Comparison of three protocols to preserve Leptospira spp. in cat urine for efficient DNA extraction and PCR amplification

Background: The pathogenic leptospira infection in mammalian species can cause a range of acute or chronic manifestations and may result in a carrier state. Previous studies have suggested that cats were resistant to acute leptospirosis however, the description of some clinical cases suggests that Leptospira spp. may also be pathogenic to this species. Recentstudies have shown that leptospires may be shed in the urine of infected cats. Endogenous substances present in urine mayinhibit PCR and allow leptospires to evade detection. This study aims to compare three protocols for sample processingto optimize the detection of pathogenic leptospires in cat urine.Materials, Methods & Results: Three protocols to optimize the detection of pathogenic leptospires in cat urine were tested.Aliquots of standard concentration of L. interrogans serovar Canicola culture were added to urine samples to achieveconcentrations of 1×105 to 1×102 leptospires/mL for each protocol. In protocols A and B the urine was neutralized by theaddition of phosphate-buffered saline (PBS), pH 7.4, in a proportion of 1 PBS: 2.5 urine (v/v). In protocol A, PBS wasadded to neutralize the urine pH for the leptospiral organisms immediately after addition of leptospires. In protocol B,PBS was added just before DNA extraction. In protocol C, no PBS was added. DNA extraction was performed at 4, 24and 48 h after addition of the leptospires using a modified protocol. Samples were incubated at 37ºC for 10 min. Sampleswere then centrifuged (850 g) for 15 min, at 25ºC. The supernatants were transferred to another tube, and the pellets werediscarded. The supernatants were centrifuged (16060 g) for 20 min at 4ºC. The supernatants were then discarded, and thepellets resuspended and washed with 1000 µL of PBS. All the samples were centrifuged at 16060 g for an additional 20min at 25ºC. The supernatants were discarded and the pellets were resuspended in 100 µL of PBS and incubated at 94ºCfor...(AU)

Comparison of three protocols to preserve Leptospira spp. in cat urine for efficient DNA extraction and PCR amplification

Background: The pathogenic leptospira infection in mammalian species can cause a range of acute or chronic manifestations and may result in a carrier state. Previous studies have suggested that cats were resistant to acute leptospirosis however, the description of some clinical cases suggests that Leptospira spp. may also be pathogenic to this species. Recentstudies have shown that leptospires may be shed in the urine of infected cats. Endogenous substances present in urine mayinhibit PCR and allow leptospires to evade detection. This study aims to compare three protocols for sample processingto optimize the detection of pathogenic leptospires in cat urine.Materials, Methods & Results: Three protocols to optimize the detection of pathogenic leptospires in cat urine were tested.Aliquots of standard concentration of L. interrogans serovar Canicola culture were added to urine samples to achieveconcentrations of 1×105 to 1×102 leptospires/mL for each protocol. In protocols A and B the urine was neutralized by theaddition of phosphate-buffered saline (PBS), pH 7.4, in a proportion of 1 PBS: 2.5 urine (v/v). In protocol A, PBS wasadded to neutralize the urine pH for the leptospiral organisms immediately after addition of leptospires. In protocol B,PBS was added just before DNA extraction. In protocol C, no PBS was added. DNA extraction was performed at 4, 24and 48 h after addition of the leptospires using a modified protocol. Samples were incubated at 37ºC for 10 min. Sampleswere then centrifuged (850 g) for 15 min, at 25ºC. The supernatants were transferred to another tube, and the pellets werediscarded. The supernatants were centrifuged (16060 g) for 20 min at 4ºC. The supernatants were then discarded, and thepellets resuspended and washed with 1000 µL of PBS. All the samples were centrifuged at 16060 g for an additional 20min at 25ºC. The supernatants were discarded and the pellets were resuspended in 100 µL of PBS and incubated at 94ºCfor...

Arterial thromboembolism secondary to subaortic stenosis in a persian kitten

Background: Aortic stenosis refers to several types of anatomic and functional obstructions of the left ventricular outflow tract. In small animals, this anomaly is most commonly documented as either a congenital lesion or an obstruction that develops soon after birth. It is a hereditary disease, caused by a dominant autosomal gene and modifying genes that interfere with phenotype expression. Even though aortic thromboembolism may be a potential complication in cats with cardiac diseases, aortic stenosis is deemed rare in that species. In this paper, we report an unusual case of a Persian kitten with aortic stenosis that eventually developed arterial thromboembolism.Case: A 7-month-old Persian kitten (3.1 kg) was admitted to a Veterinary Teaching Hospital with a history of hindlimbs paralysis over the past 48 h, as well as excessive vocalization. Also, the cat presented with inappetence, adipsia, urine incontinence, and hematuria. On physical examination, we observed hypothermia and cold paws. The footpads were cyanotic and there were no palpable femoral pulses. Cardiac auscultation disclosed a regular fast pace (200 bpm) and a grade 2/6 murmur heard best over the left cardiac base, but irradiating to the contralateral hemithorax. Prothrombin time and activated partial thromboplastin time were within the normal reference range, but the complete blood count showed microcytosis, lymphopenia, and hyperproteinemia. Also, elevated alanine aminotransferase and BUN were identified. On the echocardiogram, we observed a subvalvular aortic stenosis, which resulted in concentric remodeling of the left ventricle and a mild left atrial dilation. The stenotic lesion was classified as mild. Also, the thoracic radiography unveiled cardiomegaly. The recommended therapy included atenolol(6.25 mg/cat PO, q24h), clopidogrel (18.7 mg/cat PO, q24h), enoxaparin (1 mg/kg SC, q24 h), methadone (0.2 mg/kg IM, q8h) and amoxicillin + potassium clavulanate (20 mg/kg SC, q12h).[...]

Arterial thromboembolism secondary to subaortic stenosis in a persian kitten

Background: Aortic stenosis refers to several types of anatomic and functional obstructions of the left ventricular outflow tract. In small animals, this anomaly is most commonly documented as either a congenital lesion or an obstruction that develops soon after birth. It is a hereditary disease, caused by a dominant autosomal gene and modifying genes that interfere with phenotype expression. Even though aortic thromboembolism may be a potential complication in cats with cardiac diseases, aortic stenosis is deemed rare in that species. In this paper, we report an unusual case of a Persian kitten with aortic stenosis that eventually developed arterial thromboembolism.Case: A 7-month-old Persian kitten (3.1 kg) was admitted to a Veterinary Teaching Hospital with a history of hindlimbs paralysis over the past 48 h, as well as excessive vocalization. Also, the cat presented with inappetence, adipsia, urine incontinence, and hematuria. On physical examination, we observed hypothermia and cold paws. The footpads were cyanotic and there were no palpable femoral pulses. Cardiac auscultation disclosed a regular fast pace (200 bpm) and a grade 2/6 murmur heard best over the left cardiac base, but irradiating to the contralateral hemithorax. Prothrombin time and activated partial thromboplastin time were within the normal reference range, but the complete blood count showed microcytosis, lymphopenia, and hyperproteinemia. Also, elevated alanine aminotransferase and BUN were identified. On the echocardiogram, we observed a subvalvular aortic stenosis, which resulted in concentric remodeling of the left ventricle and a mild left atrial dilation. The stenotic lesion was classified as mild. Also, the thoracic radiography unveiled cardiomegaly. The recommended therapy included atenolol(6.25 mg/cat PO, q24h), clopidogrel (18.7 mg/cat PO, q24h), enoxaparin (1 mg/kg SC, q24 h), methadone (0.2 mg/kg IM, q8h) and amoxicillin + potassium clavulanate (20 mg/kg SC, q12h).[...](AU)

Aortic stenosis with abnormal eccentric left ventricular remodeling secondary to hypothyroidism in a Bourdeaux Mastiff / Estenose aórtica com remodelamento excêntrico anormal do ventrículo esquerdo secundário ao hipotireoidismo em um cão Dogue de Bordeaux

This paper describes a case of congenital aortic stenosis with eccentric left ventricular hypertrophy associated with hypothyroidism in a 1-year-old Bourdeaux Mastiff dog. The dog had ascites, apathy, alopecic and erythematous skin lesions in different parts of the body. A two-dimensional echocardiogram revealed aortic valve stenosis, with poststenotic dilation in the ascending aorta. The same exam showed eccentric hypertrophy and dilation of the left ventricle during systole and diastole. Aortic stenosis usually results in concentric left ventricular hypertrophy instead of eccentric hypertrophy; and therefore, this finding was very unusual. Hypothyroidism, which is uncommon in young dogs, may be incriminated as the cause of ventricular dilation, making this report even more interesting. Because hypothyroidism would only result in dilatation, the eccentric hypertrophy was attributed to pressure overload caused by aortic stenosis. Thus, cardiac alterations of this case represent a paradoxical association of both diseases.

Este trabalho descreve um caso de estenose aórtica congênita com hipertrofia excêntrica do ventrículo esquerdo associado ao hipotireoidismo em um cão Dogue de Bordeaux. O cão, de um ano de idade, apresentava ascite, apatia, lesões cutâneas alopécicas e eritematosas generalizadas. Na ecocardiografia bidimensional foi observada estenose da valva aórtica, com dilatação pós-estenótica em aorta ascendente. Foi detectado, no mesmo exame, hipertrofia excêntrica e dilatação do ventrículo esquerdo em sístole e diástole. Usualmente, como consequência, a estenose aórtica causa hipertrofia concêntrica do ventrículo esquerdo e não hipertrofia excêntrica, sendo este achado infrequente em tal cardiopatia. O hipotireoidismo, incomum em cães jovens, pode representar a causa da dilatação ventricular observada, o que torna mais relevante esse relato. Como no hipotireoidismo é esperado apenas dilatação, a hipertrofia excêntrica supostamente é atribuída à sobrecarga de pressão causada pela estenose aórtica. Dessa forma, as alterações cardíacas deste caso representam uma associação paradoxal das duas afecções.

Aortic stenosis with abnormal eccentric left ventricular remodeling secondary to hypothyroidism in a Bourdeaux Mastiff / Estenose aórtica com remodelamento excêntrico anormal do ventrículo esquerdo secundário ao hipotireoidismo em um cão Dogue de Bordeaux

This paper describes a case of congenital aortic stenosis with eccentric left ventricular hypertrophy associated with hypothyroidism in a 1-year-old Bourdeaux Mastiff dog. The dog had ascites, apathy, alopecic and erythematous skin lesions in different parts of the body. A two-dimensional echocardiogram revealed aortic valve stenosis, with poststenotic dilation in the ascending aorta. The same exam showed eccentric hypertrophy and dilation of the left ventricle during systole and diastole. Aortic stenosis usually results in concentric left ventricular hypertrophy instead of eccentric hypertrophy; and therefore, this finding was very unusual. Hypothyroidism, which is uncommon in young dogs, may be incriminated as the cause of ventricular dilation, making this report even more interesting. Because hypothyroidism would only result in dilatation, the eccentric hypertrophy was attributed to pressure overload caused by aortic stenosis. Thus, cardiac alterations of this case represent a paradoxical association of both diseases.(AU)

Este trabalho descreve um caso de estenose aórtica congênita com hipertrofia excêntrica do ventrículo esquerdo associado ao hipotireoidismo em um cão Dogue de Bordeaux. O cão, de um ano de idade, apresentava ascite, apatia, lesões cutâneas alopécicas e eritematosas generalizadas. Na ecocardiografia bidimensional foi observada estenose da valva aórtica, com dilatação pós-estenótica em aorta ascendente. Foi detectado, no mesmo exame, hipertrofia excêntrica e dilatação do ventrículo esquerdo em sístole e diástole. Usualmente, como consequência, a estenose aórtica causa hipertrofia concêntrica do ventrículo esquerdo e não hipertrofia excêntrica, sendo este achado infrequente em tal cardiopatia. O hipotireoidismo, incomum em cães jovens, pode representar a causa da dilatação ventricular observada, o que torna mais relevante esse relato. Como no hipotireoidismo é esperado apenas dilatação, a hipertrofia excêntrica supostamente é atribuída à sobrecarga de pressão causada pela estenose aórtica. Dessa forma, as alterações cardíacas deste caso representam uma associação paradoxal das duas afecções.(AU)

Síndrome hemorrágica pulmonar em cão associada à leptospirose / Pulmonary haemorrhagic syndrome in dog associated with leptospirosis

Background: Leptospirosis is probably the most widespread and prevalent zoonosis in the world, being classified as an emerging infectious disease for humans and dogs. Sporadically, dogs may present with cough and dyspnea, indicative of pulmonary involvement, associated with interstitial pneumonia and pulmonary hemorrhage with alveolar consolidation. Such changes stem from pulmonary haemorrhagic syndrome, which has emerged as a fatal complication, being documented in some areas in Europe and little described in North America. In this sense, the present study aims to report pulmonary hemorrhagic syndrome in a dog with leptospirosis.Case: A dog with no defined breed, aged 45 days, weighing 2.2 kg, with a history of apathy, anorexia, jaundice, hematochezia and vocalisation with a two day evolution was seen. Physical examination revealed a rectal temperature of 37.2°C, icteric mucosa, capillary filling time of two seconds, respiratory rate of 80 movements per minute and heart rate of 140 beats per minute, dehydration rate estimated at 8%, prostration, adequate body score, normal cardiac sounds and clean lung fields, in addition to petechiae in the abdominal region, whose palpation evidenced the presence of fluid in intestinal loops. The blood sample sent to the macroscopic serum agglutination was reagent for the serovar Icterohaemorragiae, titration of 200. Blood count revealed leukocytosis due to neutrophilia, with left-sided deviation, eosinopenia, presence of rare hypersegmentated neutrophils, rare toxic neutrophils, mild cytoplasmic basophilia and rare reactive lymphocytes. Platelet estimation demonstrated thrombocytopenia. No haemoparasites were seen. Regarding the biochemical evaluation, there were changes in liver enzymes and markers of renal failure. Fluid therapy was used with 0.9% NaCl solution, 5 mg/ kg doxycycline intravenously every 12 h and nasogastric probe for administration of nutritional support...

Síndrome hemorrágica pulmonar em cão associada à leptospirose / Pulmonary haemorrhagic syndrome in dog associated with leptospirosis

Background: Leptospirosis is probably the most widespread and prevalent zoonosis in the world, being classified as an emerging infectious disease for humans and dogs. Sporadically, dogs may present with cough and dyspnea, indicative of pulmonary involvement, associated with interstitial pneumonia and pulmonary hemorrhage with alveolar consolidation. Such changes stem from pulmonary haemorrhagic syndrome, which has emerged as a fatal complication, being documented in some areas in Europe and little described in North America. In this sense, the present study aims to report pulmonary hemorrhagic syndrome in a dog with leptospirosis.Case: A dog with no defined breed, aged 45 days, weighing 2.2 kg, with a history of apathy, anorexia, jaundice, hematochezia and vocalisation with a two day evolution was seen. Physical examination revealed a rectal temperature of 37.2°C, icteric mucosa, capillary filling time of two seconds, respiratory rate of 80 movements per minute and heart rate of 140 beats per minute, dehydration rate estimated at 8%, prostration, adequate body score, normal cardiac sounds and clean lung fields, in addition to petechiae in the abdominal region, whose palpation evidenced the presence of fluid in intestinal loops. The blood sample sent to the macroscopic serum agglutination was reagent for the serovar Icterohaemorragiae, titration of 200. Blood count revealed leukocytosis due to neutrophilia, with left-sided deviation, eosinopenia, presence of rare hypersegmentated neutrophils, rare toxic neutrophils, mild cytoplasmic basophilia and rare reactive lymphocytes. Platelet estimation demonstrated thrombocytopenia. No haemoparasites were seen. Regarding the biochemical evaluation, there were changes in liver enzymes and markers of renal failure. Fluid therapy was used with 0.9% NaCl solution, 5 mg/ kg doxycycline intravenously every 12 h and nasogastric probe for administration of nutritional support...(AU)