Development of a Potential Yeast-Based Vaccine Platform for Theileria parva Infection in Cattle.

East Coast Fever (ECF), caused by the tick-borne apicomplexan parasite Theileria parva, remains one of the most important livestock diseases in sub-Saharan Africa with more than 1 million cattle dying from infection every year. Disease prevention relies on the so-called "Infection and Treatment Method" (ITM), which is costly, complex, laborious, difficult to standardise on a commercial scale and results in a parasite strain-specific, MHC class I-restricted cytotoxic T cell response. We therefore attempted to develop a safe, affordable, stable, orally applicable and potent subunit vaccine for ECF using five different T. parva schizont antigens (Tp1, Tp2, Tp9, Tp10 and N36) and Saccharomyces cerevisiae as an expression platform. Full-length Tp2 and Tp9 as well as fragments of Tp1 were successfully expressed on the surface of S. cerevisiae. In vitro analyses highlighted that recombinant yeast expressing Tp2 can elicit IFNγ responses using PBMCs from ITM-immunized calves, while Tp2 and Tp9 induced IFNγ responses from enriched bovine CD8+ T cells. A subsequent in vivo study showed that oral administration of heat-inactivated, freeze-dried yeast stably expressing Tp2 increased total murine serum IgG over time, but more importantly, induced Tp2-specific serum IgG antibodies in individual mice compared to the control group. While these results will require subsequent experiments to verify induction of protection in neonatal calves, our data indicates that oral application of yeast expressing Theileria antigens could provide an affordable and easy vaccination platform for sub-Saharan Africa. Evaluation of antigen-specific cellular immune responses, especially cytotoxic CD8+ T cell immunity in cattle will further contribute to the development of a yeast-based vaccine for ECF.

Toxoplasma gondii profilin does not stimulate an innate immune response through bovine or human TLR5.

Toxoplasma gondii is responsible for one of the most prevalent infections in people. T. gondii profilin (TgPr) is a protein integral to parasite movement and cellular invasion. Murine TLRs has been described to bind TgPr. Furthermore, more recently, human TLR5 has been described to recognise recombinant TgPr, as well as bacterial flagellin. In addition to infections in humans, T. gondii infects farm animals, but little information is available about its innate recognition. We aimed to investigate whether, similarly to their human orthologue, bovine and porcine TLR5 could also be stimulated by TgPr by using a combination of reporter cell lines expressing full length TLR5 from each species as well as primary cells. Although human and bovine TLR5-transfected cells responded to flagellin, no response was detected upon stimulation with profilin. Furthermore, TgPr failed to elicit IL-6 secretion in human peripheral blood mononuclear cells and CD14+ monocytes. In contrast, exposure of RAW cells, known to express TLR11, to TgPr slightly increased the IL-6 response. Our data cast doubts on the possibility that profilin is a specific ligand for human TLR5 and bovine TLR5. This leaves the immunogenic properties of this potential target antigen (Ag) uncharacterised outside of the murine system.

Comparison of cellular assays for TLR activation and development of a species-specific reporter cell line for cattle.

PRRs are sentinels of the innate immune system, with TLRs being the most important. Assays for TLR ligand interactions have been used to gain insights into their function and signaling pathways. As significant differences exist between species with regard to ligand recognition, it is necessary to adapt these tools for TLRs of other species. In the present work, we describe a species-specific cell-based assay adapted for the analysis of single PRRs. Human embryonic kidney 293T cells were stably transfected with the NF-κB-inducible reporter gene secreted embryonic alkaline phosphatase (SEAP) together with bovine TLR2. We compared the SEAP response with an existing luciferase NF-κB reporter assay for correlation with IL-8 production. A dose-dependent response was detected upon stimulation using both methods with good correlation to IL-8 secretion. Lower stimulant concentrations were detected by SEAP assay than IL-8 secretion. The luciferase assay produced high non-specific background for all ligand concentrations. Of all assays tested, we found the bovine-specific SEAP reporter assay to be the most convenient and delivered results in the shortest time. The developed reporter cell line would lend well to rapid, high-throughput TLR ligand screening for cattle.

Lack of genetic diversity in newly sequenced porcine circovirus type 1 strains isolated 20 years apart.

The complete genome sequences of a porcine circovirus type 1 (PCV1) strain isolated in 1990 and one isolated in 2011 were obtained and compared to the sequences of other available PCV1 isolates. Phylogenetic analyses revealed very low genetic diversity among these viruses, indicating an advanced state in the evolution of PCV1.

Oral immunogenicity of a plant virus vector based porcine circovirus antigen - short communication.

A recombinant cucumber mosaic virus based expression system has been developed for the production of an immunogenic porcine circovirus epitope. The resulting nanoparticle was shown to elicit specific immune response in mice and pigs, when administered parenterally. To evaluate the oral applicability of this vaccine candidate, two experiments were performed. In the first one, the resistance of the vector itself to mucosal environment was tested in mice. Cucumber mosaic virus particles fed to mice were able to elicit specific mucosal and serum antibody production. In the second experiment, recombinant cucumber mosaic virus fed to piglets resulted in the appearance of porcine circovirus specific serum antibodies. The vector proved to be able to survive in the gastrointestinal tract, so that an epitope expressed on its surface could induce specific immune response. These results indicate that the developed plant virus based expression system offers an effective method for mucosal vaccine production.

Phylogeny and evolutionary genetics of porcine parvovirus in wild boars.

Porcine parvovirus (PPV) is widespread among swine and is responsible for reproductive failure of susceptible sows, characterized by embryonic and fetal death. Studies showed that PPV in domestic pig is genetically diverse and some strains differ from the ones used for vaccination. Organ samples from wild boars and domestic pigs were collected in Transylvania (Romania) and tested for the presence of PPV by polymerase chain reaction. Positive samples were grouped and 14 from the wild boar and 1 from the domestic pig PPVs were selected for VP1/VP2 sequence analysis and comparison with available GenBank data. The molecular clock analysis revealed that PPV has a relatively recent evolutionary history, originated approximately 120 years ago and the main divergence occurred in the last 20-60 years. Phylogenetic and residue substitution analysis showed that the viruses could be divided into 6 distinct clusters and that wild boar PPVs were partially different and independent from domestic pig PPVs. PPVs of wild boars proved to be more diverse than viruses of domestic pigs. The presence of the highly virulent 27a-like PPV strains in wild boars was also detected.

Detection, prevalence and analysis of emerging porcine parvovirus infections.

A number of newly identified porcine parvoviruses had been described during the last decade, but the presence and prevalence of these viruses are unknown in Hungary and only partly known for Europe. The present study was conducted to detect and measure the prevalence of these viruses, namely porcine parvovirus (PPV) 2, PPV3, PPV4, porcine bocavirus (PBoV) 1, PBoV2, PBo-likeV and the 6V and 7V parvoviruses. The prevalence of PPV1 and porcine circovirus type 2 (PCV2) was also investigated. Faecal samples, blood serum samples, organ tissues, foetuses and semen were collected from different swine herds in Hungary and tested by polymerase chain reaction methods specific for the different viruses. The results indicated that all of the examined parvoviruses were present in Hungary, hence in Europe. The prevalence was 18.1% for PCV2, 0.5 % for PPV1, 6.4% for PPV2, 9.7% for PPV3, 6.4% for PPV4, 1.5% for PBo-likeV, 4.8% for PBoV1 and PBoV2 and 1.8% for 6V and 7V. Based on the analysis of partial PPV4 and PBo-likeV sequences, these viruses showed a high degree of sequence conservation, whereas PPV3 and the majority of PPV2, PBoV1, PBoV2, 6V and 7V sequences showed higher variability. Possible sites of recombination were also identified between PBoV1 and PBoV2 genomes.

Detection of natural inter- and intra-genotype recombination events revealed by cap gene analysis and decreasing prevalence of PCV2 in wild boars.

Porcine circovirus type 2 (PCV2), the causative agent of a number of PCVAD (porcine circovirus associated diseases), is ubiquitous in domestic pig and wild boar populations. In the present study, using recombination detection program, phylogenetic analysis and base-by-base comparison of 28 PCV2 ORF2s (capsid protein coding gene) from wild boars and 8 from domestic pigs of Transylvania, recent natural intra- (PCV2b-1B/PCV2b-1C) and inter-genotype (PCV2a-2D/PCV2b-1C) recombination events were detected. Notably, one potential recombinant (F1-21) was detected in domestic pig with possible parental strains of wild boar origin. The estimated recombinant breakpoints comprised epitopes A, B and C of ORF2, without major changes in amino acid sequences. The prevalence of PCV2 in the wild boar population during the 5-year period following the first outbreaks of postweaning multisystemic wasting syndrome (PMWS) in domestic pigs in Romania showed a decrease from 13.4% to 8.3%. To our knowledge, this is the first study to show the existence of ORF2-based intra- and inter-genotype recombination in wild boar populations and the possible recombination between PCV2 strains of wild boars and domestic pigs. Our results suggest a certain independence of PCV2 infection in wild boar populations and demonstrate the possibility of infection with multiple PCV2 genotypes under natural circumstances. On the other hand, PCV2 genotypes specific for wild boars could be detected in domestic pig at lower frequency suggesting the possible spread of wild boar PCV2 to domestic swine. The recombination events described here may contribute to the genetic diversity of PCV2 and may also be the source of emergence of new PCV2 strains.

Genetic diversity of pigeon circovirus in Hungary.

Pigeon circoviruses (PiCV) had been identified worldwide and are responsible for immune suppression and a variety of diseases collectively referred to as young pigeon disease syndrome. Samples from racing pigeons were collected throughout Hungary and analyzed for the presence of PiCV by polymerase chain reaction. The capsid protein coding gene was amplified from ten PiCVs of different origins, and compared with known PiCV sequences. The results indicated that PiCV was highly variable, the viruses formed five distinct genetic groups. Differences of the 3' end of the gene suggested the possibility of genetic recombination among these groups.

A cucumber mosaic virus based expression system for the production of porcine circovirus specific vaccines.

Potential porcine circovirus type 2 (PCV2) capsid protein epitopes, suitable for expression on the surface of cucumber mosaic virus (CMV) particles were determined by a thorough analysis of the predicted PCV capsid protein structure. The ab initio protein structure prediction was carried out with fold recognition and threading methods. The putative PCV epitopes were selected on the basis of PCV virion models and integrated into the plant virus coat protein, after amino acid position 131. The recombinants were tested for infectivity and stability on different Nicotiana species and stable recombinant virus particles were purified. The particles were tested for their ability to bind to PCV induced porcine antibodies and used for specific antibody induction in mice and pigs. The results showed that PCV epitopes expressed on the CMV surface were recognized by the porcine antibodies and they were also able to induce PCV specific antibody response. Challenge experiment with PCV2 carried out in immunized pigs showed partial protection against the infection. Based on these results it was concluded that specific antiviral vaccine production for the given pathogen was feasible, offering an inexpensive way for the mass production of such vaccines.

Distribution and genetic diversity of porcine hokovirus in wild boars.

Porcine hokovirus (PHoV), a newly discovered member of the family Parvoviridae and the proposed genus Hokovirus, is considered phylogenetically distinct from other parvoviruses. Here, we report a comprehensive spatio-temporal study of PHoV infection in Romanian wild boars. The prevalence of PHoV differed significantly in samples from 2006/2007 (22.76%) and 2010/2011 (50.54%), and also increased with age. Sequence analysis of PHoVs from 2006/2007 showed a close relationship to PHoVs from pigs from England and wild boars from Germany, while the PHoVs from 2010/2011 were mostly similar to isolates from Hong Kong. The most variable regions were detected in the NS1 gene and proved to be suitable for analysis of the genetic diversity of the virus. It was observed that PHoVs from older wild boar samples differed from those collected recently. These results suggested that porcine hokovirus could be a newly emerging virus of both domestic and wild pigs with yet unknown implications.

Genetic detection and analysis of porcine bocavirus type 1 (PoBoV1) in European wild boar (Sus scrofa).

Novel porcine parvoviruses showing the genetic characteristics of bocaviruses have recently been identified. The first such porcine bocavirus (PoBoV1), described as boca-like virus (PBo-likeV), was discovered in PMWS affected pigs in Sweden. Later, several other bocaviruses with divergent genomes were reported under various names in domestic pigs. This is the first report of the presence of bocaviruses in European wild boars. 842 wild boar samples originating from the Western region of Romania (Transylvania) were collected during the 2006/2007 and the 2010/2011 hunting seasons and tested for the presence of PoBoV1 by polymerase chain reaction and sequencing. The results showed 12.94% (109/842) overall positivity, with an increasing prevalence from the 2006/2007 (9.14%, 43/470) to the 2010/2011 (17.74%, 66/372) season (P < 0.01). Differences between the prevalence of the virus in 6-12-month-old-animal (77.06%, 84/109) and 12-36-month-old-animal (22.94%, 25/109) (P < 0.01) indicated that the infection occurred mainly in younger pigs. Comparative sequence analysis of partial VP1/2 genes from wild boars and those available in the GenBank showed only minor differences, indicating that PoBoV1 circulating within the wild boar populations and domestic pigs from different geographic regions were highly similar.