Ivermectin inhibits replication of the malignant catarrhal fever virus alcelaphine herpesvirus 1.

Malignant catarrhal fever is a lymphoproliferative disease of cattle and other ungulates that is caused by genetically and antigenically related gamma herpesviruses of the genus Macavirus. Infection of the natural host species is efficient and asymptomatic but spread to susceptible hosts is often fatal with clinical signs including fever, depression, nasal and ocular discharge. There is no recognised treatment for MCF but a vaccine for one MCF virus, alcelaphine herpesvirus 1 (AlHV-1), has been described. In this paper we describe the inhibition of AlHV-1 replication and propagation by the anthelminthic drug ivermectin. Concentrations of 10 µM or greater led to significant reductions in both copy number and viable titre of virus tested in culture medium, with little replication detected at over 20 µM ivermectin. In the absence of alternative treatments, further testing of ivermectin as a candidate antiviral treatment for MCF may therefore be justified.

Development of a recombinant ELISA for ovine herpesvirus 2, suitable for use in sheep.

The minor capsid protein of ovine herpesvirus 2, identified as a potential antigen for serological testing, was over-expressed and purified to allow its assessment in ELISA. The corresponding gene sequence (OvHV-2 orf65, Ov65) was modified to incorporate epitope tags and internal restriction enzyme sites in an E. coli codon-optimised version of the gene. This codon-optimised gene was then subject to internal deletions to identify regions of the protein that could be removed while maintaining protein solubility and antigenicity. It was found that a derivative with deletion of the conserved 5'-end of the gene (Ov65delB) expressed a polypeptide that was soluble when over-expressed in bacteria and was detected by OvHV-2 specific sera. Proteomic analysis of the affinity purified Ov65delB showed that it contained multiple predicted Ov65 tryptic peptides but also showed contamination by co-purifying E. coli proteins. An indirect ELISA, based on this affinity-purified OV65delB, was optimised for use with sheep and cattle samples and cut-off values were established based on known negative serum samples. Analysis of groups of samples that were either presumed infected (UK sheep) or tested OvHV-2 positive or negative by PCR (cattle MCF diagnostic samples) showed that the assay had 95 % sensitivity and 96 % specificity for sheep serum; and 80 % sensitivity and 95 % specificity for cattle serum. The lower sensitivity with cattle samples appeared to be due to a lack of serological response in some MCF-affected cattle. This recombinant antigen therefore shows promise as the basis of an inexpensive, simple and reliable test that can be used to detect OvHV-2-specific antibody responses in both MCF-affected animals and in OvHV-2 reservoir hosts.

Analysis of immune responses to attenuated alcelaphine herpesvirus 1 formulated with and without adjuvant.

The experimental vaccine for bovine malignant catarrhal fever consists of viable attenuated alcelaphine herpesvirus 1 (AlHV-1) derived by extensive culture passage, combined with an oil-in-water adjuvant, delivered intramuscularly. This immunisation strategy was over 80% effective in previous experimental and field trials and protection appeared to be associated with induction of virus-neutralising antibodies. Whether the vaccine virus is required to be viable at the point of immunisation and whether adjuvant is required to induce the appropriate immune responses remains unclear. To address these issues two studies were performed, firstly to analyse immune responses in the presence and absence of adjuvant and secondly, to investigate immune responses to vaccines containing adjuvant plus viable or inactivated AlHV-1. The first study showed that viable attenuated AlHV-1 in the absence of adjuvant induced virus-specific antibodies but the titres of virus-neutralising antibodies were significantly lower than those induced by vaccine containing viable virus and adjuvant, suggesting adjuvant was required for optimal responses. In contrast, the second study found that the vaccine containing inactivated (>99.9%) AlHV-1 induced similar levels of virus-neutralising antibody to the equivalent formulation containing viable AlHV-1. Together these studies suggest that the MCF vaccine acts as an antigen depot for induction of immune responses, requiring adjuvant and a suitable antigen source, which need not be viable virus. These observations may help in directing the development of alternative MCF vaccine formulations for distribution in the absence of an extensive cold chain.

Immune-associated traits measured in milk of Holstein-Friesian cows as proxies for blood serum measurements.

Previous work has highlighted that immune-associated (IA) traits measurable in blood are associated with health, productivity, and reproduction in dairy cows. The aim of the present study was to determine relationships between IA traits measured in blood serum and those simultaneously measured in milk as well as their association with disease phenotypes. All animals were Holstein-Friesian cows from the Langhill research herd (n = 546) housed at the SRUC Dairy Research Centre in Scotland. Milk and serum samples were collected on 20 separate occasions between July 2010 and March 2015 and analyzed by ELISA for haptoglobin (Hp), tumor necrosis factor-α (TNF-α), and natural antibodies binding keyhole limpet hemocyanin (NAbKLH) and lipopolysaccharide (NAbLPS). Data were analyzed using mixed linear models that included pedigree information. Analyses revealed positive phenotypic correlations between milk and serum NAb (0.59 ≤ r ≤ 0.77), Hp (r = 0.37), and TNF-α (r = 0.12). Milk and serum NAb were also found to have a strong genetic correlation (0.81 ≤ r ≤ 0.94) and were genetically correlated with cow lameness (0.66 and 0.79 for milk NAbKLH and serum NAbLPS, respectively). Clinical mastitis was found to be phenotypically correlated with both milk and serum Hp (0.09 ≤ r ≤ 0.23). Serum Hp was also strongly genetically correlated with other cellular IA traits such as percent NKp46+ (a natural killer cell marker; 0.35) and percent peripheral blood mononuclear cells (PBMC; -0.90). Similarly, genetic correlations were found to exist between serum TNF-α and percent NKp46+ (0.22), percent PBMC (0.41), and percent lymphocytes (0.47). Excluding serum Hp, all milk and serum IA traits were repeatable, ranging from 0.11 (milk Hp) to 0.43 (serum NAbLPS). Between-animal variation was highest in milk and serum NAb (0.34-0.43) and significant estimates of heritability were also observed in milk and serum NAb (0.17-0.37). Our findings show that certain IA traits, such as NAbKLH and NAbLPS, found in milk and serum are strongly correlated and highlight the potential of using routinely collected milk samples as a less invasive and cost-effective source of informative data for predictive modeling of animal IA traits.

Alcelaphine herpesvirus 1 glycoprotein B: recombinant expression and antibody recognition.

The gammaherpesvirus alcelaphine herpesvirus 1 (AlHV-1) causes fatal malignant catarrhal fever (MCF) in susceptible species including cattle, but infects its reservoir host, wildebeest, without causing disease. Pathology in cattle may be influenced by virus-host cell interactions mediated by the virus glycoproteins. Cloning and expression of a haemagglutinin-tagged version of the AlHV-1 glycoprotein B (gB) was used to demonstrate that the AlHV-1-specific monoclonal antibody 12B5 recognised gB and that gB was the main component of the gp115 complex of AlHV-1, a glycoprotein complex of five components identified on the surface of AlHV-1 by immunoprecipitation and radiolabelling. Analysis of AlHV-1 virus particles showed that the native form of gB was detected by mAb 12B5 as a band of about 70 kDa, whilst recombinant gB expressed by transfected HEK293T cells appeared to be subject to additional cleavage and incomplete post-translational processing. Antibody 12B5 recognised an epitope on the N-terminal furin-cleaved fragment of gB on AlHV-1 virus particles. It could be used to detect recombinant and virus-expressed gB on western blots and on the surface of infected cells by flow cytometry, whilst recombinant gB was detected on the surface of transfected cells by immunofluorescence. Recombinant gB has potential as an antigen for ELISA detection of MCF virus infection and as a candidate vaccine antigen.

Alcelaphine Herpesvirus-1 (Malignant Catarrhal Fever Virus) in Wildebeest Placenta: Genetic Variation of ORF50 and A9.5 Alleles.

Alcelaphine herpesvirus-1 (AlHV-1), a causative agent of malignant catarrhal fever in cattle, was detected in wildebeest (Connochaetes taurinus) placenta tissue for the first time. Although viral load was low, the finding of viral DNA in over 50% of 94 samples tested lends support to the possibility that placental tissue could play a role in disease transmission and that wildebeest calves are infected in utero. Two viral loci were sequenced to examine variation among virus samples obtained from wildebeest and cattle: the ORF50 gene, encoding the lytic cycle transactivator protein, and the A9.5 gene, encoding a novel polymorphic viral glycoprotein. ORF50 was well conserved with six newly discovered alleles differing at only one or two base positions. In contrast, while only three new A9.5 alleles were discovered, these differed by up to 13% at the nucleotide level and up to 20% at the amino acid level. Structural homology searching performed with the additional A9.5 sequences determined in this study adds power to recent analysis identifying the four-helix bundle cytokine interleukin-4 (IL4) as the major homologue. The majority of MCF virus samples obtained from Tanzanian cattle and wildebeest encoded A9.5 polypeptides identical to the previously characterized A9.5 allele present in the laboratory maintained AlHV-1 C500 strain. This supports the view that AlHV-1 C500 is suitable for the development of a vaccine for wildebeest-associated MCF.

Identification of immuno-reactive capsid proteins of malignant catarrhal fever viruses.

Malignant catarrhal fever (MCF) is a fatal disease of cattle and other ungulates caused by certain gamma-herpesviruses including alcelaphine herpesvirus-1 (AlHV-1) and ovine herpesvirus-2 (OvHV-2). An attenuated virus vaccine based on AlHV-1 has been shown to induce virus-neutralising antibodies in plasma and nasal secretions of protected cattle but the targets of virus-specific antibodies are unknown. Proteomic analysis and western blotting of virus extracts allowed the identification of eight candidate AlHV-1 virion antigens. Recombinant expression of selected candidates and their OvHV-2 orthologues confirmed that two polypeptides, the products of the ORF17.5 and ORF65 genes, were antigens recognised by antibodies from natural MCF cases or from AlHV-1 vaccinated cattle. These proteins have potential as diagnostic and/or vaccine antigens.

The effect of the TLR9 ligand CpG-oligodeoxynucleotide on the protective immune response to alcelaphine herpesvirus-1-mediated malignant catarrhal fever in cattle.

We wished to determine the effect of of CpG ODN adjuvant on the magnitude and duration of protective immunity against alcelaphine herpesvirus-1 (AlHV-1) malignant catarrhal fever (MCF), a fatal lymphoproliferative disease of cattle. Immunity was associated with a mucosal barrier of virus-neutralising antibody. The results showed that CpG ODN included either with emulsigen adjuvant and attenuated AlHV-1 (atAlHV-1) or alone with atAlHV-1 did not affect the overall protection from clinical disease or duration of immunity achieved using emulsigen and atAlHV-1. This is in contrast to other similar studies in cattle with BoHV-1 or cattle and pigs with various other immunogens. In addition to this, several other novel observations were made, not reported previously. Firstly, we were able to statistically verify that vaccine protection against MCF was associated with virus-neutralising antibodies (nAbs) in nasal secretions but was not associated with antibodies in blood plasma, nor with total virus-specific antibody (tAb) titres in either nasal secretions or blood plasma. Furthermore, CpG ODN alone as adjuvant did not support the generation of virus-neutralising antibodies. Secondly, there was a significant boost in tAb in animals with MCF comparing titres before and after challenge. This was not seen with protected animals. Finally, there was a strong IFN-γ response in animals with emulsigen and atAlHV-1 immunisation, as measured by IFN-γ secreting PBMC in culture (and a lack of IL-4) that was not affected by the inclusion of CpG ODN. This suggests that nAbs at the oro-nasal-pharyngeal region are important in protection against AlHV-1 MCF.

Analysis of the genetic diversity of ovine herpesvirus 2 in samples from livestock with malignant catarrhal fever.

In order to define better virus isolates from animals with malignant catarrhal fever (MCF), segments of three genes of ovine herpesvirus-2 were amplified from diagnostic samples representing MCF cases with a range of clinical presentations in cattle, including head and eye, alimentary and neurological. The variation within each gene segment was estimated by DNA sequencing, which confirmed that the newly-annotated Ov9.5 gene was significantly more polymorphic than either of the other loci tested (segments of ORF50 and ORF75), with alleles that differed at over 60% of nucleotide positions. Despite this, the nine Ov9.5 alleles characterised had identical predicted splicing patterns and could be translated into Ov9.5 polypeptides with at least 49% amino acid identity. This multi-locus approach has potential for use in epidemiological studies and in charactering chains of infection. However there was no association between specific variants of OvHV-2 and the clinical/pathological presentation of MCF in the cattle analysed.

A novel spliced gene in alcelaphine herpesvirus 1 encodes a glycoprotein which is secreted in vitro.

Herpesviruses often contain cryptic, spliced genes that are not obvious from the initial in silico annotation. Alcelaphine herpesvirus 1 (AlHV-1) contains 72 annotated ORFs but there are also a number of gaps between these that may have protein-coding potential. Comparative analysis of coding potential between AlHV-1 and the related ovine herpesvirus 2 (OvHV-2) revealed a putative novel spliced gene that we have termed A9.5. Analysis of cDNA clones from AlHV-1-infected cells revealed three overlapping clones corresponding to A9.5 and the coding sequence was confirmed by reverse transcription PCR of RNA from AlHV-1-infected cattle tissues. The A9.5 gene was predicted to encode a secreted glycoprotein with molecular mass 19 kDa. Empirical analysis showed that a recombinant haemagglutinin-tagged A9.5 fusion protein was secreted from transfected cells and had a molecular mass of 45 kDa, which was reduced to 20 kDa by endoglycosidase F treatment, confirming that A9.5 was a secreted glycoprotein. In situ RNA hybridization showed that A9.5 was expressed in cells associated with malignant catarrhal fever (MCF) lesions in infected cattle. Detailed analysis of the available OvHV-2 sequences revealed an homologous gene (Ov9.5) with conserved splicing signals and predicted amino acid sequence features in both sequenced isolates of this related virus. We have therefore identified a novel spliced gene in two related macaviruses that is expressed in MCF lesions. Future work will determine its importance for the pathogenesis of disease.

Host gene expression changes in cattle infected with Alcelaphine herpesvirus 1.

Malignant catarrhal fever is a lymphoproliferative disease of cattle and other ungulates caused by infection with gamma-herpesviruses of the genus Macavirus. These viruses do not establish a productive infection but instead replicate in a cell-associated fashion in T lymphocytes, leading to systemic immune dysregulation and a generally fatal outcome. Despite significant progress in understanding the pathology of this disease, its pathogenesis remains unclear. To identify genes and pathways affected in clinical MCF, sixteen bovine GeneCHIP microarrays were used to assay RNA from kidney and lymph node of four MCF-affected and four control Bos taurus steers. This is the first expression study of AlHV-1-MCF in the bovine host. Over 250 genes showed significant changes in gene expression in either lymph node or kidney, while expression of 35 genes was altered in both tissues. Pathway and annotation analysis of the microarray data showed that immune response and inflammatory genes were up-regulated in the kidney while proliferation-associated transcripts were additionally increased in the lymph node. The genes that showed the largest expression rises in both diseased tissues included cytotoxic enzymes and pro-inflammatory chemokines. These data are consistent with disease-induced stimulation of inflammatory responses involving interferon-γ, including cytotoxic T cell recruitment and activation in peripheral tissues containing virus-infected cells. However it remains unclear whether the tissue damage in MCF lesions is due entirely to the activity of infected cells or whether uninfected T cells, recruited and activated at lesion sites through the action of infected cells, contribute to the pathogenesis of MCF.

Duration of protective immunity and antibody responses in cattle immunised against alcelaphine herpesvirus-1-induced malignant catarrhal fever.

Protection of cattle from alcelaphine herpesvirus-1 (AlHV-1)-induced malignant catarrhal fever (MCF) has been described previously, using an attenuated virus vaccine in an unlicensed adjuvant. The vaccine was hypothesised to induce a protective barrier of virus-neutralising antibody in the oro-nasal region, supported by the observation of high titre neutralising antibodies in nasal secretions of protected animals. Here we describe further analysis of this vaccine strategy, studying the effectiveness of the vaccine formulated with a licensed adjuvant; the duration of immunity induced; and the virus-specific antibody responses in plasma and nasal secretions. The results presented here show that the attenuated AlHV-1 vaccine in a licensed adjuvant protected cattle from fatal intranasal challenge with pathogenic AlHV-1 at three or six months. In addition, animals protected from MCF had significantly higher initial anti-viral antibody titres than animals that succumbed to disease; and these antibody titres remained relatively stable after challenge, while titres in vaccinated animals with MCF increased significantly prior to the onset of clinical disease. These data support the view that a mucosal barrier of neutralising antibody blocks infection of vaccinated animals and suggests that the magnitude of the initial response may correlate with long-term protection. Interestingly, the high titre virus-neutralising antibody responses seen in animals that succumbed to MCF after vaccination were not protective.

Novel virus-encoded microRNA molecules expressed by ovine herpesvirus 2-immortalized bovine T-cells.

A number of herpesviruses have now been shown to encode microRNAs (miRNAs) that have roles in control of both viral and cellular gene expression. Ovine herpesvirus 2 (OvHV-2) is the causative agent of sheep-associated malignant catarrhal fever, a fatal lymphoproliferative disease of cattle. Using massively parallel sequencing and Northern hybridization we have identified eight putative miRNAs encoded by OvHV-2 expressed in an OvHV-2-immortalized bovine lymphocyte cell line. These eight miRNAs are encoded in two areas of the OvHV-2 genome that contain no predicted protein coding regions and show no sequence similarity with other herpesvirus or cellular miRNAs. This represents the first report of the expression of virally encoded miRNAs in the genus Macavirus of herpesviruses.

Malignant catarrhal fever: a review.

Malignant catarrhal fever (MCF) is a fatal lymphoproliferative disease of cattle and other ungulates caused by the ruminant gamma-herpesviruses alcelaphine herpesvirus 1 (AlHV-1) and ovine herpesvirus 2 (OvHV-2). These viruses cause inapparent infection in their reservoir hosts (wildebeest for AlHV-1 and sheep for OvHV-2), but fatal lymphoproliferative disease when they infect MCF-susceptible hosts, including cattle, deer, bison, water buffalo and pigs. MCF is an important disease wherever reservoir and MCF-susceptible species mix and currently is a particular problem in Bali cattle in Indonesia, bison in the USA and in pastoralist cattle herds in Eastern and Southern Africa. MCF is characterised by the accumulation of lymphocytes (predominantly CD8(+) T lymphocytes) in a variety of organs, often associated with tissue necrosis. Only a small proportion of these lymphocytes appear to contain virus, although recent results with virus gene-specific probes indicate that more infected cells may be present than previously thought. The tissue damage in MCF is hypothesised to be caused by the indiscriminate activity of MHC-unrestricted cytotoxic T/natural killer cells. The pathogenesis of MCF and the virus life cycle are poorly understood and, currently, there is no effective disease control. Recent sequencing of the OvHV-2 genome and construction of an AlHV-1 bacterial artificial chromosome (BAC) are facilitating studies to understand the pathogenesis of this extraordinary disease. Furthermore, new and improved methods of disease diagnosis have been developed and promising vaccine strategies are being tested. The next few years are likely to be exciting and productive for MCF research.

A captured viral interleukin 10 gene with cellular exon structure.

We have characterized a novel, captured and fully functional viral interleukin (IL)-10 homologue ((OvHV)IL-10) from the gammaherpesvirus ovine herpesvirus 2. Unlike IL-10 homologues from other gammaherpesviruses, the (OvHV)IL-10 peptide sequence was highly divergent from that of the host species. The (OvHV)IL-10 gene is unique amongst virus captured genes in that it has precisely retained the original cellular exon structure, having five exons of similar sizes to the cellular counterparts. However, the sizes of the introns are dramatically reduced. The (OvHV)IL-10 protein was shown to be a non-glycosylated, secreted protein of M(r) 21 000 with a signal peptidase cleavage site between amino acids 26 and 27 of the nascent peptide. Functional assays showed that (OvHV)IL-10, in a similar way to ovine IL-10, stimulated mast cell proliferation and inhibited macrophage inflammatory chemokine production. This is the first example of a captured herpesvirus gene retaining the full cellular gene structure.

An immunisation strategy for the protection of cattle against alcelaphine herpesvirus-1-induced malignant catarrhal fever.

The aim of this study was to stimulate immunity in the oro-nasal-pharyngeal region of cattle to protect them from alcelaphine herpesvirus-1 (AlHV-1)-induced malignant catarrhal fever. Attenuated C500 strain AlHV-1 was used along with Freund's adjuvant intramuscularly (IM) in the upper neck region to immunise cattle. Virulent C500 strain AlHV-1 was used for intranasal challenge. Nine of ten cattle were protected. Protection was associated with high levels of neutralising antibody in nasal secretions. Some protected animals showed transient low levels of viral DNA in blood samples and in one lymph node sample after challenge whereas viral DNA was detected in the blood and in lymph node samples of all animals with MCF. This is the most promising immunisation strategy to date for the control of malignant catarrhal fever.

Proteomic analysis of pathogenic and attenuated alcelaphine herpesvirus 1.

The gammaherpesvirus alcelaphine herpesvirus 1 (AlHV-1) causes malignant catarrhal fever in susceptible ungulates but infects its natural host, wildebeest, without obvious clinical signs. In tissue culture, AlHV-1 is initially predominantly cell associated and virulent but on extended culture becomes cell-free and attenuated. We wanted to determine what changes in protein composition had taken place during the transition from virulent to attenuated virus in culture. Purified virus preparations were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and proteins were analyzed by liquid chromatography-electrospray ionization-tandem mass spectrometry. Peptides were identified in serial gel slices by using MASCOT software to interrogate virus-specific and nonredundant sequence databases. Twenty-three AlHV-1-encoded proteins and six cellular proteins were identified in the attenuated and virulent viruses. Two polypeptides were detected in only the virulent virus preparations, while one other protein was found in only the attenuated virus. Two of these virus-specific proteins were identified by a single peptide, suggesting that these may be low-abundance virion proteins rather than markers of attenuation or pathogenesis. The results suggest that attenuation of AlHV-1 is not the result of gross changes in the composition of the virus particle but probably due to altered viral gene expression in the infected cell.

Differential transcription of ovine herpesvirus 2 genes in lymphocytes from reservoir and susceptible species.

Ovine herpesvirus 2 (OvHV-2) is a lymphotropic gammaherpesvirus that asymptomatically infects most sheep, but causes malignant catarrhal fever in cattle, bison, pigs and deer. There is no permissive cell culture system but OvHV-2-infected T lymphocytes can be cultured from diseased animals. We showed that the OvHV-2 genome was in a circular conformation in sheep peripheral blood mononuclear cells and that the latency-associated ORF73 was transcribed, while expression of the productive cycle genes ORF9 (DNA polymerase) and ORF50 (R-transactivator) was barely detectable, suggestive of latency. Doxorubicin treatment of these cells induced the appearance of linear viral DNA and transcription of productive cycle genes along with several viral unique genes. In contrast, cultured T cells from diseased cattle and rabbits contained a mixture of circular and linear genome configurations indicative of a mixture of latently- and productively-infected cells. Most of the OvHV-2 unique genes were transcribed in these cells but ORF50 expression was only seen after doxorubicin treatment indicating a 'leaky' latent pattern of gene expression. 5-azacytidine treatment increased the proportion of circular DNA and inhibited the expression of most of the OvHV-2 unique genes except Ov2.5 (vIL-10) and Ov4.5 (Bcl-2 homologue) in the cattle cell line. These studies provide key insights into the differences in OvHV-2 gene expression in cells from reservoir and susceptible species and, for the first time, an in vitro system for studying the latent and productive phases of the OvHV-2 virus life cycle.

A novel subgroup of rhadinoviruses in ruminants.

In the course of investigating the malignant catarrhal fever (MCF) subgroup of rhadinoviruses, seven novel rhadinoviruses were identified in a variety of ruminants, including domestic sheep, bighorn sheep, bison, black-tailed deer, mule deer, fallow deer, elk and addax. Based on the DNA polymerase gene sequences, these newly recognized viruses clustered into a second distinct subgroup in ruminants with three members identified previously in cattle, goats and oryx. Phylogenetic analysis revealed that the currently known ruminant rhadinoviruses appear to comprise three distinct genetic lineages: (i) the MCF subgroup, defined by sequence identity and the presence of the 15A antigenic epitope; (ii) a second distinct subgroup, devoid of the 15A epitope, which contains the previously reported bovine lymphotropic herpesvirus and related viruses; and (iii) a third distinct subgroup represented by Bovine herpesvirus 4. Comparison of phylogenetic trees between the rhadinoviruses and their corresponding hosts further supports the gammaherpesvirus and host co-evolution theory.

Quantitative analysis of pro-inflammatory cytokine mRNA expression in Theileria annulata-infected cell lines derived from resistant and susceptible cattle.

The pathogenic mechanisms involved in tropical theileriosis, caused by the tick-borne protozoan parasite Theileria annulata, are unclear. Pathology is associated with the schizont stage of the parasite, which resides within bovine macrophages. Breed-specific differences in pathology have been observed in cattle, several Bos indicus breeds are relatively resistant to tropical theileriosis whilst Bos taurus cattle are highly susceptible. Infected cells express pro-inflammatory cytokines and it has been hypothesized that these cytokines play a major role in the pathology of the disease. Therefore, using quantitative RT-PCR we investigated the expression of the key candidates, interleukin 1 beta (IL-1beta), IL-6 and tumour necrosis factor alpha (TNF-alpha), in T. annulata low passage infected cell lines derived ex vivo from experimental infection of resistant and susceptible cattle. mRNA for each cytokine was detected in all cell lines investigated at levels higher than those observed in resting monocytes. However, the analyses did not identify any breed-specific differences. Therefore, these results are not consistent with the hypothesis that differential regulation of infected cell derived pro-inflammatory cytokines (IL-1beta, IL-6 and TNF-alpha) accounts for the breed-related differences in resistance and susceptibility to T. annulata infection. Other, currently unknown mechanisms may be of greater importance.