Longitudinal study of the short- and long-term effects of hospitalisation and oral trimethoprim-sulfadiazine administration on the equine faecal microbiome and resistome.

BACKGROUND: Hospitalisation and antimicrobial treatment are common in horses and significantly impact the intestinal microbiota. Antimicrobial treatment might also increase levels of resistant bacteria in faeces, which could spread to other ecological compartments, such as the environment, other animals and humans. In this study, we aimed to characterise the short- and long-term effects of transportation, hospitalisation and trimethoprim-sulfadiazine (TMS) administration on the faecal microbiota and resistome of healthy equids. METHODS: In a longitudinal experimental study design, in which the ponies served as their own control, faecal samples were collected from six healthy Welsh ponies at the farm (D0-D13-1), immediately following transportation to the hospital (D13-2), during 7 days of hospitalisation without treatment (D14-D21), during 5 days of oral TMS treatment (D22-D26) and after discharge from the hospital up to 6 months later (D27-D211). After DNA extraction, 16S rRNA gene sequencing was performed on all samples. For resistome analysis, shotgun metagenomic sequencing was performed on selected samples. RESULTS: Hospitalisation without antimicrobial treatment did not significantly affect microbiota composition. Oral TMS treatment reduced alpha-diversity significantly. Kiritimatiellaeota, Fibrobacteres and Verrucomicrobia significantly decreased in relative abundance, whereas Firmicutes increased. The faecal microbiota composition gradually recovered after discontinuation of TMS treatment and discharge from the hospital and, after 2 weeks, was more similar to pre-treatment composition than to composition during TMS treatment. Six months later, however, microbiota composition still differed significantly from that at the start of the study and Spirochaetes and Verrucomicrobia were less abundant. TMS administration led to a significant (up to 32-fold) and rapid increase in the relative abundance of resistance genes sul2, tetQ, ant6-1a, and aph(3")-lb. lnuC significantly decreased directly after treatment. Resistance genes sul2 (15-fold) and tetQ (six-fold) remained significantly increased 6 months later. CONCLUSIONS: Oral treatment with TMS has a rapid and long-lasting effect on faecal microbiota composition and resistome, making the equine hindgut a reservoir and potential source of resistant bacteria posing a risk to animal and human health through transmission. These findings support the judicious use of antimicrobials to minimise long-term faecal presence, excretion and the spread of antimicrobial resistance in the environment. Video Abstract.

Estimation of the basic reproduction number for Streptococcus equi spp. equi outbreaks by meta-analysis of strangles outbreak reports.

BACKGROUND: Streptococcus equi spp. equi (S. equi), the cause of strangles in horses, is considered a highly contagious pathogen affecting equines and the equine industry worldwide. Fundamental epidemiological characteristics of outbreaks, such as the basic reproduction number (R0 ), are not well described. OBJECTIVES: Estimate R0 for S. equi in equine populations from outbreak data. STUDY DESIGN: Systematic review and meta-analysis of published and unpublished data. METHODS: A literature search for outbreak reports was carried out. Depending on data available in the reports, the early epidemic growth rate or final attack rate (AR) approach was used to estimate the basic reproduction number for that outbreak. Other recorded outbreak characteristics were the type of housing (group vs. individual). An overall estimate for R0 was computed by meta-analysis. RESULTS: Data from eight outbreaks were extracted from peer-reviewed publications. Data from two additional, non-published outbreaks was also included in the meta-analysis. A conservative estimate for R0 was 2.2 (95% confidence interval [CI] 1.9-2.5). A less conservative estimate, including outbreaks with a 100% AR for which a lower limit R0 was estimated, was 2.7 (95% CI 2.1-3.3). MAIN LIMITATIONS: Few papers describing longitudinal incidence data were found so most estimates were based on the outbreaks' final size. Several outbreaks had a 100% attack rate and could therefore only be included as a lower limit estimate in the meta-analysis. The reported result therefore may be an underestimation. CONCLUSIONS: This estimate for R0 for S. equi informs parameters for future mathematical modelling, quantifies desired preventive vaccine coverage and helps evaluate the effect of prevention strategies through future modelling studies.

The Equine Faecal Microbiota of Healthy Horses and Ponies in The Netherlands: Impact of Host and Environmental Factors.

Several studies have described the faecal microbiota of horses and the factors that influence its composition, but the variation in results is substantial. This study aimed to investigate the microbiota composition in healthy equids in The Netherlands under standard housing and management conditions and to evaluate the effect of age, gender, horse type, diet, pasture access, the season of sampling and location on it. Spontaneously produced faecal samples were collected from the stall floor of 79 healthy horses and ponies at two farms. The validity of this sampling technique was evaluated in a small pilot study including five ponies showing that the microbiota composition of faecal samples collected up to 6 h after spontaneous defaecation was similar to that of the samples collected rectally. After DNA extraction, Illumina Miseq 16S rRNA sequencing was performed to determine microbiota composition. The effect of host and environmental factors on microbiota composition were determined using several techniques (NMDS, PERMANOVA, DESeq2). Bacteroidetes was the largest phylum found in the faecal microbiota (50.1%), followed by Firmicutes (28.4%). Alpha-diversity and richness decreased significantly with increasing age. Location, age, season, horse type and pasture access had a significant effect on beta-diversity. The current study provides important baseline information on variation in faecal microbiota in healthy horses and ponies under standard housing and management conditions. These results indicate that faecal microbiota composition is affected by several horse-related and environment-related factors, and these factors should be considered in future studies of the equine faecal microbiota.

Discrepancies in the bilateral intradermal test and serum tests in atopic horses.

BACKGROUND: In equine atopic patients intradermal testing (IDT) and immunoglobulin (Ig)E serology are used frequently. There is little evidence regarding the reproducibility of the IDT and IgE serology in horses. OBJECTIVES: To compare the results of a simultaneously performed IDT on the left and right side of the neck in atopic horses, and to compare these results with allergen-specific IgE serology. ANIMALS: Ten equine patients from a university hospital population with chronic urticaria and/or pruritus. METHODS AND MATERIALS: The IDT was performed using 16 allergens and the results were evaluated after 30 min, 1, 4 and 24 h. Thirteen allergens also were analysed in duplicate with two monoclonal allergen-specific IgE enzyme-linked immunosorbent assays (ELISA). RESULTS: Good agreement (Kappa > 0.6) between left and right IDT was found only for Dermatophagoides farinae, Lepidoglyphus destructor, birch pollen mixture and perennial rye at 30 min, birch pollen mixture at 1 h, and Acarus siro and nettle and common mugwort mixture at 4 h. The bilateral comparison of the other allergens and even the same allergens at other time points showed little or no concordance between left and right IDT. The interlaboratory comparison between both ELISAs, and the comparison between the ELISAs and IDT, showed a good agreement for two of 13 allergens: D. farinae and Dermatophagoides pteronyssinus. CONCLUSIONS AND CLINICAL IMPORTANCE: Based on these preliminary data, IDT and IgE serological test results should be interpreted with great care and further studies are needed to indicate the clinical relevance of these findings.

A prospective cohort study on the acute:chronic workload ratio in relation to injuries in high level eventing horses: A comprehensive 3-year study.

In human sport science, the acute:chronic workload (ACWR) ratio is used to monitor an athlete's preparedness for competition and to assess injury risks. The aim of this study was to investigate whether acute and chronic workload calculations for external and internal loads (e.g. high-speed work distance and associated exertional effort) were associated with injury risk in elite eventing horses and to identify workloads performed by horses competing in different competition and at different fitness levels. Training load and injury data were collected from 58 international eventing horses (CCI2*-CCI5* level) over 1-3 years. A total of 94 individual competition seasons were monitored. During this period, heart rate (HR; beat/min) and GPS data were collected of all their conditional training sessions and competitions. External load was determined as the distance (m) covered at high speed (HS1 ; velocity between 6.6 and 9.5 m/s), and sprint speed (SS2 ; velocity>9.5 m/s). Internal load was calculated for HS and SS, using individualized training impulses (TRIMP3 ;AU). For internal and external workload HS and SS the acute (1-week) and chronic (4-week) workloads were calculated and ACWR4 determined. The injury data in relation to ACWR was modelled with a multilevel logistic regression. Akaike's information criterion was used for model reduction. Sixty-four soft tissue injuries were registered from a total of 2300 training sessions and competitions. External and internal workload at HS and SS were significantly affected by the year and fitness level of horses. Competition level and year significantly affected the distances covered at SS. The ACWR of high-speed distance of the present week (OR; 0.133, 95 % CI; 0.032, 0.484) and the previous week (OR 3.951, 95 % CI; 1.390, 12.498) were significantly associated with injury risk. Competition level and chronic workload had no significant effect on injuries. In agreement with findings in human athletes, acute spikes of workload in eventing horses increased the risk of injury. Evaluation of horses' workload can be used to design and effectively monitor training programs and can help to improve equine welfare by reducing injury risk.

Skin disease in donkeys (Equus asinus): a retrospective study from four veterinary schools.

BACKGROUND: Donkeys are important throughout the world as work animals and occasionally as pets or a meat source. Most descriptions of skin disease in donkeys are reported in small case series, textbooks or review articles. HYPOTHESIS/OBJECTIVES: To document skin diseases and their prevalence in donkeys and to investigate predilections for the most common conditions. ANIMALS: Case populations at four veterinary schools totalling 156 donkeys. METHODS AND MATERIALS: A retrospective study was performed by searching computerized medical records, using the key word "donkey", at the School of Veterinary Medicine, University of California, Davis (UCD). Records of donkeys from the veterinary schools in Nantes, France; Utrecht, Netherlands and Ghent, Belgium were searched in a similar manner. The time periods included in the searches varied by institution. RESULTS: At UCD, 83 of 346 (24%) of donkeys had skin disease noted in their records. The most common diagnoses were insect bite hypersensitivity, sarcoid and habronemiasis. At Nantes, 36 of 144 (25%) had skin disease and the most common diagnoses were sarcoid and superficial pyoderma. At Utrecht 23 of 143 (16%) had skin disease and the most common diagnosis was dermatophytosis. At Ghent, 14 of 320 (4%) had skin disease and the most common diagnosis was sarcoid. CONCLUSIONS AND CLINICAL IMPORTANCE: Cutaneous conditions in donkeys are common. Age, sex and breed predisposition and the most common diagnoses varied with geographical location. Clinicians should include a dermatological examination regardless of the reason for presentation.

Occurrence and molecular characteristics of ESBL/AmpC-producing Escherichia coli in faecal samples from horses in an equine clinic.

Objectives: To investigate the occurrence and characteristics of ESBL/AmpC-producing Escherichia coli in faecal samples from horses at one equine clinic in the Netherlands. Methods: A total of 91 horses, including residents and patients, were sampled. ESBL/AmpC-producing E. coli were identified by a combination disc diffusion test. Phylogenetic groups and MLST were determined. ESBL/AmpC genes were analysed using PCR and sequencing. Plasmids were characterized by transformation and PCR-based replicon typing. Subtyping of plasmids was done by plasmid MLST. Results: At least one E. coli isolate with a confirmed ESBL/AmpC gene was found in samples from 76 horses (84%). Although phylogenetic group B1 E. coli bla CTX-M-1 predominated, a diverse E. coli population was found, indicating that clonal nosocomial spread was not the only reason for the high occurrence found. MLST analysis revealed the presence of 47 E. coli STs, organized in four clusters of genetically related strains. ST10, ST641, ST1079 and ST1250 were most commonly found. With regard to the genes, bla CTX-M-1 was most prevalent ( n = 91), followed by bla CTX-M-2 ( n = 26). The most frequently found plasmid type was IncHI1, but plasmids belonging to the IncF, IncI1 and IncN groups were also identified. Conclusions: A high occurrence of ESBL-producing E. coli in faecal samples was found among horses in an equine clinic and the variety of STs, ESBL genes and plasmid types suggests nosocomial transmission. ESBL E. coli can cause difficult-to-treat infections in horses and prudent use of antimicrobials is warranted. A further assessment of the risks of transmission to persons in close contact with horses, such as caretakers or veterinarians, is crucial.

Exercise testing in Warmblood sport horses under field conditions.

Regular exercise testing in Warmblood sport horses may, as in racing, potentially help to characterise fitness indices in different disciplines and at various competition levels and assist in understanding when a horse is 'fit to compete'. In this review an overview is given of the current state of the art of exercise testing in the Olympic disciplines of eventing, show jumping and dressage, and areas for further development are defined. In event horses, a simple four-step incremental exercise test measuring heart rate (HR), lactate concentration (LA) and velocity (V) is most often used. In dressage and riding horses, a wide variety of exercise tests have been developed, including incremental exercise tests, indoor riding tests and lunging tests. In show jumping, the use of a five-step incremental exercise test and exercise tests evaluating technical skills and fatigue of the horse has been reported. The velocity at a plasma LA of 4 mmol/L (VLA4) and HR recovery during submaximal exercise intensity have been shown to be the best parameters in event horses for predicting performance and impending injuries. In riding horses, the fitness level of horses is also an important determinant of injuries. Implementation of regular exercise testing and monitoring of training sessions may have important added value in the assessment of performance ability and potential future injuries in Warmblood sport horses. However, there is an urgent need to standardise methodologies and outcome parameters in order to make results comparable.

Bluetongue, Schmallenberg - what is next? Culicoides-borne viral diseases in the 21st Century.

In the past decade, two pathogens transmitted by Culicoides biting midges (Diptera: Ceratopogonidae), bluetongue virus and Schmallenberg virus, have caused serious economic losses to the European livestock industry, most notably affecting sheep and cattle. These outbreaks of arboviral disease have highlighted large knowledge gaps on the biology and ecology of indigenous Culicoides species. With these research gaps in mind, and as a means of assessing what potential disease outbreaks to expect in the future, an international workshop was held in May 2013 at Wageningen University, The Netherlands. It brought together research groups from Belgium, France, Germany, Spain, Switzerland, United Kingdom and The Netherlands, with diverse backgrounds in vector ecology, epidemiology, entomology, virology, animal health, modelling, and genetics. Here, we report on the key findings of this workshop.

Equine sarcoidosis.

Equine sarcoidosis seems to be an emerging problem. As more horses are referred for dermatologic disease, equine sarcoidosis should be considered in any case of exfoliative and/or nodular skin disease with or without systemic involvement, including generalized granulomatous disease affecting most internal organs. Multiple breeds are affected with mares being predisposed. Affected horses are typically 3 years or older. The prognosis for generalized granulomatous disease is generally poor, whereas the prognosis for the localized cutaneous form is favorable but may require lifelong treatment.

Young Friesian horses show familial aggregation in fitness response to a 7-week performance test.

The aim of this study was to monitor the fitness level of young Friesian horses and to assess whether fitness data are predictive for final performance score and whether familial aggregation of response to training could be detected. Sixty-six young Friesian horses, the offspring of six different stallions (A, B, C, D, E and F), underwent a 7-week performance test. The horses were given a performance score for their ability for dressage (in weeks 5 and 7; 0-110 points) and were evaluated for fitness using standardised exercise tests (SETs) at the beginning (week 2, SET-I) and the end (week 6, SET-II) of the period. Heart rate (HR, beats/min) was measured in both SETs, and plasma lactate concentration (LA, mmol/L) was measured only in SET-II. Fitness of the horses improved moderately but significantly between SET-I and SET-II (P=0.015). There was a large heterogeneity in responsiveness to training; some horses were high responders, whereas others were non- or low responders. There was a familial aggregation of HR canter-1 response to training (P=0.039), while the HR of stallion C's offspring decreased significantly more than those of stallions A (P=0.09), D (P=0.013) and F (P=0.009). Horses that were reluctant to exercise did not differ in HR or LA concentrations compared to those that completed the SET, which may have been a sign of overreaching. HR had no predictive value for the performance score, but horses that did not reach the anaerobic threshold in SET-II scored significantly better (73.8 ± 5.6 points) than horses that did not (69.9 ± 5.9 points, P=0.025). The findings demonstrate for the first time in the horse a familial aggregation of HR response to training, as has been reported previously in humans. Familial aggregation suggests a genetic influence on the effect of training on fitness in horses. HR could not predict final performance score, but LA concentrations during SET-II were predictive.

Cloning and expression of candidate allergens from Culicoides obsoletus for diagnosis of insect bite hypersensitivity in horses.

Insect bite hypersensitivity (IBH) is an IgE-mediated (Type I) hypersensitivity reaction induced by allergens from biting midges of the Culicoides spp. The aim of the present study was to identify, clone and express recombinant allergens from C. obsoletus, the main species found feeding on horses in the Netherlands, by sequence homology searches on the C. obsoletus specific RNA database, with previously described allergens from C. nubeculosus and C. sonorensis. BLAST searches with these described allergens resulted in similarity hits with 7 genes coding for C. obsoletus allergens. These allergens were expressed as hexahistidine tagged recombinant proteins in E. coli. Allergens were termed Cul o 1-Cul o 7. A maltase (Cul o 1) plus Cul s 1 (maltase of C. sonorensis) were additionally expressed in insect cells using the baculovirus expression system to compare homologous allergens from different species produced with different expression systems in diagnostic in vitro and in vivo tests. We demonstrate that IBH affected horses in the Netherlands show higher IgE levels to Cul o 1 than to Cul s 1, as determined by an IgE ELISA. Furthermore, we show that Cul o 1 produced in E. coli is at least as suitable for in vitro diagnosis of IBH affected horses as Cul o 1 produced in the baculovirus/insect cell expression system. The resulting proteins were evaluated for their ability to discriminate IBH affected and healthy horses by ELISA and intradermal testing. The frequency of positive test results by ELISA within IBH affected horses ranged from 38% to 67% for the different allergens. When results of IgE-binding to Cul o 1-Cul o 7 were combined the test had a sensitivity of 92% and specificity of 85%. The capability of the allergens to induce Type I hypersensitivity reaction in IBH affected horses was demonstrated by an intradermal test. The results show that E. coli expressed recombinant allergens from C. obsoletus are valuable tools to determine the allergen specific sensitisation profile (component resolved diagnosis) in horses with IBH in countries were C. obsoletus is the most abundant species and may facilitate in the development of future immunotherapy.

Equine sarcoidosis: clinical signs, diagnosis, treatment and outcome of 22 cases.

BACKGROUND: Equine sarcoidosis may present as a generalized or localized exfoliative dermatitis and/or as a granulomatous inflammation of multiple organs. OBJECTIVES: To report the clinical signs, diagnosis, treatment and outcome for 22 horses with histologically confirmed sarcoidosis. ANIMALS: Twenty-two horses of different breeds, between 3 and 17 years of age. METHODS: Diagnosis was based on clinical signs and histopathological findings. RESULTS: The following three forms of equine sarcoidosis were identified: generalized (13.6%), partially generalized (18.2%) and localized (68.2%). High-dose systemic corticosteroids were used as the initial treatment in all three forms, followed by lower doses for a variable number of weeks. No local treatment was prescribed. Case outcome was variable; one of three cases of generalized sarcoidosis was euthanized immediately and the remaining two were euthanized after 2-3 months of unsuccessful treatment; all four cases of partially generalized sarcoidosis deteriorated despite treatment and were euthanized after 3.5-12 months; two cases with localized disease showed no response or insufficient response to treatment and were euthanized; four cases recovered fully with or without treatment, and one showed partial recovery without treatment; and eight cases improved whilst receiving prednisolone but required continuous low doses to maintain remission. CONCLUSIONS AND CLINICAL IMPORTANCE: Recognition of the different forms of sarcoidosis based on history, clinical appearance and histopathology assisted in making an informed choice between treatment and euthanasia and prevented unnecessary local treatment. Equine sarcoidosis should be included in the differential diagnosis of a localized exfoliative dermatitis of unknown origin.

A prospective study on fitness, workload and reasons for premature training ends and temporary training breaks in two groups of riding horses.

Little is known about wastage in riding horses and the factors like fitness and workload that may reduce injuries and maximise welfare. To evaluate fitness, workload and reasons for premature training ends (PTEs) and temporary training breaks (TTBs) during a nine week training period, two groups of riding horses were used: Group A consisting of 58 horses used for student equitation courses (32 with training prior to admission and 26 without) and Group B consisting of 26 horses owned by two riding schools (school-I and school-II). To assess fitness, all horses performed a standardised exercise test (SET) at the start (SET-I) and end of the training period (SET-II) measuring heart rate (HR bpm) and speed (m/s). In addition, all horses were monitored daily during the training period for their health and workload. In Group A, trained horses had significantly lower HRs in SET-I (P=0.05) compared to untrained horses and in SET-II, trained horses tended to have lower HRs than untrained horses, though this was not statistically significant (P=0.057). During the training period all horses received an identical workload. A total of 19.0% of Group A horses ended the training period prematurely for veterinary reasons (PTEV); of those untrained horses had earlier a PTEV in the training period (after 2.8 ± 1.3 weeks) than trained horses (after 4.1 ± 1.5 weeks, P=0.030). In Group B, school-I and school-II horses did not differ significantly in fitness level nor in workload. More school-II horses ended the training period prematurely for veterinary reasons (n=7; 70%) compared to school-I horses (n=4; 25%, P=0.032), although seven (63.6%) of these horses were still continuously used in riding lessons. In both groups (A and B), small injuries (without a temporary training break) were significantly associated with premature training ends for veterinary reasons later on: in Group A small injuries preceded 27.3% of the PTEVs (P=0.005) and in Group B small injuries preceded 54.5% of the PTEVs (P=0.030). In conclusion, as all horses in each subgroup had the same workload, the occurrence of PTEV seemed not associated with the workload. In Group A horses, level of fitness seems to be an important factor for the point in time injuries will occur during the training period. In all horses, injuries were more likely when a temporary training break was not taken following seemingly minor injuries. Since a lot of injured Group B horses were used in riding lessons against veterinary advice, this may indicate that riding school owners have different perception on welfare and if true this may cause serious welfare problems.

Sublingual administration of detomidine in horses: sedative effect, analgesia and detection time.

A single dose of 40 µg/kg bodyweight (BW) of oromucosal detomidine gel was administered sublingually to 10 healthy Dutch Warmblood mares aged 7 ± 4 years (mean ± SD) and BW 580 ± 69 kg. Blood and urine samples were collected before and for 8 days following administration and evaluated qualitatively in an FEI Reference Laboratory and quantitatively in a research laboratory. Clinical effects were evaluated at baseline and for 24 h after administration. Sedation was determined using head height and scores of reaction to auditory and mixed auditory/sensory stimuli. Mechanical nociceptive thresholds (MNTs) were assessed using pressure algometry to evaluate analgesia. Heart rate (HR) was measured and ataxia scored. All horses were considered negative for detomidine in blood samples by 48 h post-administration and in urine by 60 h. These results indicated that a safe withdrawal time for detomidine oromucosal gel may be 72 h following a single sublingual administration of 40 µg/kgBW. Decreases in HR and head height were maximal at 40 and 60 min post-administration, respectively. The maximal decrease in response to stimuli was observed at 100 min. Ataxia was maximal at 60 min. At 40 and 80 min MNTs were significantly increased compared to baseline. All parameters, except the MNTs of two locations, which were decreased, returned to baseline values within 24 h post-administration.

Schmallenberg virus in Culicoides spp. biting midges, the Netherlands, 2011.

To determine which species of Culicoides biting midges carry Schmallenberg virus (SBV), we assayed midges collected in the Netherlands during autumn 2011. SBV RNA was found in C. scoticus, C. obsoletus sensu stricto, and C. chiopterus. The high proportion of infected midges might explain the rapid spread of SBV throughout Europe.

Prevalence of the causative agents of equine piroplasmosis in the South West of The Netherlands and the identification of two autochthonous clinical Theileria equi infections.

Equine piroplasmosis (EP) has not been considered indigenous in The Netherlands. However, following the detection of an apparently indigenous subclinical Babesia caballi infection in a horse on Schouwen-Duiveland (an island in the Zeeland Province), a survey was undertaken between May and September 2010 to assess the prevalence of the causative agents of EP in the South-West of The Netherlands. Blood samples from 300 randomly selected horses were tested for specific antibodies against Theileria equi and B. caballi using an indirect fluorescence antibody test (IFAT), and for parasite DNA using a specific polymerase chain reaction combined with reverse line blotting (PCR-RLB). Twelve of the horses (4%) were seropositive for EP. Of these, nine (75%) were positive (titre⩾1:160) for B. caballi alone and three (25%) were also positive for T. equi. PCR-RLB detected T. equi DNA in five horses (1.6%), two of which were seronegative. Four (1.3%) of the positive horses (three positive for T. equi and one for both B. caballi and T. equi) were considered truly indigenous. During the study, two indigenous ponies from a farm situated outside the sampling area were diagnosed with acute clinical piroplasmosis characterized by severe anaemia and pyrexia. Blood smears showed T. equi - like inclusions in red blood cells, and T. equi infection was confirmed in both ponies by PCR-RLB. The initial subclinical B. caballi infection, the survey results and the two acute clinical EP cases confirmed the autochthonous transmission of B. caballi and T. equi infections in The Netherlands.

The influence of challenging objects and horse-rider matching on heart rate, heart rate variability and behavioural score in riding horses.

A good horse-rider 'match' is important in the context of equine welfare. To quantify the influence of repetition and horse-rider matching on the stress of horses encountering challenging objects, 16 Warmblood horses were ridden in a test-setting on three occasions. On each occasion the horse was ridden by a different rider and was challenged by three objects (A-C). Heart rate (HR), heart rate variability (HRV) of horse and rider, and behaviour score (BS) of the horse were obtained for each object and as a total for each test. The horse-rider interaction was evaluated with each combination and assessed as 'matching' or 'mismatching', and the horses were categorised as 'compliant', 'partly-compliant' or 'non-compliant'. Horses exhibited a decreased HR (P=0.015) and a decreased BS (P=0.004) within and across different tests. 'Matching' horse-rider combinations exhibited less stress as indicated by reduced HR ('match' 69±10 vs. 'mismatch' 72±9, P=0.001) and BS ('match' 1.9±1.1 vs. 'mismatch' 3.8±1.4, P=0.017) of the horse. 'Compliant' (68±8, P<0.001) and 'partly-compliant' (71±9, P=0.002) horses had significantly lower HR than 'non-compliant' (75±9) animals. The findings of the study indicate that HR and BS measurements support a subjective 'match' diagnosis and HR measurement may be a valuable tool in assessing horse compliance.

Culicoides species associated with sheep in the Netherlands and the effect of a permethrin insecticide.

Culicoides spp. act as vectors for a number of viral diseases of animals including bluetongue in sheep. The aims of this study were to determine: (1) which Culicoides spp. are associated with sheep in The Netherlands; (2) the time of the day when they are most active; and (3) the effect of treatment of animals with a permethrin insecticide. Two pairs of sheep were each housed within mosquito tents of either one or two layers of netting and all trapped Culicoides spp. were identified microscopically. For the permethrin insecticide study, one of two pairs of sheep was treated with 3.6% permethrin and all animals were housed in tents of similar design. Of the 6210 midges captured, 54.1% were identified as C. chiopterus and 42.7% as C. obsoletus. C. imicola was not identified. The average insect feeding rate was 35-40% and midge activity was greatest around sunset. Permethrin treatment reduced the number of midges captured by 50% and also resulted in a decrease in the percentage of midges that had fed. The findings provide useful information on the behaviour and distribution of Culicoides spp. that will facilitate the development of appropriate control strategies to minimise the risk of insect-vector borne virus diseases such as bluetongue.

Experimental infection with neuropathogenic equid herpesvirus type 1 (EHV-1) in adult horses.

Equid herpesvirus type 1 (EHV-1)-associated myeloencephalopathy (EHM) may follow an infection with the virus in horses. This study tested three hypotheses: (1) a large inhaled dose of a neuropathogenic EHV-1 strain would induce a cell-associated viraemia in all infected horses; (2) neurological disease will only occur in viraemic horses, and (3) the cerebrospinal fluid (CSF) composition following EHV-1 viraemia will be an indicator for EHM. Four EHV-1 seronegative horses were inoculated with EHV-1 by inhalation. Three developed clinical signs of neurological disease, which were mild in two horses and lacking typical EHM histopathological findings, but moderately severe in the third horse. This latter animal was the only one found to be viraemic, with xanthochromic CSF and spinal cord histopathology findings characteristic of EHM. This study showed that cell-associated viraemia was not guaranteed, despite a large-dose inoculation with EHV-1, yet viraemia was probably a pre-requisite for subsequent development of EHM. The histopathological changes used to confirm EHM may be predicted from CSF analysis.