Multistrain genome analysis identifies candidate vaccine antigens of Anaplasma marginale

Anaplasmosis in domestic livestock is an impediment to animal health and production worldwide, especially in developing countries in Africa, Asia, and South America. Vaccines have been developed and marketed against the causative organism, Anaplasma marginale; however, these have not been widely used because of breakthrough infections caused by heterologous strains and because of the risk of disease induced by live vaccine strains themselves. Recently, molecular studies have enabled progress to be made in understanding the causes for breakthrough infections and in defining new vaccine targets. A. marginale has a system for antigenic variation of the MSP2 and MSP3 outer membrane proteins which are members of the pfam01617 gene superfamily. In this study, we used high throughput genome sequencing to define conservation of different superfamily members in ten U.S. strains of A. marginale and also in the related live vaccine strain A. marginale subspecies centrale. The comparisons included the pseudogenes that contribute to antigenic variation and other superfamily-encoded outer membrane proteins. Additionally, we examined conservation of other proteins proposed previously as vaccine candidates. These data showed significantly increased numbers of SNPs in A. marginale subspecies centrale when compared to all U.S. A. marginale strains. We defined a catalog of 19 conserved candidate vaccine antigens that may be suitable for development of a multi-component recombinant vaccine. The methods described are rapid and may be suitable for other prokaryotes where repeats comprise a substantial portion of their genomes.     

First molecular survey and novel genetic variants’ identification of Anaplasma marginale,A. centrale and A. bovis in cattle from Tunisia

Few data are available about the presence and distribution of Anaplasma species in cattle in North African countries. In this study prevalence, co-infections, risk factors and genetic diversity of Anaplasma species were evaluated in bovines from Northern Tunisia. A total of 232 cattle from 36 randomly selected farms in three Tunisian localities were investigated for the presence of Anaplasma species in blood by Real-time PCR and/or nested PCR. Overall infection rates of Anaplasma spp., Anaplasma marginale, Anaplasma centrale and Anaplasma bovis were 34.9%, 25.4%, 15.1%, and 3.9%, respectively. Anaplasma phagocytophilum was not detected in cattle. The most common co-infection pattern was an association of A. marginale and A. centrale (11.2%). Five cattle (2.1%) all reared in the sub-humid bioclimatic area, were co-infected by the three Anaplasma species. Molecular prevalence of Anaplasma infection varied significantly according to locality, bioclimatic area, tick infestation and type of breeding. Animals of the Holstein breed were less infected by A. marginale and A. centrale than other breeds. Genetic analysis of A. marginale msp4 gene indicated a high sequence diversity of Tunisian strains, suggesting a multiple introduction of infected cattle from different origins. Phylogenetic studies based on the 16S rRNA gene showed that the most prevalent A. centrale strains were closely related to the A. centrale vaccine strain. Moreover, all A. bovis variants clustered with other A. bovis sequences obtained from domestic and wild ruminant strains. This is the first molecular investigation on Anaplasma species in Tunisian cattle providing pivotal background for designing epidemiological studies and to develop control strategies in the country.     

Control of Salmonella Enteritidis and Salmonella Gallinarum in birds by using live vaccine candidate containing attenuated Salmonella Gallinarum mutant strain

The ideal live vaccine to control Salmonella in commercial chicken flocks should engender protection against various strains. The purpose of the present study was to confirm the attenuation of a Salmonella Gallinarum (SG) mutant strain with deletion on genes cobS and cbiA, that are involved in the biosynthesis of cobalamin. Furthermore, evaluate its use as a live vaccine against Salmonella. For the evaluation of the vaccine efficacy, two experiments were conducted separately. Birds from a commercial brown line of chickens were used to perform challenge with SG wild type strain and birds from a commercial white line of chickens were used to perform challenge with Salmonella Enteritidis (SE) wild type strain. In both experiments, the birds were separated in three groups (A, B and C). Birds were orally vaccinated with the SG mutant as the following programme: group A, one dose at 5 days of age; group B, one dose at 5 days of age and a second dose at 25 days of age; and group C, birds were kept unvaccinated as controls. At 45 days of age, birds from all groups, including the control, were challenged orally by SG wild type (brown line) or SE wild type (white line). Lastly, another experiment was performed to evaluate the use of the SG mutant strain to prevent caecal colonization by SE wild type on 1-day-old broiler chicks. Mortality and systemic infection by SG wild type strain were assessed in brown chickens; faecal shedding and systemic infection by SE wild type were assessed in white chickens and caecal colonization was assessed in broiler chicks. Either vaccination with one or two doses of SG mutant, were capable to protect brown chickens against SG wild type. In the experiment with white chickens, only vaccination with two doses of SG mutant protected the birds against challenge with SE wild type. Although, SG mutant could not prevent caecal colonization in 1-day-old broiler chicks by the challenge strain SE wild type. Overall, the results indicated that SG mutant is a promising Salmonella live vaccine candidate that demonstrated good efficacy to control the infection by two serotypes of major importance to the poultry industry.     

The immunization-induced antibody response to the Anaplasma marginale major surface protein 2 and its association with protective immunity

Many vector-borne pathogens evade clearance via rapid variation in their immunogenic surface expressed proteins. This is exemplified by Anaplasma marginale, a tick-borne bacterial pathogen that generates major surface protein 2 (Msp2) variants to provide for immune escape and allow long-term pathogen persistence. In contrast to persistence following infection, immunization with a surface protein complex, which includes Msp2, induces a response that prevents infection upon challenge. We hypothesized that the immune response induced by immunization altered the anti-Msp2 antibody repertoire as compared to that induced during infection, shifting the immune response toward conserved and thus broadly protective epitopes. The antibody response to the conserved (CR) and hypervariable (HVR) regions encoded by the full set of msp2 variant alleles was determined for immunized animals prior to challenge and non-immunized, infected animals. While both groups of animals had a similar antibody repertoire in terms of breath and magnitude, the titers to the Msp2 CR were strongly correlated (p < 0.005) with control of bacteremia only in the infected animals. Among the immunized animals, there was no correlation between the breadth or magnitude of the anti-Msp2 antibody response and either complete protection from infection or control of bacteremia. This is consistent with separate immunologic mechanisms being responsible for control of bacteremia in infected animals as compared to immunized animals and suggests that conserved outer membrane proteins other than Msp2 are responsible for the complete clearance observed following challenge of vaccinees.     

Preclinical in vitro and in vivo profile of a highly-attenuated,broadly efficacious pneumolysin genetic toxoid

Pneumolysin is a highly conserved, cholesterol-dependent cytolysin that is an important Streptococcus pneumoniae virulence factor and an attractive target for vaccine development. To attenuate pneumolysin toxicity, a genetic toxoid was constructed with two amino acid changes, G293S and L460D, termed PLY-D, that reduced cytolytic activity > 125,000-fold. In mice, PLY-D elicited high anti-PLY IgG antibody titers that neutralized the cytolytic activity of the wild-type toxin in vitro. To evaluate the protective efficacy of PLY-D, mice were immunized intramuscularly and then challenged intranasally with a lethal dose of 28 clinical isolates of S. pneumoniae originating from different geographical locations, disease states (i.e. bacteremia, pneumonia), or body sites (i.e. sputum, blood). PLY-D immunization conferred significant protection from challenge with 17 of 20 serotypes (85%) and 22 of 28 strains (79%). Further, we demonstrated that immunization with PLY-D provided statistically significant improvement in survival against challenge with serotype 4 and 18C strains compared to mice immunized with a pneumococcal conjugate vaccine Prevnar 13® (PCV13). Co-administration of PLY–D and PCV13 conferred greater protection against challenge with a serotype 6B strain than immunization with either vaccine alone. These data indicate that PLY-D is a broadly protective antigen with the potential to serve as a serotype-independent vaccine against invasive pneumococcal disease either alone or in combination with PCVs.     

Anaplasma marginale outer membrane protein vaccine candidates are conserved in North American and South African strains

Bovine anaplasmosis is a globally economically important tick-borne disease caused by the obligate intraerythrocytic rickettsia, Anaplasma marginale. A live Anaplasma centrale blood-based vaccine is available, but it does not protect against all A. marginale field strains and may also transmit other blood-borne pathogens. Five potential outer membrane protein (OMP) vaccine candidates have been well-characterised in A. marginale strains from the USA, however, their levels of conservation in other countries must be ascertained in order to inform their use in a vaccine with regional or global efficacy. This study assessed the amino acid variation in vaccine candidate OMPs in South African strains of A. marginale, and also compared the immunogenic properties between South African and US strains. OMP genes Am779, Am854, omp7, omp8 and omp9 were amplified and sequenced from a set of genetically diverse South African samples with different msp1α-genotypes. OMPs Am854 and Am779 were highly conserved, with 99–100 % amino acid identity, while Omp7, Omp8 and Omp9 had 79–100 % identity with US strains. As has been shown previously, Omp7–9 possess conserved N- and C- termini, a central variable region, and a highly conserved CD4 T-cell epitope, FLLVDDA(I/V)V, in the N-terminal region. Western blot analysis of recombinant OMPs indicates strong antigenic conservation between South African and US strains of A. marginale, suggesting that they are good candidates for use in a novel global vaccine cocktail, although further work on the best formulation and delivery methods will be necessary.     

Comparative evaluation of ELPylated virus-like particle vaccine with two commercial PCV2 vaccines by experimental challenge

Porcine circovirus type 2 (PCV2) is an economically important swine pathogen and vaccination is the primary tool for the disease control. Previously, we developed a more cost-effective PCV2 virus-like particle (VLP) vaccine by using ELPylation technology. In the present study, we compared the ELPylated VLP (ELP-VLP) PCV2 vaccine efficacy with commercial inactivated Yuanlijia vaccine and VLP-based Circoflex vaccine by experimental challenge. After one dose of vaccination with the three different vaccines, ELP-VLP vaccine group showed significantly (p < 0.05) stronger virus neutralizing antibody and interferon-γ responses than the two commercial vaccine groups. All vaccinated pigs showed significant (p < 0.05) improvement in average daily weight gain (ADWG) before challenge. After challenge with PCV2, however, only ELP-VLP-vaccinated pigs showed significant (p < 0.05) improvement in ADWG. All vaccinated pigs showed significant (p < 0.05) reductions in PCV2 loads in the blood, nasal secretion and lymph nodes, ELP-VLP-vaccinated pigs in particular. In addition, vaccination with ELP-VLP vaccine provided stronger protection against pulmonary and lymphoid pathologies than that with the two commercial vaccines. Therefore, ELP-VLP vaccine is more effective to control PCV2 infection than the two commercial vaccines based on clinical, immunological, virological and pathological evaluations.     

A commercial vaccine based on PCV2a and an experimental vaccine based on a variant mPCV2b are both effective in protecting pigs against challenge with a 2013 U.S. variant mPCV2b strain

During 2012 and 2013, an apparent increase in porcine circovirus associated disease occurred in the USA. A variant PCV2b strain designated mPCV2b was recovered from many of these cases. This raised concerns of a decrease in efficacy of commercially available PCV2 vaccines. The objective of this study was to compare the ability of a commercial PCV2a-based vaccine and an experimental mPCV2b-based vaccine to control mPCV2b-associated disease, lesions, and viremia in a challenge model. Twenty-six caesarian-derived, colostrum-deprived pigs were randomly assigned to one of four groups: (1) vaccinated with a commercial PCV2a-based vaccine and challenged (PCV2a-VAC; n = 7), (2) vaccinated with an experimental mPCV2b-based vaccine and challenged (mPCV2b; n = 7), (3) sham-vaccinated with saline and challenged (positive controls; n = 7), and (4) sham-vaccinated with saline without challenge (negative controls; n = 5). Vaccination was done on D0 and D14, challenge was done on D28 using a tissue homogenate containing PRRSV and mPCV2b and the experiment was terminated on D49. Among the challenged pigs, 47.6% (10/21) developed severe clinical disease and either died or had to be humanely euthanized between D39 and D48 (11–20 days after challenge). PCV2 viremia was almost completely absent in the vaccinated groups regardless of vaccine type except for two PCV2a-vaccinated pigs which had detectable PCV2 DNA levels on individual days after challenge. Microscopic lesions typical of PCV2 infection were limited to the positive control group which developed mild-to-severe lesions associated with low-to-abundant PCV2 antigen. Under the conditions of this study, PCV2 vaccines regardless of PCV2 type were effective against mPCV2b challenge.     

Cellular and humoral immunity are synergistic in protection against types A and B Francisella tularensis

Herein we report studies with a novel combination vaccine that, when administered to mice, conferred protection against highly virulent strains of Francisella tularensis by stimulating both arms of the immune system. Our earlier studies with Ft.LVS::wbtA, an O-polysaccharide (OPS)-negative mutant derived from the available live vaccine strain of F. tularensis (Ft.LVS), elucidated the role of antibodies to the OPS – a key virulence determinant – in protection against virulent type A organisms. However, when expressed on the organism, the OPS enhances virulence. In contrast, in purified form, the OPS is completely benign. We hypothesized that a novel combination vaccine containing both a component that induces humoral immunity and a component that induces cellular immunity to this intracellular microbe would have an enhanced protective capacity over either component alone and would be much safer than the LVS vaccine. Thus we developed a combination vaccine containing both OPS (supplied in an OPS–tetanus toxoid glycoconjugate) to induce a humoral antibody response and strain Ft.LVS::wbtA (which is markedly attenuated by its lack of OPS) to induce a cell-mediated protective response. This vaccine protected mice against otherwise-lethal intranasal and intradermal challenge with wild-type F. tularensis strains Schu S4 (type A) and FSC 108 (type B). These results represent a significant advance in our understanding of immunity to F. tularensis and provide important insight into the development of a safer vaccine effective against infections caused by clinical type A and B strains of F. tularensis.     

A novel subunit vaccine co-expressing GM-CSF and PCV2b Cap protein enhances protective immunity against porcine circovirus type 2 in piglets

Porcine circovirus type 2 (PCV2) causes porcine circovirus-associated disease. Capsid (Cap) protein of PCV2 is the principal immunogenic protein that induces neutralizing antibodies and protective immunity. GM-CSF is an immune adjuvant that enhances responses to vaccines. In this study, recombinant baculoviruses Ac-Cap and Ac-Cap-GM-CSF expressing the Cap protein alone and co-expressing the Cap protein and porcine GM-CSF, respectively, were constructed successfully. The target proteins were analyzed by western blotting and IFA. Further, these proteins were confirmed by electron microscopy, which showed that Cap proteins could self-assemble into virus-like particles having diameters of 17–25 nm. Animal experiments showed that pigs immunized with Cap-GM-CSF subunit vaccine showed significantly higher levels of PCV2-specific antibodies and neutralizing antibodies than pigs immunized with the Cap subunit vaccine and a commercial vaccine (Ingelvac CircoFLEX; P < 0.05). After PCV2 wild strain challenged, Pigs receiving the Cap-GM-CSF subunit vaccine showed significantly higher average daily weight gain after wild-type PCV2 challenge than pigs receiving the other three vaccines (P < 0.05). None of PCV2 DNA was detected in all immunized animals, except control animals immunized with phosphate-buffered saline. These results indicated that GM-CSF was a powerful immunoadjuvant for PCV2 subunit vaccines because it enhanced humoral immune response and improved immune protection against PCV2 infection in pigs. Thus, the novel Cap-GM-CSF subunit vaccine has the potential to be used as an effective and safe vaccine candidate against PCV2 infection.     

Evaluation of an attenuated strain of Ehrlichia canis as a vaccine for canine monocytic ehrlichiosis

Canine monocytic ehrlichiosis is an important tick-borne disease worldwide. No commercial vaccine for the disease is currently available and tick control is the main preventive measure against the disease. The aim of this study was to evaluate the potential of a multi-passaged attenuated strain of Ehrlichia canis to serve as a vaccine for canine monocytic ehrlichiosis, and to assess the use of azithromycin in the treatment of acute ehrlichiosis. Twelve beagle dogs were divided into 3 groups of 4 dogs. Groups 1 and 2 were inoculated (vaccinated) with an attenuated strain of E. canis (#611A) twice or once, respectively. The third group consisted of naïve dogs which served as controls. All 3 groups were challenged with a wild virulent strain of E. canis by administering infected dog-blood intravenously. Transient thrombocytopenia was the only hematological abnormality observed following inoculation of dogs with the attenuated strain. Challenge with the virulent strain resulted in severe disease in all 4 control dogs while only 3 of 8 vaccinated dogs presented mild transient fever. Furthermore, the mean blood rickettsial load was significantly higher in the control group (27–92-folds higher during days 14–19 post challenge with the wild the strain) as compared to the vaccinated dogs. The use of azithromycin was assessed as a therapeutic agent for the acute disease. Four days treatment resulted in further deterioration of the clinical condition of the dogs. Molecular comparison of 4 genes known to express immunoreactive proteins and virulence factors (p30, gp19, VirB4 and VirB9) between the attenuated strain and the challenge wild strain revealed no genetic differences between the strains. The results of this study indicate that the attenuated E. canis strain may serve as an effective and secure future vaccine for canine ehrlichiosis.     

Development and field evaluation of a nested polymerase chain reaction-restriction fragment length polymorphism (nPCR-RFLP) analysis to identify A. marginale-infected and A. centrale-vaccinated cattle

A nested polymerase chain reaction-restriction fragment length polymorphism (nPCR-RFLP) assay based on the amplification of the Anaplasma spp. highly conserved msp5 gene and posterior digestion with HindIII endonuclease was developed and evaluated in field samples. Results were compared using an nPCR specific for Anaplasma marginale (nPCR-Am) based on the msp1β gene and an nPCR specific for A. centrale (nPCR-Ac) based on the msp2 operon (msp2-o) gene. Amplicons dilutions of msp1β and msp5 of A. marginale and msp2-o and msp5 of A. centrale and dilutions of parasited erythrocytes (PE) with A. marginale and A. centrale were used to determine the detection limits. The results were 20 DNA copies/reaction and 30 PE for A. marginale and A. centrale by nPCR-RLFP and nPCR-Am/Ac. A mix of msp5-Am and msp5-Ac was used to evaluate the interference of msp5 from one species for the detection of the other. Co-amplification of the DNA from both species was observed up to a 1:7 ratio of one species to the other. Field samples positive for Anaplasma spp. antibodies (n = 260) from 32 herds were evaluated. Strength of agreement between results by nPCR-RFLP and nPCR-Am or nPCR-Ac was 78% (κ = 0.44) and 94% (κ = 0.85), respectively. Thirty-four samples were positive for A. marginale by nPCR-RFLP but negative by nPCR-Am. msp1β amplicons of 10 samples from 5 herds with discrepancies between nPCR-Am and nPCR-RFLP results were cloned and sequenced. The analysis of the msp1β sequence showed several mutations in the target region of the internal forward primer that would explain the failure in the amplification. Only 10 of the 20 coinfections identified by nPCR-Ac/nPCR-Am were detected by nPCR-RFLP. nPCR-RFLP is a sensitive, low-cost and accessible molecular method for low-complexity laboratories. More studies are needed to establish in which circumstances coinfections can be underestimated.     

Yersinia enterocolitica ghost with msbB mutation provides protection and reduces proinflammatory cytokines in mice

Yersinia enterocolitica is an important human pathogen. Yersiniosis, caused by Y. enterocolitica, has become more prevalent globally in recent years. Prevention of yersiniosis still remains a challenge, and an efficacious and safe vaccine that confers protection against this enteric pathogen needs to be developed. In this study, a novel vaccine based on the bacterial ghost, in combination with mutation of the Y. enterocolitica msbB gene, was developed and the immunopotency of this vaccine was evaluated in mice. Significant levels of IgG1/IgG2a antibodies and IL-4/IFN-γ cytokines were detected after mice were administered this vaccine intragastrically, indicating that a Th1/Th2-mediated mixed immune response was stimulated. Importantly, mutation of the msbB gene efficiently reduced secretion of the proinflammatory cytokines IL-1β, IL-6 and TNF-α, suggesting a reduction in inflammatory reaction caused by lipopolysaccharide. In addition, when challenged with a dose that was 100-fold the minimal lethal dose of the virulent wild strain of Y. enterocolitica, this mutated ghost vaccine was capable of eliciting the same effective protection (80%) in comparison with the non-mutated ghost strain, and the survival time was extended by at least two days. Together, our results demonstrated that this novel ghost bacterial strain could be used as a safe and effective vaccine against Y. enterocolitica.     

Clonal and clinical profile of Streptococcus pneumoniae serotype 19A causing pediatric invasive infections: a 2-year (2007-2009) laboratory-based surveillance in Madrid

Studies of clonality and clinical profile of serotype 19A invasive pneumococcal disease in children (IPD-19A) are worthy after PCV7 introduction. A prospective, hospital-based surveillance of IPD-19A, culture and/or PCR confirmed, was performed in 2007-2009 in Madrid (all 22 hospitals with pediatric departments). Sixty-two cases were found: 90.3% in children <5 years, 87.1% in <36 months, and 74.2% in ≤18 months. Clinical presentations: meningitis (22.6%), primary bacteremia (19.4%), secondary bacteremia to otic foci (SBOF; 17.7%), bacteremic pneumonia (17.7%), pediatric parapneumonic empyema (PPE; 17.7%) and others (4.8%). Presentations by age: meningitis (35.7%), SBOF (28.6%) and primary bacteremia (21.4%) in children <12 months, bacteremic pneumonia, PPE and primary bacteremia (26.3% each) in 12-23 months, and bacteremic pneumonia (33.3%) and PPE (26.6%) in ≥24 months. Sequence types ST276 and ST320 represented 83.0% isolates, all oral-penicillin/erythromycin non-susceptible. In nonmeningeal isolates, non-susceptibility to parenteral penicillin/cefotaxime was 0%/17.6% (ST276) and 93.8%/75.0% (ST320). Non-susceptibility in ST276 and ST320, prevalence of these STs among 19A isolates, and serotype 19A prevalence among IPDs, indicate the importance of 19A inclusion in PCV13 for IPD-19A prevention and for reducing 19A nasopharyngeal carriage, thus preventing 19A otitis (one-third of 19A bacteremia in this study were from otic origin).     

Efficacy of a new bivalent vaccine of porcine circovirus type 2 and Mycoplasma hyopneumoniae (Fostera™ PCV MH) under experimental conditions

The objective of this study was to evaluate the efficacy of a new bivalent vaccine (Fostera™ PCV MH, Zoetis) of porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae in growing pigs under experimental conditions. A total of 80 pigs were randomly divided into 8 groups (10 pigs per group). The pigs were administered the bivalent vaccine intramuscularly as a 2.0 mL dose at 21 days of age based on the manufacturer's instructions. Three weeks after vaccination, the pigs were inoculated with either PCV2 (intranasal route) or M. hyopneumoniae (intratracheal route) or both. Regardless of the type of inoculation, vaccinated pigs after challenge exhibited effective reduction of clinical signs, PCV2 viremia levels and mycoplasma nasal shedding, and lung and lymphoid lesion when compared to unvaccinated challenged pigs. Vaccinated challenged pigs had significantly higher (P < 0.05) levels of PCV2-specific neutralizing antibodies, and numbers of PCV2-and M. hyopneumoniae-specific interferon-γ secreting cells compared to unvaccinated challenged pigs. This study demonstrates that the bivalent vaccine is able to protect pigs against either PCV2 or M. hyopneumoniae infection or both based on clinical, microbiological, immunological, and pathological evaluation.     

AasP autotransporter protein of Actinobacillus pleuropneumoniae does not protect pigs against homologous challenge

Actinobacillus pleuropneumoniae is a major respiratory pathogen of pigs; current vaccines provide only limited protection. AasP, a subtilisin-like serine protease, is a conserved outer membrane-localised autotransporter protein. We hypothesized that AasP would induce protective immunity and may thus constitute a useful component of a vaccine against A. pleuropneumoniae infection. Here we confirm experimentally that AasP is an antigenic in vivo-expressed protein. In pig protection studies, a detectable specific antibody response was induced in response to recombinant AasP. However, the vaccine was not capable of protecting pigs from colonization, infection or severe clinical disease resulting from challenge with the homologous A. pleuropneumoniae strain.     

Efficacy of vaccination strategies against intranasal challenge with Brucella abortus in BALB/c mice

Brucellosis is a zoonotic disease affecting 500,000 people worldwide annually. Inhalation of aerosol containing a pathogen is one of the major routes of disease transmission in humans. Currently there are no licensed human vaccines available. Brucella abortus strain RB51 is a USDA approved live attenuated vaccine against cattle brucellosis. In a mouse model, strain RB51 over-expressing superoxide dismutase (SOD) administered intraperitoneally (IP) has been shown to be more protective than strain RB51 against an IP challenge with B. abortus pathogenic strain 2308. However, there is lack of information on the ability of these vaccine strains to protect against intranasal challenge. With the long-term goal of developing a protective vaccine for animals and people against respiratory challenge of Brucella spp., we tested a number of different vaccination strategies against intranasal infection with strain 2308. We employed strains RB51 and RB51SOD to assess the efficacy of route, dose, and prime-boost strategies against strain 2308 challenge. Despite using multiple protocols to enhance mucosal and systemic protection, neither rough RB51 vaccine strains provided respiratory protection against intranasal pathogenic Brucella infection. However, intranasal (IN) administration of B. abortus vaccine strain 19 induced significant (p ≤ 0.05) pulmonary clearance of strain 2308 upon IN challenge infection compared to saline. Further studies are necessary to address host-pathogen interaction in the lung microenvironment and elucidate immune mechanisms to enhance protection against aerosol infection.     

Recombinant Leishmania tarentolae expressing the A2 virulence gene as a novel candidate vaccine against visceral leishmaniasis

Visceral leishmaniasis is the most severe form of leishmaniasis. To date, there is no effective vaccine against this disease. Many antigens have been examined so far as protein- or DNA-based vaccines, but none of them conferred complete long-term protection. The use of live attenuated vaccines has recently emerged as a promising vaccination strategy. In this study, we stably expressed the Leishmania donovani A2 antigen in Leishmania tarentolae, a non-pathogenic member of the genus Leishmania, and evaluated its protective efficacy as a live vaccine against L. infantum challenge. Our results show that a single intraperitoneal administration of the A2-recombinant L. tarentolae strain protects BALB/c mice against L. infantum challenge and that protective immunity is associated with high levels of IFN-γ production prior and after challenge. This is accompanied by reduced levels of IL-5 production after challenge, leading to a potent Th1 immune response. In contrast, intravenous injection elicited a Th2 type response, characterized by higher levels of IL-5 and high humoral immune response, resulting in a less efficient protection. All together, these results indicate the promise of A2-expressing L. tarentolae as a safe live vaccine against visceral leishmaniasis.     

Evaluation of humoral and cellular immune response of BALB/c mice immunized with a recombinant fragment of MSP1a from Anaplasma marginale using carbon nanotubes as a carrier molecule

Bovine anaplasmosis is a disease caused by the intraerythrocytic rickettsia Anaplasma marginale. Surface proteins (MSPs) of A. marginale are important in the interaction of the pathogen with the host and constitute potential vaccine targets against this pathogen. Currently, there is no commercial inactivated vaccine against bovine anaplasmosis that can generate a protective immune response that effectively prevents the development of clinical disease. The objective of this study was to evaluate the humoral and cellular immune responses of BALB/c mice immunized with the recombinant fragment of rMSP1a from A. marginale using carbon nanotubes as a carrier molecule. The fragment of rMSP1a comprising the N-terminal region of the protein was expressed in Escherichia coli BL21, purified by nickel affinity chromatography and covalently linked to multiwalled carbon nanotubes (MWNTs). After this functionalization, thirty BALB/c mice were divided into five groups, G1 (rMSP1a), G2 (MWNT + rMSP1a), G3 (MWNT), G4 (adjuvant) and G5 (unimmunized). The mice were immunized subcutaneously at days 0, 21 and 42. Blood samples were collected on day 11 after immunization. The spleens were collected, and the splenocytes were cultured for cell proliferation assays and cell immunophenotyping. Mice immunized with rMSP1a (G1 and G2) produced high levels of anti-rMSP1a IgG, demonstrating that the functionalization to carbon nanotubes did not interfere with protein immunogenicity. Immunization with MWNT + rMSP1a significantly induced higher percentages of CD4⁺CD44⁺ and CD4⁺CD62L⁺ lymphocytes, high levels of TNF-α, and a higher proliferative rate of splenocytes compared to mice immunized with rMSP1a alone (G1 group). Therefore, additional experiments using cattle should be performed to determine the efficacy, safety, immunogenicity and protection induced by rMSP1a associated with MWNT.     

Development of a Multilocus Sequence Typing scheme for the study of Anaplasma marginale population structure over space and time

Bovine Anaplasmosis caused by Anaplasma marginale is a worldwide disease prevalent in tropical and subtropical regions where Rhipicephalus microplus is considered the most significant biological vector. Molecular markers previously applied for A. marginale typing are efficient for isolate discrimination but they are not a suitable tool for studying population structure and dynamics. Here we report the development of an MLST scheme based on the study of seven genes: dnaA, ftsZ, groEl, lipA, recA, secY and sucB. Five annotated genomes (Saint Maries, Florida, Mississippi, Puerto Rico and Virginia) and 53 bovine blood samples from different world regions were analyzed. High nucleotide diversity and a large proportion of synonymous substitutions, indicative of negative selection resulted from DnaSP 5.00.02 package application. Recombination events were detected in almost all genes, this evidence together with the coexistence of more than one A. marginale strain in the same sample might suggest the superinfection phenomena as a potential source of variation. The allelic profile analysis performed through GoeBURST shown two main CC that did not support geography. In addition, the AMOVA test confirmed the occurrence of at least two main genetically divergent groups. The composition of the emergent groups reflected the impact of both historical and environmental traits on A. marginale population structure. Finally, a web-based platform “Galaxy MLST-Pipeline” was developed to automate DNA sequence editing and data analysis that together with the Data Base are freely available to users.The A. marginale MLST scheme developed here is a valuable tool with a high discrimination power, besides PCR based strategies are still the better choice for epidemiological intracellular pathogens studies. Finally, the allelic profile describe herein would contribute to uncover the mechanisms in how intracellular pathogens challenge virulence paradigm.