Milk antibodies against Ostertagia ostertagi: relationships with milk IgG and production parameters in lactating dairy cattle

The present study was carried out to evaluate the relationship between milk optical density ratios (ODRs) from an indirect Ostertagia ostertagi ELISA, total milk IgG levels and milk production and then establish a correction factor to adjust ODR. Five hundred and sixty composite milk samples collected from 358 cows on four dairy herds in June and August 2002 were used in this analysis. The average ODR was 0.34. A positive correlation was found between ODR and IgG values in milk, days in milk, age and log transformed somatic cell counts (SCC). However, ODR was negatively correlated with milk production. The IgG levels and ODR values were constant from 30 to 200 days in milk. However, ODRs increased from 200 days until the end of the lactation. After controlling for age, season, herd and SCC, an increase in milk production of 13 kg/day was associated with a reduction in ODR values of 0.052. The results of the present study suggest that ODR values are not greatly influenced by production factors. ODR follow the same pattern as the IgG variation across lactation and could be adjusted in order to compare ODR values obtained from high producing cows with those obtained from low producing animals.     

The effect of an experimentally induced acute mastitis on the test results of an Ostertagia ostertagi milk ELISA

Antibody levels against Ostertagia ostertagi in the milk are a promising parameter to identify dairy cows or herds with production losses due to gastrointestinal nematodes. However, milk antibody levels can be influenced by udder-related factors. In this study, the effect of an experimentally induced mastitis on the test results (ODR) of a milk O. ostertagi ELISA was investigated. Twenty-five cows that were naturally infected with gastrointestinal nematodes, were inoculated in their left udder quarters with Escherichia coli P4:O32 and quarter milk samples were collected at several intervals from 24 h before until 144 h after experimental infection. The effect of the contribution of milk from one or more infected quarters on the bulk tank milk ODR was estimated, based on a titration experiment. The mean O. ostertagi antibody levels of the infected udder quarters were significantly (P < 0.001) higher than those of the uninfected udder quarters at each sampling time post-infection. The largest difference was observed at 24 h post-infection with a mean difference of 0.251 ODR (95%CI: 0.172; 0.330). There was also a significant increase (P < 0.001) in total IgG levels with the largest difference being observed at 24 h post-infection. Highly significant (P < 0.005) correlation coefficients were observed between O. ostertagi ODR, total IgG ODR, Na+ and Cl- ion concentration and log transformed somatic cell counts at 24 h post-infection. The results demonstrate that an acute mastitis causes a flow of specific and non-specific antibodies from serum to milk with a subsequent increase in the O. ostertagi ODR values. The effect of the contribution of milk from infected quarters on the bulk tank milk O. ostertagi ODR was estimated to be minor if the relative number of infected quarters is small (< 3%).     

Evaluation of the repeatability of a crude adult indirect Ostertagia ostertagi ELISA and methods of expressing test results

An indirect enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against Ostertagia ostertagi using a crude adult worm antigen was evaluated using serum and milk samples from adult cows, as well as from bulk tank milk. Within and between plate repeatabilities were determined. In addition, the effects of factors such as antigen batch, freezing, preserving of the samples and somatic cell counts (SCCs) of the samples were evaluated. Raw optical densities (ODs) and normalized values were compared using the concordance correlation coefficient (CCC), the coefficient of variation (CV), Bland-Altman plots (BA). Based on raw OD values, there was a high repeatability within a plate (CCC approximately 0.96 and CV<10%). Repeatability between plates was evaluated following normalization of OD values by four methods. Computing normalized values as (OD-Nt)/(Pst-Nt), gave the most repeatable results, with the CCC being approximately 0.95 and the CV approximately 11%. When the OD values were higher than 1.2 and 0.3 for the positive and the negative controls, respectively, none of the normalization methods evaluated provided highly repeatable results and it was necessary to repeat the test. Two batches of the crude antigen preparation were evaluated for repeatability, and no difference was found (CCC=0.96). The use of preservative (bronopol) did not affect test results, nor did freezing the samples for up to 8 months. A significant positive relationship between ELISA OD for milk samples and SCC score was found. Therefore, the use of composite milk samples, which have less variable SCC than samples taken from each quarter, would be more suitable when the udder health status is unknown. The analytical methods used to evaluate repeatability provided a practical way to select among normalization procedures.     

Recovery of Infective 3Rd Stage Larvae of Haemonchus Contortus and Ostertagia Ostertagi Ry Migration in Agar Gel

A laboratory technique is described for the recovery of Haemonchus contortus and Ostertagia ostertagi infective larvae by migration in agar gel. The addition of bile increased the recovery rate of the haemonchus larvae, but had a somewhat depressive effect on the number of ostertagia larvae recovered. Similarly, storage at 4°G lowered the yield of larvae of both species, compared to freshly harvested larvae. However, the recovery rates for both species were sufficiently high to recommend the technique for isolation of the infective stages from field samples.     

Bulk milk ELISA and the diagnosis of parasite infections in dairy herds: a review

The bulk milk enzyme-linked immune sorbent assay (ELISA) is a rapid and inexpensive method of assessing herd exposure to pathogens that is increasingly being used for the diagnosis of parasite infections in dairy herds. In this paper, with the dairy herd health veterinarian in mind, we review the principles of the assay and the recent literature on the potential role of bulk milk ELISA for the diagnosis of ostertagiosis, fasciolosis, parasitic bronchitis due to cattle lung worm and neosporosis. It is generally accepted that assay results reflect exposure to the parasite rather than the presence of active infection. Bulk milk ELISA can be a useful tool for the veterinary practitioner as a component of a herd health monitoring programme or in the context of a herd health investigation. It can also play a role in regional or national surveillance programmes. However, the results need to be interpreted within the context of the herd-specific health management, the milk production pattern and the parasite life cycle.     

Evaluation of the stability of Ostertagia ostertagi ELISA microtitre plates over time using cow milk samples

The objective of this study was to assess the stability of ELISA plates prepared with one of three blocking agents and used with one of two conjugates at various time intervals after preparation of the plates. Two of the blocking agents used were commercially available: one termed stabilgaurd (stab) and one manufactured by SVANOVA Biotek AB Inc. (svan). The third blocking agent used was bovine serum albumin (bsa). A polyclonal rabbit anti-bovine IgG (poly) and an anti-bovine IgG monoclonal (mono) conjugate were used. Eighteen composite individual cow milk samples collected late in lactation (200-400 days in milk) were used in this study. An indirect microtitre plate ELISA that used the Ostertagia ostertagi antigen was used to quantify antibodies against the parasite, present in the milk samples. Each of six blocking agent/conjugate combinations (called systems) were used to test 18 milk sub-samples at 1, 4 and 24 weeks after blocking the plates. Plates blocked with stab and svan were kept at room temperature and an additional set were incubated at 37 degrees C so as to mimic long term storage (about 1 year) and tested only once at 4 weeks. Those blocked with bsa were frozen at -20 degrees C. Concordance correlation coefficients (CCC) and reproducibility were used to assess the agreement between test results conducted on the same milk sample at the various test-times using a particular system. Generally, there was good agreement between tests conducted at different times for all systems. However, the svan-mono and bsa-poly systems had the best agreement with overall CCC values of 96% and 93%, respectively. The svan-poly system had the lowest CCC of 75%. The CCC and reproducibility ranked the systems in a similar way. The high CCC between tests done using plates kept at room temperature and ones incubated at 37 degrees C, suggested that plates would be stable up to a year after blocking. The storage of plates blocked with svan and stab agents under room temperature, makes them more convenient to use and transport relative to bsa-blocked plates that have to be frozen.     

Immune modulation by Ostertagia ostertagi and the effects of diet

IgG1 antibody responses to Ostertagia ostertagi third stage larvae (L3) and the third party antigen, keyhole limpet haemocyanin (KLH), and faecal egg counts were determined in calves infected with a single dose of O. ostertagi and in uninfected, pair-fed calves. The infected and uninfected calves were given diets either high (H) or low (L) in protein and energy. The diets were within the normal range of husbandry practice in the UK. IgG1 antibody responses to L3 antigen were significantly greater from 6 weeks post-infection in infected calves given the L diet than in infected calves given the H diet (P less than 0.05). The effects of diet and infection on anti-KLH IgG1 responses were independent of each other. IgG1 responses to KLH were decreased by infection and by the L diet compared with the H diet.     

Milk and milk components reduce the motility of Ostertagia circumcincta larvae in vitro

AIM: To examine the effects in vitro of bovine milk and milk products and soymilk on the motility of sheathed and ex- sheathed L3 Ostertagia circumcincta (also known as Teladorsagia circumcincta) as a measure of larval viability and infectivity.

METHODS: L3 were exsheathed in 0.2% sodium hypochlorite, resuspended in Hank’s Balanced Salt Solution (HBSS) pH 7.4 and incubated with test solutions at 37°C for up to 48 h. The motility of 50 larvae from each incubate was assessed at selected times using a McMaster slide. Larvae were considered immotile only if straight and not moving. Fresh bovine milk, homogenised milk (3.3% fat), low-fat milk (0.2% fat) and lamb milk replacer were diluted with HBSS pH 7.4 to concentrations from 1.6–100%, and incubated with exsheathed L3 for 1, 24 or 48 h. Bovine whey protein was tested in concentrations of 5–15% at pH 2.5–6.5, casein at 5 or 7.5%, and skim milk powder from 5–15% at pH 5.5 or 6.5, all for 2, 4 or 24 h. Soymilk was tested in concentrations of 1.6–100% for 1, 2, 24 or 48 h. HBSS was used as the control solution. Sheathed L3 were incubated in HBSS pH 7.4, 50% homogenised milk in HBSS, or 50% soymilk in HBSS. Each solution was incubated for 1, 2, 24 or 48 h.

RESULTS: The motility of exsheathed L3 was reduced by fresh bovine milk, homogenised milk, low-fat milk, lamb milk replacer, whey, casein and skim milk solutions, but not by soymilk. The mean percentage (and SE) immotile at 48 h were: fresh milk 38% (SE 20); homogenised milk 65% (SE 7); low-fat milk 57% (SE 5); lamb milk replacer 43% (SE 7); and soymilk 7% (SE 0.5). Larval immotility increased in whey protein solutions from 5–15%, from pH 2.5–6.5 and from 2 to 24 h (all p<0.001); in skim milk from 5–15% (p<0.001), and was greater at pH 6.5 than at pH 5.5 (p<0.001); in casein from 5–7.5% (p<0.001), but was no different at pH 5.5 and 6.5. The motility of sheathed L3 was reduced at 24 h (p=0.009) and 48 h (p<0.001) by 50% homogenised milk, but not by 50% soymilk or HBSS.

CONCLUSIONS: Bovine milk proteins, or components associated with the proteins, reduced the motility of both sheathed and exsheathed L3 O. circumcincta. Soymilk had no effect on nematode motility. Lower larval motility may reduce worm establishment and be a contributing factor to the smaller burdens of gastrointestinal nematodes in milk-fed animals compared with animals after weaning.     

A longitudinal survey of anti-Ostertagia ostertagi antibody levels in individual and bulk tank milk in two dairy herds in Normandy

The Ostertagia-specific antibody levels in milk were monitored in 2 dairy herds to investigate seasonal variations and the relationship between individual and bulk tank milk antibody levels. Bulk tank and individual milk samples from all lactating animals were collected over a 1-year period at weekly and monthly intervals, respectively. The Ostertagia-specific antibody levels were measured with an indirect ELISA and the test results were expressed as optical density ratios (ODR). A clear seasonal pattern that followed the expected intake of infectious larvae was observed in the individual and bulk tank milk antibody levels of both herds. Within each herd, there was a large variation in the individual ODRs. This variation remained large when the distribution of individual ODRs was plotted according to high and low bulk tank milk ODR categories. The results suggest that the effect of seasonal variations on cut-off levels that predict production responses after anthelmintic control, needs to be assessed.     

The use of an indirect Ostertagia ostertagi ELISA to predict milk production response after anthelmintic treatment in confined and semi-confined dairy herds

An indirect Ostertagia ostertagi ELISA was used in late lactation milk samples from cows in confined and semi-confined dairy herds to determine if it could predict milk production response after endectocide treatment at calving. Holstein cows from 9 dairy farms from Prince Edward Island (PEI), 5 from central Nova Scotia and 16 from southern Ontario that were participating in a clinical trial of endectocide treatment around calving were used in this study. The cows were randomly treated with either eprinomectin pour-on endectocide or a placebo solution. Milk samples were obtained from cows late in the lactation before treatment was applied. These samples were tested for antibodies to gastrointestinal nematodes (GIN) using the indirect ELISA with the results expressed as optical density ratios (ODR). Production records were obtained from a computerized database of dairy herd improvement data. Pre-calving ODR showed a seasonal pattern. They were higher in the summer and fall and lower during the winter months. Older animals had higher pre-calving ODR values compared with younger cows. Similarly, cows from semi-confined herds had higher parasite antibody levels compared with cows from confined herds. The endectocide treatment did not affect the milk production response in the overall study population. However, the interaction effect between treatment and pre-calving ODR on milk production response after endectocide treatment was significant (P = 0.02), with some evidence of positive treatment response in cows with an ODR > 0.4. The relationship between pre-calving ODR and production response appeared to be quadratic rather than linear.     

Ostertagia ostertagi antibodies in milk samples: Relationships with herd management and milk production parameters in two Mediterranean production systems of Spain

The present study analyzed Ostertagia ostertagi antibodies by indirect ELISA in milk samples in two cattle systems in Mediterranean Spain to indirectly monitor gastrointestinal nematode (GI) parasitism effects on production. Individual samples from 10 animals and the corresponding milk herd samples were collected from 133 herds in Girona (intensive management) and 123 herds in Minorca (extensive management). Both locations showed high and significant positive relationships between average optical density ratios (ODR) of individual animals and ODR in their milk tank. Although antibodies levels were low, there were significantly higher in Minorca. Negative correlations between ODR values and milk production were found in both systems. Importantly, in Minorca, average herd milk production was higher in the herds that treated their animals against GI nematodes compared to those that did not treat. The ELISA technique was valuable to indirectly assess differences in the level of GI nematode infection even in cattle production systems with low levels of infection.     

Ostertagia ostertagi infection in the calf: effects of a trickle challenge on the hormonal control of digestive and metabolic function

Metabolic effects of a trickle challenge with the equivalent of 10,000 infective Ostertagia ostertagi larvae per day were investigated in 12 calves allocated to infected, pair-fed control or ad libitum-fed control groups. Changes in hormone levels reflecting abomasal, pituitary and pancreatic function were monitored using radioimmunoassay techniques previously validated for use in cattle. A range of metabolic profile parameters and blood metabolites was also measured. Feed intake of the infected calves began to decline as blood gastrin and pepsinogen levels reached a peak. The depression in appetite recorded in this group was responsible for significant increases in plasma urea and non-esterified fatty acid levels and associated with an increase in growth hormone/insulin ratio. No significant difference in glucagon levels was recorded between groups. A decline in blood albumin values was also shown in the infected group and associated with a drop in nitrogen digestibility. A significant depression in circulating calcium levels was related to either the hypoalbuminaemia or impaired mineral absorption in the intestine. A decrease in plasma cholesterol values in the infected group was associated with changes in digestive function.     

Validation of a competitive ELISA for diagnosis of Brucella melitensis infection in sheep and goats

A competitive enzyme-linked immunosorbent assay (cELISA) was validated for the serodiagnosis of Brucella melitensis infection in small ruminants using 2108 positive and 2154 negative reference sera from sheep and goats. The optimum cut-off values, offering the highest diagnostic sensitivity (DSn) and diagnostic specificity (DSp), determined by receiver operating characteristic analysis, were at 23.6%, 21.8% and 25.0% inhibition of the conjugate control for sheep, goats and both species, respectively. The DSns of the cELISA for sheep, goats and both species at these cut-off values were 89.2% (95% confidence interval 87.1-91.1%), 74.0% (95% CI 71.4-76.5%) and 77.9% (95% CI 76.1-79.7%), whereas DSps were 96.4% (95% CI 95.2-97.4%), 92.9% (95% CI 91.1-94.3%) and 97.2% (95% CI 96.4-97.8%), respectively. Compared to cELISA, indirect ELISA and fluorescence polarisation assay have higher DSns and DSps. However, the results obtained with the cELISA were in good agreement with those of the complement fixation test (CFT) under field conditions using 5735 sheep and goat sera. The cELISA can be used as an alternative to the CFT for diagnosing B. melitensis infection in small ruminants.     

Ostertagia ostertagi infection in the calf: effects of a trickle challenge on appetite, digestibility, rate of passage of digesta and liveweight gain

The effects of a trickle challenge with the equivalent of 10,000 infective Ostertagia ostertagi larvae per day on appetite, digestibility, rate of passage of digesta and liveweight gain were investigated in 12 calves assigned to infected, pair-fed control and ad libitum-fed control groups. Digestibility of cellulose, nitrogen, organic matter and dry matter was determined using insoluble acid detergent fibre as a marker on two occasions during the study: (i) Between days 31 and 38, when abomasal dysfunction was greatest; and (ii) between days 52 and 58, beginning approximately one week after anthelmintic treatment (day 46). Rate of passage of digesta was measured using chromium mordanted hay, fed to each calf after each digestibility study period. Voluntary feed intake of the infected group was significantly reduced from day 37 with the greatest depression (77 per cent) occurring just before anthelmintic treatment. The drop in appetite was responsible for nearly 73 per cent of the difference in liveweight gain between the infected and ad libitum fed control groups. The apparent digestibility coefficient of nitrogen was significantly depressed (22 per cent) in the infected group though was restored to control levels by anthelmintic treatment. The rate of passage of digesta was significantly reduced in both pair-fed control (50 per cent) and infected (74 per cent) groups. Anthelmintic treatment increased the latter though only to pair-fed control group levels. It is suggested that the marked hypergastrinaemia seen in the infected calves may have been in part responsible for the decreased rate of passage of digesta and in turn for the drop in appetite.     

Ostertagia ostertagi infection in the calf: effects of a trickle challenge on appetite, digestibility, rate of passage of digesta and liveweight gain

The effects of a trickle challenge with the equivalent of 10,000 infective Ostertagia ostertagi larvae per day on appetite, digestibility, rate of passage of digesta and liveweight gain were investigated in 12 calves assigned to infected, pair-fed control and ad libitum-fed control groups. Digestibility of cellulose, nitrogen, organic matter and dry matter was determined using insoluble acid detergent fibre as a marker on two occasions during the study: (i) Between days 31 and 38, when abomasal dysfunction was greatest; and (ii) between days 52 and 58, beginning approximately one week after anthelmintic treatment (day 46). Rate of passage of digesta was measured using chromium mordanted hay, fed to each calf after each digestibility study period. Voluntary feed intake of the infected group was significantly reduced from day 37 with the greatest depression (77 per cent) occurring just before anthelmintic treatment. The drop in appetite was responsible for nearly 73 per cent of the difference in liveweight gain between the infected and ad libitum fed control groups. The apparent digestibility coefficient of nitrogen was significantly depressed (22 per cent) in the infected group though was restored to control levels by anthelmintic treatment. The rate of passage of digesta was significantly reduced in both pair-fed control (50 per cent) and infected (74 per cent) groups. Anthelmintic treatment increased the latter though only to pair-fed control group levels. It is suggested that the marked hypergastrinaemia seen in the infected calves may have been in part responsible for the decreased rate of passage of digesta and in turn for the drop in appetite.     

Evaluation of a blocking ELISA for the detection of bovine viral diarrhoea virus (BVDV) antibodies in serum and milk

The performance characteristics of a blocking ELISA test applied to serum and individual milk for the detection of antibodies to bovine viral diarrhoea virus (BVDV) were assessed using 1189 matched milk/serum samples collected from cows of 42 dairy herds located in Brittany (west of France). This test was based on a monoclonal antibody directed against non-structural protein NS2-3 of pestiviruses. All tests were performed blind. For each type of sample, negative/positive cut-off values were determined using receiver operating characteristic (ROC) analysis. Sensitivity and specificity were estimated using the virus neutralisation test as a reference. For sera, the ROC analysis provided a negative/positive inhibition percentage cut-off value of 50% giving a sensitivity and a specificity of 96.9 and 97.8%. For individual milk samples, the cut-off was fixed at 30%, leading to a sensitivity and a specificity of 96.9 and 97.3%. Using this test, a good overall agreement was found between results obtained on matched milk/serum samples (Kappavalue=0.95). The present results indicate that this blocking ELISA test is reliable enough for use in a mass screening and control scheme on BVDV.     

Test-retest repeatability of the National Animal Health monitoring system dairy heifer health report in New York and Pennsylvania, USA

We did a test-retest study on 21 farms to assess the repeatability of the Dairy Heifer Health Report of the National Dairy Heifer Evaluation Project. The median retest interval was 42 days (range 14–63 days), retest interviewers were blinded as to responses on the first visits, and all date-specific questions were anchored to the dates of the first tests. The overall discrepancy percentage was 8.5% (95% CI 8.0–9.0). Individual farms had discrepancy percentages of 3.0–15.2%; 240 out of the 539 questions had no discrepancies at all, but 81 questions had more than four (out of 21 possible) discrepancies (lowest percentages among the 81: 24%; 95% lower confidence limit on 24%: 6%). Combining ‘no’ and ‘not applicable’ responses for six similarly worded questions in the Biosecurity section would reduce the overall questionnaire discrepancy percentage to 7.8% and would bring that section's discrepancy percentage down to 8.6%. The other discrepancy percentages by section were: Maternity Hygiene, 14.1 %; Preweaning Hygiene, 4.8%; Disease Agents, 3.6%; Vaccination Practices, 12.1%; Preventive Practices, 16.8%. Discrepancy percentages did not differ importantly by type of question (independent, filter, or subordinate) or by type of response required (categorical, count, or measurement). Discrepancy percentages were independent of retest interviewer, test-retest interval and order of test within state (a proxy for time since training of the interviewers) but were correlated with herd size (r_sp = 0.59).     

Investigation of a Commercial ELISA for the Detection of Canine Procalcitonin

Background

Rapid identification of sepsis enables prompt administration of antibiotics and is essential to improve patient survival. Procalcitonin (PCT) is a biomarker used to diagnose sepsis in people. Commercial assays to measure canine PCT peptide have not been validated.

Objective

To investigate the validity of a commercially available enzyme‐linked immunosorbent assay (ELISA) marketed for the measurement of canine PCT.

Animals

Three dogs with sepsis, 1 healthy dog, 1 dog with thyroid carcinoma.

Methods

Experimental study. The ELISA''s ability to detect recombinant and native canine PCT was investigated and intra‐assay and interassay coefficients of variability were calculated. Assay validation including mass spectrometry of the kit standard solution was performed.

Results

The ELISA did not consistently detect recombinant canine PCT. Thyroid lysate yielded a positive ELISA signal. Intra‐assay variability ranged from 18.9 to 77.4%, while interassay variability ranged from 56.1 to 79.5%. Mass spectrometry of the standard solution provided with the evaluated ELISA kit did not indicate presence of PCT.

Conclusions and Clinical Importance

The results of this investigation do not support the use of this ELISA for the detection of PCT in dogs.     

快速ELISA诊断猪旋毛虫病的研究

在快速间接ELISA中将被检血清样品和酶结合物一起保温。既简化了操作步骤,又比常规ELISA缩短了30～90分钟。通过对猪旋毛虫病阴阳性血清检测,表明该方法敏感性为93.62％,特异性为96.88％。为猪旋毛虫病捉供了一种快速诊断方法。