Evaluation of three qPCR for the detection of pathogenic leptospires in domestic animals in Nicaragua / [Evaluación de tres PCR cuantitativas para la detección de leptospiras patógenas en animales domésticos en Nicaragua].

Introduction: Molecular biology diagnostic methods such as real-time PCR should be used in Nicaragua to improve the diagnosis of leptospirosis in humans and animals. Objective: To evaluate three qPCR methods for pathogenic Leptospira detection in domestic animals. Materials and methods: Real-time PCR primers were designed for the amplification of specific regions from the Lip 32 gene of Leptospira in SYBER Green (SYBER Green-A) and TaqMan, as well in SYBER Green-B as previously published. The sequences of 12 strains obtained from the database of the National Center for Biotechnology Information (NCBI) were aligned to select probes and primers. The analytical sensitivity was determined by calculating the detectable genomic equivalent while 18 pathogenic references strains and 28 negative controls were used to evaluate the sensitivity and specificity of each one of the three sets in 129 urine samples of domestic animals. Results: The detection limit of four genomic equivalents per reaction was obtained from SYBR Green-A. The specificities were 94.4% (95% CI: 81.1-100.0) for TaqMan, 77.8% (95% CI: 55.8-99.8) for SYBR Green-A, while for SYBR Green-B it was 61.1% (95% CI: 35.8-86.4). In the three tests, we obtained a specificity of 100% (95% CI: 98.2-100.0). In the field samples, 26.4% were positive with SYBR Green-A and 6.1% with SYBR Green-B. Conclusion: SYBR Green-A presented the lowest detection limit while the three techniques under evaluation showed high specificity while TaqMan was the most sensitive.

Introducción. En Nicaragua es necesario estandarizar pruebas moleculares como la PCR en tiempo real (quantitative Polymerase Chain Reaction, qPCR) que mejoren el diagnóstico de leptospirosis en humanos y animales. Objetivo. Evaluar tres qPCR para la detección de leptospiras patógenas en animales domésticos de Nicaragua. Materiales y métodos. Se diseñaron cebadores para la amplificación del gen LipL32 en SYBR Green (SYBR Green-A) y TaqMan, y en otros descritos previamente (SYBR Green-B). Las secuencias de 12 cepas obtenidas de la base de datos del National Center for Biotechnology Information (NCBI) se alinearon para la búsqueda de sondas y cebadores. La sensibilidad analítica se determinó calculando el equivalente genómico detectable, se utilizaron 18 cepas de referencia para la sensibilidad diagnóstica y 28 controles negativos para la especificidad. Los métodos se aplicaron en 129 muestras de orina de animales domésticos. Resultados. En SYBR Green-A se obtuvo un límite de detección de cuatro equivalentes genómicos; en TaqMan, la sensibilidad fue del 94,4 % (IC95% 81,1-100,0). Con SYBR Green-A, se obtuvo una sensibilidad del 77,8 % (IC95% 55,8-99,8), en tanto que con SYBR Green-B fue del 61,1 % (IC95% 35,8-86,4). En las tres pruebas se logró una especificidad del 100 % (IC95% 98,2-100,0). El 26,4 % de las muestras de animales domésticos fueron positivas con SYBR Green-A y el 6,2 % con SYBR Green-B. Conclusiones. El SYBR Green-A presentó un límite de detección bajo, en tanto que las tres técnicas evaluadas mostraron alta especificidad, en tanto que la TaqMan tuvo la mayor sensibilidad.

Resorcylic Acid Lactones in Urine Samples of Croatian Farm Animals.

Metabolic transformation of zearalenone (ZEA), a mycotoxin which can contaminate both food and feed, results in the formation of five metabolites, one of them being zeranol (α-ZAL), which can be abused in farm animals as a growth promoter. To the best of our knowledge, there is no analytical method that can distinguish whether α-ZAL is present in an animal urine sample as a result of ZEA biotransformation or as a result of anabolic abuse. This study aimed at monitoring resorcylic acid lactones (RALs) concentration in urine of farm animals over several years. Six hundred and three cattle and pig urine samples were collected on farms in different Croatian regions and analyzed for RAL presence. Based on the testing results, all RAL-positive samples were considered to be consequential to feed contamination. The difference in primary ZEA metabolites' ratio (α-zearalenol/ß-zearalenol) was observed between cattle (0.03-0.41) and pig (2.05-17.39) urine samples. If the animals are treated with α-ZAL and fed on ZEA-contaminated feed, α-ZAL and taleranol found in their organisms could come from two sources, so that the reliability of the statistical model might be questionable. Based on these findings, there exists the need for improving the approach to the distinction between α-ZAL abuse and ZEA feed contamination.

Analysis of naturally occurring zearalenone in feeding stuffs and urine of farm animals in Croatia.

The aim of this study was to determine feed and urinary levels of zearalenone. A total of 114 samples, 64 feeding stuffs (commodities, pig and cattle feed), and 50 urine samples were analyzed by the use of enzyme-linked immunosorbent assay (ELISA). Zearalenone was detected in 68.7% of feeding stuffs, while all urine samples except for four yearling samples were positive for zearalenone. The maximum zearalenone concentration in feeding stuffs and urine was 577 ng/g and 241.1 ng/mL, respectively. Although zearalenone concentrations in some samples were high, the risk for humans was negligible since the calculated concentrations in meat were below the tolerable daily intake (TDI).

High prevalence of pathogenic Leptospira in wild and domesticated animals in an endemic area of China.

OBJECTIVE: To assess the prevalence of Leptospira detected in wildlife and domesticated animals in Jiangxi Province, China, in. METHODS: Urine samples from 28 buffaloes and kidney samples from 50 pigs, 50 dogs and 38 rats were collected from Fuliang and Shangrao County, Jiangxi Province, China, in October 2009. Polymerase chain reaction(PCR)and culture analyses were used to detect Leptospira. The cultured isolates were typed using the microscopic agglutination test(MAT). RESULTS: The results showed that rats potentially serve as the main reservoir of leptospiral infection, followed by dogs. Although 16% of rats (6/38) were positive using culture analysis, PCR analysis using the diagnostic primers G1/G2 and B64I/B64II or lipL32 showed identification as 50% and 24%, respectively, of the rat samples as positive for the presence of leptospiral DNA. CONCLUSIONS: PCR-based detection of leptospiral DNA in infected kidney tissues of reservoirs is more efficient when using G1/G2 primers than lipL32 primers. However, the latter primers have a potential application for detection in urine samples. The alarmingly high prevalence of leptospiral DNA in the wild rat population near human habitation underscores the utility of routine Leptospira surveillance, preferably using PCR methods, which are more sensitive than traditional culture-based methods.

Environmental tobacco smoke and canine urinary cotinine level.

Epidemiologic studies of companion animals such as dogs have been established as models for the relationship between exposure to environmental tobacco smoke (ETS) and cancer risk in humans. While results from these studies are provocative, pet owner report of a dog's ETS exposure has not yet been validated. We have evaluated the relationship between dog owner's report of household smoking by questionnaire and dog's urinary cotinine level. Between January and October 2005, dog owners presenting their pet for non-emergency veterinary care at the Foster Hospital for Small Animals at Cummings School of Veterinary Medicine, Tufts University, were asked to complete a 10-page questionnaire measuring exposure to household ETS in the previous 24 h and other factors. A free-catch urine sample was also collected from dogs. Urinary cotinine level was assayed for 63 dogs, including 30 whose owners reported household smoking and 33 unexposed dogs matched on age and month of enrollment. Urinary cotinine level was significantly higher in dogs exposed to household smoking in the 24 h before urine collection compared to unexposed dogs (14.6 ng/ml vs. 7.4 ng/ml; P=0.02). After adjustment for other factors, cotinine level increased linearly with number of cigarettes smoked by all household members (P=0.004). Other canine characteristics including age, body composition and nose length were also associated with cotinine level. Findings from our study suggest that household smoking levels as assessed by questionnaire are significantly associated with canine cotinine levels.

Clinical pathology in veterinary geriatrics.

Alterations in an individual analyte rarely provide an indication of the initiating circumstances that caused the abnormality. It is obvious from the previous discussion that multiple organs or organ systems can cause abnormal results in the same analyte. This fact underscores the importance of evaluating a biochemical profile in an integrated fashion, relating abnormalities of a particular analyte with the rest of the profile as well as with the signalment, history, and physical findings in the patient. Furthermore, assessment of abnormalities should be approached with some degree of skepticism because they may not be indicative of an actual disease.

Biochemical composition of urine from farmed ostriches (Struthio camelus) in Botswana.

Biochemical and qualitative evaluation of the supernatant of urine from hydrated farmed ostriches (Struthio camelus) indicated that the urine was comparable to that described by other workers. The disparities obtained between the biochemical constituents in the present and previous studies were partly attributed to the state of hydration influenced by climatic factors. Results of the cytological examination of the supernatant and the sediment concurred with the observations of other workers. It was therefore concluded that parameters from both the quantitative and qualitative analyses could be used as a guideline to monitor the health status of farmed ostriches in Botswana.

Gas and liquid chromatography coupled to tandem mass spectrometry for the multiresidue analysis of beta-agonists in biological matrices.

beta-Agonists are substances used in veterinary and human medicine for the treatment of pulmonary disorders. They have found a use as growth promoters to improve meat-to-fat ratios in cattle but they are not authorized for use in the European Union. Due to their presence in trace levels (usually less than 1 microgram/kg), to the diversity of the illegally used compounds and to the complexity of the biological matrices analysed, the detection of these residues requires a very sensitive and specific method of determination. This work describes the strategy of analysis we developed for five beta-agonists in urine and liver. The combination of improved solid- or liquid-phase extraction methods and LC or GC-MS-MS (in the multiple reaction monitoring mode) has shown to provide a system suitable for the control of these substances. The efficiency of extraction and the sensitivity and selectivity allow this multiresidue detection down to, and below, the UK regulatory level of 0.5 microgram/kg. Moreover, the use of LC removes the need for the derivatisation step (cyclic methylboronate derivatives) which is required prior to GC-MS-MS analysis.

Effect of an interfering substance on determination of potassium by ion-specific potentiometry in animal urine.

Analytical characteristics of photometry and ion-specific potentiometry for urine from sheep, horses, cows, dogs, and cats were determined, using solutions of sodium and potassium chloride. The performance of both methods were acceptable, but the ion-specific potentiometer (in the mode for urine analysis) was superior in terms of linearity of response and correlation between actual vs measured concentrations. Coefficients of variation of either method for repeated analyses of various concentrations of sodium and potassium were always less than 2.5%. The measurement of sodium concentration in urine samples correlated well between both methods for samples from sheep, horses, cows, dogs, and cats. In contrast, measurement of potassium concentrations in urine samples from sheep, horses, cows, and cats was underestimated consistently by ion-specific potentiometry. The magnitude of the apparent error was variable between species and was often increased with greater urine potassium concentrations. These phenomena were not seen in urine samples from dogs. Sequential dilution of urine samples from sheep before analysis reduced the magnitude of the error observed by ion-specific potentiometry. Seemingly, an equilibrium process existed in which potassium was bound by an anionic or zwitterionic chemical and was sequestered from interaction with the ion-specific electrode. Ultrafiltration experiments indicated the putative potassium chelator was a low molecular weight compound.