PCR-RLFP characterization of Leishmania spp. in domestic animals from the south-western border of Brazil.

The aim of this study was to characterize Leishmania spp. from canine and feline samples using Polymerase Chain Reaction (PCR)- Restriction Fragment Length Polymorphism (RFLP). It was conducted in the southern region of Brazil, located at border crossings to Argentina and Uruguay. Samples were collected from 116 dogs (Canis lupus familiaris) and 89 cats (Felis catus). The PCR was performed to screen for an LT1 fragment from kinetoplast DNA (kDNA) target gene, and positive samples were subjected to a second PCR for an internal transcribed spacers (ITS1) region from ribosomal DNA (rDNA) target. RFLP was performed using the Haemophilus aegyptius (HAE III) restriction endonuclease (Fermentas ®). Positive samples by PCR ITS1 were sequenced and deposited in NCBI GenBank, and a phylogenetic analysis was developed. We found that 12.9% (15/116) of the samples from dogs were positive. All the 89 cat samples were negative. Positive samples were tested against Leishmania reference strains presenting different patterns in PCR-RFLP, and these samples showed bands denoting similarity to the standard species of Leishmania infantum, proven through sequencing and phylogenetic analysis. The RFLP technique, alone, was shown to be feasible for practical application and confirmation of the involved Leishmania spp.

Risk and protective factors of Leishmaniasis in the rural area of the western border region of Rio Grande do Sul, Brazil.

BACKGROUND: The Leishmaniases are on the top of the global list of tropical neglected diseases. The number of infected dogs in South America is estimated in millions and correlated to disease cases in humans, especially in Brazil. Equines may get infected too and can play a role in the epidemiological chain. Thus, the aim of the present study was to evaluate risk and protective factors of leishmaniasis in rural areas of the western border region of Rio Grande do Sul state, Brazil by Leishmania spp. protozoa molecular detection and serological evaluation (ELISA) in equine and canine blood samples. This work included nine farms around the city of Uruguaiana. Epidemiologic information regarding farm characteristics and biologic material collection of canine (22) and equine (91), totalizing 113 samples was collected. The polymerase chain reaction (PCR) technique was used to detect Leishmania spp. in biological samples. Variables related to the farm were collected and evaluated through descriptive analysis followed by chi-square test and a logistic regression analysis. RESULTS: Nineteen positive samples (19/113 - 16,81%) were detected, being 18 equines and 1 canine, in six of the nine farms included in the study. No animal showed clinical signs of the disease. According to the variables analyzed, when compared each characteristic separately, the presence of abundant vegetation and poor hygiene demonstrated to be risk factors to Leishmania infection in rural areas. The logistic regression showed that excellent general hygiene, proximity to the weir and trimmed grass were protective factors (p=0.038, p=0.001 and p=0.014, respectively). Having excellent hygiene represents a 70% lower chance of getting infected, keeping the grass cut protects the animal by more than 90% and the proximity of the weir represents a protective factor of 96%. CONCLUSIONS: The presence of Leishmania infection in the western border region of Rio Grande do Sul was 16,81% and it was influenced by farm characteristics. The role of the excellent general hygiene as a protective factor is extremely relevant in the leishmaniases prevention.

Influence of morphometric parameters on the size of collateral ligaments of the distal interphalangeal joint of equines.

The physiological measurements of collateral ligaments of distal interphalangeal joint (CL-DIPJ) differ in the literature. The factors that influence these differences are not well described. The aims of this study are to compare CL-DIPJ sizes in equines with different withers height, as well as to correlate body weight and hoof size to the size of these ligaments. In total, 52 horses were used in the study. They were divided into two groups according to wither height: Group 1 (G1) - 21 animals with up to 147 cm - and group 2 (G2) - 21 animals with greater than 148 cm. CL-DIPJ was ultrasonographically measured in order to find the mean of dorso-palmar (DPD) and latero-medial (LMD) diameters and the cross-sectional area (CSA). Hoof width and length were measured, and the results were used to calculate the hoof surface area. Withers height and body weight were also measured. Groups were statistically compared by Student's t test and Pearson's correlation application to each group. Groups were different in body size, HS and CL-DIPJ size when p < .05, except for the LMD of the medial collateral ligament of left thoracic limb. Withers height and body weight did not show significant correlations to CL-DIPJ size in G1, but they had little influence on the size of CL-DIPJ on G2. No correlation between the hoof size and the CL-DIPJ was noted in any of the two groups. In conclusion, the CL-DIPJ were larger in taller horses and their sizes were correlated to their height and weight, but they were not correlated to hoof size.

Parâmetros sanguíneos de potros da raça Crioula / Blood parameters of crioulo breed horse

Com o propósito de estabelecer valores de hematócrito, proteínas plasmáticas totais, fibrinogênio, creatina quinase , aspartato transferase e lactato em potros da raça Crioula, do nascimento até os dois anos, utilizaram-se amostras sanguíneas de 85 animais, divididos pela estratificação etária: Grupo 1 (G1) Até 15 dias de vida (n=70); grupo 2 (G2), entre 16 dias até um mês (n=67); grupo 3 (G3), entre 1 e 3 meses (n=75); grupo 4 (G4), entre 3 e 6 meses (n=64); grupo 5 (G5), entre 6 e 9 meses (n=59); grupo 6 (G6), entre 9 e 18 meses (n=39); e grupo 7 (G7), entre 18 meses até 2 anos (n=17). Foi realizado estudo estatístico entre os grupos pela análise de variância unidirecional (one-wayANOVA), complementada pelo teste de Tukey. Para comparação das médias entre os sexos utilizou-se o teste t de Student. O hematócrito foi significativamente mais elevado até os 90 dias e nas fêmeas do G7. Para proteínas plasmáticas totais, notou-se aumento significativo nos grupos 3, 4, 6 e 7. Os valores de fibrinogênio foram maiores no G1. A CK apresentou maior concentração no G5 e a AST no G7. A AST assumiu valores semelhantes dos 30 dias até os 2 anos. A concentração de lactato foi mais elevada no G3. Conclui-se que na interpretação dos exames laboratoriais de potros da raça crioula, o gênero não interfere significativamente nos resultados, porém a idade deve ser considerada devido à ocorrência de variações relevantes. Recomenda-se que para interpretação sejam consultadas tabelas específicas para cada análise.

Plasma levels of hematocrit, total plasma protein, fibrinogen, creatine phosphokinase, aspartate transferase, and lactate were analyzed in blood samples of 85 Crioula breed foals, from birth to two years of age. The animals were divided into age groups: G1 (up to 15 days of age; n=70), G2 (from 16 days to one month of age; n=67), G3 (between one and three months of age; n=75), G4 (between three and six months of age; n=64), G5 (between six and nine months of age; n=59), G6 (between nine and 18 months of age; n=39), and G7 (between 18 months and two years of age; n=17). These groups were statistically analyzed by one-way variance analysis (ANOVA) and Tukey’s test. Male and female means were compared by Student’s t-test. Hematocrit levels were significantly higher up to 90 days of age and in G7 females. Total plasma proteins increased significantly in groups 3, 4, 6, and 7. The highest fibrinogen levels were found in G1. Yet for creatine phosphokinase, the highest concentrations were detected in G5, whereas those of aspartate aminotransferase in G7. The levels of this enzyme remained similar from 30 days to two years of age. Lactate concentrations were higher in G3. We concluded that the sex of the animal had no significant effect on laboratory test interpretations. By contrast, the age of the animal should be considered since relevant variations were observed with time. Nevertheless, specific tables for each analysis should be consulted for interpretation of results.

Blood parameters of crioulo breed horse / Parâmetros sanguíneos de potros da raça Crioula

Plasma levels of hematocrit, total plasma protein, fibrinogen, creatine phosphokinase, aspartate transferase, and lactate were analyzed in blood samples of 85 Crioula breed foals, from birth to two years of age. The animals were divided into age groups: G1 (up to 15 days of age; n=70), G2 (from 16 days to one month of age; n=67), G3 (between one and three months of age; n=75), G4 (between three and six months of age; n=64), G5 (between six and nine months of age; n=59), G6 (between nine and 18 months of age; n=39), and G7 (between 18 months and two years of age; n=17). These groups were statistically analyzed by one-way variance analysis (ANOVA) and Tukey's test. Male and female means were compared by Student's t-test. Hematocrit levels were significantly higher up to 90 days of age and in G7 females. Total plasma proteins increased significantly in groups 3, 4, 6, and 7. The highest fibrinogen levels were found in G1. Yet for creatine phosphokinase, the highest concentrations were detected in G5, whereas those of aspartate aminotransferase in G7. The levels of this enzyme remained similar from 30 days to two years of age. Lactate concentrations were higher in G3. We concluded that the sex of the animal had no significant effect on laboratory test interpretations. By contrast, the age of the animal should be considered since relevant variations were observed with time. Nevertheless, specific tables for each analysis should be consulted for interpretation of results.

Com o propósito de estabelecer valores de hematócrito, proteínas plasmáticas totais, fibrinogênio, creatina quinase , aspartato transferase e lactato em potros da raça Crioula, do nascimento até os dois anos, utilizaram-se amostras sanguíneas de 85 animais, divididos pela estratificação etária: Grupo 1 (G1) Até 15 dias de vida (n=70); grupo 2 (G2), entre 16 dias até um mês (n=67); grupo 3 (G3), entre 1 e 3 meses (n=75); grupo 4 (G4), entre 3 e 6 meses (n=64); grupo 5 (G5), entre 6 e 9 meses (n=59); grupo 6 (G6), entre 9 e 18 meses (n=39); e grupo 7 (G7), entre 18 meses até 2 anos (n=17). Foi realizado estudo estatístico entre os grupos pela análise de variância unidirecional (one-wayANOVA), complementada pelo teste de Tukey. Para comparação das médias entre os sexos utilizou-se o teste t de Student. O hematócrito foi significativamente mais elevado até os 90 dias e nas fêmeas do G7. Para proteínas plasmáticas totais, notou-se aumento significativo nos grupos 3, 4, 6 e 7. Os valores de fibrinogênio foram maiores no G1. A CK apresentou maior concentração no G5 e a AST no G7. A AST assumiu valores semelhantes dos 30 dias até os 2 anos. A concentração de lactato foi mais elevada no G3. Conclui-se que na interpretação dos exames laboratoriais de potros da raça crioula, o gênero não interfere significativamente nos resultados, porém a idade deve ser considerada devido à ocorrência de variações relevantes. Recomenda-se que para interpretação sejam consultadas tabelas específicas para cada análise.

Ceftiofur Side Effect in a Mare-Case Report.

Adverse drug reactions in horses are rare. The antimicrobials are in the list of the most common drugs associated with reaction in horses. The aim of this report is to describe the clinical presentation of an adverse drug reaction after the intramuscular administration of ceftiofur hydrochloride. A 5-year-old crioulo broodmare at one day postpartum presented signs of difficulty to walk, positive pulse in the four limbs, and heart rate of 80 beats per minute (bpm), with a history of fighting with another mare. The clinical suspicions were rhabdomyolysis and laminitis. Initially a dose of flunixin meglumine was administrated intravenous to prevent an endotoxemia. On the same day, the blood collection showed an elevated number of white blood cells, predominantly neutrophils and monocytes. An antimicrobial treatment with ceftiofur hydrochloride at a dose of 2.2 mg/kg intramuscular was applied. At the first day, the mare does not show signs of drug side effect. However, 24 hours later, in the second application, the patient presents incoordination, dizziness, and loss of equilibrium. At the same time, dexamethasone was administrated. The signs were reverted, and the mare was normal after the reaction. The treatment with ceftiofur was changed to enrofloxacin, and the animal had a complete recovery. These drug reactions are not common in the routine of equine clinical practitioners, but they cause a great preoccupation for the owner and the veterinarian.

Digit innervation of the thoracic limb of Criollo horses: Anatomical description and consequences to perineural blocks.

Criollo horse breeding is an important economic activity in South America. Because of their athletic performance, these animals tend to show great incidence of musculoskeletal disorders, many of them diagnosed by means of perineural blocks. However, incorrect interpretation of these blocks may be due to anatomical differences in nerve distribution. The objective of this study was to describe the innervation of the digit region of thoracic limbs in Criollo horses, in order to improve the interpretation of tests for claudication diagnosis based on nerve block. Thirty thoracic limbs from Criollo horses were dissected. It could be observed that in 90% of the limbs, dorsal branches of the palmar nerve originated proximally to the proximal sesamoid bone. In 93% of the cases, the palmar digital nerve and the dorsal branches communicated; in 87% of the cases, communication between branches of the dorsal branch was observed; and in 27% (8/30) of the limbs, the palmar metacarpal nerve and the dorsal branch presented communications. None of the specimens showed complete symmetry in the distribution of nerves in contralateral limbs. The high frequency of communication between the nerves may be a particularity of the Criollo breed that may interfere with the interpretation of perineural blocks. Based on the anatomical position, it may be inferred that divergent results in Criollo horses may occur when abaxial sesamoid nerve block is used. Palmar digital nerve block may be less influenced by these variations, provided it is performed as distal as possible from the ungular cartilage.

Molecular detection of Leishmania spp. in Brazilian cross-border south region mammalian hosts.

Visceral leishmaniasis is an endemic zoonotic disease identified especially in developing territories. Brazil's northeast, southeast and midwest have been endemic for several years; currently, the infection is spreading to the south. Dogs are the main reservoirs; however, other mammal species have also been infected. Herein, we have identified the infecting Leishmania species in dogs and horses from the south of Brazil, a new outbreak of the infection. Blood samples were collected in the urban area of Uruguaiana city. DNA was extracted from peripheral blood, kinetoplast DNA (kDNA) and ribosomal DNA (rDNA) fragments were obtained by polymerase chain reaction (PCR) and sequenced. Out of 123 samples, 25 of them (14 dogs and 11 horses) were positive for Leishmania spp. Sequence alignment and phylogenetic analysis revealed that the kDNA in positive samples was similar to four species previously reported: L. infantum/L. chagasi, L. donovani, L. major. Despite kDNA minicircles regions are very useful due to high sensitivity to Leishmania spp. DNA detection, the sequence polymorphism among minicircles can be an obstacle to interspecific differentiation. Our results suggest that these strains are circulating in Brazil south region cross-border and indicate the susceptibility of new outbreak for visceral leishmaniasis infection in horses domiciled in endemic region for canine and human visceral leishmaniasis.

Assessment of Leishmania infantum infection in equine populations in a canine visceral leishmaniosis transmission area.

BACKGROUND: Leishmaniosis, zoonosis that produces significant public health impacts, is caused by Leishmania infantum. Canines are the main domestic reservoir and, besides humans, other species of mammals could be infected when living in endemic areas. In this study, we detected equine Leishmania infantum infections in a canine visceral leishmaniosis transmission area and evaluated the clinical, haematological, biochemical and oxidative stress disorders. This study was conducted in Uruguaiana, Rio Grande do Sul, south of Brazil. Peripheral blood samples were collected from 124 animals (98 horses and 26 dogs) of both genders and several breeds after they underwent general and dermatologic examinations. RESULTS: Twenty five Leishmania infantum infected animals (20.16%), 14 horses and 11 dogs were detected by PCR (Polymerase Chain Reaction) amplification of kinetoplast DNA regions with 96% homology to Leishmania infantum (GenBank Accession No. L 19877.1). The clinical and haematological alterations of infected equines were skin lesions, nodules, lymphadenopathy, decreased levels in red blood cells and haematocrit (p < 0.05) and increase in urea serum concentration (p < 0.05), while CVL presented a decrease in red blood cells counts (p < 0.05), increase in lymphocytes (p < 0.05), and decrease in neutrophil-lymphocyte ratio (p < 0.05). Oxidative stress markers of plasma protein carbonyl and plasma lipid peroxidation were not statistically significant (p > 0.05) in both species. CONCLUSIONS: To our knowledge, this has been the first leishmaniosis equine survey performed in south of Brazil, caused by Leishmania infantum that were able to initially identify haematological and biochemical changes in the species, even in asymptomatic animals. We present evidence supporting those findings of haematological and biochemical changes could be related to infection. Surprisingly, the clinical manifestations of equine infection were similar to those found in canine visceral leishmaniosis. The equine population could be play an important role in the cycle of leishmaniosis in south Brazil and consequently indicates a great risk of public health. This evaluation of infected animals is important to establish the clinical and laboratory parameters involved in the disease progression.

Casearia sylvestris na permeabilidade gástrica à sacarose em equinos submetidos a protocolo de indução de úlcera gástrica / Casearia sylvestris on gastric permeability to sucrose in horses submitted to gastric ulcer induction protocol

Estudos em animais de laboratório sugerem um efeito antiulcerogênico do extrato de Casearia sylvestris. Esse extrato ainda não foi estudado para a profilaxia e/ou o tratamento de úlceras gástricas em equinos. Para avaliar a influência do extrato de C. sylvestris na permeabilidade gástrica à sacarose, seis equinos adultos foram submetidos a modelo de indução de úlceras gástricas. Os animais foram submetidos ao teste de permeabilidade à sacarose antes e ao término do protocolo de restrição alimentar intermitente, para detecção de ulceração gástrica. Durante os sete dias da indução, os animais foram submetidos a tratamentos diários via sondagem nasogástrica com extrato de C. sylvestris (9mg kg-1 de peso corpóreo) ou veículo (ágar). Após intervalo de 32 dias em piquete, para permitir a cicatrização das úlceras induzidas, cada animal foi submetido novamente ao protocolo de indução de úlcera gástrica, e os tratamentos foram alternados. Dessa forma, cada animal foi submetido a ambos os tratamentos em períodos distintos. A concentração de sacarose na urina foi determinada para cada amostra obtida, por cromatografia líquida de alto desempenho e detecção amperométrica pulsátil. Não foram observadas alterações nos exames clínicos e hemogramas. O tratamento com o extrato de C. sylvestris evitou o aumento da concentração de sacarose urinária (P<0,05) quando comparado ao veículo, sugerindo um efeito antiulcerogênico gástrico em equinos. Estudos mais amplos incluindo gastroscopia são necessários para avaliar a possibilidade de usar o extrato para a profilaxia e/ou o tratamento das úlceras gástricas em equinos.

Studies on laboratory animals suggest an antiulcergonic effect of Casearia sylvestris extract. This extract has not yet been tested for the prophylaxis and/or treatment of gastric ulcers in horses. In order to evaluate the influence of C. sylvestris extract on gastric sucrose permeability, six adult horses underwent a protocol of gastric ulcer induction. All animals were submitted to sucrose permeability testing before and at the end of gastric ulcers induction by intermittent feed deprivation, for detection of gastric ulcers. During the seven days of induction, the animals were submitted to daily treatment by nasogastric tubing with C. sylvestris extract (9mg kg-1 b.w.) or vehicle (Agar). After 32 days of pasture turnout, in order to allow healing of induced ulcers, each animal underwent a second induction protocol, in which treatments were alternated. By this manner, each animal was submitted to both treatments in distinct periods. The urine sucrose concentration was determined for each sample obtained, by high performance liquid chromatography and pulsed amperometric detection. No alterations in clinical examination and hemograms were detected. Treatment with C. sylvestris extract avoided the increase on urine sucrose concentration (P<0.05) when compared to the vehicle, suggesting an antiulcer preventive effect for equine gastric ulcers. More extensive studies including gastroscopy are necessary to evaluate the possibility of employing this extract for the prophylaxis and/or treatment of gastric ulcers in horses.

Enterorrafias em plano aposicional convencionale com adesivo à base de cianoacrilato no cólon descendente de eqüinos / Conventional appositional pattern and cyanoacrylate tissue adhesive in descending colon enterorrhaphy in horses

Em eqüinos, as enterotomias no cólon descendente säo necessárias para remover enterólitos, corpos estranhos e material alimentar compactado que näo podem ser removidos por técnicas conservativas. Este segmento intestinal possui um suprimento sangüíneo pobre, além de predisposiçäo a complicaçöes pós-operatórias relacionadas à contaminaçäo bacteriana. Assim, este estudo foi conduzido com o objetivo de efetuar avaliaçöes clínicas e anatomopatológicas do emprego de adesivo tecidual à base de cianoacrilato no cólon descendente de eqüinos, comparando-o a uma técnica de sutura aposicional convencional. A intervençäo cirúrgica foi realizada em 15 animais posicionados em decúbito lateral direito, através de laparotomia pelo flanco esquerdo, sob anestesia geral inalatória. Após a exteriorizaçäo do cólon descendente, foram realizadas duas enterotomias de cinco centímetros de extensäo cada, distanciadas 20cm uma da outra. Os animais foram aleatoriamente distribuídos em cinco tempos de observaçäo de três animais cada e sacrificados aos três, sete, 14, 35 e 70 dias de pós-operatório. O adesivo butil-2-cianoacrilato e o fio de poliglactina 910 mostraram-se igualmente apropriados para uso em enterorrafias desse órgäo, sendo que o fio de poliglactina 910 provocou inflamaçäo inicial mais intensa que o cianoacrilato e, mais tardiamente, ambos causaram inflamaçäo granulomatosa do tipo corpo estranho observada, primeiramente, junto ao fio de poliglactina 910 (7º dia) e, a partir do 35º dia, ao redor do cianoacrilato