Serologic survey of West Nile virus in horses from Central-West, Northeast and Southeast Brazil

Since the emergence of West Nile virus (WNV) in North America in 1999, there have been several reports of WNV activity in Central and South American countries. To detect WNV in Brazil, we performed a serological survey of horses from different regions of Brazil using recombinant peptides from domain III of WNV. Positive samples were validated with the neutralisation test. Our results showed that of 79 ELISA-positive horses, nine expressed WNV-specific neutralising antibodies. Eight of the infected horses were from the state of Mato Grosso do Sul and one was from the state of Paraíba. Our results provide additional evidence for the emergence of WNV in Brazil and for its circulation in multiple regions of the country.

Senecio spp. POISONING OF HORSES IN SOUTHERN BRAZIL

Cases of seneciosis in horses occurring in four farms in the state of Santa Catarina and in another in the state of Rio Grande do Sul, southern Brazil, are reported. S. brasiliensis or S. oxyphyllus or both were detected in four of the five properties. Five horses (one on each property) were necropsied, and tissues for histopathological examination were collected from four horses. Neurological signs, such as depression, ataxia, aimeless walking, circling, head pressing, faulty prehension of food, dysphagia and blindness were consistently observed. Other signs included inappetence, loss of weight, colic, subcutaneous edema, icterus and photodermatitis. At necropsy the livers were firmer and darker than normal and had accentuation of lobular pattern. Edema of the mesentery and ascites were observed in one horse. Main histopathological changes consisted of hepatic chiefly periportal fibrosis, hepatomegalocytosis and biliary hyperplasia. Marked cholestasis and morphological evidence of hepatic encephalopathy were seen respectively in the liver and brain of one of the horses.

Serodiagnosis of Babesia equi in horses submitted to exercise stress

A complement fixation test (CFT), performed in microtitre plates, based upon the use of crude antigenic preparation of Babesia equi was adapted for the detection of antibodies in serum of infected horses. The indirect fluorescent antibody test (IFAT) and enzyme-linked immunosorbent assay (ELISA) were also used for the immunodiagnosis of B. equi. Serum samples from 15 apparently healthy horses, previously conditioned to a high-speed equine treadmill, were taken before and after exercise. All the samples analyzed were positive for B. equi infection. There were no significant differences (P<0.01) between these 3 tests, or the condition of rest or stress. The combined use of CFT and IFAT or ELISA should be recommended in order to enable veterinary services to more efficiently prevent introduction of infected horses into disease-free areas.

Development of IgY antibodies in chickens and IgG in rabbits immunized against proteins of Pythium insidiosum isolated from horses in the state of Rio de Janeiro

Pythiosis is caused by Pythium insidiosum and the occurrence of disease in horses was described in the North and Northwest State of Rio de Janeiro, Brazil. The disease was described in cattle, sheep, humans, and horses in different states and regions across the country. This paper describes the development of IgY and IgG polyclonal antibodies, in chicken and rabbits, respectively against proteins extracted from kunkers and hyphae of P. insidiosum from affected horses. The proteins were recognized by chicken, rabbit and horse antibodies by immunodiffusion and Western blot against majority bands of 27 and 43 KDa, and titrated by ELISA. The antibodies IgY developed by the first time against Brazilian strains of P. insidiosum may represent a valuable tool in the detection of antigens of the pathogen and contribute to further studies aimed at immunotherapy and knowledge about this disease in endemic areas in Rio de Janeiro and in Brazil.

Ophthalmic alterations in horses with leptospirosis by serovar Icterohaemorrhagiae in Rio de Janeiro, Brazil

The objective of the study was to determine the association between clinical ophthalmic alterations and seroreactivity to leptospirosis by serovar Icterohaemorrhagiae in horses in Rio de Janeiro, Brazil. A total of 199 horses were studied. A microscopic agglutination test (MAT) was used to detect specific anti-Leptospira antibodies in blood serum. A total of 107 (53.8%) horses were seroreactive (titres > 200); 54 had high (> 800) titres, of which 44 were against serovar Icterohaemorrhagiae. Forty-two out of these 44, plus 40 seronegative horses (titers < 100) were given detailed ophthalmic examinations. Epiphora, ocular congestion, blepharospasm, photophobia, and peripapillary focal depigmentation were the most frequent alterations in seroreactive horses. Most ocular alterations were significantly more frequent in seroreactive horses. Horses seroreactive for leptospirosis (serovar Icterohaemorrhagiae) had a significantly higher prevalence of ophthalmic alterations than seronegative horses, providing additional evidence for an association between leptospirosis and equine uveitis.

Peritoneal fluid changes in horses subjected to small colon distension

Intestinal devitalization in cases of small colon obstruction may be difficult to detect based only in clinical signs. The purpose was to serially evaluate blood and peritoneal fluid of horses subjected to small colon distension. Seventeen adult horses were allotted in three groups. In the small colon-distended group (DG, n=7) a surgically-implanted latex balloon was inflated to promote intraluminal small colon distension. In the shamoperated group (SG, n=5), the balloon was implanted but not inflated, and no surgery was done in the control group (CG, n=5). Blood and peritoneal fluid were sampled before and after (6 samples with a 30-minute interval) intestinal obstruction for cytological and biochemical analyses. No significant changes in clinical signs occurred within groups or across time during the experimental period. There were no statistical differences among SG and SG groups in hematologic and blood chemistry variables. Although total protein concentration and lactate dehydrogenase (LDH) activity in peritoneal fluid remained most of the time within reference values during the experimental period in all groups, increases from baseline values were detected in SG and DG groups. Such increases occurred earlier, progressively and with greater magnitude in the DG when compared with the SG (P<0.05). Increases from baselines values were also observed in total nucleated cells and neutrophils counts in the DG (P<0.05). In conclusion, distension of the equine small colon induced progressive subtle increases in total protein and LDH concentrations in the peritoneal fluid during the first hours. Serial evaluation of these variables in peritoneal fluid may be useful for early detection of intestinal devitalization in clinical cases of equine small colon obstruction.

Pulmonary hemorrhage in horses seropositive to leptospirosis

The aim of this study was to verify the occurrence and severity of pulmonary hemorrhage (PH) in horses seropositive or not to leptospirosis. A total of 84 training horses were tested for anti-Leptospira antibodies and submitted to endoscopy of the upper airways 30-60 minutes after exercise. Forty-nine (46.4%) horses were seropositive (titer>100) to serovar Copenhageni. Seventeen horses (20.2%) presented PH in different grades, significantly more commonly observed in seropositives (34.7%) than seronegatives (8.6%) (p<0.05), and also more severe in the first. Additionally, a seropositive horse is 4.26 times more likely (OR) to present PH than a seronegative one. Fifteen days after specific antibiotic treatment for leptospirosis a significant improvement in grade of hemorrhage was observed. Therefore it is suggested that leptospirosis may potentiate the severity of PH and that specific antibiotic treatment directed towards leptospirosis may help to control the disease.

Hydrocortisone decreases apoptosis in jejunum of horses subjected to experimental ischemia and reperfusion

In order to evaluate the effect of hydrocortisone on apoptosis in the jejunum of horses subjected to ischemia and reperfusion, ten horses were paired and grouped into two groups - treated (n=5) and non treated (n=5). Segments of the jejunum were used as controls (C), or as venous ischemia (VIsc), which were subjected to 2h of ischemia followed by 2 or 12h of reperfusion. C samples were collected at time zero (prior to ischemia) and VIsc samples were collected at 2h of ischemia and at 2 and 12h of reperfusion. TUNEL positive apoptotic cells were counted in 10 microscopical fields in deep mucosa from each horse throughout the time course. After 12h of reperfusion, the number of apoptotic cells in treated group were significantly lower than in untreated animals, indicating that hydrocortisone inhibits apoptosis. These results indicate that hydrocortisone has a beneficial effects favoring the maintenance of jejunal integrity in horses with ischemia and reperfusion injuries by preventing apoptotic cell death.

Platelet activation: ultrastructure and morphometry in platelet-rich plasma of horses

This study was conducted to investigate the activation ability of the platelet-rich plasma (PRP) by pharmacological agents, as well as to verify the need or not of this activation for therapeutic use. The PRP was obtained from four healthy crossbred geldings aged 13 to 16 years (15±1years), and was processed for observation and quantification of the platelet morphology by using the transmission electron microscopy. All PRP samples were activated with 10% calcium chloride (CaCl2) solution, pure bovine thrombin or associated with CaCl2. The control (pure PRP) was not pharmacologically activated. In the pure PRP samples, 49% of the platelets were classified as state of activation uncertain, 41% as resting, 9% as fully activated and 1% as irreversibly damaged. Treatment with 10% CaCl2 provided a distribution of 54% platelets in state of activation uncertain, 24% as fully activated, 20% as resting, and 2% as irreversibly damaged. The platelet morphology of the bovine thrombin treated samples did not fit into classification adopted, as showing irregular shape with emission of large filamentous pseudopods, appearance of ruptured and whole granules in the remaining cytoplasm and extracellular environment. There was effect of the treatment on the platelet morphology (P=0.03). The 10% CaCl2 is an adequate platelet-activating agent. However, in cases the use of PRP under its liquid form is necessary, the use of pure PRP is recommended, since besides presenting an adequate percentage of fully activated platelets it also has significant amount of the resting type, which can be activated by substances found in the injured tissue.

Do hematologic constituents really increase due to endurance exercise in horses?

The aim of this study was to evaluate the behavior of blood constituents in a group of horses that successfully completed long endurance rides in tropical conditions. Jugular vein puncture was done to collect blood before, during and after rides. Data were analyzed using a mathematic approach, based on the hematocrit and blood volume where the percentual change in plasma volume was used to correct the values of each variable analyzed. Significance was inferred when P<0.05. The proposed mathematical model to assess blood constituents concentrations allowed the observation of a different pattern of the variables behavior, pointing out that the approach followed by the authors could be more sensitive than ones that did not take this routine. In conclusion, the method used in this study enabled to monitor the physiological processes that actually occur during endurance effort in tropical conditions.

Corrective shoeing in horses: analysis of the adaptation period to the new condition imposed

Corrective shoeing is a method commonly used to alter the locomotion pattern in animals or as therapy for various disorders of the locomotive system of horses. However, to date, there have been no scientific studies discussing the period during which animals adapt to this type of intervention. The goal of this study was to evaluate the horseshoe adaptation period with the toe or heel elevated by six degrees at 0, 48, and 96 hours after each type of shoeing. For this analysis, the horses were recorded while walking on a treadmill. Stride length and gait qualitative analyzes were performed using Dvideow software. The level of significance adopted was 5%. In the present study, there was no significant difference between the evaluation times; elevating the toe or heel by six degrees do not generates discomfort during locomotion, therefore, horses are able to return to a regular exercise or training routine immediately after shoeing.

Low dose of dichloroacetate infusion reduces blood lactate after submaximal exercise in horses

The acute administration of an indirect activator of the enzyme pyruvate dehydrogenase (PDH) in human athletes causes a reduction in blood lactate level during and after exercise. A single IV dose (2.5m.kg-1) of dichloroacetate (DCA) was administered before a submaximal incremental exercise test (IET) with five velocity steps, from 5.0 m.s-1 for 1 min to 6.0, 6.5, 7.0 and 7.5m.s-1 every 30s in four untrained mares. The blood collections were done in the period after exercise, at times 1, 3, 5, 10, 15 and 20 min. Blood lactate and glucose (mM) were determined electro-enzymatically utilizing a YSI 2300 automated analyzer. There was a 15.3% decrease in mean total blood lactate determined from the values obtained at all assessment times in both trials after the exercise. There was a decrease in blood lactate 1, 3, 5, 10, 15 and 20 min after exercise for the mares that received prior DCA treatment, with respective mean values of 6.31±0.90 vs 5.81±0.50, 6.45±1.19 vs 5.58±1.06, 6.07±1.56 vs 5.26±1.12, 4.88±1.61 vs 3.95±1.00, 3.66±1.41 vs 2.86±0.75 and 2.75±0.51 vs 2.04±0.30. There was no difference in glucose concentrations. By means of linear regression analysis, V140, V160, V180 and V200 were determined (velocity at which the rate heart is 140, 160, 180, and 200 beats/minute, respectively). The velocities related to heart rate did not differ, indicating that there was no ergogenic effect, but prior administration of a relatively low dose of DCA in mares reduced lactatemia after an IET.

Serial superficial digital flexor tendon biopsies for diagnosing and monitoring collagenase-induced tendonitis in horses

The purpose of this investigation was to demonstrate the feasibility of a biopsy technique by performing serial evaluations of tissue samples of the forelimb superficial digital flexor tendon (SDFT) in healthy horses and in horses subjected to superficial digital flexor tendonitis induction. Eight adult horses were evaluated in two different phases (P), control (P1) and tendonitis-induced (P2). At P1, the horses were subjected to five SDFT biopsies of the left forelimb, with 24 hours (h) of interval. Clinical and ultrasonographic (US) examinations were performed immediately before the tendonitis induction, 24 and 48 h after the procedure. The biopsied tendon tissues were analyzed through histology. P2 evaluations were carried out three months later, when the same horses were subjected to tendonitis induction by injection of bacterial collagenase into the right forelimb SDFT. P2 clinical and US evaluations, and SDFT biopsies were performed before, and after injury induction at the following time intervals: after 24, 48, 72 and 96 h, and after 15, 30, 60, 90, 120 and 150 days. The biopsy technique has proven to be easy and quick to perform and yielded good tendon samples for histological evaluation. At P1 the horses did not show signs of localised inflammation, pain or lameness, neither SDFT US alterations after biopsies, showing that the biopsy procedure per se did not risk tendon integrity. Therefore, this procedure is feasible for routine tendon histological evaluations. The P2 findings demonstrate a relation between the US and histology evaluations concerning induced tendonitis evolution. However, the clinical signs of tendonitis poorly reflected the microscopic tissue condition, indicating that clinical presentation is not a reliable parameter for monitoring injury development. The presented method of biopsying SDFT tissue in horses enables the serial collection of material for histological analysis causing no clinical signs and tendon damage seen by US images. Therefore, this technique allows tendonitis to be monitored and can be considered an excellent tool in protocols for evaluating SDFT injury.

Concentrações de ferro, cobre, zinco e manganês em equinos da raça Puro-sangue Lusitano, antes e após exercício

O presente trabalho objetivou determinar a atividade sérica dos microminerais ferro, cobre, zinco e manganês em 30 equinos atletas da raça Puro-sangue Lusitano (PSL), antes e depois de exercícios, atestados por avaliações clínicas e laboratoriais. Amostras de sangue foram colhidas, antes e imediatamente após 20 minutos de exercício físico de trote e galope suaves realizados em pista de areia, para a realização das dosagens dos microminerais. A concentração dos microelementos foi determinada por espectrofotometria de absorção atômica. Observou-se que após o exercício, a atividade sérica de ferro não sofreu variações (P=0,2365), enquanto os valores de cobre se elevaram significativamente após o treinamento (P<0,001). Já o zinco e o manganês diminuíram após o exercício (P<0,001). Pode-se concluir que o exercício físico de curta duração pode gerar sudorese capaz de alterar as concentrações séricas de ferro, cobre, zinco e manganês em cavalos atletas da raça Puro-sangue Lusitano.

Metabolic evaluation of Crioulo horses participating in competitions of 750 km

The Crioulo breed of horses performs in one of the most physically demanding equestrian competitions, the Marcha de Resistência, which is a contest in which the horses run 750 km in 15 days. The study's aim was to characterize the metabolic responses during this period. We evaluated eleven Crioulo horses in the competition, specifically, two males and nine females. Blood samples were collected 24 hours before the contest and on the 4th, 9th, 11th, 14th and 15th days of competition. We evaluated CK, AST, LDH, glucose, lactate, urea, creatinine, sodium, potassium, chloride, magnesium, total calcium, ionized calcium, total protein, hematocrit and the white blood cell count. At the end of the competition, the mean values of serum AST were 1151±358 IU/ L the mean LDH values were 7418±1695 IU/L and CK was 13,867±3998UI /L. There was a significant increase in urea, creatinine and lactate (p<0.0001). A decrease in the mean values of chloride, sodium, potassium, and total and ionized calcium was observed (p≤0.0002). An evaluation of the total leukocytes and segmented neutrophils (p≤0.0002) revealed their increased values, and decreased values were observed for hematocrit, plasma protein and total lymphocytes (p≤0.0003). The values of glucose, on average, remained constant. Based on these data, we conclude that the Marcha de Resistência competition necessitated a high muscular demand and the depletion of energy and electrolytes, suggesting an inflammatory process in the animals evaluated.