A locus conferring tolerance to Theileria infection in African cattle.

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.

Inherited Tolerance in Cattle to the Apicomplexan Protozoan Theileria parva is Associated with Decreased Proliferation of Parasite-Infected Lymphocytes.

Theileria parva is the causative agent of East Coast fever and Corridor disease, which are fatal, economically important diseases of cattle in eastern, central and southern Africa. Improved methods of control of the diseases are urgently required. The parasite transforms host lymphocytes, resulting in a rapid, clonal expansion of infected cells. Resistance to the disease has long been reported in cattle from T. parva-endemic areas. We reveal here that first- and second-generation descendants of a single Bos indicus bull survived severe challenge with T. parva, (overall survival rate 57.3% compared to 8.7% for unrelated animals) in a series of five field studies. Tolerant cattle displayed a delayed and less severe parasitosis and febrile response than unrelated animals. The in vitro proliferation of cells from surviving cattle was much reduced compared to those from animals that succumbed to infection. Additionally, some pro-inflammatory cytokines such as IL1ß, IL6, TNFα or TGFß which are usually strongly expressed in susceptible animals and are known to regulate cell growth or motility, remain low in tolerant animals. This correlates with the reduced proliferation and less severe clinical reactions observed in tolerant cattle. The results show for the first time that the inherited tolerance to T. parva is associated with decreased proliferation of infected lymphocytes. The results are discussed in terms of whether the reduced proliferation is the result of a perturbation of the transformation mechanism induced in infected cells or is due to an innate immune response present in the tolerant cattle.

Clinical Evaluation of Corridor Disease in Bos indicus (Boran) Cattle Naturally Infected With Buffalo-Derived Theileria parva.

Corridor disease (CD) is a fatal condition of cattle caused by buffalo-derived Theileria parva. Unlike the related condition, East Coast fever, which results from infection with cattle-derived T. parva, CD has not been extensively studied. We describe in detail the clinical and laboratory findings in cattle naturally infected with buffalo-derived T. parva. Forty-six cattle were exposed to buffalo-derived T. parva under field conditions at the Ol Pejeta Conservancy, Kenya, between 2013 and 2018. The first signs of disease observed in all animals were nasal discharge (mean day of onset was 9 days post-exposure), enlarged lymph nodes (10 days post-exposure), and pyrexia (13.7 days post-exposure). Coughing and labored breathing were observed in more than 50% of animals (14 days post-exposure). Less commonly observed signs, corneal edema (22%) and diarrhea (11%), were observed later in the disease progression (19 days post-exposure). All infections were considered clinically severe, and 42 animals succumbed to infection. The mean time to death across all studies was 18.4 days. The mean time from onset of clinical signs to death was 9 days and from pyrexia to death was 4.8 days, indicating a relatively short duration of clinical illness. There were significant relationships between days to death and the days to first temperature (chi2 = 4.00, p = 0.046), and days to peak temperature (chi2 = 25.81, p = 0.001), animals with earlier onset pyrexia died sooner. These clinical indicators may be useful for assessing the severity of disease in the future. All infections were confirmed by the presence of macroschizonts in lymph node biopsies (mean time to parasitosis was 11 days). Piroplasms were detected in the blood of two animals (4%) and 20 (43%) animals seroconverted. In this study, we demonstrate the successful approach to an experimental field study for CD in cattle. We also describe the clinical progression of CD in naturally infected cattle, including the onset and severity of clinical signs and pathology. Laboratory diagnoses based on examination of blood samples are unreliable, and alternatives may not be available to cattle keepers. The rapid development of CD requires recognition of the clinical signs, which may be useful for early diagnosis of the disease and effective intervention for affected animals.

Simultaneous and Staggered Foot-and-Mouth Disease Virus Coinfection of Cattle.

Foot-and-mouth disease (FMD) field studies have suggested the occurrence of simultaneous infection of individual hosts by multiple virus strains; however, the pathogenesis of foot-and-mouth disease virus (FMDV) coinfections is largely unknown. In the current study, cattle were experimentally exposed to two FMDV strains of different serotypes (O and A). One cohort was simultaneously infected with both viruses, while additional cohorts were initially infected with FMDV A and subsequently superinfected with FMDV O after 21 or 35 days. Coinfections were confirmed during acute infection, with both viruses concurrently detected in blood, lesions, and secretions. Staggered exposures resulted in overlapping infections as convalescent animals with persistent subclinical FMDV infection were superinfected with a heterologous virus. Staggering virus exposure by 21 days conferred clinical protection in six of eight cattle, which were subclinically infected following the heterologous virus exposure. This effect was transient, as all animals superinfected at 35 days post-initial infection developed fulminant FMD. The majority of cattle maintained persistent infection with one of the two viruses while clearing the other. Analysis of viral genomes confirmed interserotypic recombination events within 10 days in the upper respiratory tract of five superinfected animals from which the dominant genomes contained the capsid coding regions of the O virus and nonstructural coding regions of the A virus. In contrast, there were no dominant recombinant genomes detected in samples from simultaneously coinfected cattle. These findings inculpate persistently infected carriers as potential FMDV mixing vessels in which novel strains may rapidly emerge through superinfection and recombination. IMPORTANCE Foot-and-mouth disease (FMD) is a viral infection of livestock of critical socioeconomic importance. Field studies from areas of endemic FMD suggest that animals can be simultaneously infected by more than one distinct variant of FMD virus (FMDV), potentially resulting in emergence of novel viral strains through recombination. However, there has been limited investigation of the mechanisms of in vivo FMDV coinfections under controlled experimental conditions. Our findings confirmed that cattle could be simultaneously infected by two distinct serotypes of FMDV, with different outcomes associated with the timing of exposure to the two different viruses. Additionally, dominant interserotypic recombinant FMDVs were discovered in multiple samples from the upper respiratory tracts of five superinfected animals, emphasizing the potential importance of persistently infected FMDV carriers as sources of novel FMDV strains.

Duration of protection and humoral immunity induced by an adenovirus-vectored subunit vaccine for foot-and-mouth disease (FMD) in Holstein steers.

Foot-and-mouth disease (FMD) is a highly contagious viral infection of cloven hooved animals that continues to cause economic disruption in both endemic countries or when introduced into a formally FMD free country. Vaccines that protect against clinical disease and virus shedding are critical to control FMD. The replication deficient human adenovirus serotype 5 (Ad5) vaccine vector expressing empty FMD virus (FMDV) capsid, AdtFMD, is a promising new vaccine platform. With no shedding or spreading of viral vector detected in field trials, this vaccine is very safe to manufacture, as there is no requirement for high containment faciitites. Here, we describe three studies assessing the proportion of animals protected from clinical vesicular disease (foot lesions) following live-FMDV challenge by intradermolingual inoculation at 6 or 9 months following a single vaccination with the commercial AdtFMD vaccine, provisionally licensed for cattle in the United States. Further, we tested the effect of vaccination route (transdermal, intramuscular, subcutaneous) on clinical outcome and humoral immunity. Results demonstrate that a single dose vaccination in cattle with the commercial vaccine vector expressing capsid proteins of the FMDV strain A24 Cruzeiro (Adt.A24), induced protection against clinical FMD at 6 months (100% transdermal, 80% intramuscular, and 60% subcutaneous) that waned by 9 months post-vaccination (33% transdermal and 20% intramuscular). Post-vaccination serum from immunized cattle (all studies) generally contained FMDV specific neutralizing antibodies by day 14. Anti-FMDV antibody secreting cells are detected in peripheral blood early following vaccination, but are absent after 28 days post-vaccination. Thus, the decay in antibody mediated immunity over time is likely a function of FMDV-specific antibody half-life. These data reveal the short time span of anti-FMDV antibody secreting cells (ASCs) and important performance characteristics of needle-free vaccination with a recombinant vectored subunit vaccine for FMDV.

Similar levels of diversity in the gene encoding the p67 sporozoite surface protein of Theileria parva are observed in blood samples from buffalo and cattle naturally infected from buffalo.

Theileria parva is a tick-transmitted, apicomplexan protozoan found in buffalo (Syncerus caffer) and cattle in eastern, central and southern Africa. The parasite causes a fatal, lymphoproliferative disease in susceptible cattle. Previous studies have shown that the parasites in buffalo comprise a more heterogeneous population than those in cattle, which has led to the concept that the population of parasites circulating in cattle represents a restricted subpopulation of those in buffalo. The present study was undertaken to identify if and where this restriction may occur in cattle naturally infected with parasites from buffalo, by sequencing the T. parva p67 antigen gene from eight buffalo and 12 acutely infected cattle from the same endemic site in Kenya. From 103 sequences, we detected 44 different alleles. Nine alleles were found in both cattle and buffalo, and 17 and 18 found only in the cattle and buffalo populations respectively. Nucleotide and amino acid sequence analyses revealed a similar level of diversity of parasites in both hosts. Principal coordinates and phylogenetic tree analyses did not reveal any clustering associated with the host animals, and the number and degree of mixed T. parva infections was similar in the respective populations. The results suggest that any restriction in the ability of T. parva from buffalo to survive and be transmitted from cattle occurs after entry into and initial transformation of bovine lymphocytes.

Theileria parva antigens recognized by CD8+ T cells show varying degrees of diversity in buffalo-derived infected cell lines.

The extent of sequence diversity among the genes encoding 10 antigens (Tp1-10) known to be recognized by CD8+ T lymphocytes from cattle immune to Theileria parva was analysed. The sequences were derived from parasites in 23 buffalo-derived cell lines, three cattle-derived isolates and one cloned cell line obtained from a buffalo-derived stabilate. The results revealed substantial variation among the antigens through sequence diversity. The greatest nucleotide and amino acid diversity were observed in Tp1, Tp2 and Tp9. Tp5 and Tp7 showed the least amount of allelic diversity, and Tp5, Tp6 and Tp7 had the lowest levels of protein diversity. Tp6 was the most conserved protein; only a single non-synonymous substitution was found in all obtained sequences. The ratio of non-synonymous: synonymous substitutions varied from 0.84 (Tp1) to 0.04 (Tp6). Apart from Tp2 and Tp9, we observed no variation in the other defined CD8+ T cell epitopes (Tp4, 5, 7 and 8), indicating that epitope variation is not a universal feature of T. parva antigens. In addition to providing markers that can be used to examine the diversity in T. parva populations, the results highlight the potential for using conserved antigens to develop vaccines that provide broad protection against T. parva.

Ancient diversity and geographical sub-structuring in African buffalo Theileria parva populations revealed through metagenetic analysis of antigen-encoding loci.

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.

Corrigendum to "Exposure of vaccinated and naive cattle to natural challenge from buffalo-derived Theileria parva" [Int. J. Parasitol. Parasites Wildl. 4(2) (2015) 244-251].

[This corrects the article DOI: 10.1016/j.ijppaw.2015.04.006.].

Rapid identification of bovine MHCI haplotypes in genetically divergent cattle populations using next-generation sequencing.

The major histocompatibility complex (MHC) region contains many genes that are key regulators of both innate and adaptive immunity including the polymorphic MHCI and MHCII genes. Consequently, the characterisation of the repertoire of MHC genes is critical to understanding the variation that determines the nature of immune responses. Our current knowledge of the bovine MHCI repertoire is limited with only the Holstein-Friesian breed having been studied in any depth. Traditional methods of MHCI genotyping are of low resolution and laborious and this has been a major impediment to a more comprehensive analysis of the MHCI repertoire of other cattle breeds. Next-generation sequencing (NGS) technologies have been used to enable high throughput and much higher resolution MHCI typing in a number of species. In this study we have developed a MiSeq platform approach and requisite bioinformatics pipeline to facilitate typing of bovine MHCI repertoires. The method was validated initially on a cohort of Holstein-Friesian animals and then demonstrated to enable characterisation of MHCI repertoires in African cattle breeds, for which there was limited or no available data. During the course of these studies we identified >140 novel classical MHCI genes and defined 62 novel MHCI haplotypes, dramatically expanding the known bovine MHCI repertoire.

Longitudinal evaluation of leukocyte transcripts in killer whales (Orcinus Orca).

Early identification of illness and/or presence of environmental and/or social stressors in free-ranging and domestic cetaceans is a priority for marine mammal health care professionals. Incorporation of leukocyte gene transcript analysis into the diagnostic tool kit has the potential to augment classical diagnostics based upon ease of sample storage and shipment, inducible nature and well-defined roles of transcription and associated downstream actions. Development of biomarkers that could serve to identify "insults" and potentially differentiate disease etiology would be of great diagnostic value. To this end, a modest number of peripheral blood leukocyte gene transcripts were selected for application to a domestic killer whale population with a focus on broad representation of inducible immunologically relevant genes. Normalized leukocyte transcript values, longitudinally acquired from 232 blood samples derived from 26 clinically healthy whales, were not visibly influenced temporally nor by sex or the specific Park in which they resided. Stability in leukocyte transcript number during periods of health enhances their potential use in diagnostics through identification of outliers. Transcript levels of two cytokine genes, IL-4 and IL-17, were highly variable within the group as compared to the other transcripts. IL-4 transcripts were typically absent. Analysis of transcript levels on the other genes of interest, on an individual animal basis, identified more outliers than were visible when analyzed in the context of the entire population. The majority of outliers (9 samples) were low, though elevated transcripts were identified for IL-17 from 2 animals and one each for Cox-2 and IL-10. The low number of outliers was not unexpected as sample selection was intentionally directed towards animals that were clinically healthy at the time of collection. Outliers may reflect animals experiencing subclinical disease that is transient and self-limiting. The immunologic knowledge derived from longitudinal immunologic studies in killer whales, as was the target of the present study, has the potential to improve diagnostics and health related decision making for this and other domestic and free-ranging cetacean species.

Exposure of vaccinated and naive cattle to natural challenge from buffalo-derived Theileria parva.

Integrative management of wildlife and livestock requires a clear understanding of the diseases transmitted between the two populations. The tick-borne protozoan parasite Theileria parva causes two distinct diseases in cattle, East Coast fever and Corridor disease, following infection with parasites derived from cattle or buffalo, respectively. In this study, cattle were immunized with a live sporozoite vaccine containing three T. parva isolates (the Muguga cocktail), which has been used extensively and successfully in the field to protect against cattle-derived T. parva infection. The cattle were exposed in a natural field challenge site containing buffalo but no other cattle. The vaccine had no effect on the survival outcome in vaccinated animals compared to unvaccinated controls: nine out of the 12 cattle in each group succumbed to T. parva infection. The vaccine also had no effect on the clinical course of the disease. A combination of clinical and post mortem observations and laboratory analyses confirmed that the animals died of Corridor disease. The results clearly indicate that the Muguga cocktail vaccine does not provide protection against buffalo-derived T. parva at this site and highlight the need to evaluate the impact of the composition of challenge T. parva populations on vaccine success in areas where buffalo and cattle are present.

Differential levels of mRNA transcripts encoding immunologic mediators in mammary gland secretions from dairy cows with subclinical environmental Streptococci infections.

Dry-off, and the period around parturition, are associated with increased susceptibility to intramammary infections in dairy cows. The immunological profiles of mammary gland secretions during these periods are not well described. The objective of the present study was to better characterize association(s) between chronic subclinical Environmental Streptococci infections at dry-off and relative levels of mRNA transcripts encoding multiple immunologic mediators present in cells derived from mammary gland secretions at dry-off and continuing through parturition. The chronic subclinical bacterial infections in the present study were characterized by multiple isolations of Streptococcus species and elevated SSC for a minimum of three weeks prior to dry-off. The majority of differences between principal and control quarters were identified at dry-off. Transcript levels of IL-17, IL2Rα and iNOS were increased while pro-inflammatory cytokine IL-6, and the regulatory cytokine IL-10, were reduced. Following antibiotic treatment of mammary glands, IL-17 transcripts remained elevated over the course of the study, indicative of a persistent insult. IL-4 transcript levels were modestly elevated at 7 days following dry-off and significantly elevated at 14 days, consistent with activated T(H)1 and T(H)2 lymphocytes in the principal quarters, respectively. From a temporal perspective, transcript levels of IL-8 decreased in all animals through the dry-off period animals and returned to pre-dry-off levels at parturition; levels of iNOS peaked at parturition. Five of the six principal cows experienced recurrent bacterial mastitis during the subsequent lactation; four were in the same quarter as was initially infected with Streptococcus and three of these four were due to coliforms. Taken together, this apparent chronic susceptibility of select mammary glands to bacterial infection would suggest a physiologic and/or immunologic dysfunction. Identification of factor(s) that contribute to the predisposition of mammary glands to developing mastitis should facilitate development of new control strategies.

Cellular immune responses in cetaceans immunized with a porcine erysipelas vaccine.

Clinical erysipelas represents a significant health problem in managed cetacean species. Vaccination was suspended in many oceanariums in the past due to losses associated with vaccine-induced hypersensitivities which were deemed to be a greater threat than clinical erysipelas. A perceived shift in clinical presentation of erysipelas from a chronic dermatologic form to an acute systemic form in dolphins sparked interest in re-initiating vaccination with improved subunit vaccines of Erysipelothrix rhusiopathiae. This manuscript describes the development and application of in vitro correlates of immunity (T(H)1, T(H)2 and T(REG)) in Tursiops truncatus induced by immunization with a commercial porcine 65 kDa subunit E. rhusiopathiae vaccine. Variable degrees of pre-existing T cell memory were identified prior to vaccination. Vaccine-induced IFN gamma responses were consistent with a T(H)1 response and associated with elimination of erysipelas in all vaccinated animals. Comparative analysis between six-month and 12-month vaccination booster regimes demonstrated maintenance of superior memory in the six-month group; however, anamnestic responses induced by booster were only identified in the 12-month group. To our knowledge, this is the first study to develop and apply advanced immunologic analyses for assessing vaccine efficacy in captive or free-ranging wildlife.

Quantitation of leukocyte gene expression in cetaceans.

Real-time quantitation of cytokine mRNA is a routine immunologic technique, especially fitting for use in those species for which monoclonal antibodies are not available. Quantitative gene expression assays were developed to assist in the immunologic assessment of three cetacean species including bottlenosed dolphins, Pacific white-sided dolphins and beluga whales. Nine cytokine genes (IL-2, -4, -10, -12, -13, -18, TNFalpha, TGFbeta and IFNgamma) and Cox-2 were selected for analysis. Most mitogen-induced mononuclear leukocyte responses were similar between the three cetacean species with either up- or down-regulation of cytokine genes. IL-10 expression was highly variable between species. No TH/1TH2 polarization was evident. Cytokine gene analysis has the potential to identify immune system perturbations induced by environmental insult as well as providing diagnostic tools for characterizing immune responses to environmental antigens and vaccines.