Serological patterns, antibody half-life and shedding in urine of Leptospira spp. in naturally exposed sheep.

AIMS: To determine within-farm prevalence, longitudinal pattern of exposure measured by serology, antibody titre longevity and point prevalence of shedding in urine of Leptospira borgpetersenii serovar Hardjo and L. interrogans serovar Pomona in naturally infected sheep on a sample of commercial farms in New Zealand. METHODS: On eight commercial sheep farms, between September 2011 and January 2014, blood samples were collected from 115-217 ewe lambs on each farm, at intervals of 2-11 months. They were analysed by microscopic agglutination test (MAT) for antibodies to L. borgpetersenii serovar Hardjo and L. interrogans serovar Pomona, using a titre cut-point of 48. Urine from 98 animals was tested by quantitative PCR (qPCR). The half-life of antibodies was estimated in 185 sheep for serovar Hardjo and 21 for Pomona, and the seroprevalence and mean titre of animals lost to follow-up was compared with those remaining in the study. RESULTS: Within-flock seroprevalence for serovar Hardjo reached a maximum at 17-22 months of age, ranging from 79 to 100%. Seroprevalence for serovar Pomona rose above 10% on three farms and increased to 21-54% by 4-14 months. Seroconversions occurred mainly from late autumn to early summer at 7-15 months of age. Seroprevalences ranging from 3 to 76% for serovar Hardjo and 0.5 to 15% for serovar Pomona were observed up to 3 months of age, likely due to maternally derived immunity. The half-life of antibody in response to infection was estimated to be 6.7 (95% CI=5.8-7.9) months for serovar Hardjo and 6.3 (95% CI=4.8-9.0) months for Pomona. The prevalence of sheep with urine positive for leptospires on qPCR on each farm ranged from 11 to 88%. All but one of the qPCR-positive animals were seropositive for serovar Hardjo. On two farms where Pomona exposure was observed, animals that were lost to follow-up had a higher geometric mean titre for serovar Pomona than those remaining in the study. CONCLUSIONS: This study demonstrated seasonal exposure from autumn to early summer in young sheep, a wide range of within-flock serological and shedding prevalence, and gives an estimation of the half-life of MAT titres in sheep. More extensive data are needed to fully understand the epidemiology of leptospirosis in sheep flocks across New Zealand and, along with economic analysis, to justify and design cost-effective and efficient control measures to protect human and animal health.

Interlaboratory and between-specimen comparisons of diagnostic tests for leptospirosis in sheep and cattle.

A study was performed to investigate interlaboratory test agreement between a research and a commercial veterinary diagnostic laboratory on blood and urine samples, and to investigate test agreement between blood, urine, and kidney samples (research laboratory) for leptospirosis diagnosis. Samples were sourced from 399 sheep and 146 beef cattle from a local abattoir. Interlaboratory agreement for real-time quantitative polymerase chain reaction (qPCR) results on urine samples was almost perfect (kappa = 0.90), despite the use of different amplification targets (DNA gyrase subunit B gene vs. 16s ribosomal RNA gene), chemistries (SYTO9 vs. TaqMan probe), and pre-PCR processing. Interlaboratory agreement for microscopic agglutination test (MAT) positivity was almost perfect (kappa = 0.93) for Leptospira borgpetersenii serovar Hardjo subtype Hardjobovis (Hardjobovis) but moderate (kappa = 0.53) for Leptospira interrogans serovar Pomona (Pomona). Among animals that had different titers recorded, higher Hardjobovis and lower Pomona titers were reported by the commercial laboratory than by the research laboratory (P < 0.005). These interlaboratory comparisons can assist researchers and diagnosticians in interpreting the sometimes discrepant test results. Within the research laboratory, the comparison of qPCR results on urine and kidney showed almost perfect agreement (kappa = 0.84), suggesting that the qPCR on these 2 specimens can be used interchangeably. The agreement between MAT positivity and urine and kidney qPCR results was fair (kappa = 0.32 and kappa = 0.33, respectively). However, the prevalence ratio of urine and kidney qPCR positivity in Hardjobovis-seropositive versus Hardjobovis-seronegative sheep indicated that Hardjobovis seropositivity found in sheep may be able to predict shedding or renal carriage.

Identification of factors associated with increased excretion of foot-and-mouth disease virus.

We investigated which variables possibly influence the amount of foot-and-mouth disease virus (FMDV) shed in secretions and excretions by FMDV infected animals, as it is likely that the amount of FMDV shed is related to transmission risk. First, in a separate analysis of laboratory data, we showed that the total amount of FMDV in secretions and excretions from infected animals is highly correlated with maximum titres of FMDV. Next, we collected data from 32 published scientific articles in which FMDV infection experiments were described. The maximum titres of FMDV reported in different secretions and excretions (the response variable) and the experimental conditions in which they occurred (the explanatory variables), were recorded in a database and analyzed using multivariate regression models with and without random effects. In both types of models, maximum titres of FMDV were significantly (p<0.05) associated with types of secretions and excretions, animal species, stage of the disease and days post infection. These results can be used to prioritize biosecurity measures in contingency plans.

Early detection of ketoprofen-induced acute kidney injury in sheep as determined by evaluation of urinary enzyme activities.

OBJECTIVE: To evaluate early indicators of renal tissue destruction and changes in urinary enzyme activities in sheep during the first hours after acute kidney injury induced by administration of an overdose of an NSAID. ANIMALS: 12 adult female sheep. PROCEDURES: Acute kidney injury was induced in 6 sheep by administration of ketoprofen (30 mg/kg, IV) and detected by evaluation of urinary protein concentration, iohexol clearance, and results of histologic examination. Six sheep served as control animals. Blood and urine samples were collected for up to 24 hours after administration of ketoprofen. Plasma concentrations of urea, creatinine, albumin, and total protein; plasma activities of alkaline phosphatase, acid phosphatase, γ-glutamyl transpeptidase (GGT), matrix metalloproteinase (MMP)-2, and MMP-9; and urinary creatinine and protein concentrations, specific gravity, and activities of alkaline phosphatase, acid phosphatase, GGT lactate dehydrogenase, N-acetyl-ß-D-glucosaminidase (NAG), MMP-2, and MMP-9 were measured. Urinary protein concentration and enzyme activities were normalized on the basis of urinary creatinine concentrations and reported as ratios. RESULTS: Many urinary enzyme-to-creatinine ratios increased before the plasma creatinine concentration exceeded the reference value. Urine NAG, lactate dehydrogenase, and acid phosphatase activities were increased beginning at 2 hours after ketoprofen administration, and alkaline phosphatase, GGT, and MMP-2 activities were increased beginning at 4 hours after ketoprofen administration. Most peak urinary enzyme-to-creatinine ratios were detected earlier than were the highest plasma creatinine and urea concentrations. CONCLUSIONS AND CLINICAL RELEVANCE: Urinary enzyme activities were sensitive early indicators of acute kidney injury induced by an overdose of an NSAID in sheep.

Detection of specific hydatid antigens and antibodies in serum and urine of experimentally infected sheep.

Immunodiagnostic confirmation of cystic human hydatidosis is frequently required before surgical intervention or of chemotherapy. However, it remains inadequate to detect specific antibodies or antigens in some confirmed cases of echinococcosis. This study was carried out to investigate the accuracy of three different immunodiagnostic tests for detection of specific circulating antigens or antibodies in the serum and urine of 13 experimentally infected sheep. For this purpose, Echinococcus granulosus were collected from small intestine of experimentally infected dogs, and 2000 taenid eggs were orally administered to each of the 13 sheep. There were six other sheep, which were kept as the control group. Biweekly serum and urine samples were collected from all the sheep for 4 months after infection. The sera were subjected to indirect hemagglutination test and the concentrated urine samples were subjected to coagglutination and counter immunoelectrophoresis tests. The results revealed that the sensitivity of these tests in detecting the hydatid antigens in the urine or antihydatid antibodies in the serum of the infected sheep reached their maximum in 12th and 13th week after infection; then it decreased in the following weeks. Examination of the non-infected sheep samples throughout the experiment showed that the aforesaid findings were specific only to the infected sheep. It seems that the appearance of specific hydatid antigen in urine and its antibodies in the serum were simultaneous. Although these tests are highly specific, false negative outcomes were encountered in their detection of cystic echinococcosis. In general, it seems rational to establish some series of diagnostic procedures in order to reveal antibodies and antigen of metacestode in serum and urine of the patients.

Clinicopathological features in ovine AA amyloidosis.

This paper describes the clinicopathological findings in sheep with AA amyloidosis. Serum samples from 12 AA amyloid-affected sheep and urine samples from 5 of these ewes were analyzed. In sera, the most important alteration was reflected in hypoalbuminemia, high concentration in beta and gamma-globulins and high levels of serum BUN, phosphorous and potassium. Serum creatinine, cholesterol and calcium concentrations showed no alterations. Urinary analysis showed proteinuria and a high protein/creatinine ratio. In three urine samples, high activities of urinary enzymes gamma-glutamyl transferase (GGT), beta-glucuronidase (GRS) and N-acetyl-beta-D-glucosaminidase (NAG) were observed, their ratios with urinary creatinine being increased for GGT and NAG and decreased for GRS. In conclusion, important alterations in biochemical and urinary parameters were observed in ovine affected by systemic AA amyloidosis. Those related to the activities of urinary enzymes could constitute reliable parameters for assessing renal injury in ovine AA amyloidosis.

Effects of D-penicillamine on urinary copper excretion in high-copper supplemented sheep.

The effects of a single oral application of D-penicillamine (DPA, mean dosage 28 mg/kg body weight) on urinary copper (Cu) excretion and general renal function in six high-Cu supplemented sheep (Cu intake of 3.7 mg/day per kg body weight for 84 days) and four controls (Cu intake of 0.16 mg/day per kg body weight) were investigated to quantify induced cupruresis and the therapeutic effect of DPA as a decoppering agent. Changes in liver Cu concentration were examined before and after DPA treatment by liver biopsies. The influence of DPA treatment on general renal function was low. A 10-fold increase in renal Cu excretion was induced in both groups of sheep. Maximal Cu excretion was observed 4 h after DPA treatment, with mean values of 280 pmol/min per kg body weight in the high Cu group and 145 pmol/min per kg body weight in the controls. In the high Cu sheep, urinary Cu excretion within 24 h after DPA application was equivalent to only 0.42 +/- 0.26% of liver Cu content (mean concentration 347 +/- 124 mg/kg wet weight). Moreover, no effect of DPA on liver Cu concentration was evident. These findings demonstrate that a single application of DPA is not effective in inducing sufficient Cu loss from the bodies of Cu-loaded sheep.

Epididymitis by Brucella ovis: experimental infection in virgin ram lambs.

Ten sexually immature rams were experimentally infected with Brucella ovis, to verify the antibody kinetics and its localization in urinary and genital tracts. All animals became positive to the complement fixation test from the 2nd post infection (p.i.) week and reached the maximum titre (1:256) on the 4th p.i. week. Bacteriemia was demonstrated on 3rd, 4th and 5th p.i. weeks. Two animals, respectively slaughtered 11 and 13 weeks after the infection, showed macroscopic and microscopic genital lesions and the etiologic agent was cultured from their urine and genital organs.

Acute intra-abomasal toxicity of tannic acid in sheep.

Acute tannic acid toxicity was studied in 6 adult Merino ewes by administering 7-10% (w/v) tannic acid solution once into the abomasum via an abomasal fistula at dose rates of 0.5, 1.0 or 2.0 g tannic acid/kg body weight. Samples of blood, urine and abomasal fluid were collected over a 48-h period before the sheep were necropsied 48 h after dosing. Dose rates of 1.0 and 2.0 g tannic acid/kg body weight produced abomasal, liver and kidney lesions. The abomasum had superficial mucosal erosions, hemorrhage and submucosal edema; the liver showed midzonal or periacinar coagulative necrosis; and the kidneys had focal tubular vacuolation and necrosis. In sheep dosed with 1.0 g tannic acid/kg body weight, there was a significant (P < 0.01) decrease in plasma phosphate and an increase in plasma aspartate aminotransferase and urea levels. Plasma total protein concentrations significantly (P < 0.05) decreased 24 h after dosing, but returned to normal by 48 h. By 4 h after dosing, abomasal fluid pH was significantly (P < 0.01) higher than predosing value, whereas urine pH significantly (P < 0.05) decreased. Both abomasal fluid and urine pH returned to normal 48 h after dosing. The liver and kidney damage seen in this study, in contrast to the methemoglobinemia without liver and kidney lesions previously reported when sheep were given tannic acid by oral gavage (1), suggest that, in cases of natural poisoning, tannins are not released from plant material until it enters the abomasum.

Evaluating a fluorosis hazard after a volcanic eruption.

The August, 1991 eruption of Mt. Hudson (Chile) deposited ash across southern Argentina and contributed to the deaths of thousands of grazing sheep. Early ash analysis revealed high levels of fluoride, a potential ash constituent toxic to humans and animals. In order to evaluate fluorosis as the cause of sheep deaths and to examine the possibility that similar ash and airborne toxins could also have an effect on the human population, we conducted an investigation that included health provider interviews, hospital record review, physical examination of sheep, determination of sheep urine fluoride levels, and complete constituent analysis of ash samples collected at proscribed distances from the volcano. Ash deposited farthest from the volcano had highest fluoride levels; all fluoride measurements were normal after rainfall. There were no signs or symptoms of fluorosis observed in sheep or humans. Sheep deaths resulted from physical, rather than chemical properties of the ash.

Physicochemical alterations in blood, cerebrospinal fluid and urine in experimental lactic acidosis in sheep.

Ruminal acidosis was induced in six adult sheep by oral feeding of wheat grain at 90 g kg-1 body-weight. Ruminal fluid, blood, cerebrospinal fluid (CSF) and urine samples were collected before grain feeding (0 h) and thereafter at 12, 24, 48, 72, 96 and 120 hour intervals. Decrease in the rumen pH was associated with decreased pH in blood, CSF and urine and increased total lactic acid concentrations in rumen liquor, blood, CSF and urine. Significantly (P < 0.05) increased levels of sodium, inorganic phosphorus and decreased levels of potassium and calcium were observed in the serum of acidotic sheep. CSF analysis revealed decreased potassium and chloride levels and increased glucose concentration and total leucocyte count within 12 hours of the grain feeding. Pandy's test was positive in half of the surviving sheep at 12 hours and in all the sheep at 96 and 120 hours of observation. In urine, sodium and chloride levels decreased while potassium and inorganic phosphorus increased. The changes in the biochemical parameters started within 12 hours and lasted up to 48 to 120 hours of the observation period.

Urinary excretion of purines in sheep with experimental orotic aciduria.

Urinary excretion of uric acid, hypoxanthine, allantoin, and urea was measured in sheep (Polish Merino, about 20 kg b.w.) with experimental orotic aciduria. The 240 min infusion of 6-azauridine solution into the jugular vein induced a highly significant increase of urinary orotic acid, uric acid and hypoxanthine excretion. No differences were found in relation to excretion of allantoin and urea in examined sheep. It was calculated that renal clearance of uric acid and hypoxanthine increased significantly in response to 6-azauridine infusion. Intravenous infusion of sodium orotate evoked a highly significant elevation of renal urate clearance. No significant change in renal urea clearance was observed. The data suggest that competition between the renal transport of orotate and actively transported purine compounds (uric acid and hypoxanthine) occurs.

Renal net acid and electrolyte excretion in an experimental model of hypochloremic metabolic alkalosis in sheep.

Renal electrolyte and net acid excretion were characterized during generation and maintenance of hypochloremic metabolic alkalosis in a ruminant model. Two phases of renal response with regard to sodium and net acid excretion were documented. An initial decrease in net acid excretion was attributable to increase in bicarbonate excretion with associated increase in sodium excretion. As the metabolic disturbance became more advanced, a second phase of renal excretion was observed in which sodium and bicarbonate excretion were markedly decreased, leading to increase in net acid excretion and development of aciduria. Throughout the metabolic disturbance, chloride excretion was markedly decreased; potassium excretion also decreased. These changes were accompanied by increase in plasma renin and aldosterone concentrations. There was apparent failure to concentrate the urine optimally during the course of the metabolic disturbance, despite increasing plasma concentration of antidiuretic hormone.

[The significance of pregnancy-induced ketonuria in sheep and goats]. / Zur Bedeutung der graviditätsbedingten Ketonurie bei Schaf und Ziege.

The last third of pregnancy in sheep and goats is characterized by a considerable reduction in the volume of the rumen. When the animal is carrying more than one fetus there is thus a latent ketosis caused by starvation. A discrete hypoglycemia is in close correlation to this. If other special factors like increasing age of the pregnant animal, chronic liver disease, unbalanced nutrition containing too little carbohydrates are also present, the latent starvation ketosis can give rise to an acute gestation ketosis. The clinical picture is described in detail and contrasted with the gestation hypocalcemia. Furthermore, the parameters relevant to laboratory diagnosis will be discussed.

Urinary indices of renal function in sheep with induced aminoglycoside nephrotoxicosis.

Aminoglycoside nephrotoxicosis (AGNT) was induced in ewes by daily SC administration of gentamicin. Changes in urinary indices of renal function during the development of AGNT are reported. Measurements from timed, volume-measured urine samples were made on days 0, 7, and 8 and included creatinine clearance, total excretion (TE) rates of electrolytes (Na, K, Cl, P) and urine volume. Measurements from free-catch urine samples (without volume measurement) were made daily and included fractional excretion (FE) rate of electrolytes, urine osmolality, and urine-to-serum osmolality and urine-to-serum creatinine ratios. With the onset of AGNT, FE rates of Na, K, Cl, and P- increased many fold above baseline values (200x, 4 to 5x, 6 to 9x, and 70 to 95x, respectively, on days 7 and 8), indicating decreased tubular reabsorption or increased tubular secretion. The increased FE rates were not representative of increases in total electrolyte excretion rates. The total excretion of Na (TENa) was mildly increased, TEK was decreased, TECl was unchanged, and TEP was significantly increased on days 7 and 8. Abnormal urinalysis results, glucosuria, and increased FEP preceded appreciable increase in serum creatinine concentration. Other abnormal urinary indices of renal function coincided with or followed the increase in serum creatinine concentration. Urinary indices may help characterize renal function associated with the disease state, but did not provide early indication of AGNT.

Enzymuria as an index of renal damage in sheep with induced aminoglycoside nephrotoxicosis.

Acute nephrotoxicosis was induced in ewes by daily SC administration of gentamicin. Activity of 3 urine enzymes, gamma-glutamyltransferase (GGT), beta-N-acetylglucosaminidase (AGS), and beta-glucuronidase (GRS), were measured during the development of aminoglycoside nephrotoxicosis. Measurements from timed, volume-measured urine samples were performed on days 0, 7, and 8. Measurements from urine samples obtained without volume measurement (spot samples) were performed daily. Urine GGT and AGS activities were high 3 days prior to detection of high serum creatinine concentration and 1.5 days before the appearance of casts in the urine sediment; values consistently remained in the abnormal range until termination of the study. High urine GRS activity was inconsistent and transient; serum GGT activity did not change during the course of the study. Urine GGT and AGS activities expressed as total excretion per unit time and body weight, enzyme activity per unit volume, and as ratio of urine enzyme activity to urine creatinine concentration were strongly correlated. Urine GGT and AGS, but not GRS activities, are suitable indicators of renal tubular cell damage in sheep with aminoglycoside nephrotoxicosis. Urine GGT and AGS activities indicate cellular changes occurring several days prior to the first indications of renal functional change.

Comparison of serum and renal gentamicin concentrations with fractional urinary excretion tests as indicators of nephrotoxicity.

Gentamicin was given to six sheep at a dosage rate of 80 mg/kg/day divided into three daily doses to cause nephrotoxicity. Peak serum gentamicin concentrations rose significantly throughout dosing (P less than 0.05), but trough serum gentamicin concentrations increased dramatically (P less than 0.01) from initial concentrations of 3.2-9.1 micrograms/ml to final trough concentrations of 31.5-195 micrograms/ml by 6-10 days on gentamicin. The serum gentamicin elimination half-life (t1/2) was doubled in each animal by approximately 6 days on therapy, with the sheep that were the most clinically affected by the nephrotoxic effects of gentamicin showing increases in t1/2 earlier than those sheep that remained less intoxicated. These changes occurred before many other clinical indicators of nephrotoxicity, with only urinary enzyme excretions preceding the changes in gentamicin elimination. Thus, alterations in the elimination of gentamicin may be one of the first clinical indicators of the occurrence of gentamicin-induced nephrotoxicity.