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African swine fever virus (ASFV) causes a severe hemorrhagic disease in pigs, leading to up to 100% case fatality. The virus May persist on solid surfaces for long periods; thus, fomites, such as contaminated clothing, footwear, farming tools, equipment, and transport vehicles, May contribute to the indirect transmission of the virus. Here, a plastic surface functionalized with tall oil rosin was tested against ASFV. The rosin-functionalized plastic reduced ASFV infectious virus titers by 1.3 log 10 after 60 min of contact time and killed all detectable viruses after 120 min, leading to a ~ 6 log 10 reduction. In contrast, the infectious virus titer of ASFV in contact with low-density polyethylene (LDPE) plastic reduced <1 log 10 after 120 min. Transmission electron microscopy (TEM) showed significant morphological changes in the virus after 2 h of contact with the rosin-functionalized plastic surface, but no changes were observed with the LDPE plastic. The use of antiviral plastic in the farming sector could reduce the spread of ASFV through fomites and could thus be part of an integrated program to control ASFV., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Hemmink, Shroff, Chege, Haapakoski, Dixon and Marjomäki.)

The African buffalo (Syncerus caffer) is a wild bovid with a historical distribution across much of sub-Saharan Africa. Genomic analysis can provide insights into the evolutionary history of the species, and the key selective pressures shaping populations, including assessment of population level differentiation, population fragmentation, and population genetic structure. In this study we generated the highest quality de novo genome assembly (2.65 Gb, scaffold N50 69.17 Mb) of African buffalo to date, and sequenced a further 195 genomes from across the species distribution. Principal component and admixture analyses provided little support for the currently described four subspecies. Estimating Effective Migration Surfaces analysis suggested that geographical barriers have played a significant role in shaping gene flow and the population structure. Estimated effective population sizes indicated a substantial drop occurring in all populations 5-10,000 years ago, coinciding with the increase in human populations. Finally, signatures of selection were enriched for key genes associated with the immune response, suggesting infectious disease exert a substantial selective pressure upon the African buffalo. These findings have important implications for understanding bovid evolution, buffalo conservation and population management., (© 2024. The Author(s).)

The development of a contamination-free and on-site nucleic acid detection platform with high sensitivity and specificity but low-cost for the detection of pathogenic nucleic acids is critical for infectious disease diagnosis and surveillance. In this study, we combined the recombinase-aided amplification (RAA) with the exonuclease III ( Exo III)-assisted signal amplification into a platform for sensitive and specific detection of nucleic acids of African swine fever virus (ASFV). We found that this platform enabled a naked eye visual detection of ASFV at a detection limit as low as 2 copies/μL in 30 min. As expected, no cross-reactivity was observed with other porcine viruses. In addition, to avoid aerosol contamination, a one-tube RAA- Exo III colorimetric assay was also established for the accurate detection of ASFV in clinical samples. Taken together, we developed a rapid, instrument-free, and low-cost Exo III-assisted RAA colorimetric-assay-based nucleic acid detection platform.

Background: Understanding the variation between well and poorly adapted cattle breeds to local environments and pathogens is essential for breeding cattle with improved climate and disease-resistant phenotypes. Although considerable progress has been made towards identifying genetic differences between breeds, variation at the epigenetic and chromatin levels remains poorly characterized. Here, we generate, sequence and analyse over 150 libraries at base-pair resolution to explore the dynamics of DNA methylation and chromatin accessibility of the bovine immune system across three distinct cattle lineages., Results: We find extensive epigenetic divergence between the taurine and indicine cattle breeds across immune cell types, which is linked to the levels of local DNA sequence divergence between the two cattle sub-species. The unique cell type profiles enable the deconvolution of complex cellular mixtures using digital cytometry approaches. Finally, we show distinct sub-categories of CpG islands based on their chromatin and methylation profiles that discriminate between classes of distal and gene proximal islands linked to discrete transcriptional states., Conclusions: Our study provides a comprehensive resource of DNA methylation, chromatin accessibility and RNA expression profiles of three diverse cattle populations. The findings have important implications, from understanding how genetic editing across breeds, and consequently regulatory backgrounds, may have distinct impacts to designing effective cattle epigenome-wide association studies in non-European breeds., (© 2023. The Author(s).)

African swine fever virus (ASFV) is the causative agent of African swine fever (ASF), resulting in up to 100% mortality in pigs. Although endemic in most sub-Saharan African countries, where all known ASFV genotypes have been reported, the disease has caused pandemics of significant economic impact in Eurasia, and no vaccines or therapeutics are available to date. In endeavors to develop live-attenuated vaccines against ASF, deletions of several of the ~170 ASFV genes have shown contrasting results depending on the genotype of the investigated ASFV. Here, we report the in vivo outcome of a single deletion of the A238L (5EL) gene and double deletions of A238L (5EL) and EP402R (CD2v) genes from the genome of a highly virulent genotype IX ASFV isolate. Domestic pigs were intramuscularly inoculated with (i) ASFV-Ke-ΔA238L to assess the safety of A238L deletion and (ii) ASFV-Ke-ΔEP402RΔA238L to investigate protection against challenge with the virulent wildtype ASFV-Ke virus. While A238L (5EL) gene deletion did not yield complete attenuation, co-deletion of A238L (5EL) and EP402R (CD2v) improved the safety profile of the single deletions, eliciting both humoral and cellular immune responses and conferred partial protection against challenge with the virulent wildtype ASFV-Ke virus.

Infection of pigs with the African swine fever virus (ASFV) leads to a devastating hemorrhagic disease with a high mortality of up to 100%. In this study, a CD2v gene deletion was introduced to a genotype IX virus from East Africa, ASFV-Kenya-IX-1033 (ASFV-Kenya-IX-1033-∆CD2v), to investigate whether this deletion led to reduced virulence in domestic pigs and to see if inoculation with this LA-ASFV could induce protective immunity against parental virus challenge. All pigs inoculated with ASFV-Kenya-IX-1033-ΔCD2v survived inoculation but presented with fever, reduced appetite and lethargy. ASFV genomic copies were detected in only one animal at one time point. Seven out of eight animals survived subsequent challenge with the pathogenic parental strain (87.5%) but had mild to moderate clinical symptoms and had a gross pathology compatible with chronic ASFV infection. All mock-immunised animals developed acute ASF upon challenge with ASFV-Kenya-IX-1033 and were euthanised upon meeting the humane endpoint criteria. ASFV genome copy numbers after challenge were similar in the two groups. ASFV-Kenya-IX-1033-∆CD2v is therefore a useful tool to investigate the development of immunity to ASFV genotype IX, but safety concerns preclude its use as a candidate vaccine without further attenuation.

We describe the characterization of an African swine fever genotype IX virus (ASFV-Kenya-IX-1033), which was isolated from a domestic pig in western Kenya during a reported outbreak. This includes the efficiency of virus replication and in vivo virulence, together with genome stability and virulence, following passage in blood macrophages and in a wild boar lung cell line (WSL). The ASFV-Kenya-IX-1033 stock retained its ability to replicate in primary macrophages and retained virulence in vivo, following more than 20 passages in a WSL. At the whole genome level, a few single-nucleotide differences were observed between the macrophage and WSL-propagated viruses. Thus, we propose that the WSL is suitable for the production of live-attenuated ASFV vaccine candidates based on the modification of this wild-type isolate. The genome sequences for ASFV-Kenya-IX-1033 propagated in macrophages and in WSL cells were submitted to GenBank, and a challenge model based on the isolate was developed. This will aid the development of vaccines against the genotype IX ASFV circulating in eastern and central Africa., Competing Interests: The authors declare that they have no conflicts of interest.

East Coast fever, a tick-borne cattle disease caused by the Theileria parva parasite, is among the biggest natural killers of cattle in East Africa, leading to over 1 million deaths annually. Here we report on the genetic analysis of a cohort of Bos indicus (Boran) cattle demonstrating heritable tolerance to infection with T. parva (h2 = 0.65, s.e. 0.57). Through a linkage analysis we identify a 6 Mb genomic region on bovine chromosome 15 that is significantly associated with survival outcome following T. parva exposure. Testing this locus in an independent cohort of animals replicates this association with survival following T. parva infection. A stop gained variant in a paralogue of the FAF1 gene in this region was found to be highly associated with survival across both related and unrelated animals, with only one of the 20 homozygote carriers (T/T) of this change succumbing to the disease in contrast to 44 out of 97 animals homozygote for the reference allele (C/C). Consequently, we present a genetic locus linked to tolerance of one of Africa's most important cattle diseases, raising the promise of marker-assisted selection for cattle that are less susceptible to infection by T. parva., Competing Interests: We have read the journal’s policy and the authors of this manuscript have the following competing interests: The Centre for Tropical Livestock Genetics and Health (CTLGH) has filed a patent application regarding the use of this locus in improving cattle through methods such as marker assisted breeding.

Despite only 8% of cattle being found in Europe, European breeds dominate current genetic resources. This adversely impacts cattle research in other important global cattle breeds, especially those from Africa for which genomic resources are particularly limited, despite their disproportionate importance to the continent's economies. To mitigate this issue, we have generated assemblies of African breeds, which have been integrated with genomic data for 294 diverse cattle into a graph genome that incorporates global cattle diversity. We illustrate how this more representative reference assembly contains an extra 116.1 Mb (4.2%) of sequence absent from the current Hereford sequence and consequently inaccessible to current studies. We further demonstrate how using this graph genome increases read mapping rates, reduces allelic biases and improves the agreement of structural variant calling with independent optical mapping data. Consequently, we present an improved, more representative, reference assembly that will improve global cattle research., (© 2022. The Author(s).)

Rapid diagnosis based on naked-eye colorimetric detection remains challenging, but it could build new capacities for molecular point-of-care testing (POCT). In this study, we evaluated the performance of 16 types of single-stranded DNA-fluorophore-quencher (ssDNA-FQ) reporters for use with clusters of regularly spaced short palindrome repeats (CRISPR)/Cas12a-based visual colorimetric assays. Among them, nine ssDNA-FQ reporters were found to be suitable for direct visual colorimetric detection, with especially very strong performance using ROX-labeled reporters. We optimized the reaction concentrations of these ssDNA-FQ reporters for a naked-eye read-out of assay results (no transducing component required for visualization). In particular, we developed a convolutional neural network algorithm to standardize and automate the analytical colorimetric assessment of images and integrated this into the MagicEye mobile phone software. A field-deployable assay platform named RApid VIsual CRISPR (RAVI-CRISPR) based on a ROX-labeled reporter with isothermal amplification and CRISPR/Cas12a targeting was established. We deployed RAVI-CRISPR in a single tube toward an instrument-less colorimetric POCT format that required only a portable rechargeable hand warmer for incubation. The RAVI-CRISPR was successfully used for the high-sensitivity detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and African swine fever virus (ASFV). Our study demonstrates this RAVI-CRISPR/MagicEye system to be suitable for distinguishing different pathogenic nucleic acid targets with high specificity and sensitivity as the simplest-to-date platform for rapid pen- or bed-side testing.

East Coast fever (ECF) in cattle is caused by the Apicomplexan protozoan parasite Theileria parva , transmitted by the three-host tick Rhipicephalus appendiculatus . The African buffalo ( Syncerus caffer ) is the natural host for T. parva but does not suffer disease, whereas ECF is often fatal in cattle. The genetic relationship between T. parva populations circulating in cattle and buffalo is poorly understood, and has not been studied in sympatric buffalo and cattle. This study aimed to determine the genetic diversity of T. parva populations in cattle and buffalo, in an area where livestock co-exist with buffalo adjacent to the Serengeti National Park, Tanzania. Three T. parva antigens (Tp1, Tp4, and Tp16), known to be recognized by CD8 + and CD4 + T cells in immunized cattle, were used to characterize genetic diversity of T. parva in cattle ( n = 126) and buffalo samples ( n = 22). Long read (PacBio) sequencing was used to generate full or near-full length allelic sequences. Patterns of diversity were similar across all three antigens, with allelic diversity being significantly greater in buffalo-derived parasites compared to cattle-derived (e.g., for Tp1 median cattle allele count was 9, and 81.5 for buffalo), with very few alleles shared between species (8 of 651 alleles were shared for Tp1). Most alleles were unique to buffalo with a smaller proportion unique to cattle (412 buffalo unique vs. 231 cattle-unique for Tp1). There were indications of population substructuring, with one allelic cluster of Tp1 representing alleles found in both cattle and buffalo (including the TpM reference genome allele), and another containing predominantly only alleles deriving from buffalo. These data illustrate the complex interplay between T. parva populations in buffalo and cattle, revealing the significant genetic diversity in the buffalo T. parva population, the limited sharing of parasite genotypes between the host species, and highlight that a subpopulation of T. parva is maintained by transmission within cattle. The data indicate that fuller understanding of buffalo T. parva population dynamics is needed, as only a comprehensive appreciation of the population genetics of T. parva populations will enable assessment of buffalo-derived infection risk in cattle, and how this may impact upon control measures such as vaccination., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Allan, Jayaraman, Paxton, Sindoya, Kibona, Fyumagwa, Mramba, Torr, Hemmink, Toye, Lembo, Handel, Auty, Morrison and Morrison.)

Despite the role of pigs as a source of new Influenza A Virus viruses (IAV) potentially capable of initiating human pandemics, immune responses to swine influenza virus (SwIV) in pigs are not fully understood. Several SwIV epitopes presented by swine MHC (SLA) class I have been identified using different approaches either in outbred pigs or in Babraham large white inbred pigs, which are 85% identical by genome wide SNP analysis. On the other hand, some class II SLA epitopes were recently described in outbred pigs. In this work, Babraham large white inbred pigs were selected to identify SLA II epitopes from SwIV H1N1. PBMCs were screened for recognition of overlapping peptides covering the NP and M1 proteins from heterologous IAV H1N1 in IFNγ ELISPOT. A novel SLA class II restricted epitope was identified in NP from swine H1N1. This conserved novel epitope could be the base for further vaccine approaches against H1N1 in pigs., (Copyright © 2020 Baratelli, Morgan, Hemmink, Reid, Carr, Lefevre, Montaner-Tarbes, Charleston, Fraile, Tchilian and Montoya.)

The tick-borne protozoan parasite Theileria parva causes an acute, often fatal disease in cattle throughout a large part of eastern and southern Africa. Infection of African buffalo (Syncerus caffer) is also widespread in this region but does not cause clinical disease in this species. This difference most likely reflects the evolutionary history of the parasites in these species, in that cattle were only introduced into Africa within the last 8000 years. In both hosts, T. parva establishes a carrier state, involving persistence of small numbers of parasites for many months following the acute phase of infection. This persistence is considered important for maintaining the parasite populations. Although cattle and buffalo parasites both produce severe disease when transmitted to cattle, the buffalo-derived parasites are usually not transmissible from infected cattle. Recent studies of the molecular and antigenic composition of T. parva, in addition to demonstrating heterogeneity in the populations in both host species, have revealed that infections in individual animals are genotypically mixed. The results of these studies have also shown that buffalo T. parva exhibit much greater genotypic diversity than the cattle population and indicate that cattle parasites represent a subpopulation of T. parva that has adapted to maintenance in cattle. The parasites in cattle and buffalo appear to be maintained largely as separate populations. This insight into the genotypic composition of T. parva populations has raised important questions on how host adaptation of the parasite has evolved and whether there is scope for further adaptation of buffalo-maintained populations to cattle., (Crown Copyright © 2020. Published by Elsevier Ltd. All rights reserved.)

There is increasing evidence that induction of local immune responses is a key component of effective vaccines. For respiratory pathogens, for example tuberculosis and influenza, aerosol delivery is being actively explored as a method to administer vaccine antigens. Current animal models used to study respiratory pathogens suffer from anatomical disparity with humans. The pig is a natural and important host of influenza viruses and is physiologically more comparable to humans than other animal models in terms of size, respiratory tract biology and volume. It may also be an important vector in the birds to human infection cycle. A major drawback of the current pig model is the inability to analyze antigen-specific CD8+ T-cell responses, which are critical to respiratory immunity. Here we address this knowledge gap using an established in-bred pig model with a high degree of genetic identity between individuals, including the MHC (Swine Leukocyte Antigen (SLA)) locus. We developed a toolset that included long-term in vitro pig T-cell culture and cloning and identification of novel immunodominant influenza-derived T-cell epitopes. We also generated structures of the two SLA class I molecules found in these animals presenting the immunodominant epitopes. These structures allowed definition of the primary anchor points for epitopes in the SLA binding groove and established SLA binding motifs that were used to successfully predict other influenza-derived peptide sequences capable of stimulating T-cells. Peptide-SLA tetramers were constructed and used to track influenza-specific T-cells ex vivo in blood, the lungs and draining lymph nodes. Aerosol immunization with attenuated single cycle influenza viruses (S-FLU) induced large numbers of CD8+ T-cells specific for conserved NP peptides in the respiratory tract. Collectively, these data substantially increase the utility of pigs as an effective model for studying protective local cellular immunity against respiratory pathogens., Competing Interests: AMT is named on a European patent (publication no. EP2758525 A2, published July 30, 2014) concerning the use of S-FLU as a vaccine. AKS. is an inventor of patent WO 2010032022, “Use of protein kinase inhibitor to detect immune cells, such as T-cells. The other authors have no financial conflicts of interest.

Influenza virus infection is a significant global health threat. Because of the lack of cross-protective universal vaccines, short time window during which antivirals are effective and drug resistance, new therapeutic anti-influenza strategies are required. Broadly, cross-protective antibodies that target conserved sites in the hemagglutinin (HA) stem region have been proposed as therapeutic agents. FI6 is the first proven such monoclonal antibody to bind to H1-H16 and is protective in mice and ferrets. Multiple studies have shown that Fc-dependent mechanisms are essential for FI6 in vivo efficacy. Here, we show that therapeutic administration of FI6 either intravenously or by aerosol to pigs did not reduce viral load in nasal swabs or broncho-alveolar lavage, but aerosol delivery of FI6 reduced gross pathology significantly. We demonstrate that pig Fc receptors do not bind human IgG1 and that FI6 did not mediate antibody-dependent cytotoxicity (ADCC) with pig PBMC, confirming that ADCC is an important mechanism of protection by anti-stem antibodies in vivo . Enhanced respiratory disease, which has been associated with pigs with cross-reactive non-neutralizing anti-HA antibodies, did not occur after FI6 administration. Our results also show that in vitro neutralizing antibody responses are not a robust correlate of protection for the control of influenza infection and pathology in a natural host model.

The Babraham pig is a highly inbred breed first developed in the United Kingdom approximately 50 years ago. Previous reports indicate a very high degree of homozygosity across the genome, including the major histocompatibility complex (MHC) region, but confirmation of homozygosity at the specific MHC loci was lacking. Using both direct sequencing and PCR-based sequence-specific typing, we confirm that Babraham pigs are essentially homozygous at their MHC loci and formalise their MHC haplotype as Hp-55.6. This enhances the utility of the Babraham pig as a useful biomedical model for studies in which controlling for genetic variation is important., (© 2018 The Authors. HLA published by John Wiley & Sons Ltd.)

An infection and treatment protocol involving infection with a mixture of three parasite isolates and simultaneous treatment with oxytetracycline is currently used to vaccinate cattle against Theileria parva. While vaccination results in high levels of protection in some regions, little or no protection is observed in areas where animals are challenged predominantly by parasites of buffalo origin. A previous study involving sequencing of two antigen-encoding genes from a series of parasite isolates indicated that this is associated with greater antigenic diversity in buffalo-derived T. parva. The current study set out to extend these analyses by applying high-throughput sequencing to ex vivo samples from naturally infected buffalo to determine the extent of diversity in a set of antigen-encoding genes. Samples from two populations of buffalo, one in Kenya and the other in South Africa, were examined to investigate the effect of geographical distance on the nature of sequence diversity. The results revealed a number of significant findings. First, there was a variable degree of nucleotide sequence diversity in all gene segments examined, with the percentage of polymorphic nucleotides ranging from 10% to 69%. Second, large numbers of allelic variants of each gene were found in individual animals, indicating multiple infection events. Third, despite the observed diversity in nucleotide sequences, several of the gene products had highly conserved amino acid sequences, and thus represent potential candidates for vaccine development. Fourth, although compelling evidence for population differentiation between the Kenyan and South African T. parva parasites was identified, analysis of molecular variance for each gene revealed that the majority of the underlying nucleotide sequence polymorphism was common to both areas, indicating that much of this aspect of genetic variation in the parasite population arose prior to geographic separation., (Copyright © 2018 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Animal model systems for human and animal influenza virus infection and transmission have been established to address research questions which cannot be addressed using in vitro models. Several animal models have been established, such as mice, guinea pig, ferret, pig, poultry, nonhuman primates, and others. Each animal model has its own strength and weaknesses, which should be taken into consideration to select the appropriate animal model to use. This chapter will describe standard protocols relevant for in vivo experiment, including procedures required prior to the start of the animal experiment and sample processing. The animal models described in this chapter are mice, guinea pigs, ferrets, pigs, and chickens.

Influenza virus infection in pigs is a major farming problem, causing considerable economic loss and posing a zoonotic threat. In addition the pig is an excellent model for understanding immunity to influenza viruses as this is a natural host pathogen system. Experimentally, influenza virus is delivered to pigs intra-nasally, by intra-tracheal instillation or by aerosol, but there is little data comparing the outcome of different methods. We evaluated the shedding pattern, cytokine responses in nasal swabs and immune responses following delivery of low or high dose swine influenza pdmH1N1 virus to the respiratory tract of pigs intra-nasally or by aerosol and compared them to those induced in naturally infected contact pigs. Our data shows that natural infection by contact induces remarkably high innate and adaptive immune response, although the animals were exposed to a very low virus dose. In contacts, the kinetics of virus shedding were slow and prolonged and more similar to the low dose directly infected animals. In contrast the cytokine profile in nasal swabs, antibody and cellular immune responses of contacts more closely resemble immune responses in high dose directly inoculated animals. Consideration of these differences is important for studies of disease pathogenesis and assessment of vaccine protective efficacy.

An infection and treatment protocol is used to vaccinate cattle against Theileria parva infection. Due to incomplete cross-protection between different parasite isolates, a mixture of three isolates, termed the Muguga cocktail, is used for vaccination. While vaccination of cattle in some regions provides high levels of protection, some animals are not protected against challenge with buffalo-derived T. parva. Knowledge of the genetic composition of the Muguga cocktail vaccine is required to understand how vaccination is able to protect against field challenge and to identify the potential limitations of the vaccine. The aim of the current study was to determine the extent of genetic and antigenic diversity within the parasite isolates that constitute the Muguga cocktail. High throughput multi-locus sequencing of antigen-encoding loci was performed in parallel with typing using a panel of micro- and mini-satellite loci. The former focused on genes encoding CD8(+) T cell antigens, believed to be relevant to protective immunity. The results demonstrate that each of the three component stocks of the cocktail contains limited parasite genotypic diversity, with single alleles detected at many gene/satellite loci and, moreover, that two of the components show a very high level of similarity. Thus, the vaccine incorporates very little of the genetic and antigenic diversity observed in field populations of T. parva. The presence of alleles at low frequency (<10%) within vaccine component populations also points to the possibility of variability in the content of vaccine doses and the potential for loss of allelic diversity during tick passage. The results demonstrate that there is scope to modify the content of the vaccine in order to enhance its diversity and thus its potential for providing broad protection. The ability to accurately quantify genetic diversity in vaccine component stocks will facilitate improved quality control procedures designed to ensure the long-term efficacy of the vaccine., (Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Influenza A viruses are a major health threat to livestock and humans, causing considerable mortality, morbidity, and economic loss. Current inactivated influenza vaccines are strain specific and new vaccines need to be produced at frequent intervals to combat newly arising influenza virus strains, so that a universal vaccine is highly desirable. We show that pandemic H1N1 influenza virus in which the hemagglutinin signal sequence has been suppressed (S-FLU), when administered to pigs by aerosol can induce CD4 and CD8 T cell immune responses in blood, bronchoalveolar lavage (BAL), and tracheobronchial lymph nodes. Neutralizing Ab was not produced. Detection of a BAL response correlated with a reduction in viral titer in nasal swabs and lungs, following challenge with H1N1 pandemic virus. Intratracheal immunization with a higher dose of a heterologous H5N1 S-FLU vaccine induced weaker BAL and stronger tracheobronchial lymph node responses and a lesser reduction in viral titer. We conclude that local cellular immune responses are important for protection against influenza A virus infection, that these can be most efficiently induced by aerosol immunization targeting the lower respiratory tract, and that S-FLU is a promising universal influenza vaccine candidate., (Copyright © 2016 The Authors.)

African Cape buffalo (Syncerus caffer) is the wildlife reservoir of multiple species within the apicomplexan protozoan genus Theileria, including Theileria parva which causes East coast fever in cattle. A parasite, which has not yet been formally named, known as Theileria sp. (buffalo) has been recognized as a potentially distinct species based on rDNA sequence, since 1993. We demonstrate using reverse line blot (RLB) and sequencing of 18S rDNA genes, that in an area where buffalo and cattle co-graze and there is a heavy tick challenge, T. sp. (buffalo) can frequently be isolated in culture from cattle leukocytes. We also show that T. sp. (buffalo), which is genetically very closely related to T. parva, according to 18s rDNA sequence, has a conserved orthologue of the polymorphic immunodominant molecule (PIM) that forms the basis of the diagnostic ELISA used for T. parva serological detection. Closely related orthologues of several CD8 T cell target antigen genes are also shared with T. parva. By contrast, orthologues of the T. parva p104 and the p67 sporozoite surface antigens could not be amplified by PCR from T. sp. (buffalo), using conserved primers designed from the corresponding T. parva sequences. Collectively the data re-emphasise doubts regarding the value of rDNA sequence data alone for defining apicomplexan species in the absence of additional data. 'Deep 454 pyrosequencing' of DNA from two Theileria sporozoite stabilates prepared from Rhipicephalus appendiculatus ticks fed on buffalo failed to detect T. sp. (buffalo). This strongly suggests that R. appendiculatus may not be a vector for T. sp. (buffalo). Collectively, the data provides further evidence that T. sp. (buffalo). is a distinct species from T. parva.

Infection with Theileria parva is asymptomatic in African buffalo but results in severe disease in cattle. Currently, vaccination relies on infection and treatment, using a mixture of three parasite isolates to overcome the strain specificity of immunity. Genotypic analyses of field populations of T. parva indicate a panmictic population structure, reflecting frequent sexual recombination. Profound immunodominance of protective CD8 T cell responses, together with polymorphism of the target antigens and frequent genetic recombination, contribute to the strain-restricted immunity. The dominant CD8 target antigens are highly polymorphic, but the live vaccine appears to contain limited diversity. A model to explain the ability of the vaccine to confer immunity against highly diverse field parasite challenge is discussed. Parasites in cattle exhibit much more limited antigenic diversity than parasites in buffalo, consistent with other evidence that the cattle-maintained population represents a subset of T. parva recently adapted to cattle.