IL-1β, IL-8, NKEF, Mx, IgD, IgM, TNFα, MHC I α, MHC IIα, and CD8α. Fish vaccinated with rTAT-Sia10 exhibited significantly higher levels of survival rates (98% at 1 month and 92% at 2 months) compared to fish vaccinated with rSia10 (57% at 1 month and 53% at 2 months). Taken together, these results indicate that TAT-derived peptide has a great potential in the application of bacterial vaccines., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Avian Pasteurella multocida is a causative agent of fowl cholera. Two proteins OmpH and OmpA are the major immunogenic antigens of avian P. multocida, which play an important role in inducing immune responses that confer resistance against infections. In the present study, we used pcDNA3.1(+) as a vector and constructed DNA vaccines with the genes encoding the two antigens mentioned above. These DNA vaccines include monovalent (pcDNA-OMPH, pOMPH and pcDNA-OMPA, pOMPA), divalent combination (pcDNA-OMPH+pcDNA-OMPA, pOMPH+pOMPA) and fusion of two gene vaccines (pcDNA-OMPH/OMPA, pOMPHA). The immune responses to these DNA vaccines were evaluated by serum antibody titers, lymphocyte proliferation assay and titers of a cytokines, IFN-γ. The protective efficacy after challenging with a virulent avian P. multocida strain, CVCC474, was evaluated by survival rate. A significant increase in serum antibody levels was observed in chickens vaccinated with divalent combination and fusion DNA vaccines. Additionally, the lymphocyte proliferation (SI value) and the levels of IFN-γ were both higher in chickens immunized with divalent combination and fusion DNA vaccines than in those vaccinated with monovalent DNA vaccines (P<0.05). Furthermore, the protection provided by divalent combination and fusion DNA vaccines was superior to that provided by monovalent DNA vaccines after challenging with the avian P. multocida strain CVCC474. And the protective efficacy in chickens immunized three times with the fusion DNA vaccine was equivalent to the protective efficacy in chickens vaccinated once with the attenuated live vaccine. This suggests that divalent combination and fusion DNA vaccines represent a promising approach for the prevention of fowl cholera., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Actinobacillus pleuropneumoniae is an infective agent that leads to porcine pleuropneumonia, a disease that causes severe economic losses in the swine industry. Based on the fact that the respiratory tract is the primary site for bacterial infection, it has been suggested that bacterial exclusion in the respiratory tract through mucosal immune induction is the most effective disease prevention strategy. ApxIIA is a vaccine candidate against A. pleuropneumoniae infection, and fragment #5 (aa. 439-801) of ApxIIA contains the major epitopes for effective vaccination. In this study, we used mice to verify the efficacy of intranasal immunization with fragment #5 in the induction of protective immunity against nasal challenge with A. pleuropneumoniae and compared its efficacy with that of subcutaneous immunization. Intranasal immunization of the fragment induced significantly higher systemic and mucosal immune responses measured at the levels of antigen-specific antibodies, cytokine-secreting cells after antigen exposure, and antigen-specific lymphocyte proliferation. Intranasal immunization not only efficiently inhibited the bacterial colonization in respiratory organs, but also prevented alveolar tissue damage in infectious condition similar to that of a contaminated pig. Moreover, intranasal immunization with fragment #5 provided acquired protective immunity against intranasal challenge with A. pleuropneumoniae serotype 2. In addition, it conferred cross-protection against serotype 5, a heterologous pathogen that causes severe disease by ApxI and ApxII secretion. Collectively, intranasal immunization with fragment #5 of ApxIIA can be considered an efficient protective immunization procedure against A. pleuropneumoniae infection., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Aeromonas hydrophila is an important fish pathogen responsible for huge economic losses in aquaculture sector. The bacterial outer membrane proteins (OMPs), especially adhesins play a key role in the virulence of the bacteria and are considered potential vaccine candidates. We evaluated the immunogenicity of two important outer membrane proteins namely Aha1 and OmpW of A. hydrophila. These proteins were over-expressed in Escherichia coli, purified and used for the vaccination of common carp. Sequence analysis predicted that, Aha1 and OmpW are adhesins and antigenic. Common carp immunized with recombinant Aha1 and OmpW proteins showed significant antibody production and a relative percentage survival of 52 and 71 respectively indicating their protective efficacy against A. hydrophila infection., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The selection of an optimal adjuvant to enhance the potency and longevity of antigen specific immune responses has always been imperative for the development of more effective and safer vaccines. A balanced type of immune enhancing ability of a new adjuvant known as polyphosphazene (PCEP) has been demonstrated in mice. In the present study we have compared the humoral and cellular immune responses to vaccine formulations containing Actinobacillus pleuropneumoniae outer membrane antigen (OmlA) with PCEP or Emulsigen as adjuvants. Our data showed a significant improvement and a shift toward more balanced immune responses when OmlA antigen was formulated with PCEP and CpG ODN. Moreover, in contrast to Emulsigen, immunization with PCEP did not result in local injection site reactions highlighting its potential as a safe and effective adjuvant for pigs. Further, the ease of formulation, administration and relatively low per dose cost of PCEP make it a promising adjuvant for pigs., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The efficacy of a novel, live delivery vaccine was examined for protection against post-weaning diarrhea in pigs. An expression/secretion plasmid harboring genes encoding enterotoxigenic Escherichia coli K88ab, K88ac, FedA and FedF fimbriae was constructed and harbored in an attenuated Salmonella, which was used as the vaccine candidate. Groups A (n=3) and B (n=3) sows were orally immunized with the candidate vaccine and PBS as a control, respectively, at 8 and 11 weeks of pregnancy. All group piglets were challenged with two challenge strains at 5-week-old. All immunized sows had significantly increased IgG and IgA levels in both serum and colostrum to individual adhesins compared to the control (p ≤ 0.05). Immune response in Group A piglets were significantly increased (p ≤ 0.05). Furthermore, no clinical signs were observed in Group A piglets after the challenge and no challenge strains were detected in rectal swabs, while diarrhea was observed in 47.8% control piglets and challenge strains were isolated from all the diarrheic piglets. These results show that immune response of sucking piglets can maintain at higher levels through the milk of the immunized sows and vaccination of sows with the candidate may protect colibacillosis in weaned piglets., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Pneumonia caused by Rhodococcus equi remains a significant problem in foals. The objective of this study was to develop a safe and efficacious attenuated strain of R. equi for eventual use in oral immunization of foals. The approach involved expression of vapA in a live, virulence plasmid-negative, strain of R. equi (strain 103-). PCR-amplified fragments of the vapA gene, with and without the upstream genes virR, orf5, vapH, orf7 and orf8 (orf4-8), were cloned into a shuttle vector pNBV1. These plasmids, named pAW48A and pAWVapA respectively, were electroporated into strain 103-. The presence of the recombinant vectors in the attenuated strain (103-) and the integrity of the inserted genes were confirmed, and both constructs expressed VapA. The virulence of the two strains was compared to that of wild type R. equi 103+ and negative controls by their intravenous inoculation into mice, followed by examination of liver clearance 4 days later. Mice inoculated with R. equi 103-, 103-/pAWVapA and 103-/pNBV1 completely cleared infection, whereas strain 103-/pAW48A persisted in 47% of mice., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Mycobacterium avium subsp. paratuberculosis (Map), the etiological agent of chronic enteritis of the small intestine in domestic and wild ruminants, causes substantial losses to livestock industry. Control of this disease is seriously hampered by the lack of adequate diagnostic tools and vaccines. Here we report on the immunogenicity of eight Map specific antigens, i.e. MAP1693c, Ag3, MAP2677c (identified by post-genomic and immunoproteomic analysis of Map secretome) and Ag5, Ag6, MAP1637c, MAP0388 and MAP3743 (identified by bioinformatic in silico screening of the Map genome). Strong, antigen-specific IFN-γ responses were induced in mice vaccinated with plasmid DNA encoding MAP1693c, MAP1637c, MAP0388 and MAP3743. In contrast, T cell responses in Map infected mice were directed preferentially against Ag5 and to a lesser extent against MAP3743. None of the tested DNA vaccines conferred protection against subsequent challenge with Map., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Lyme disease is a zoonotic, vector-borne disease affecting humans, dogs, horses and other species. It is caused by infection with spirochetes of the Borrelia burgdorferi sensu lato group which are transmitted to the mammalian host by infected ticks (Ixodes). Exposure to B. burgdorferi is commonly diagnosed by serological testing. The gold standard for the detection of antibodies to B. burgdorferi is a two-step procedure of an ELISA followed by confirmatory Western blotting (WB). Here, we developed and validated a new bead-based multiplex assay for the detection of antibodies to B. burgdorferi in canine serum which combined the testing by ELISA and WB in a single quantitative test. B. burgdorferi outer surface protein A (OspA), OspC and OspF were expressed in E. coli. The recombinant proteins were coupled to fluorescent beads providing the matrix of the assay. Two sets of canine sera were used for validation of the multiplex assay. First, sera from 79 dogs with known ELISA and WB results were used to establish the conditions of the assay. These samples were selected to provide similar numbers of pre-tested sera ranging from negative to high positive results and included sera from vaccinated and/or naturally infected dogs. A high correlation was observed for detection of antibodies to B. burgdorferi in the single and multiplex assays (n=79). Spearman's rank correlations were 0.93, 0.88 and 0.96 for OspA, OspC and OspF, respectively. Second, a total of 188 canine serum samples that were not tested previously were used for further multiplex assay validation. All samples were also blindly analyzed for antibodies to B. burgdorferi antigens by WB. The WB results provided a 'relative gold standard' for each antigen and were used to perform a receiver operating curve analysis. The areas under the curves were 0.93 for OspA, 0.82 for OspC, and 0.89 for OspF. Multiplex assay interpretation ranges for antibodies to all three B. burgdorferi antigens in canine serum were established by likelihood analysis. The diagnostic sensitivities of the individual OspA, OspC and OspF bead-based assays were 83%, 62% and 82%, respectively, and the diagnostic specificities were 90%, 89% and 86%, respectively. The new multiplex assay provides a sensitive and fully quantitative platform for the simultaneous evaluation of antibodies to B. burgdorferi OspA, OspC and OspF antigens and distinguishes between antibodies that originated from vaccination or natural exposure to B. burgdorferi., (Copyright © 2011 Elsevier B.V. All rights reserved.)

In Europe, Streptococcus suis serotypes 2 and 9 are very important causative agents of meningitis, arthritis and septicemia in piglets. So far, no vaccine has been described which elicits protection against both serotypes. The working hypothesis of this study was that an isogenic serotype 2 mutant attenuated in virulence but not in colonization of the respiratory tract might induce protective immunity against both serotypes. Piglets were immunized with the attenuated S. suis serotype 2 strain 10Deltaofs2/12, which is deficient in expression of the serum opacity factor OFS. Three weeks after intranasal application of 10Deltaofs2/12 piglets were challenged intravenously with two strains representing the most important S. suis pathotypes in Europe, the homologous MRP+ EF+ SLY+ serotype 2 strain and a heterologous MRP* SLY+ serotype 9 strain. Application of the live vaccine elicited significant serum IgG responses against muramidase-released protein (MRP), extracellular factor (EF) and, most prominently, suilysin. Seroconversion against suilysin was accompanied with an increase of suilysin-neutralization titers in the vaccinated group. Though mortality was lower in the vaccinated groups, significant protection was not observed, neither against the homologous nor against the heterologous challenge. This was in agreement with the finding that the vaccinated piglets had low opsonizing antibody titers insufficient to mediate elimination of the two challenge strains by porcine neutrophils. In conclusion, a single intranasal application of the S. suis serotype 2 strain 10Deltaofs2/12 elicited humoral immune responses against different S. suis antigens but failed to induce sufficient opsonizing antibody titers and protective immunity against systemic serotypes 2 and 9 challenges.

This study was done to determine if intranasal vaccination of weaned beef calves with a chimeric protein containing the immunodominant surface epitope of Mannheimia haemolytica PlpE (R2) and the neutralizing epitope of leukotoxin (NLKT) covalently linked to truncated cholera toxin (CT) subunit B (CTB) could stimulate secretory and systemic antibodies against M. haemolytica while enhancing resistance of cattle against M. haemolytica intrabronchial challenge. Sixteen weaned beef calves were intranasally vaccinated with CTB-R2-NLKT chimeric (SAC102) or with R2-NLKT-R2-NLKT chimeric (SAC89) protein with or without native CT on days 0 and 14 and were challenged intrabronchially on day 28. In vitro, SAC102 bound the CT receptor molecule, GM(1)-ganglioside. Mean IgA antibodies to M. haemolytica whole cells (WC) and to LKT were high on day 0. A small, yet significant increase (p<0.05) was found in mean nasal antibodies to M. haemolytica WC for the SAC89+CT and SAC102 vaccinates after the second vaccination. SAC102 stimulated significant (p<0.05) mean serum antibody responses to all three antigens by day 28. Following challenge, mean antibodies to WC and LKT significantly increased (p<0.05) for the SAC102, SAC89 and SAC89+CT groups with the mean antibody responses to rPlpE stimulated by SAC102 vaccination being significantly higher (p<0.05) than for the other vaccinated and control groups. On day 1 after challenge, mean clinical score for the control group was significantly higher (p<0.05) than for the SAC102 and SAC89+CT vaccinates, and by day 2 after challenge, clinical score for the control group was significantly higher (p<0.05) than for all three chimeric vaccinated groups. Therefore, intranasal vaccination with CTB-R2-NLKT (SAC102) and R2-NLKT-R2-NLKT (SAC89) chimeric proteins enhanced resistance against intrabronchial challenge with the bacterium as well as stimulating antibody responses to M. haemolytica antigens.

Selection of potent cytokine adjuvants is important for the development of Staphylococcus aureus DNA vaccines. Several potential cytokines have been proven to induce enhanced immune responses in animal models and clinical tests. There is still no reported use of IL18 as an adjuvant to design DNA vaccines against S. aureus. In this study, we cloned the main fibronectin binding protein gene (a fragment from clumping factor A, ClfA(221-550)) of S. aureus and bovine interleukin 18 (bIL18). Then recombinant plasmids were constructed based on the eukaryotic expression vector pVAX1 with or without bIL18. Indirect immunofluorescence assays in transfected HeLa cells indicated that the recombinant DNAs (rDNAs) could be expressed correctly and had antigenicity. BALB/c mice were used as experimental models to examine the immunogenicity of rDNAs in vivo. The ClfA(221-550) rDNA provoked antibody production. The bIL18 rDNA induced production of the Th1 type cytokines IL2 and IFNgamma, and ClfA(221-550) and bIL18 synergistically stimulated T-lymphocyte proliferation. The data demonstrated that bIL18 is a potent adjuvant that could be used to enhance cellular immunity.

The gene fragment coding for bovine C3d gene (boC3d) was cloned and expressed as a component of fusion proteins destined for use in vaccine studies in cattle, and for in vitro experiments. This fragment of complement protein C3 (C3d) has been shown to enhance B cell responses when complexed with antigen. Three potential vaccine constructs were engineered to contain one, two or three boC3d units linked to a fragment of the leukotoxin of Mannheimia haemolytica A1, an economically important pathogen of cattle that causes a fibrinous pneumonia in calves. A recombinant biotinylated boC3d protein (for use in in vitro studies) was generated by endogenous biotinylation in Escherichia coli by means of the BirA holoenzyme synthetase. All recombinant proteins incorporated polyhistidine tags and were purified by nickel-agarose chromatography, then analyzed by SDS-PAGE and Western immunoblot. The identity of boC3d was confirmed by mass spectrometry, since monoclonal antibodies to boC3d were not available. To date, published research into the adjuvant activities of C3d has been limited to experiments in mice and rabbits, using antigens unrelated to diseases occurring naturally in these species. The boC3d fusion proteins expressed in this study will provide the basis for immunization trials in cattle and studies of receptor binding and cell activation of bovine lymphocytes.

There is a need to produce a vaccine against Rhodococcus equi pneumonia in foals in which immunity against infection is largely based on a type 1, cell-mediated, immune response. The VapA protein of the virulence plasmid of R. equi is highly immunogenic. To assess the potential of vapA-DNA to produce immunity, C57BL/6 and BALB/c mice were immunized with a DNA vaccine constructed from vapA incorporated into pcDNA3.1. The plasmid construct expressed VapA in a COS-7 cell line. Intramuscular immunization of mice resulted in enhanced clearance of R. equi from the liver of intravenously challenged mice compared to non-immunized controls. This effect was more marked when pORF-IL-12, a plasmid expressing murine IL12, was included with the vaccine. Antibody developed to VapA, with an IgG2a response being more marked in mice immunized with pcDNA-vapA than in non-immunized or in mice immunized with the mixed vapA and IL-12 plasmid constructs. In conclusion, this study has shown for the first time that DNA immunization with vapA enhances the immune responses of mice against R. equi infection, that the IgG subisotype response is consistent with a type 1-based immune response, and that this can be enhanced by injection of the IL-12 gene.

Rhodococcus equi is a significant intracellular bacterial pathogen in foals. However, at present there is no commercially available vaccine for the prevention of R. equi-induced disease in these animals. Studies have shown that GroEL based vaccines can afford protection against some intracellular pathogens. In this study, the R. equi gene encoding the heat shock protein GroEL2 was cloned and sequenced, with a view to using it as a vaccine candidate. The promoter region of the gene contained two copies of controlling inverted repeat of chaperone expression (CIRCE) motifs, which are well-recognised transcriptional regulators of bacterial heat shock proteins. The R. equi GroEL2 was expressed in E. coli BL21 DE3 with a C-terminal His-tag and sequenced to confirm its identity. The R. equi purified His-tagged GroEL2 protein and a groEL2-based DNA vaccine were used in separate experiments to immunise BALB/c mice. The recombinant protein-based vaccine elicited a mixed Th1/Th2 response whereas the DNA vaccine was found to elicit a predominantly Th1 biased immune response. However, when vaccinated mice were challenged intravenously with 1.5 x 10(7) R. equi neither vaccine elicited enhanced bacterial clearance from the spleen or liver in this model. The reasons for this apparent lack of success are discussed.

The capacity of a quimeric protein, formed by the genetic fusion of five antigenic determinants from four Leishmania proteins, formulated with BCG, to protect dogs against Leishmania infantum infection is described. The data showed that after i.v. administration of 500,000 parasites of the L. infantum M/CAN/ES/96/BCN150 strain, zymodeme MON-1, the animals became infected as suggested by the humoral response against the parasite antigens. All control unvaccinated dogs had parasites in the lymph nodes at day 150 post-infection. One of these unvaccinated infected dog was parasite negative at day 634 behaving, thus, as resistant. In contrast, only 50% of the immunized dogs had parasites in the lymph nodes at day 150 post-infection. Four of these dogs became parasite negative by day 634 post-infection. The control animals developed at various times during the follow-up period clinical symptoms associated with Leishmaniasis. The control diseased dogs developed also in the liver and spleen some of the abnormal histological features associated with natural visceral Leishmaniasis. The immunized dogs, however, were not only normal at the clinical but also at the anatomo-pathological level. A positive delayed type hypersensitivity (DTH) response was observed in nine of the immunized protected dogs. The data indicated that Q+BCG confers 90% protection against infection and at least 90% protection at the clinical level.