Infectious disease surveillance of apparently healthy horses at a multi-day show using a novel nanoscale real-time PCR panel

In the United States, horses are used for a variety of purposes including recreation, exhibition, and racing. As farm, performance, and companion animals, horses are a unique species from a zoonotic disease risk perspective, and the risks of subclinical infections spreading among horses can pose challenges. Using a nanoscale real-time PCR platform, we investigated the prevalence of 14 enteric pathogens, 11 Escherichia coli genes, and 9 respiratory pathogens in fecal samples from 97 apparently healthy horses at a multi-day horse event. In addition, sugar flotation test was performed for fecal parasites. E. coli f17 was commonly detected, prevalent in 59% of horses, followed closely by Streptococcus equi subsp. zooepidemicus (55%). Additional pathogens recognized included betacoronavirus, Campylobacter jejuni, Cryptosporidium sp., E. coli O157, equine adenovirus 1, equine rhinitis B virus, and others. The use of PCR data may overestimate the true prevalence of these pathogens but provides a sensitive overview of common pathogens present in healthy horses. Our results prompt the continued need for practical biosecurity measures at horse shows, both to protect individuals interacting with these horses and to minimize transmission among horses.     

Myofibroblastic fibrosarcoma with multifocal osseous metaplasia at the site of equine influenza vaccination

We describe a fibrosarcoma in a 12-year-old Quarterhorse × Arabian gelding as a sequela to equine influenza vaccination. Shortly after the second vaccination, swelling at the site was noticed by the owner and it continued to increase in size over the following 6 months. Biopsy of the mass indicated a fibrosarcoma had developed at the vaccination site. It was approximately 20 cm in diameter and elevated well above the level of the skin. There was no clinical evidence of metastases to the lungs or local lymph nodes. Surgical resection of the mass was performed and the wound healed by first and second intention. Histopathological examination and immunohistochemical staining confirmed a myofibroblastic fibrosarcoma with multifocal osseous metaplasia. To the authors' knowledge, this is the first equine case of a vaccine-associated fibrosarcoma.     

Antibody responses after vaccination against equine influenza in the Republic of Korea in 2013

In this study, antibody responses after equine influenza vaccination were investigated among 1,098 horses in Korea using the hemagglutination inhibition (HI) assay. The equine influenza viruses, A/equine/South Africa/4/03 (H3N8) and A/equine/Wildeshausen/1/08 (H3N8), were used as antigens in the HI assay. The mean seropositive rates were 91.7% (geometric mean antibody levels (GMT), 56.8) and 93.6% (GMT, 105.2) for A/equine/South Africa/4/03 and A/equine/Wildeshausen/1/08, respectively. Yearlings and two-year-olds in training exhibited lower positive rates (68.1% (GMT, 14) and 61.7% (GMT, 11.9), respectively, with different antigens) than average. Horses two years old or younger may require more attention in vaccination against equine influenza according to the vaccination regime, because they could be a target of the equine influenza virus.     

Effect of vaccination against leptospirosis on the frequency, days to recurrence and progression of disease in horses with equine recurrent uveitis

OBJECTIVE: To evaluate the effect of vaccination against leptospirosis on frequency and days to recurrence of uveitis and progression of disease in horses with equine recurrent uveitis (ERU). ANIMALS: Forty-one horses with ERU. PROCEDURE: Horses were randomly assigned to experimental (vaccinated) or control groups. Vaccine containing six serovars of Leptospira or placebo was administered, an ophthalmic examination performed and blood samples drawn on days 0, 28, 180 and 365. Antibody titers were measured against each serovar. Recurrence of uveitis was verified by ophthalmic examination. Results of the initial and final ophthalmic examinations were compared and progression of disease defined as an increase in extent of synechiae, or development of new or progression of an existing cataract. RESULTS: Vaccination increased the average geometric mean serum antibody titer from 225 on day 0, to 4077 and 593 on days 28 and 180, respectively. After the second vaccination, days to first recurrence was significantly longer (median 126 days; range 24-231 days) when compared with controls (median 86 days; range 14-192 days, P=0.04). Recurrence of ERU was observed among 7/20 (35%) vaccinated horses and 12/21 (57%) controls; however, this difference was not statistically significant (P=0.061, OR 0.25, 95% CI 0.06, 1.07). More horses in the experimental group 13/20 (65%) experienced progression of disease when compared with controls 12/21(57%); however, this difference was statistically nonsignificant (P=0.35). CONCLUSIONS: Vaccine significantly increased days to recurrence, but failed to slow the progression of disease. These data do not support the use of vaccination against leptospirosis as adjunct therapy for the routine treatment of horses with ERU.     

Suppression of behavioural and physiological oestrus in the mare by vaccination against GnRH

OBJECTIVE: To examine the immunogenicity of an equine immunocontraceptive vaccine and its efficacy in controlling hormone-related behaviour. DESIGN: A total of 24 mares at two sites in Australia were vaccinated with an immunocontraceptive vaccine comprising gonadotrophin releasing hormone (GnRH) conjugated to a carrier protein in immunostimulating complex as an adjuvant. Twelve animals at each site received a placebo of adjuvant alone and served as controls for seasonal oestrus, hormonal and behaviour patterns. Animals were observed for injection site reactions, ovarian and follicular activity, and serum levels of antibody, 17beta-oestradiol and progesterone in the weeks following vaccination. Mares were also examined for oestrous behaviour by teasing with a stallion. RESULTS: All mares responded to vaccination. Two weeks following the second vaccination there was a peak in antibody response to GnRH that declined gradually over the following weeks. Commensurate with the elevated anti-GnRH antibody there was a marked effect on ovarian activity with a reduction in 17beta-oestradiol and progesterone levels in the 24 vaccinated mares. There was also a reduction of oestrus-related behaviour as determined by a teaser stallion. This effect lasted a minimum of 3 months and correlated with the initial level of antibody response. CONCLUSION: Following a conventional two-dose immunisation regime this commercially available equine immunocontraceptive vaccine was effective at inhibiting oestrous behaviour for at least 3 months. This vaccine has a high level of safety since there were no significant local reactions nor were there any adverse systemic responses to vaccination.     

Serological responses to two serovar‐independent ELISA antigens of Actinobacillus pleuropneumoniae in Australian commercial pig herds

Objective To compare serological responses in pig herds classified as low or high risk for disease caused by Actinobacillus pleuropneumoniae, using two ELISA tests based on serovar‐independent antigens. Procedure Cross‐sectional sampling was undertaken in 13 commercial herds, the clinical and slaughter histories of which indicated either freedom from (n = 5) or prior confirmed cases of A. pleuropneumoniae (n = 8). In nine herds, approximately 40 pigs each were sampled at 4, 8, 12, 16 and 20 weeks. Three of the remaining four herds were sampled between 6 and 30 weeks of age, and the last was sampled only prior to slaughter, at approximately 24 weeks. Sera were tested in ELISA based on two antigens common among A. pleuropneumoniae serovars: a 39‐kDa outer membrane protein and a recombinant ApxIVA‐N terminus protein. Results Sampling of 1 and 5 to 6‐month‐old pigs provided the most useful information on herd status. The 39‐kDa ELISA was sensitive in detecting infected herds, but had evidence of cross‐reactivity with high seroreactivity rates in older pigs in some low‐risk herds. The ApxIVA‐N ELISA was less seroreactive in high‐risk herds and had higher specificity in low‐risk herds. Conclusion ELISA based on the 39‐kDa subunit are of limited use, because of possible cross‐reactivity, but a high negative predictive value may be useful for risk assessment in suspect herds. Maternal antibody to ApxIVA‐N may be of value in detecting high‐risk herds, but 5% of 4‐week‐old pigs in low‐risk herds were also seropositive in this assay.     

Rinderpest vaccination responses among calves and adult cattle in Uganda monitored using ELISA assay

Responses to the Plowright Rinderpest vaccine by 43 calves and 70 adult cattle in Uganda in 1990, through the production of IgG antibodies, were monitored for 4 weeks using the ELISA assay. 80% of the calves were seronegative before vaccination of which 32% remained seronegative and 68% subsequently seroconverted at 2 weeks postvaccination. 20% of the calves were seropositive before vaccination but registered a decline during the 2nd and 4th weeks postvaccination. 50% of adult Ankole cattle seroconverted after 3 weeks postvaccination, while the other 50% of them, which were seropositive before vaccination, showed a decline in seronegative levels during the first 2 weeks postvaccination, but increased again at the 4th week. 90% of Friesian adult cattle were seronegative before vaccination; however, they all seroconverted within the 2nd week of postvaccination. The other 10% remained seropositive, with a slight decline of antibody levels during the 4 weeks after vaccination.     

Preliminary evaluation of the effects of a 1:1 inspiratory-to-expiratory ratio in anesthetized and ventilated horses

ObjectiveTo describe some cardiorespiratory effects of an inspiratory-to-expiratory (IE) ratio of 1:1 compared with 1:3 in ventilated horses in dorsal recumbency.Study designRandomized crossover experimental study.AnimalsA total of eight anesthetized horses, with 444 (330–485) kg body weight [median (range)].MethodsHorses were ventilated in dorsal recumbency with a tidal volume of 15 mL kg^–1 and a respiratory rate of 8 breaths minute^–1, and IE ratios of 1:1 (IE1:1) and 1:3 (IE1:3) in random order, each for 25 minutes after applying a recruitment maneuver. Spirometry, arterial blood gases and dobutamine requirements were recorded in all horses during each treatment. Electrical impedance tomography (EIT) data were recorded in four horses and used to generate functional EIT variables including regional ventilation delay index (RVD), a measure of speed of lung inflation, and end-expiratory lung impedance (EELI), an indicator of functional residual capacity (FRC). Results were assessed with linear and generalized linear mixed models.ResultsCompared with treatment IE1:3, horses ventilated with treatment IE1:1 had higher mean airway pressures and respiratory system compliance (p < 0.014), while peak, end-inspiratory and driving airway pressures were lower (p < 0.001). No differences in arterial oxygenation or dobutamine requirements were observed. PaCO₂ was lower in treatment IE1:1 (p = 0.039). Treatment IE1:1 resulted in lower RVD (p < 0.002) and higher EELI (p = 0.023) than treatment IE1:3.Conclusions and clinical relevanceThese results suggest that IE1:1 improved respiratory system mechanics and alveolar ventilation compared with IE1:3, whereas oxygenation and dobutamine requirements were unchanged, although differences were small. In the four horses where EIT was evaluated, IE1:1 led to a faster inflation rate of the lung, possibly the result of increased FRC. The clinical relevance of these findings needs to be further investigated.     

Immune response following a vaccination campaign against rabies in dogs from northwestern Spain

A cross-sectional study was carried out to determine the seroprevalence of antibodies to the rabies virus in 156 vaccinated dogs from two provinces in the Castilla y León Autonomous Community (northwest Spain). An obligatory anti-rabies programme is currently in place in this region. Seroprevalence was established by an indirect enzyme-linked immunosorbent assay. Of the 156 animals tested, 91 (58.3%) were positive (titres of 0.5 IU ml⁻¹ or above). However, Soria province showed a significantly higher seroprevalence (77.1%) than Leon province (50%). Age, sex, habitat and use were evaluated with regard to the response obtained after vaccination: no significant differences were discovered for any of these factors. However, guard and herding dogs in León province tended to have lower seroprevalence than dogs not used in these ways. In general, there is a limited response to the vaccination programme in dogs from Castilla y León—especially in León province.     

Age at first start and racing career of a cohort of Australian Standardbred horses

OBJECTIVE Compare the career profiles of a cohort of Standardbred horses that first raced as 2-year-olds with those that started their racing careers at a later age. METHOD Retrospective analysis of the racing records of all foals born in New South Wales in the 2000 foaling season. RESULTS The career records of 999 horses were analysed. Almost half (43.9%) first raced as 2-year-olds and one-third (33.9%) as 3-year-olds. The median career duration for horses that first raced as 2-year-olds was 2.93 years (interquartile range (IQR) 2.70-3.16), which was significantly greater than the median for horses that first raced at 3, 4 or ≥5 years old (P < 0.001). Males, and horses that first raced as 2-year-olds, earned significantly more prize money than females or horses that started racing aged ≥3 years (P < 0.001). The population median number of career starts was 28.0 (IQR 8-64). Males, and horses that first raced as 2-year-olds, had significantly more career starts than females or horses that started racing aged ≥3 years (P < 0.001). CONCLUSION This study found no evidence suggesting that racing as a 2-year-old had a deleterious effect on a horse's racing career.     

Detection of antibodies against Anaplasma marginale in milk using a recombinant MSP5 indirect ELISA

An indirect enzyme linked immunosorbent assay (iELISA) for diagnosis of anaplasmosis using undiluted individual milk samples from dairy cows was developed. The recombinant 19 kDa major surface protein 5 (rMSP5) of Anaplasma marginale was used as antigen. A monoclonal antibody against bovine IgG1 conjugated with peroxidase and the chromogen 3,5,3',5'-tetramethylbenzidine were used in the test. Strong and weak, positive and negative milk samples were set up as reference controls. Results were expressed as percentage of positivity (PP) contrasting with the strongest positive control. The test was evaluated in two groups (G1 and G2) of lactating dairy cows from herds located in A. marginale non-endemic areas of Argentina. The infection status of both groups, G1 (n=128) sampled after anaplasmosis outbreak, and G2 (n=216) free of anaplasmosis was established by polymerase chain reaction (PCR). Serum samples of cows from G1 and G2 were analyzed by card agglutination test (CAT) and competitive ELISA (cELISA), while the novel iELISA was evaluated in their corresponding milk samples. At a cutoff of 42 PP, the ELISA has 98% sensitivity and 95% specificity. A significant difference (P<0.0001) was found between the mean PP value of negative samples from G1 (17.4+/-14.9), and G2 (8.6+/-7.1). The agreement and kappa (kappa) value between iELISA and PCR was 96%, kappa=0.919; between iELISA and CAT was 97%, kappa=0.880; and between iELISA and cELISA was 97%, kappa=0.899. These results strongly support the usefulness of iELISA to detect A. marginale antibodies in milk. Additional studies are necessary to define the ability of the milk iELISA to detect not only acutely infected, but also carrier cattle.     

Physiological responses of horses to 24 hours of transportation using a commercial van during summer conditions

Fifteen mature horses (mares, n = 6); geldings, n = 9) were used to assess the physiological responses of 24 h of transport in a commercial van under California summer conditions. The study was conducted on four consecutive days, and data were collected on d 1 and d 2 to obtain baseline values and to determine any diurnal variation in the individual measurements. Travel commenced on d 3 at 0800 for 24 h, with a total of 1,622 km traveled. Blood samples were collected at 0800, 1100, 1400, 2000, and 0200 each day. Horses were weighed and rectal temperatures recorded at 0800 each day and at 2000 each day except d 3. Body weight, rectal temperature, serum cortisol, serum lactate, and white blood cell (WBC) counts exhibited diurnal variation (P = .0001) on d 1 and d 2. Body weight immediately after unloading showed a 6% loss. At 24 h following transit, a 3% deficiency in body weight loss remained. The WBC counts showed a progressive increase with duration of travel and peaked at the termination of transport. Dehydration measures of hematocrit and total protein increased during transport and returned to baseline during the posttransport period. Serum concentrations of lactate, creatine kinase and aspartate aminotransferase increased during transport and in the early posttransit period, but returned to baseline values at the conclusion of the 24-h posttransport period. Glucose concentration increased with the initiation of transport and did not decrease to baseline concentration at the end of the 24-h posttransport period. Plasma cortisol and neutrophil:lymphocyte ratio increased with duration of transit and returned to baseline during the posttransport period. These data clearly showed physiological responses of horses undergoing 24 h of transport including changes in muscle metabolism, stress indices, dehydration and immune parameters, and body weight. These responses may increase disease susceptibility and influence energy availability for athletic performance following long-term transport of horses.     

Persistence of virus-neutralizing antibodies in horses inoculated with two doses of a live equine herpesvirus type 1 vaccine with different vaccination intervals

The antibody response in horses inoculated with 2 doses of a live equine herpesvirus type 1 vaccine with different vaccination intervals (1 to 3 months) was evaluated with regard to the persistence of virus-neutralizing (VN) antibodies. The durations for which the geometric mean VN titers were maintained significantly higher than those before the first vaccination (P<0.05) were up to 5 months in horses that received the vaccination with a 1-month interval (n=17) and 7 months for those that received it with a 2-month (n=17) or 3-month interval (n=14 or 17). The vaccination program with the 2-month interval was the most effective in maintaining VN antibodies for a long duration with the smallest gap of antibody decline between the first and second vaccinations.     

Preliminary evaluation of a behaviour-based system for assessment of post-operative pain in horses following arthroscopic surgery

Objective To develop a method for objective assessment of equine post‐operative pain. Study design Prospective nonblinded clinical study. Animals Twelve adult horses: Group 1 (G1, n = 6), admitted for arthroscopy (under general anaesthesia, with multimodal analgesia); Group 2 (G2, n = 6), ‘pain free’ controls. Materials and methods Horses were filmed continuously (CVI, time‐lapse video recorder) over 72 hours, from 24 hours pre‐surgery (PS) to 48 hours post‐recovery (PR) (G1), and over 24 hours (G2). Activity budgets were determined from 24 to 0 hours PS, 0–24 and 24–48 hours PR (G1) and for 24 hours (G2). Using direct observation (DO), active behaviours and postures were recorded at set time points PS and PR (G1) and at two time points (morning/evening) (G2). Heart rate (HR) and respiration rate (RR) were recorded simultaneously. Statistical analysis investigated within‐group and between‐group time‐related changes in behaviour, HR and RR. Results There was no difference in HR or RR between G1 and G2 at any time point. Anaesthetic ‘hangover’ and hunger‐related activity modulated behaviour from 0 to 6 hours PR, when abnormal postures and locomotion occurred in all G1 horses, but no G2 horses. Compared with G1 (0–24 hours PR), G2 spent more time eating (t = ?3.34, p < 0.01), more time at the front of the stable (t = ?2.42, p < 0.05), and less time exhibiting ‘abnormal’ behaviour (U = 56, p < 0.01). Comparing PS and PR behaviour, G1 spent less time exploring from 24 to 48 hours PR (t = 3.49, p < 0.05), less time in sternal recumbency (t = ?3.8, p < 0.05) and less time moving (t = 3.19, p < 0.05). Horses tended (p < 0.07) to spend less time positioned in the front of the stable PR (less from 24 to 48 hours PR than from 0 to 24 hours PR). Comparing PR (evening) behaviour, G2 spent more time with head above withers (U = 21.5, p < 0.01), and ears forwards (U = 22, p < 0.01). G1 showed time‐related changes (all p < 0.05) in time with lower lip tense (S = 15.8), eating (S = 17.08) and with head positioned above withers (S = 18.04). No differences in event behaviours were observed between G1 and G2. Within G1, only olfactory behaviour varied significantly with time (S = 14.52, p < 0.05). Conclusions Changes in equine behaviour suggestive of post‐operative discomfort were identified using both DO and CVI. Analysis of activity budgets may be a more sensitive method of identifying behavioural changes indicative of equine discomfort than repeated DO of specific events and postures.     

Effect of adjuvants on in ovo vaccination against Newcastle disease on hatchability,performance and antibody titres in commercial pullets

This study was conducted to investigate the efficacy of in ovo administration of aluminium hydroxide (AH) and/or mannan oligosaccharide (MOS) adjuvants along with lentogenic VG/GA strain‐Avinew to alleviate the embryonic pathogenicity of Newcastle disease virus. Six hundred and thirty fertilized Bovans eggs were divided into nine groups of 70 each incubated in a commercial hatchery and administered with eight types of in ovo injections in a factorial design of 2 × 2 × 2 including with/without AH, MOS and Newcastle disease vaccine (NDV), and one uninjected group on day 18 of incubation. Hatchability was higher in the eggs received MOS and/or AH adjuvants plus NDV compared those injected with NDV alone which confirmed the attenuation of NDV. However, the average daily feed intake and feed conversion ratio of pullets hatched from NDV‐injected eggs were significantly reduced, but did not affect growth performance during 0‐42 days of age. The performance of pullets hatched from eggs injected with AH, MOS or their mixture with NDV was not significantly different during all growth periods. Pullets from MOS + vaccine injected eggs had significantly higher antibody titres against NDV compared to those hatched from either injected with saline or uninjected on d 28 (p < .05). In addition, AH plus vaccine and MOS significantly improved total anti‐SRBC and IgG respectively. Histological observation revealed that injection of MOS adjuvant into eggs led to increase crypt depth, whereas AH injection caused a reduction in villus surface area of jejunum in chicks on d 14 post‐hatch. It is concluded that in ovo MOS injection as compared to AH may be more effective to attenuate the embryonic pathogenicity of in ovo NDV injection.