16S rRNA gene profiling of bacterial communities mediating production of tsetse attractive phenols in mammalian urine.

Several types of odours are involved in the location of host animals by tsetse (Diptera: Glossinidae), a vector of animal African trypanosomiasis. Host animals' ageing urine has been shown to be the source of a phenolic blend attractive to the tsetse. Nevertheless, limited research has been performed on the microbial communities' role in the production of phenols. This study aimed at profiling bacterial communities mediating the production of tsetse attractive phenols in mammalian urine. Urine samples were collected from African buffalo (Syncerus caffer), cattle (Bos taurus) and eland (Taurotragus oryx) at Kongoni Game Valley Ranch and Kenyatta University in Kenya. Urine samples, of each animal species, were pooled and left open to age in ambient conditions. Bacteriological and phenols analyses were then carried out, at 4 days ageing intervals, for 24 days. Phenols analysis revealed nine volatile phenols: 4-cresol, ortho-cresol, 3-cresol, phenol, 3-ethylphenol, 3-propylphenol, 2-methyloxyphenol, 4-ethylphenol and 4-propylphenol. Eight out of 19 bacterial isolates from the ageing urine revealed the potential to mediate production of phenols. 16S rRNA gene characterisation of the isolates closely resembled Enterococcus faecalis KUB3006, Psychrobacter alimentarius PAMC 27887, Streptococcus agalactiae 2603V, Morganella morganii sub.sp. morganii KT, Micrococcus luteus NCTC2665, Planococcus massiliensis strain ES2, Ochrobactrum pituitosum AA2 and Enterococcus faecalis OGIRF. This study established that some of the phenols emitted from mammalian urine, which influence the tsetse's host-seeking behaviour, are well characterised by certain bacteria. These results may allow the development of biotechnological models in vector control that combines the use of these bacteria in the controlled release of semiochemicals.

Evaluation of pheromone-based kit: A noninvasive approach of estrus detection in buffalo.

In view of the silent nature of estrus in buffalo, a noninvasive assay kit has long been felt necessary for easy and effective estrus detection. This study was designed to detect estrus in buffalo using a kit formulated in our laboratory based on pheromone compound. Group I: Urine samples collected at estrus phase and group II: randomly collected urine samples were subjected to the test using the kit. No colour developed (i.e., positive reaction) in estrus urine after adding the kit solution. By contrast, pale and/or dark pink colour developed (i.e., negative reaction) in urine from the proestrus and diestrus buffaloes, respectively. Field evaluation of the kit in groups I and II revealed that 60.87% and 71.43% of urine samples were correctly identified as estrus and nonestrus (i.e., proestrus and diestrus), respectively. Therefore, the first of its kind estrus detection kit formulated based on urinary pheromone can as well be used as a simple device to detect estrus in buffalo.

Exploration of Luteinizing hormone in murrah buffalo (Bubalus bubalis) urine: Extended surge window opens door for estrus prediction.

Estrus detection in buffaloes has been a major concern for decades, and lack of reliable methods affects their effective reproductive management. Luteinizing hormone (LH) detection in urine is in practice for several mammals for timed insemination, whereas very few reports are available on buffalo urinary LH. The focus of this study is to detect the presence of LH in buffalo urine, quantitate variation in urinary LH during different estrous cycle phases and examine the duration of mid-cycle LH window. Nearly hundred buffaloes were examined, longitudinal urine samples (n=42) were collected from seventeen animals and classified into respective phases based on several estrus detection parameters. The urinary LH was detected using bovine LH ELISA kit validated for serum/plasma/tissue homogenate. Detection of buffalo LH in the neat urine convincingly proved the competence of the bovine LH kit. Variation in the LH range was observed between different phases of estrous cycle and significant fold variation (P<0.05) was noticed during estrus phase (1.01±0.23) with average baseline value of 46.73±3.36mIU/mL. Interestingly, an extended window (A1-A3) of mid-cycle LH surge was observed due to its lingering excretion in urine. The results, altogether, revealed that LH can be detected in buffalo urine with noticeable fold variation during estrus phase and the extended LH window intensifies the chance of ovulation prediction for timed insemination.

A nationwide survey of pathogenic leptospires in urine of cattle and buffaloes by Loop-mediated isothermal amplification (LAMP) method in Thailand, 2011-2013.

Leptospirosis is a worldwide distributed zoonosis which has long been endemic in Thailand. Cattle and buffaloes are important livestock species that live in close contact with humans, especially in rural areas. These animals may, therefore, act as long-term carriers of leptospirosis for humans and other livestock species. The present study employed loop-mediated isothermal amplification (LAMP) method to detect pathogenic leptospiral 16S rDNA in the urine of cattle and buffaloes for assessing associations between uroprevalence and species, sex, age and spatial distribution. A total of 3,657 urine samples were collected for laboratory diagnosis, and 312 of which turned positive to the test (true prevalence 5.90%; 95% CI 4.98-6.91). The highest true uroprevalence was found in lower northern region at 19.80% (95% CI 15.83-24.32) followed by upper and lower northeastern regions at 15.22% and 6.25%, respectively. However, the highest true uroprevalence in beef cattle, the majority of cattle in Thailand, was recorded in northeastern region which is the endemic area of human leptospirosis. The uroprevalence was not statistically different among species and types of examined animals. Male animals were over twice more likely to be infected compared to females. Excluding animals younger than one year of age due to small sample size, the uroprevalence upraised with increasing age. A collaborative investigation between veterinary and public health sectors is required to holistically explore the link between leptospirosis in humans and livestock, especially in high prevalent areas.

Gas chromatographic-mass spectrometric analysis of chemical volatiles in buffalo (Bubalus bubalis) urine.

Isolation of active fraction and characterization of chemosignals from urine have been attempted in several mammalian species in the recent years. The objective of this study was to identify the urinary volatiles across various reproductive stages of buffalo cow, namely, estrus, diestrus, and pregnancy, and in bull, by chemical extraction followed by gas chromatography-linked mass spectrometry (GC-MS). Urine samples were collected from six buffalo cows at two different phases of estrous cycle, namely, estrus and diestrus. Besides, urinary samples were collected from five pregnant buffalo cows (60-75 days after artificial insemination (AI)) and six adult bulls. Thin-layer chromatography was performed as a preliminary test for qualitative comparison of different compounds extracted by organic solvents. Identification of the urinary compounds was carried out in a gas chromatograph (Perkin Elmer, Autosystem XL) linked to a mass spectrometer (Turbomass). The results of GC-MS analysis indicated the presence of 21 compounds with varying molecular weights and retention time, which were further categorized as diestrus-specific, pregnancy-specific, and bull-specific urinary compounds. No compound, however, could be identified as estrus-specific. We concluded that qualitative differences do exist in estrus, diestrus, and pregnant buffalo cow urine and in bull urine, as evidenced by GC-MS.

Pharmacokinetics, urinary excretion and plasma protein binding of ofloxacin in water buffalo calves (Bubalus bubalis).

Pharmacokinetics and urinary excretion of an intravenous dose of 5 mg.kg-1 ofloxacin were investigated in water buffalo calves. Plasma concentrations of ofloxacin were determined by high-performance liquid chromatography. Ofloxacin was rapidly distributed from the central to the peripheral compartment as evidenced by a short distribution half-life (0.09 h ± 0.003 h) and high K12 (4.7 h(-1) ± 0.1 h(-1)), and was detected in plasma for 8 h. The large volume of distribution (2.48 L.kg(-1) ± 0.18 L.kg(-1)) obtained in this study indicated high distribution of ofloxacin in water buffalo calves. The elimination half-life, the area under the plasma drug concentration-time curve and total body clearance were 2.11 h ± 0.13 h, 6.20 µg.mL(-1) ± 0.23 µg.mL(-1).h and 0.81 mL.kg(-1).h(-1) ± 0.03 mL.kg(-1).h(-1), respectively. About 18.7% of administered drug was bound to plasma proteins and approximately 32.5% of the administered dose was recovered in urine within 48 h. The results of the study indicated a favourable pharmacokinetic profile of ofloxacin in water buffalo calves, which suggests that ofloxacin may be effective against urinary pathogens in this species.

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.

Identification of urinary sex pheromones in female buffaloes and their influence on bull reproductive behaviour.

The present study was carried out to identify the urinary sex pheromones of buffaloes and their role in relation to bull reproductive behaviour. Urinary samples were collected from 12 buffalo heifers at four different phases of estrous cycles. Fourteen compounds were identified throughout the cycle, which included phenol, ketone, alkane, alcohol, amide, acid and aldehyde. Among the 14 chemical profiles, three compounds were only found on the day of estrus, viz. 1-chlorooctane, 4-methylphenol and 9-octadecenoic acid. Behavioural investigation clearly showed that bulls were attracted and exhibited repeated flehmen behaviour towards the 4-methyl phenol. The bulls displayed penile erection and mounting behaviour while exposed to 9-octadecenoic acid. However, the other compound, 1-chlorooctane, did not influence such sexual behaviours. The present results provide evidence that the estrus-specific urinary volatile compounds appear to be sex pheromones which initiate the bull's reproductive behaviour.

Disposition kinetics and urinary excretion of cefpirome after intravenous injection in buffalo calves.

We investigated the disposition kinetics and urinary excretion of cefpirome in buffalo calves after a single intravenous administration of 10 mg/kg. Also, an appropriate dosage regimen was calculated. At 1 min after injection, the concentration of cefpirome in the plasma was 57.4 +/- 0.72 microg/ml, which declined to 0.22 +/- 0.01 microg/ml at 24 h. The cefpirome was rapidly distributed from the blood to the tissue compartment as shown by the high distribution coefficient values (8.67 +/- 0.46/h), and by the drug's rate of transfer constant from the central to the peripheral compartment, K(12) (4.94 +/- 0.31/h). The elimination halflife and the volume of distribution were 2.14 +/- 0.02 h and 0.42 +/- 0.005 l/kg, respectively. Once the distribution equilibrium was reached between the tissues and plasma, the total body clearance (Cl(B)) and the ratio of the drug present in the peripheral to the central compartment (T/P ratio) were 0.14 +/- 0.002 l/kg/h and 1.73 +/- 0.06, respectively. Based on the pharmacokinetic parameters we obtained, an appropriate intravenous cefpirome dosage regimen for treating cefpiromesensitive bacteria in buffalo calves would be 8.0 mg/kg repeated at 12 h intervals for 5 days, or until persistence of the bacterial infection occurred.

Plasma concentrations, pharmacokinetics and urinary excretion of gatifloxacin after single intravenous injection in buffalo calves.

The pharmacokinetics and urinary excretion of gatifloxacin were investigated after a single intravenous injection of 4 mg/kg body weight in buffalo calves. The therapeutic plasma drug concentration was maintained for up to 12 h. Gatifloxacin rapidly distributed from blood to tissue compartments, which was evident from the high values of the distribution rate constant, alpha1 (11.1 +/- 1.06 h(-1)) and the rate constant of transfer of drug from central to peripheral compartment, k12 (6.29 +/- 0.46 h(-1)). The area under the plasma drug concentration-time curve and apparent volume of distribution were 17.1 +/- 0.63 (microg.h)/ml and 3.56 +/- 0.95 L/kg, respectively. The elimination half-life (t (1/2 beta)), total body clearance (ClB) and the ratio of drug present in tissues and plasma (T/P) were 10.4 +/- 2.47 h, 235.1 +/- 8.47 ml/(kg.h) and 10.1 +/- 2.25, respectively. About 19.7% of the administered drug was excreted in urine within 24 h. A satisfactory intravenous dosage regimen for gatifloxacin in buffalo calves would be 5.3 mg/kg at 24 h intervals.

Isolation of leptospira santarosai, serovar guaricura from buffaloes (Bubalus bubalis) in Vale do Ribeira, São Paulo, Brazil

In April 1998 urine samples from adult female buffaloes were collected in a farm located in Registro, Vale do Ribeira, São Paulo State, Brazil. The urine samples obtained after furosemide injection were immediately transported to the laboratory in liquid modified EMJH medium and seeded, by the serial dilution technique, into Fletcher's or modified EMJH-0.2 (per cent) agar, both of them with 5-fluorouracil 100mg/mL. The intraperitoneoum inoculation of 0.5 mL was also performed with each urine sample in young, adult hamsters (Mesocricetus auratus). All samples seeded directly in culture medium were contaminated. The hamsters did not show any sign of disease and were killed at the 21st post inoculation day. At this time kidney cultures of these animals were performed and from one of them, one leptospira strain (M04-98) was isolated, identified as belonging to serogroup Sejroe by Microscopic Agglutination Test (MAT) with a panel of 36 rabbit sera against serovars representative for the pathogenic serogroups. Subsequently, MAT was carried out with antisera against the 19 reference strains of serogroup Sejroe, revealing a close relationship with serovar guaricura. Afterwards the MAT was done with a panel of 18 monoclonal antibodies representative for serovars of serogroup Sejroe. The histogram closely resembled that of serovar guaricura. So Cross Agglutination Absorption Test (CAAT) was carried out with the buffalo isolate and guaricura, supporting the relationship between the buffalo isolate and serovar guaricura.

The disposition kinetics, urinary excretion and dosage regimen of ciprofloxacin in buffalo calves (Bubalus bubalis).

The disposition kinetics, urinary excretion and a dosage regimen for ciprofloxacin after a single intravenous administration of 5 mg/kg was investigated in 5 healthy buffalo calves. The disposition kinetics were best fitted to a three-compartment open model. After 1 min, the concentration of ciprofloxacin in plasma was 8.50 +/- 0.39 microg/ml and the minimum therapeutic concentration was maintained for 10 h. The elimination half-life and volume of distribution were 3.88 and 0.08 h and 3.97 +/- 0.22 L/kg, respectively. The total body clearance and T/P ratio were 0.709 +/- 0.025 L/kg per h and 6.13 +/- 0.54, respectively. Approximately 28.3% of the total administered dose of ciprofloxacin was recovered in urine within 24 h of administration. To maintain a minimum therapeutic plasma concentration of 0.10 microg/ml, a satisfactory intravenous dosage regimen of ciprofloxacin, computed on the basis of disposition kinetic data obtained in healthy buffalo calves, would be 3 mg/kg repeated at 12 h intervals.

Urinary excretion of purine derivatives and tissue xanthine oxidase (EC 1.2.3.2) activity in buffaloes (Bubalis bubalis) with special reference to differences between buffaloes and Bos taurus cattle.

The urinary excretion of purine derivatives (PD) was measured in six buffaloes (Bubalis bubalis) during fasting and in fourteen buffaloes given four restricted levels of roughage (2.5-4.8 kg DM/d). Only allantoin and uric acid, not xanthine and hypoxanthine, were present in the urine, the pattern of excretion being similar to that in cattle. The fasting PD excretion amounted to 0.20 (SD 0.06) mmol/kg metabolic weight (W)0.75 per d, and the rate of PD excretion as a linear function of feed intake was 5.2 mmol/kg digestible organic matter intake. Both values were considerably lower than the values for cattle reported in the literature. Creatinine excretion values were 0.33 (SD 0.06) and 0.44 (SD 0.09) mmol/kg (W)0.75 per d determined in fasting and feeding periods respectively. Fasting N excretion was 257 (SD 49) mg N/kg (W)0.75 per d. Both creatinine and fasting N excretions were also lower than in cattle. The activities of xanthine oxidase (EC 1.2.3.2) in plasma, liver and intestinal mucosa were determined in buffaloes, cattle and sheep. Xanthine oxidase activities in buffaloes were 24.5 (SD 2.7) unit/l plasma and 0.44 (SD 0.02) and 0.31 (SD 0.10) unit/g fresh tissue in liver and intestinal mucosa respectively. These activities were higher than those in cattle and sheep. Xanthine oxidase was practically absent from plasma and intestine of sheep. It is suggested that the differences in PD excretion between buffaloes and cattle were probably due to the smaller proportion of plasma PD that was disposed of in the urine of buffaloes.

Pharmacokinetics and urinary excretion of sulfadiazine in buffalo calves.

Pharmacokinetics and urinary excretion of sulfadiazine were determined in buffalo calves following single oral administration (150 mg/kg). Kinetic evaluation of plasma levels was performed using a 2-compartment model. The absorption half-life and elimination half-life were 3.41 +/- 0.63 and 13.75 +/- 1.94 h, respectively. Based on this study, an optimal dosage regimen of sulfadiazine in buffalo calves would be 165 mg/kg, followed by 75 mg/kg at 12-h intervals. Sulfadiazine was mainly excreted in the urine as free amine, while the percentage of acetylated sulfadiazine was comparatively low.

Pharmacokinetics and urinary excretion of gentamicin in Bubalus bubalis calves following intramuscular administration.

The pharmacokinetics and urinary excretion of gentamicin was studied in buffalo calves after a single intramuscular administration (10 mg kg-1). Kinetic determinants were calculated by using a two compartment open model. The absorption (t1/2Ka) and biological half lives (t1/2 beta) were calculated to be 0.43 +/- 0.08 and 3.79 +/- 0.23 h, respectively. The value of the apparent volume of distribution (VdB) was found to be 0.38 +/- 0.07 litre kg-1. The satisfactory intramuscular dosage regimen of gentamicin for buffalo calves would be 3.23 mg kg-1 as priming dose and 2.88 mg kg-1 as maintenance dose to be repeated at 12 hour intervals to achieve and maintain the therapeutic plasma levels within safe limits. Urinary excretion of gentamicin was very rapid during the first 12 hours as 48.07 +/- 1.39 per cent of the total administered dose was excreted unchanged during this period.

Comparative studies on the effect of continuous intravenous infusion of some sulphonamides on blood and urine glucose levels in buffaloes.

Three groups of five clinically healthy buffaloes each were injected intravenously with sulphadiazine, sulphadimidine and sulphamerazine in a dose of 100 mg/kg b. wt. (as a singly initial dose of 40 mg/kg b. wt. an subsequently the plasma level kept constant by a continuous intravenous infusion of a maintenance dose of 20 mg/kg per hour over a period of 3 hours). It was found that, 1) sulphadiazine, sulphadimidine and sulphamerazine increase the plasma glucose levels at 1, 2, 2.5 and 3.5 hours from the start of i.v. infusion. 2) The glucose concentration in urine increased in the buffaloes infused i.v. with sulphadiazine. 3) The glucose level in urine of buffaloes infused i.v. with sulphadimidine and sulphamerazine was slightly increased. 4) The concentrations of sulphadiazine, sulphadimidine and sulphamerazine in plasma reached its highest level, 2.5, 2 and 2.5 hours during the i.v. infusion, respectively, then declined rapidly. 5) The concentrations of sulphadiazine, sulphadimidine and sulphamerazine in urine reached their highest concentrations 3.5 hours after i.v. infusion.