Development of enzyme immunoassay for chromogranin A (CgA) and profiling of plasma CgA concentrations in the cow

The objectives of this study were to establish and characterize a homologous immunoassay for bovine chromogranin A (bCgA) and to profile plasma bCgA concentrations during early pregnancies. We synthesized oligopeptide corresponding to the amino acid sequence 341–355 of bCgA for immunizing rabbits and peptide corresponding to the amino acid sequence 336–365 of bCgA for both a biotinylated tracer and reference standards. Recombinant bCgA protein was also generated in Escherichia coli lysate. Dose-dependent displacement curves were obtained from 1 to 1,000 nM of the reference standards. The displacement curves showed a good relationship between the reference standards of the synthetic peptide and the serially diluted plasma sample or recombinant bCgA protein generated in the present study. The assay sensitivity defined as the value of two standard deviations below the zero standard was calculated as 0.46 nM. The intraassay and interassay coefficients of variation were 6.48% and 13.4%, respectively. Changes in the plasma bCgA concentrations in early pregnancies undulated in nonpregnant animals. The results of the present study suggest that assaying plasma bCgA concentrations could be utilized as measures to evaluate the physiological status of cattle.     

Time lag between peak concentrations of plasma and salivary cortisol following a stressful procedure in dairy cattle

Background

Measurement of salivary cortisol has been used extensively as a non-invasive alternative to blood sampling to assess adrenal activity in ruminants. However, there is evidence suggesting a considerable delay in the transfer of cortisol from plasma into saliva. Previous studies in cattle have used long sampling intervals making it difficult to characterise the relationship between plasma and salivary cortisol (PLCort and SACort, respectively) concentrations at different time points and determine whether or not such a time lag exist in large ruminants. Therefore, the objective of this study was to characterise the relationship between plasma and salivary cortisol and determine if there is a significant time lag between reaching peak cortisol concentrations in plasma and saliva across a 4.25 h time-period, using short sampling intervals of 10–15 min, following social separation in dairy cattle.Five cows were separated from their calves at 4 days after calving, and six calves were separated from a group of four peers at 8 weeks of age. Following separation, the animals were moved to an unfamiliar surrounding where they could not see their calves or pen mates. The animals were catheterised with indwelling jugular catheters 1 day before sampling. Blood and saliva samples were obtained simultaneously before and after separation.

Results

In response to the stressors, PLCort and SACort increased reaching peak concentrations 10 and 20 min after separation, respectively. This suggested a 10 min time lag between peak cortisol concentrations in plasma and saliva, which was further confirmed with a time-series analysis. Considering the 10 min time lag, SACort was strongly correlated with PLCort (P < 0.0001).

Conclusions

Salivary cortisol correlates well with plasma cortisol and is a good indicator of the time-dependent variations in cortisol concentrations in plasma following acute stress. However, there is a time lag to reach peak cortisol concentrations in saliva compared to those in plasma, which should be considered when saliva samples are used as the only measure of hypothalamic-pituitary-adrenal axis response to stress in cattle.     

Validation of a human progesterone enzyme immunoassay (EIA) kit for use on serum of cattle in Cameroon

A study aimed at validating a human progesterone enzyme immunoassay kit was carried out on cattle at Bambui, Cameroon. Progesterone ELISA Kits (EH-511) were obtained from Clinpro International. Forty-one cows were selected, of which 19 were pregnant and 22 within 14 days post partum. Blood samples were analysed and progesterone levels were deduced from a curve obtained from standard absorbance values (A ₄₅₀). Results show that 95.5% of postpartum cows had progesterone levels below 1 ng/ml, with the highest level being 0.75 ng/ml. The mean level was 0.5 ± 0.26 ng/ml. The cows in the ‘pregnant group’ had progesterone levels ranging from 3.5 to 17.5 ng/ml. This kit can be used for measuring progesterone levels in cattle. Levels of 1 ng/ml for two consecutive samples or one sample at or above 3 ng/ml are an indication of the presence of corpus luteum, while cows below 1 ng/ml will be in anoestrus.     

Interday variation and effect of transportation on indirect blood pressure measurements,plasma endothelin-1 and serum cortisol in Standardbred and Icelandic horses

Background

Systemic hypertension is a prominent feature in humans with metabolic syndrome (MS) and this is partly caused by an enhanced endothelin-1 (ET-1) mediated vasoconstriction. There are indications that systemic hypertension might be a feature in equine metabolic syndrome (EMS) but if ET-1 is involved in the development of hypertension in horses is not known. Increased levels of cortisol have also been found in humans with MS but there are no reports of this in horses. Before blood pressure, plasma ET-1 and serum cortisol can be evaluated in horses with EMS, it is necessary to investigate the interday variation of these parameters on clinically healthy horses. The aims of the present study were therefore to evaluate the interday variation and influence of transportation on systemic blood pressure, plasma ET-1 and serum cortisol in healthy Standardbred and Icelandic horses, and to detect potential breed differences.

Methods

Nine horses of each breed were included in the study. Blood pressure was measured and blood samples were collected between 6 and 9 am on two separate days. Eight of the horses (four of each breed) were transported to a new stable were they stayed overnight. The next morning, the sampling procedure was repeated.

Results

The interday variation was higher for plasma ET-1 (37%) than for indirect pressure measurements (8-21%) and serum cortisol (18%). There were no differences in systemic blood pressure between the two breeds. The Icelandic horses had significantly lower serum cortisol and significantly higher plasma ET-1 concentrations compared to the Standardbred horses. Plasma ET-1 was significantly elevated after transportation, but systemic blood pressure and serum cortisol did not differ from the values obtained in the home environment.

Conclusions

Indirect blood pressure, plasma ET-1 and serum cortisol are of interest as markers for cardiovascular dysfunction in horses with EMS. The elevated plasma ET-1 concentrations recorded after transportation was likely caused by a stress response. This needs to be considered when evaluating plasma ET-1 in horses after transportation. The differences detected in plasma ET-1 and serum cortisol between the two breeds might be related to differences in genetic setup, training status as well as management conditions.     

Plasma melatonin in domestic female Mediterranean sheep (Comisana breed) and goats (Maltese and Red Syrian)

The plasma melatonin nychtemeral profiles in Mediterranean ewes and goats were evaluated six times throughout the year. Melatonin levels were high throughout the night and generally below the assay detection limit during daytime. However, during long days, 30% of the last daytime samples had high melatonin concentrations. Plasma melatonin levels were higher in Comisana sheep than in goats, and higher in Maltese than in Red Syrian goats, with highly significant effect of the individual animal and high repeatability. Plasma melatonin was higher in April than in August. When there was a large difference between the duration of day and night, the plasma melatonin pattern and the light/dark cycle did not always match exactly, suggesting some form of superimposition and/or the prevalence of an endogenous rhythm. The difference found at similar scotoperiods with increasing or decreasing day length may be involved in the perception of the photoperiodic changes.     

Serological survey of Neospora caninum and Toxoplasma gondii in cattle (Bos indicus) and water buffaloes (Bubalus bubalis) in ten provinces of Brazil

The objective of this study was to determine the prevalence of antibodies to Neospora caninum and Toxoplasma gondii among 500 cattle (Bos indicus) and 500 buffaloes (Bubalus bubalis) using the indirect fluorescent antibody test (IFAT) technique. Blood samples from were collected from water buffalo and cattle in 10 municipalities in the northern region of Brazil. The frequency of cattle and water buffaloes seropositive for Neospora caninum in Pará state, Brazil, was 55% and 44%, respectively, and the frequency of cattle and water buffaloes seropositive for Toxoplasma gondii was 52% and 39%, respectively. Seropositivity for both N. caninum and T. gondii was detected in 10.6% of the cattle samples and 14.8% of the buffalo samples. The frequency of cattle positive for N. caninum and T. gondii was significantly (p < 0.05) higher than that of buffalo in two and three provinces, respectively. Buffaloes had a lower seroprevalence for N. caninum or T. gondii in all of the provinces studied. These results suggest that both species, when exposed to the same risks for N. caninum and T. gondii infection, have a high serological prevalence. Cattle showed a higher probability of being seropositive when exposed to the same risks for N. caninum and T. gondii. Our study, which included an extensive number of blood samples, provides important epidemiological information pertinent to buffalo production in tropical countries that can be used as a basis for disease-management practices in Latin America.     

雌二醇与孕酮奶样纸片酶免法的建立及应用

乳汁雌二醇(E2)与孕酮(P4)含量是判断母牛卵巢机能状态的重要指标。本试验建立了E2与P4奶样纸片酶免法,P4检测灵敏度达到2.1 pg,回收率91%,板内和板间变异系数分别为5.3%(n=8)和10.4%(n=10);E2检测灵敏度达到1.7 pg,回收率89%,板内和板间变异系数分别为4.8%(n=7)和9.6%(n=10)。将该方法用于测定定时排卵程序中母牛乳汁E2与P4含量,结果表明所建立的方法简便实用,排卵程序中母牛乳汁E2、P4变化规律与理论分析相符,可以用来判定母牛发情、妊娠及卵巢功能。     

Comparison of the VIDAS and IMMULITE‐2000 methods for cortisol measurement in canine serum

Background: Serum cortisol concentration is often measured in dogs for the diagnosis and monitoring of adrenal disease. An enzyme‐linked fluorescent assay (VIDAS method) on the MiniVidas analyzer has been validated for the measurement of cortisol concentration in human serum and could have applications for canine samples. Objective: The aim of this study was to compare canine cortisol results obtained using the VIDAS method with those obtained using the IMMULITE‐2000 immunoassay, which has previously been validated for canine serum. Methods: The concentration of cortisol in 40 canine serum samples was determined concurrently with the VIDAS and IMMULITE methods, the latter as the reference method. Pearson's correlation coefficient, linear, and Deming regression analyses and Bland–Altman analysis were used to compare the 2 methods. Acceptability of the new method was judged using a medical decision chart (MEDx chart). Results: Cortisol concentrations obtained with the IMMULITE method ranged from 23.1 to 1380 nmol/L. Correlation (r=.977) and simple linear regression (slope=1.0722, confidence interval [CI] 0.996–1.148; intercept=?4.799, CI ?42.838 to 33.240) revealed no proportional or constant error. Based on Deming regression and a Bland–Altman plot the 2 methods gave comparable results. The MEDx chart indicated that performance of the new method was good at decision limits of 40, 132, and 480 nmol/L. Conclusion: Results of the VIDAS method were comparable to those of the IMMULITE‐2000 reference method such that the VIDAS may be used as an alternative assay to evaluate serum cortisol concentration in dogs for the diagnosis of adrenal disease.     

Development of a simple, direct, microtitre plate enzymeimmunoassay (EIA) for progesterone determination in whole milk of buffaloes

A method of estimating progesterone in buffalo whole milk by EIA using progesterone 6 beta-OH-hemisuccinate-horseradish peroxidase as the enzyme label and an antiserum raised against progesterone-7 alpha-carboxyethyl-thioether-BSA was developed. The microtitration plates used in the assay were first coated with affinity purified sheep IgG developed against rabbit IgG. The immune reaction was performed by incubating a mixture of 1 microliter of whole milk (diluted to 20 microliters with assay buffer), 100 microliters of enzyme label and 100 microliters of antiserum for 90 min in the dark. After washing the plates, 150 microliters of the substrate solution was added. The mixture was incubated in the dark for 40 min before the reaction was stopped and the optical density was measured at 450 nm. The calibration curve was sensitive in the range 0.8-40 pg/well, corresponding to 0.8-40 ng/ml. Milk samples from cycling buffaloes were tested for progesterone concentration by running parallel EIA and RIA. A good correlation of 0.91 was obtained and the estimated values were similar using both techniques. The method has demonstrated about 10 times greater sensitivity than RIA in buffalo milk.     

Epidemiology of Oestrus ovis (Linneo, 1761) infestation in goats in Spain

This survey was conducted to determine the chronobiology and seroprevalence of nasal bot infestation (Oestrus ovis) in Spain and to identify the risk factors associated with this disease in caprine herds.

A total of 1590 sera from adult goats were collected at random on 175 farms in southwestern Spain. Sera were tested by ELISA, using crude protein from second stage larvae as antigen. The mean seroprevalence was 46.04% and mean percentage of optical densities was 41.83. These data indicate a high prevalence of this parasite in the investigated areas. The serological survey revealed that goats managed at higher altitudes, at meridians latitudes and on farms with small herds had a smaller probability of infestation. Eighty goat heads, obtained from abattoirs in the central region of Spain, were collected and examined for nasal botflies from February to October 2002. O. ovis larval stages were recovered from the nasal-sinus cavities of 23 goats, reaching a prevalence of 34.94%. The mean larval burden was 3.9 larvae per infested head. No first instars were found during February and March, when the second instar reached its larger count. The third instar was observed in very small number during the whole period of study, with one peak occurring in July–August. These data show the existence of a favourable period for the development of larval instars of O. ovis in goats that starts in February and finishes in September.     

Effect of age and sex on plasma cortisol and dehydroepiandrosterone concentrations in the dog (Canis familiaris)

Limited data exist on age-related physiological variations in plasma concentrations of cortisol and dehydroepiandrosterone (DHEA) in dogs, despite their potential role in the pathophysiology of ageing. This study examined plasma cortisol and DHEA concentrations and cortisol/DHEA ratio variations, according to age and sex in 311 dogs, aged from two months to 16 years. Before adulthood, DHEA concentrations were higher in peri-pubertal males. During adulthood, cortisol and DHEA were higher in males than females. Among females, DHEA was lower in older dogs, but the decrease was observed at an older age in intact than ovariectomised females. Variations in the cortisol/DHEA ratio inversely reflected those of DHEA. Results indicate that testicles are an important source of DHEA in males, and that DHEA is mainly secreted by the adrenal glands in females. The ovaries’ contribution to circulating DHEA appears to be limited, although it may partially compensate an age-related decrease in adrenal secretion.     

Evaluation of an automated,homogeneous enzyme immunoassay for serum thyroxine measurement in dog and cat serum

A homogenous enzyme immunoassay (EIA) for measurement of serum thyroxine (T4) concentration was evaluated for use with canine and feline serum. The EIA method was linear from 0 to 150 nmol T4/L for human serum, 0 to 94 nmol T4/L for feline serum and 10 to 60 nmol T4/L for canine serum. Intra- and interassay precision studies yielded coefficients of variation 相似文献   

Development and evaluation of a rapid absorbed enzyme immunoassay test for the diagnosis of Johne''s disease in cattle

An absorbed enzyme immunoassay (EIA) test for Johne's disease in cattle was developed in which absorption of cross-reacting antibodies occurred as a rapid reaction in solution rather than overnight with whole organisms and a subsequent centrifugation step. Total test time was reduced to less than 2 h with a minimum of manipulations. The test was evaluated in cattle herds from Johne's disease-endemic and Johne's disease-free regions of Australia. Specificity was 99.8%. Calculations of sensitivity were affected by the history of the herd under test. However, the EIA detected in excess of 80% of animals before onset of clinical disease and 65% of faecal shedders were EIA positive on, or before, first detection of Mycobacterium paratuberculosis in their faeces. The test should aid epidemiological studies and be a useful tool in the management and control of Johne's disease.     

Development and validation of a radioimmunoassay for thyrotropin in cattle.

In mammals, thyrotropin, or thyroid-stimulating hormone (TSH), assay is used for the diagnosis of primary hypothyroidism. Hypothyroidism is the most common type of thyroid disorder in cattle. The aim of this study was to develop and validate, under physiologic and pathologic conditions, a radioimmunoassay (RIA) for bovine TSH (bTSH). Double RIA was performed with purified bTSH and specific bovine antiserum. Laboratory validation included research of minimal detection limit, accuracy, and reproducibility. The physiologic validation included a thyrotropin-releasing hormone (TRH) challenge performed on euthyroid cows and a follow-up of bTSH concentration over a 24-hour period. Furthermore, bTSH concentration was assayed in a large population of healthy dairy and beef cows to define reference interval. The pathologic validation was made by assaying bTSH and thyroid hormones on healthy and goitrous newborn calves. The minimum detection limit (MDL) for bTSH assay was 1.3 microU/ml. The recovery was 101% to 106%. The intra- and interassay coefficients of variation (CVs) ranged from 5% to 11% and 11% to 15%, respectively. The RIA covered the whole range of physiologic bTSH values, as shown by bTSH values induced by TRH-challenge. A pulsatile secretion of bTSH was observed, accompanied by a diurnal variation with lower night values than day values. Reference intervals of bTSH ranged from 1.3 to 13.0 microU/ml for beef and dairy breeds. Finally, bTSH easily discriminated goitrous newborn calves from healthy ones, leading to the definition of a cutoff value of 35 microU/ml. The bTSH assay positively reacted to physiologic and pathologic conditions. The accuracy and precision of the RIA were satisfying.     

Fluorescence spectra and measurement of phylloerythrin (phytoporphyrin) in plasma from clinically healthy sheep,goats, cattle and horses

AIM: To measure the background concentration of phylloerythrin in plasma from clinically healthy sheep, goats, cattle and horses on pasture.

METHODS: Blood samples were taken from 34 sheep of the Dala breed, 20 female Norwegian dairy goats, 35 Norwegian Red cows and 34 horses of different breeds. All animals were grazing green pasture when blood samples were taken. Blood samples were collected from each of four clinically healthy newborn lambs, goats, calves and foals, and pooled into one sample per species. Plasma samples were analysed for phylloerythrin by fluorescence spectroscopy, using a Perkin-Elmer LS-50B luminescence spectrometer equipped with a red-sensitive photo- multiplier. The fluorescence spectra of phylloerythrin in plasma from the adult ruminants were compared with those in plasma from the neonatal ruminants, to which a known concentration of phylloerythrin had been added.

RESULTS: Plasma obtained from the adult ruminants had spectral characteristics similar to those of phylloerythrin, namely weak emission peaks at 650 and 711 nm, when excited at 425 nm. Emission spectra obtained from plasma from the neonatal ruminants showed no fluorescence at these wavelengths. On average, 0.012 (SD 0.004), 0.06 (SD 0.04), and 0.05 (SD 0.03) μmol/l phylloerythrin were present in plasma samples from the sheep, goats, and cattle, respectively. The fluorescence spectra of plasma from the newborn foals were similar to spectra of plasma from adult horses, with weak emission at 669 nm.

CONCLUSION: Small concentrations of phylloerythrin were detected in plasma from clinically healthy sheep, goats and cattle, but none could be detected in plasma from clinically healthy horses.     

Fluorescence spectra and measurement of phylloerythrin (phytoporphyrin) in plasma from clinically healthy sheep, goats, cattle and horses

AIM: To measure the background concentration of phylloerythrin in plasma from clinically healthy sheep, goats, cattle and horses on pasture. METHODS: Blood samples were taken from 34 sheep of the Dala breed, 20 female Norwegian dairy goats, 35 Norwegian Red cows and 34 horses of different breeds. All animals were grazing green pasture when blood samples were taken. Blood samples were collected from each of four clinically healthy newborn lambs, goats, calves and foals, and pooled into one sample per species. Plasma samples were analysed for phylloerythrin by fluorescence spectroscopy, using a Perkin-Elmer LS-50B luminescence spectrometer equipped with a red-sensitive photomultiplier. The fluorescence spectra of phylloerythrin in plasma from the adult ruminants were compared with those in plasma from the neonatal ruminants, to which a known concentration of phylloerythrin had been added. RESULTS: Plasma obtained from the adult ruminants had spectral characteristics similar to those of phylloerythrin, namely weak emission peaks at 650 and 711 nm, when excited at 425 nm. Emission spectra obtained from plasma from the neonatal ruminants showed no fluorescence at these wavelengths. On average, 0.012 (SD 0.004), 0.06 (SD 0.04), and 0.05 (SD 0.03) micromol/l phylloerythrin were present in plasma samples from the sheep, goats, and cattle, respectively. The fluorescence spectra of plasma from the newborn foals were similar to spectra of plasma from adult horses, with weak emission at 669 nm. CONCLUSION: Small concentrations of phylloerythrin were detected in plasma from clinically healthy sheep, goats and cattle, but none could be detected in plasma from clinically healthy horses.     

Development and preliminary assessment of a polyclonal antibody-based enzyme immunoassay for the detection of Tritrichomonas foetus antigen in breeding cattle

More sensitive tests are required for the diagnosis of Tritrichomonas foetus infection in cattle and an antigen-detecting enzyme immunoassay (EIA) has been applied to this purpose. An affinity purified immunoglobulin fraction obtained from rabbits immunised with cultured T. foetus served as both capture antibody and as biotinylated indicator antibody. While highly sensitive in the detection of antigen derived from cultured organisms, the assay showed poor sensitivity in the detection of antigen in the cervico-vaginal mucus of artificially infected heifers, with only 75% of culture-positive samples being considered positive for antigen. In a direct comparison, 23/122 samples from a naturally infected dairy herd gave positive cultures, while only 10/122 samples were considered antigen positive by EIA.     

Pharmacokinetics and distribution of ampicillin in plasma,milk and uterine fluid of female buffaloes

A pharmacokinetic study of ampicillin (6 mg/kg intravenous) revealed that the peak concentrations of 17.81±1.25, 5.64±2.24 and 1.09±0.10 g/ml of the drug were attained at 15 min, 30 min and 2 h in plasma, milk and uterine fluid respectively. A therapeutic concentration of 0.1 g/ml was maintained from 15 min–8 h, 15 min–6 h and 30 min–6 h in plasma, milk and uterine fluid. Hence, the drug may be used effectively in mammary gland and uterine infections apart from its use in other systemic infections.     

Prevalence and genetic profiles of Shiga toxin-producing Escherichia coli strains isolated from buffaloes, cattle, and goats in central Vietnam

We investigated the prevalence of Shiga toxin-producing Escherichia coli (STEC) in 568 healthy domestic animals (buffaloes, cattle, and goats) from 98 farms in the central region of Vietnam. The aims of this study were to determine if the prevalence of STEC in South East Asia is similar to that in other parts of the world, to characterize the virulence gene profiles from the recovered STEC and to determine if the recovered STEC belong to serotypes commonly associated with human disease. STEC and intimin-positive strains were recovered from 27% of buffaloes, 23% of cattle, and 38.5% of goats. Seventy percent of buffalo farms, 60% of cattle farms and 100% goat farms were positive for STEC. Of 170 STEC strains, 99 carried both stx1 and stx2 genes, 36 carried the stx2 gene, and 35 carried the stx1 gene. The eae gene was found in six caprine isolates, but not in buffalo or bovine isolates. Among 173 E. coli strains (170 STEC and 3 intimin-positive), 110 carried the ehxA gene, 106 possessed the saa gene. Further characterization of stx subtypes demonstrated that among 134 stx1-containing isolates, 107 belonged to the stx1c subtype and 27 were the stx1 subtype. Of the 132 stx2-containing isolates, 36 were stx2, 34 were stx2c, 43 were stx2d subtype, 3 belonged to stx2g, and 16 strains were stx2d(act). The stx2c variant was dominant in strains isolated from buffalo while the stx2d variant occurred more frequently in caprine isolates. Only 9 (5%) STEC strains contained genes encoding for serotypes O26, O91, O121, O145, and O157 LPS, which are more frequently associated with human infections. The results of this study provide data for understanding of epidemiology of STEC among domestic animals in Vietnam and indicate that buffaloes are also an important reservoir of STEC.