Dynamics and risk of transmission of bovine tuberculosis in the emerging dairy regions of Ethiopia.

The Ethiopian government has several initiatives to expand and intensify the dairy industry; however, the risk of bovine tuberculosis (bTB) spread is a challenge. To assess the rate of expansion and risk factors for transmission of bTB within-herds, we carried out a repeated cross-sectional survey at two time points, 2016/17 and 2018, in three regional cities, namely, Gondar, Hawassa and Mekelle, representing the emerging dairy belts of Ethiopia. The total number of herds involved was 128, comprising an average of 2303 cattle in each round. The Single Intradermal Comparative Cervical Tuberculin (SICCT) test was used to identify reactor status and data on herd-level risk factors were collected using a structured questionnaire. In the first survey, the apparent prevalence of bTB, as measured by the SICCT test, was 4.5% (95% CI 3.7-5.4%) at the individual animal-level and 24% (95% CI 17.5-32%) at the herd-level. There was no statistically significant change in the overall apparent prevalence or regional distribution at the second survey, consistent with the infection being endemic. The incidence rate was estimated at 3.6 (95% CI 2.8-4.5) and 6.6 (95% CI 3.0-12.6) cases/100 cattle (or herd)-years at the animal- and herd-levels, respectively. Risk factors significantly associated with the within-herd transmission of bTB were age group and within-herd apparent prevalence at the start of the observation period. We noted that farmers voluntarily took steps to remove reactor cattle from their herds as a consequence of the information shared after the first survey. Removal of reactors between surveys was associated with a reduced risk of transmission within these herds. However, with no regulatory barriers to the sale of reactor animals, such actions could potentially lead to further spread between herds. We therefore advocate the importance of setting up regulations and then establishing a systematic bTB surveillance programme to monitor the impact prior to implementing any control measures in Ethiopia.

Antigenic variation of foot-and-mouth disease virus serotype A.

The current measures to control foot-and-mouth disease (FMD) include vaccination, movement control and slaughter of infected or susceptible animals. One of the difficulties in controlling FMD by vaccination arises due to the substantial diversity found among the seven serotypes of FMD virus (FMDV) and the strains within these serotypes. Therefore, vaccination using a single vaccine strain may not fully cross-protect against all strains within that serotype, and therefore selection of appropriate vaccines requires serological comparison of the field virus and potential vaccine viruses using relationship coefficients (r1 values). Limitations of this approach are that antigenic relationships among field viruses are not addressed, as comparisons are only with potential vaccine virus. Furthermore, inherent variation among vaccine sera may impair reproducibility of one-way relationship scores. Here, we used antigenic cartography to quantify and visualize the antigenic relationships among FMD serotype A viruses, aiming to improve the understanding of FMDV antigenic evolution and the scope and reliability of vaccine matching. Our results suggest that predicting antigenic difference using genetic sequence alone or by geographical location is not currently reliable. We found co-circulating lineages in one region that were genetically similar but antigenically distinct. Nevertheless, by comparing antigenic distances measured from the antigenic maps with the full capsid (P1) sequence, we identified a specific amino acid substitution associated with an antigenic mismatch among field viruses and a commonly used prototype vaccine strain, A22/IRQ/24/64.

The effect of seasonal birth pulses on pathogen persistence in wild mammal populations.

The notion of a critical community size (CCS), or population size that is likely to result in long-term persistence of a communicable disease, has been developed based on the empirical observations of acute immunizing infections in human populations, and extended for use in wildlife populations. Seasonal birth pulses are frequently observed in wildlife and are expected to impact infection dynamics, yet their effect on pathogen persistence and CCS have not been considered. To investigate this issue theoretically, we use stochastic epidemiological models to ask how host life-history traits and infection parameters interact to determine pathogen persistence within a closed population. We fit seasonal birth pulse models to data from diverse mammalian species in order to identify realistic parameter ranges. When varying the synchrony of the birth pulse with all other parameters being constant, our model predicted that the CCS can vary by more than two orders of magnitude. Tighter birth pulses tended to drive pathogen extinction by creating large amplitude oscillations in prevalence, especially with high demographic turnover and short infectious periods. Parameters affecting the relative timing of the epidemic and birth pulse peaks determined the intensity and direction of the effect of pre-existing immunity in the population on the pathogen's ability to persist beyond the initial epidemic following its introduction.

Co-circulation of diverse paramyxoviruses in an urban African fruit bat population.

Bats constitute a reservoir of zoonotic infections and some bat paramyxoviruses are capable of cross-species transmission, often with fatal consequences. Determining the level of viral diversity in reservoir populations is fundamental to understanding and predicting viral emergence. This is particularly relevant for RNA viruses where the adaptive mutations required for cross-species transmission can be present in the reservoir host. We report the use of non-invasively collected, pooled, neat urine samples as a robust sample type for investigating paramyxoviruses in bat populations. Using consensus PCR assays we have detected a high incidence and genetic diversity of novel paramyxoviruses in an urban fruit bat population over a short period of time. This may suggest a similarly unique relationship between bats and the members of the family Paramyxoviridae as proposed for some other viral families. Additionally, the high rate of bat-human contact at the study site calls for the zoonotic potential of the detected viruses to be investigated further.

Estimating reassortment rates in co-circulating Eurasian swine influenza viruses.

Swine have often been considered as a mixing vessel for different influenza strains. In order to assess their role in more detail, we undertook a retrospective sequencing study to detect and characterize the reassortants present in European swine and to estimate the rate of reassortment between H1N1, H1N2 and H3N2 subtypes with Eurasian (avian-like) internal protein-coding segments. We analysed 69 newly obtained whole genome sequences of subtypes H1N1-H3N2 from swine influenza viruses sampled between 1982 and 2008, using Illumina and 454 platforms. Analyses of these genomes, together with previously published genomes, revealed a large monophyletic clade of Eurasian swine-lineage polymerase segments containing H1N1, H1N2 and H3N2 subtypes. We subsequently examined reassortments between the haemagglutinin and neuraminidase segments and estimated the reassortment rates between lineages using a recently developed evolutionary analysis method. High rates of reassortment between H1N2 and H1N1 Eurasian swine lineages were detected in European strains, with an average of one reassortment every 2-3 years. This rapid reassortment results from co-circulating lineages in swine, and in consequence we should expect further reassortments between currently circulating swine strains and the recent swine-origin H1N1v pandemic strain.

Antigenic and genetic evolution of equine influenza A (H3N8) virus from 1968 to 2007.

Equine influenza virus is a major respiratory pathogen in horses, and outbreaks of disease often lead to substantial disruption to and economic losses for equestrian industries. The hemagglutinin (HA) protein is of key importance in the control of equine influenza because HA is the primary target of the protective immune response and the main component of currently licensed influenza vaccines. However, the influenza virus HA protein changes over time, a process called antigenic drift, and vaccine strains must be updated to remain effective. Antigenic drift is assessed primarily by the hemagglutination inhibition (HI) assay. We have generated HI assay data for equine influenza A (H3N8) viruses isolated between 1968 and 2007 and have used antigenic cartography to quantify antigenic differences among the isolates. The antigenic evolution of equine influenza viruses during this period was clustered: from 1968 to 1988, all isolates formed a single antigenic cluster, which then split into two cocirculating clusters in 1989, and then a third cocirculating cluster appeared in 2003. Viruses from all three clusters were isolated in 2007. In one of the three clusters, we show evidence of antigenic drift away from the vaccine strain over time. We determined that a single amino acid substitution was likely responsible for the antigenic differences among clusters.

Endemic Lagos bat virus infection in Eidolon helvum.

Phylogenetic analyses suggest lyssaviruses, including Rabies virus, originated from bats. However, the role of bats in the maintenance, transmission and evolution of lyssaviruses is poorly understood. A number of genetically diverse lyssaviruses are present in Africa, including Lagos bat virus (LBV). A high seroprevalence of antibodies against LBV was detected in Eidolon helvum bats. Longitudinal seroprevalence and age-specific seroprevalence data were analysed and capture-mark-recapture (CMR) analysis used to follow 98 bats over 18 months. These data demonstrate endemic infection, with evidence of horizontal transmission, and force of infection was estimated for differing age categories. The CMR analysis found survival probabilities of seronegative and seropositive bats were not significantly different. The lack of increased mortality in seropositive animals suggests infection is not causing disease after extended incubation. These key findings point towards acute transmission of bat lyssaviruses in adapted bat hosts that occurs at a far higher rate than the occurrence of disease.

Bartonella species in bat flies (Diptera: Nycteribiidae) from western Africa.

Bat flies are obligate ectoparasites of bats and it has been hypothesized that they may be involved in the transmission of Bartonella species between bats. A survey was conducted to identify whether Cyclopodia greefi greefi (Diptera: Nycteribiidae) collected from Ghana and 2 islands in the Gulf of Guinea harbour Bartonella. In total, 137 adult flies removed from Eidolon helvum, the straw-coloured fruit bat, were screened for the presence of Bartonella by culture and PCR analysis. Bartonella DNA was detected in 91 (66·4%) of the specimens examined and 1 strain of a Bartonella sp., initially identified in E. helvum blood from Kenya, was obtained from a bat fly collected in Ghana. This is the first study, to our knowledge, to report the identification and isolation of Bartonella in bat flies from western Africa.

The potential distribution of Bacillus anthracis suitability across Uganda using INLA.

To reduce the veterinary, public health, environmental, and economic burden associated with anthrax outbreaks, it is vital to identify the spatial distribution of areas suitable for Bacillus anthracis, the causative agent of the disease. Bayesian approaches have previously been applied to estimate uncertainty around detected areas of B. anthracis suitability. However, conventional simulation-based techniques are often computationally demanding. To solve this computational problem, we use Integrated Nested Laplace Approximation (INLA) which can adjust for spatially structured random effects, to predict the suitability of B. anthracis across Uganda. We apply a Generalized Additive Model (GAM) within the INLA Bayesian framework to quantify the relationships between B. anthracis occurrence and the environment. We consolidate a national database of wildlife, livestock, and human anthrax case records across Uganda built across multiple sectors bridging human and animal partners using a One Health approach. The INLA framework successfully identified known areas of species suitability in Uganda, as well as suggested unknown hotspots across Northern, Eastern, and Central Uganda, which have not been previously identified by other niche models. The major risk factors for B. anthracis suitability were proximity to water bodies (0-0.3 km), increasing soil calcium (between 10 and 25 cmolc/kg), and elevation of 140-190 m. The sensitivity of the final model against the withheld evaluation dataset was 90% (181 out of 202 = 89.6%; rounded up to 90%). The prediction maps generated using this model can guide future anthrax prevention and surveillance plans by the relevant stakeholders in Uganda.

Quantifying antigenic relationships among the lyssaviruses.

All lyssaviruses cause fatal encephalitis in mammals. There is sufficient antigenic variation within the genus to cause variable vaccine efficacy, but this variation is difficult to characterize quantitatively: sequence analysis cannot yet provide detailed antigenic information, and antigenic neutralization data have been refractory to high-resolution robust interpretation. Here, we address these issues by using state-of-the-art antigenic analyses to generate a high-resolution antigenic map of a global panel of 25 lyssaviruses. We compared the calculated antigenic distances with viral glycoprotein ectodomain sequence data. Although 67% of antigenic variation was predictable from the glycoprotein amino acid sequence, there are in some cases substantial differences between genetic and antigenic distances, thus highlighting the risk of inferring antigenic relationships solely from sequence data at this time. These differences included epidemiologically important antigenic differences between vaccine strains and wild-type rabies viruses. Further, we quantitatively assessed the antigenic relationships measured by using rabbit, mouse, and human sera, validating the use of nonhuman experimental animals as a model for determining antigenic variation in humans. The use of passive immune globulin is a crucial component of rabies postexposure prophylaxis, and here we also show that it is possible to predict the reactivity of immune globulin against divergent lyssaviruses.

Uncovering the fruit bat bushmeat commodity chain and the true extent of fruit bat hunting in Ghana, West Africa.

Harvesting, consumption and trade of bushmeat are important causes of both biodiversity loss and potential zoonotic disease emergence. In order to identify possible ways to mitigate these threats, it is essential to improve our understanding of the mechanisms by which bushmeat gets from the site of capture to the consumer's table. In this paper we highlight the previously unrecognized scale of hunting of the African straw-colored fruit bat, Eidolon helvum, a species which is important in both ecological and public health contexts, and describe the commodity chain in southern Ghana for its trade. Based on interviews with 551 Ghanaians, including bat hunters, vendors and consumers, we estimate that a minimum of 128,000 E. helvum bats are sold each year through a commodity chain stretching up to 400 km and involving multiple vendors. Unlike the general bushmeat trade in Ghana, where animals are sold in both specialized bushmeat markets and in restaurants, E. helvum is sold primarily in marketplaces; many bats are also kept by hunters for personal consumption. The offtake estimated in this paper raises serious conservation concerns, while the commodity chain identified in this study may offer possible points for management intervention. The separation of the E. helvum commodity chain from that of other bushmeat highlights the need for species-specific research in this area, particularly for bats, whose status as bushmeat is largely unknown.

Making use of equine population demography for disease control purposes: preliminary observations on the difficulties of counting and locating horses in Great Britain.

Detailed knowledge of horse populations can better facilitate effective control of equine diseases. Preliminary studies were undertaken to ascertain the type of information held on the UK's National Equine Database (NED) and to determine the geographical resolution at which mandatorily recorded owner addresses might be a suitable proxy for predicting horse locations. Results indicated that relatively few UK passport-issuing organisations requested details of where horses were kept in addition to owner address details. Examination of data on 1440 horses held on an Animal Health Trust syndromic surveillance database showed that 90% of them were kept within 10 km of their owners. While owner location may provide an indication of where most horses are kept, further work is also needed to evaluate the usefulness of NED as an epidemiological resource in future equine disease control measures.

Long-term follow-up of owned, free-roaming dogs in South Africa naturally exposed to Babesia rossi.

Babesia rossi is an important, tick-borne intraerythrocytic protozoan parasite; however, its natural history and epidemiology is poorly understood. Babesia rossi is the most virulent Babesia sp. in domestic dogs and is generally considered to cause severe babesiosis, which is fatal if left untreated. However, subclinical infections and mild disease from B. rossi have been reported, although the clinical progression of these cases was not reported. Therefore, to better understand B. rossi under field conditions, we evaluated its clinical progression and seroprevalence in an owned, free-roaming dog population in Zenzele, South Africa, where the parasite is endemic and prevention is not routine. The entire dog population in Zenzele was monitored intensively at the individual level from March 2008 until April 2014, primarily for a longitudinal study on rabies control. Subsequent evaluation of B. rossi comprised analyses of clinical and laboratory data collected from the Zenzele dog population during the 6 year study period. A substantial proportion (31% (n = 34)) of 109 dogs (randomly selected from every available dog in February/March 2010 older than ~6-8 weeks (n = 246)) tested by Indirect Fluorescent Antibody Test had seroconverted strongly to B. rossi. All 34 dogs were generally consistently healthy adults, determined from regular clinical examinations between March 2008 and April 2014. Blood smear examinations at multiple time points between July 2009 and February 2011 were also undertaken for almost all of these (34) seropositive dogs and all those tested were consistently negative for Babesia spp. Subclinical infections and mild disease were also the main findings for a separate group of 18 dogs positive for Babesia spp. on blood smear examination and confirmed to be infected with B. rossi by Polymerase Chain Reaction - Reverse Line Blot. Almost all of these dogs were positive at only one time point from repeat blood smear examinations between July 2009 and February 2011. We suggest that these observations are consistent with immunity acquired from repeated, low-level exposure to the parasite, generating transient subclinical infections or mild disease. Should this be the case, the use of tick control, particularly in adult dogs in free-roaming populations in B. rossi endemic regions, should be carefully considered.

Ultrasonographic assessment of the superficial digital flexor tendons of National Hunt racehorses in training over two racing seasons.

REASONS FOR PERFORMING STUDY: It is important to ascertain the prevalence of superficial digital flexor tendon (SDFT) injuries and to improve methods of predicting injury in National Hunt (NH) racehorses. OBJECTIVES: To establish: 1) the prevalence of SDFT tendinopathy in NH horses; 2) whether routine ultrasonography can be used to predict SDFT injuries; 3) whether previous tendinopathy predisposes to reinjury; 4) a normal range for the SDFT cross-sectional area (CSA); and 5) the effects of gender, age, background (ex-flat or ex-store), limb, training and rest periods on SDFT CSA. METHODS: Routine ultrasound assessment of the palmar metacarpal soft tissues of 263 NH racehorses was performed on up to 6 occasions over 2 NH racing seasons. RESULTS: The prevalence of SDFT pathology detected using ultrasonography was 24% (n = 148), with a nonsignificant variation between yards of 10-40%. No changes in SDFT CSA or ultrasonographic appearance were detected prior to injury. Older horses had a significantly higher prevalence of SDFT pathology compared to younger horses, and horses with tendinopathy were more likely to suffer an acute injury compared to horses with no evidence of pathology. A reference range for normal CSA measurements was established as 77-139 mm2 at level 4, from 142 horses with no ultrasonographic evidence of SDFT pathology. The CSA of normal horses did not vary significantly with age, limb or over 2 racing seasons, but did with sex and background. CONCLUSIONS: The study confirms that SDFT tendinopathy is common in NH horses, with substantial variation between training yards. Ultrasonography at 3 month intervals did not seem to predict acute SDFT injuries. POTENTIAL RELEVANCE: Variation in the prevalence of tendinopathy between yards suggests that training methods may influence injury rate. It was not possible to predict injury using routine ultrasonography and therefore other methods must be identified. A normal reference range for SDFT CSA is provided.

Descriptive epidemiology of fracture, tendon and suspensory ligament injuries in National Hunt racehorses in training.

REASONS FOR PERFORMING STUDY: While fractures and tendon injuries are known to be important diseases in National Hunt (NH) racehorses during racing, there are no accurate estimates of their incidence in NH training yards. OBJECTIVES: To estimate the incidence of fractures and tendon and suspensory ligament injuries (TLIs) in NH racehorses in training; to describe the injuries incurred and to compare injury incidence rates by horse age, trainer, gender and background (ex-flat vs. ex-store horses). METHODS: Cohort data were collected from 14 UK NH training yards for 2 racing seasons. Daily exercise regimens and details of fractures and TLIs occurring in study horses were recorded. RESULTS: Data were gathered from 1223 horses that spent 9466 months at risk of injury. The fracture incidence rate was 1.1/100 horse months and varied significantly by trainer (P<0.001) but not by gender, age or background. The pelvis and third metacarpal bone (MCIII) were the most common fracture sites, although this varied between racing and training. The TLI incidence rate was 1.9/100 horse months and varied significantly by trainer (P = 0.05) and age (P<0.001) but not by gender or background. However, ex-store horses were significantly more likely to have a TLI on the racecourse than ex-flat horses (P = 0.01). Superficial digital flexor injuries accounted for 89% of all TLIs, the remainder being suspensory ligament injuries. CONCLUSIONS AND POTENTIAL RELEVANCE: Fractures and TLIs are important causes of morbidity and mortality in NH racehorses in training in England. This study provides accurate estimates of their incidence in this population and provides a baseline against which to monitor the effect of future interventions.

Risk factors for time to diagnosis of feline upper respiratory tract disease in UK animal adoption shelters.

Feline upper respiratory tract disease (URTD), mainly caused by feline calicivirus (FCV) and feline herpesvirus, is a major cause of disease outbreaks in feline accommodation such as animal shelters, catteries and multi-cat households. We conducted a longitudinal, yearlong study in five UK feline animal shelters to identify risk factors for the time to diagnosis of URTD. We were especially interested in risk factors that could be identified at the time the cat entered the shelter. Shelter staff recorded data for 1,434 cats during 2002-2003. Most of the cats were domestic shorthair cats and were from private households, or were stray or abandoned. Sixty cats without clinical signs of URTD at entry had URTD diagnosed (typically within the first month at the centre). We used two multivariable models: one was a Cox proportional-hazards model, and the other a regression analyses with complementary log-log model. The hazard varied substantially between shelters and was considerably lower for the shelter that had a purpose-built admissions unit with its own isolation facilities. The hazard was greater for purebred cats (HR 4.3-5.0) and for neutered cats (HR 2.0). The hazard was also typically greater if the centre had a greater proportion of cats present with URTD. The analyses suggested that the centre-level risk factors were more important in determining hazard than cat-level risk factors.

Molecular epidemiology of Streptococcus zooepidemicus infection in naturally occurring equine respiratory disease.

The objective of the study was to characterise the molecular epidemiology of Streptococcus zooepidemicus infection among isolates collected sequentially from recently weaned, pasture maintained Welsh mountain ponies with naturally occurring respiratory disease. Weekly nasopharyngeal and tracheal lavage samplings over a 10-week period were conducted in 29 ponies. Two PCR typing methods based on characterisation of the M-protein hypervariable (HV) region and the 16S-23S rRNA gene intergenic spacer were then applied to isolates of S. zooepidemicus recovered from nasopharyngeal swab and tracheal wash samples. S. zooepidemicus infection was highly prevalent during the study, being isolated from 94% of tracheal washes and 88% of nasopharyngeal swabs. Among 39 different S. zooepidemicus types isolated, more were isolated from the trachea (n=33) than the nasopharynx (n=27). There was evidence from temporal patterns of infection for clonal succession over time by the more prevalent S. zooepidemicus types. Novel S. zooepidemicus types were identified, including previously untyped HV regions and intra-strain multiples of both the HV region and intergenic spacer types.

Heart murmurs and valvular regurgitation in thoroughbred racehorses: epidemiology and associations with athletic performance.

BACKGROUND: Cardiac murmurs associated with valvular regurgitation occur commonly in conditioned performance horses, but their association with athletic performance is unknown. HYPOTHESIS: Cardiac valvular regurgitation has a negative association with race performance. ANIMALS: Five hundred and twenty-six "race fit" Thoroughbred racehorses engaged in either flat (race distance 1,000-2,500 m) or jump racing (race distance 3,200-6,400 m). METHODS: Cardiac auscultation and color flow Doppler (CFD) echocardiography were performed on 777 occasions. The associations between the presence and severity of either an audible cardiac murmur or valvular regurgitation assessed by CFD, and published, objective measures of race performance were determined using a standard regression approach. RESULTS: The prevalence of murmurs and of regurgitation varied significantly between racetypes (P<.02), generally increasing from 2-year olds to chasers. There were no consistent associations between racing performance and either grade of murmur or regurgitation, whether the presence or absence of regurgitation or murmur, or only murmurs > or =3/6 or regurgitation > or =6/9, were considered. CONCLUSIONS AND CLINICAL IMPORTANCE: There were differences in prevalence and severity of murmurs of atrioventricular and aortic valve regurgitation between racehorses in different disciplines. Data also showed that neither regurgitation nor murmurs were negatively associated with Timeform rating, an index of UK racehorse quality, in any of the groups of racehorses studied.

Risk factors for anaesthetic-related death in cats: results from the confidential enquiry into perioperative small animal fatalities (CEPSAF).

BACKGROUND: Cats are commonly anaesthetized in veterinary practice, but recent figures describing the frequency of or risk factors for anaesthetic-related death are not available. The aims of this study were to address these deficiencies. METHODS: A nested case-control study was undertaken in 117 UK veterinary centres. All anaesthetic and sedation procedures and anaesthetic and sedation-related deaths (i.e. 'cases') occurring within 48 h were recorded. Details of patient, procedure, and perioperative management were recorded for all cases and randomly selected non-deaths (controls). A detailed statistical model of factors associated with anaesthetic and sedation-related death was constructed. RESULTS: Between June 2002 and June 2004, 175 deaths were classified as anaesthetic and sedation-related and 14 additional deaths (with insufficient information to be excluded) were included for the estimation of risk. During the study, 79 178 anaesthetic and sedation procedures were recorded and the overall risk of anaesthetic and sedation-related death was 0.24% (95% CI 0.20-0.27). Factors associated with increased odds of anaesthetic-related death were poor health status (ASA physical status classification), increasing age, extremes of weight, increasing procedural urgency and complexity, endotracheal intubation, and fluid therapy. Pulse monitoring and pulse oximetry were associated with reduced odds. CONCLUSIONS: The risk of anaesthetic-related death in cats appears to have decreased since the last published study in the UK. The results should aid the preoperative identification of cats at greatest risk. Greater care with endotracheal intubation and fluid administration are recommended, and pulse and pulse oximetry monitoring should be routinely implemented in cats.

Equine herpesvirus-1-specific interferon gamma (IFNgamma) synthesis by peripheral blood mononuclear cells in thoroughbred horses.

REASONS FOR PERFORMING STUDY: An assay has been developed that measures EHV-1 specific interferon gamma synthesis (IFNgamma), a cytokine produced following the activation of memory T lymphocytes and therefore a measure of cell mediated immunity. The method requires validation in the field. OBJECTIVES: To measure the frequency of EHV-1 specific, IFNgamma synthesising peripheral blood mononuclear cells (PBMC) in a population of Thoroughbred horses, and examine its relationship with age, gender, premises and history of vaccination or field infection with EHV-1. METHODS: Lymphocytes from 200 Thoroughbred horses were stimulated with EHV-1 in vitro, and IFNgamma detected using a monoclonal antibody and indirect immunofluorescence. Percent positive cells were enumerated by flow cytometric analysis and the results described and compared statistically between groups. RESULTS: The frequency of IFNgamma+ PBMC was significantly higher in animals age >5 years compared with 2-4 years, in females vs. males, on stud farms vs. training yards and following vaccination of 2-year-olds with inactivated virus compared with nonvaccinates. Age strongly confounded all these associations and care must therefore be taken interpreting these results. Mares exposed to a field infection with EHV-1 also had higher frequencies of IFNgamma+ PBMC than other vaccinated horses. CONCLUSIONS: The frequency of EHV-1 specific, IFNgama+ PBMC among the sample Thoroughbred population was diverse but lowest in young, unvaccinated horses-in-training. POTENTIAL RELEVANCE: The frequency of EHV-1 specific lymphocytes synthesising IFNgamma in this population may be associated with its susceptibility to infection with this virus. This easy technique may be applied to monitor the antigenicity of vaccines and their effectiveness at stimulating cellular immunity.