Comparison of two serum and bulk-tank milk ELISAs for diagnosing natural (sub)clinical Dictyocaulus viviparus infection in dairy cows

Lungworm antibody ELISAs developed in Germany (DE) and The Netherlands (NL) were compared using four sets of serum (S) and bulk-tank milk (BTM) samples from adult dairy cows. The samples originated from 37 farms with or without a suspected clinical lungworm infection during August–October 2010 (Dataset 1), from cows excreting lungworm larvae or not during August–October 2010 (n = 59) or May–June 2011 (n = 164) (Dataset 2), from 305 farms in a national survey during October 2010 (Dataset 3), and 14 zero-grazing farms during February–April 2011 (Dataset 4).During August–October 2010, covering the second half of the grazing season, the NL-S and NL-BTM ELISA outperformed the DE-S and DE-BTM ELISAs in terms of sensitivity. For at least the NL-S and DE-S ELISA the opposite was found during May–June 2011, covering the end of the winter housing period and the early grazing season. Of the 305 farms in the survey 62.6% were found positive with the NL-BTM ELISA, whereas 2.6% was found positive with the DE-BTM ELISA. ODR values for the zero-grazing farms indicated that a cut-off value of 30% for the DE-BTM ELISA might be more appropriate than the currently used 41%. Results suggest that the NL ELISAs also respond to lungworm antigens other than Major Sperm Protein as used in the DE ELISAs.It is concluded that the generally higher sensitivity of the NL-BTM ELISA makes it better suited for large-scale prevalence studies and herd health monitoring programmes than the DE-BTM ELISA, positivity of which is more associated with the presence of clinical lungworm infection. Reducing the cut-off value of the DE-BTM ELISA from the original 49.3% to the current 41% or the possibly more appropriate 30% increased its sensitivity for detecting lungworm infections, but did not lead to similar sensitivity estimates as found for the NL-BTM ELISA.     

Lungworm infection (Dictyocaulus viviparus) on dairy cattle farms in tropical highlands of Tanzania

Tropical Animal Health and Production -     

Validation of a commercial ELISA for the detection of bluetongue virus (BTV)-specific antibodies in individual milk samples of Dutch dairy cows

A recently developed indirect ELISA for the detection of bluetongue virus (BTV)-specific antibodies in bovine milk samples was compared to that of the routinely used competitive ELISA on serum samples. During the bluetongue outbreak in the Netherlands in 2006, caused by BTV serotype 8, coupled serum and milk samples were obtained from 470 individual cows from 10 BTV-infected farms with an average seroprevalence of 57%. In addition, bulk milk samples of the same farms, and historically BT-negative samples were tested. Compared to the ELISA for sera, the relative specificity and sensitivity of the ELISA for milk samples is 96.5% and 98.9%, respectively when using a S/P% cut-off value of 50% as advised by the manufacturer. The optimal cut-off value was found at S/P% of 90% revealing an optimal specificity (99.0%) combined with an optimal sensitivity (98.1%). Titres in positive individual milk samples ranged from 1 to 2048 with a peak titre of 128. Bulk milk samples contained antibodies with titres ranging from 64 to 512. The ELISA for milk samples was found to be a reliable and robust test. This diagnostic tool is very useful, and may replace the ELISA for serum samples as first choice in order to get insight into the status of lactating individual animals and therewith of the entire herd with respect to BTV infection.     

Optimization of in-house ELISA based on recombinant major sperm protein (rMSP) of Dictyocaulus viviparus for the detection of lungworm infection in cattle

The aim of this study was to optimize an in-house ELISA based on a recombinant version of the major sperm protein (MSP) of Dictyocaulus viviparus for routine diagnosis of lungworm infection in cattle. A recombinant MSP (rMSP) was cloned into pGEX-6P-1 vector and expressed as a glutathione-S-transferase (GST) fusion protein in Escherichia coli BL21 (DE3) chemically competent cells. The product was then employed as capture antigen in an ELISA, and validated against 304 samples of known status (216 negative and 88 positive) in which the antibody levels in sera had also been measured earlier with a commercial ELISA kit (Ceditest® lungworm ELISA). The receiver operating characteristic (ROC) curve analysis of the ELISA results estimated the optimized diagnostic sensitivity and specificity as 97.7% (95% confidence interval [CI]: 91.9–99.7%) and 98.1% (CI: 95.3–99.5%), respectively. The results from the in-house rMSP-based ELISA were compared with results obtained on both fecal examination and the Ceditest® lungworm ELISA. Rising antibody levels in sera of experimentally infected calves were observed between 21 and 28 days post infection, when patency was also confirmed by the presence of larvae in feces. Notably, using the in-house rMSP-based ELISA infection was confirmed in calves shedding larvae approximately 3–4 weeks post inoculation, while using the Ceditest® lungworm ELISA those animals remained negative. Additionally, 251 sera samples from calves naturally exposed to the parasites on pasture were used to evaluate the test. In in-house rMSP-based ELISA no cross-reactions were observed with sera from calves infected with the gastrointestinal nematodes (Ostertagia ostertagi and Cooperia oncophora), even though the presence of eggs in the feces was confirmed. Overall, the in-house rMSP-based ELISA optimized in this study showed excellent diagnostic performance for detection of lungworm infection in cattle.     

Intra- and inter-observer agreement of a protocol for clinical examination of dairy cows

In 2004,five veterinarians examined 283 dairy cows from four Danish dairy herds and independently assigned scores for the clinical signs lameness, hock lesions and other cutaneous lesions to each cow using a clinical protocol. We evaluated the inter-observer and the intra-observer agreement using prevalence-adjusted, bias-adjusted kappa (PABAK). We chose two different cut-offs between the ordinal scores for classifying cows as healthy or diseased for each of the clinical signs, and we compared the ability of the observers to discriminate between healthy and diseased cows for the two cut-offs. Without any formal training of the observers we found PABAK in the range 0.36-0.88 and we concluded that the choice of cut-off had an effect on the observed agreement among the observers.     

Prevalence of Fasciola hepatica in dairy herds in England and Wales measured with an ELISA applied to bulk-tank milk

An ELISA developed to diagnose Fasciola hepatica infection in cattle by detecting serum antibodies was adapted and validated for use with samples of bulk-tank milk. The prevalence of the infection in 61 dairy herds was established by using serum antibody levels or faecal egg counts measured in a proportion of the cows in each herd. The correlation between the results of the ELISA and the herd seroprevalence was 0.83. Using a cut-off value of 27 per cent positive, the bulk-tank ELISA identified herds in which more than 25 per cent of the cows were infected with a diagnostic sensitivity of 96 per cent (95 per cent confidence interval 89 to 100 per cent) and a diagnostic specificity of 80 per cent (95 per cent confidence interval 66 to 94 per cent). By applying the ELISA to 623 herds in England and 445 herds in Wales, the prevalence of F hepatica infection in England was estimated to be 48 per cent (95 per cent confidence interval 46 to 54 per cent), and in Wales 86 per cent (95 per cent confidence interval 84 to 90 per cent).     

Prevalence of Neospora caninum infection in Australian (NSW) dairy cattle estimated by a newly validated ELISA for milk

AIM: To determine the performance characteristics of an Institut Pourquier (IP) enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against Neospora caninum in bovine milk and subsequent determination of the prevalence of N. caninum infection in New South Wales (NSW) dairy cattle. METHODS: Matching serum and milk samples from 93 cattle were assayed in two commercially available ELISAs for the detection of anti-N. caninum antibodies. Serum test results of one ELISA (IDEXX) were used to determine the N. caninum infection status of the cattle. Optimised cut-off values for the IP ELISA using milk samples were determined by two-graph receiver operating characteristic (TG-ROC) analysis and then applied to a representative sample of 398 milk samples from dairy herds around NSW. RESULTS: When this ELISA was applied to a representative collection of 398 milk samples from dairy cattle across NSW it demonstrated a 21.1% prevalence of N. caninum infection in those cattle. From the TG-ROC analysis an IP ELISA protocol was derived which suggested a cut-off threshold that would allow milk testing with 97% sensitivity and specificity, respectively, relative to serum testing. CONCLUSIONS: The prevalence of N. caninum in NSW dairy cattle was higher than previously believed. When used on individual milk samples this ELISA demonstrated high sensitivity and specificity and so could be used to accurately identify N. caninum infection. TG-ROC analysis of the IP ELISA optimised the protocol and prescribed cut-off values enabling the ELISA to be used for the screening of N. caninum antibodies in the milk of dairy cattle.     

Validation of a Dictyocaulus viviparus MSP-ELISA and cut-off adjustment in a one-year longitudinal field study in dairy cattle herds

A one-year field study analysing lungworm seropositivity by use of the MSP-ELISA was performed (1) to investigate the antibody dynamics in individual milk samples following field (re-)infections of dairy cows with the bovine lungworm Dictyocaulus viviparus, (2) to investigate the correlation between individual and bulk tank milk (BTM) antibody titres and (3) to review the current individual as well as BTM cut-off value, which was extrapolated from dilution experiments (Fiedor et al., 2009). Over a one-year period individual and BTM samples were collected monthly on 15 dairy farms. Following a critical review of previous cut-off values, individual and BTM samples were subjected to different cut-off thresholds. Following Receiver-Operating-Characteristics (ROC) analysis, individual milk samples were assessed with the cut-off value 0.573, previously shown to be associated with each 100% sensitivity and specificity. In addition, the present study enabled BTM cut-off adjustment based on field data. To ensure reliable detection of herds with an in-herd prevalence of ≥20% the BTM cut-off was lowered from 0.493 to 0.410, corresponding to 100% sensitivity and 97.32% specificity. Regression analysis showed that the percentage of seropositive animals related to the corresponding BTM ODR correlated moderately (r=0.581, P<0.001), whereas a strong correlation (r=0.764, P<0.001) was found between mean individual and BTM ODR per herd and sampling month. Seasonal antibody pattern became obvious in a single-peaked antibody curve in late summer/early autumn for individual milk whilst BTM showed a two-peaked distribution with an additional spring peak besides the late summer/early autumn peak. This leads to the conclusion that the BTM-ELISA could be a useful tool to detect and control pasture contamination in the spring, following sexual maturation of hypobiotic lungworm larvae harboured by clinically asymptomatic carrier animals. In addition to the knowledge gained on antibody patterns in dairy herds and the relationship of individual and BTM, the present study enabled sensitivity and specificity calculations for the obsolete BTM cut-off value 0.493 to be performed.     

Reduction in milk yield associated with Mycobacterium avium subspecies paratuberculosis (Map) infection in dairy cows

To assess the profitability of control schemes for Mycobacterium avium subspecies paratuberculosis (Map)-infection implemented in dairy herds, accurate estimates of its production effects are needed. This study aimed at quantifying the variation in milk yield of dairy cows according to their Map-infection status. The cow-status was determined by combining (i) its testing(s)-result(s) (serum ELISA, faecal culture (FC), PCR, Ziehl staining), (ii) the Map-status of its herd, and (iii) its possible vaccination against Map. A total of 15 490 cows in 569 herds located in western France was considered. The effect on test-day milk yield (TDMY) of the cow-status to Map was assessed separately in parity 1, 2 and 3 or more, using mixed linear models, after adjustment for herd-season (random), days in milk and breed. Average TDMY was significantly lower in cows from herds with at least one Map-infected cow (defined as positive herds). Individual TDMY showed a reduction ranging from 1.58 to 3.30, 2.03 to 2.51, 5.36 to 7.20 kg/day (P < 0.001) depending on parity for unvaccinated cows and testing ELISA-positive, PCR- or FC-positive, and Ziehl-positive, respectively, compared to cows in Map-free herds. The loss in milk yield increased with increased parity in ELISA-positive and Ziehl-positive cows. Cows that were both tested ELISA-positive and vaccinated had a smaller loss in TDMY than those that were unvaccinated. The estimates from this study can be used to further assess the economic impact associated with Map-infection in dairy herds or to help in the culling decisions regarding infected cows.     

A retrospective evaluation of a Bovine Herpesvirus-1 (BHV-1) antibody ELISA on bulk-tank milk samples for classification of the BHV-1 status of Danish dairy herds

Bulk-tank milk samples analysed in a Bovine Herpesvirus-1 (BHV-1) blocking ELISA are still in use in the Danish BHV-1 programme as a tool to classify dairy herds as BHV-1 infected or BHV-1 free herds. In this retrospective study, we used data from the Danish BHV-1 eradication campaign to evaluate performance characteristics of the BHV-1 blocking ELISA in 1039 BHV-1-seropositive and 502 repeatedly BHV-1-negative dairy herds using the results of blood testing of the individual animals as the true infection status. At a cut-off value of 30% blocking reaction, the herd-level relative sensitivity and relative specificity were 82 and 100%, respectively. The herd-level relative sensitivity depended on the within-herd prevalence of seropositive cows and the cut-off value in the assay, but not on the time interval (up to 90 days) between the collection of the bulk-tank milk sample and the individual serum samples. The BHV-1 blocking ELISA on bulk-tank milk could detect seropositive herds (few), with prevalence proportions as low as one seropositive cow out of 70 cows.     

Prevalence of Coxiella burnetii infection in Dutch dairy herds based on testing bulk tank milk and individual samples by PCR and ELISA

A study using an ELISA and a real-time PCR assay based on the detection of the repetitive transposon-like gene of Coxiella burnetii revealed that infection with the bacterium was widespread among Dutch dairy herds, with antibodies detected in bulk tank milk (BTM) from 268 of 341 herds (78.6 per cent) and bacterial DNA detected in 193 of 341 herds (56.6 per cent). The BTM samples were taken in November and December 2007. Serological and molecular studies in young and adult cattle selected from 100 herds showed that antibodies were present in the blood of 470 of 2936 (16.0 per cent) lactating cows but only in 19 of 1831 (1.0 per cent) young animals. Bacterial DNA was detected in the milk of 254 of 2925 (8.7 per cent) lactating cows; bacterial DNA was not detected in any of the faecal samples obtained from youngstock. The blood and milk samples were taken from the cattle in the period January to April 2008.     

The use of the enzyme-linked immunosorbent assay (ELISA) in serological diagnosis of Mycoplasma bovis in dairy cattle

Between December 1985 and March 1987 an enzyme-linked immunosorbent assay (ELISA) was used with 3774 sera to estimate the prevalence of antibodies to Mycoplasma bovis in sera from three age groups of cattle in four dairies in California and to test for possible associations between the presence of M. bovis antibodies and the age or breed of the cattle and the farm. Unadjusted and adjusted associations were evaluated using the -square test for associations and multiple logistic regression analysis, respectively.There was a tendency for the proportion of cattle seropositive for M. bovis to increase steadily and approximately linearly with age (p<0.05). There was also a statistically significant relationship bbetween a M. bovis seropositive test and being from Farm IV (p<0.05). Farm IV was the largest of the four dairies and this association may be due to the effect of herd size.These findings confirm the ubiquitous distribution of antibodies to M. bovis in dairy cattle in California and also support previous reports of herd size as an important factor in mycoplasmal mastitis.     

Teat endoscopy (theloscopy) for diagnosis and therapy of milk flow disorders in dairy cows.

Teat endoscopy (theloscopy) is a useful technique for diagnosis and therapy of covered teat injuries. Minimal invasive theloscopic surgery may help to restore milk flow, milk yield, and SCC of the affected quarter.Infection with pathogens may not change significantly, however. Cows treated as described may yield as much milk as their herdmates at a slightly increased udder SCC and stay as long in the herd as their herdmates. Theloscopy also may be used for diagnosis and therapy of various other teat disorders.     

Herd-level prevalence of Mycobacterium avium subspecies paratuberculosis by bulk-tank milk PCR in Fars province (southern Iran) dairy herds

A cross-sectional study was conducted from March to August 2006 in dairy herds in Fars province, southern Iran to determine the herd-level prevalence of Mycobacterium avium subspecies paratuberculosis (MAP) infection. Bulk-tank milk samples were collected from 110 dairy herds in the 3 districts (Shiraz, Marvdasht and Sepidan) of the province. Among study populations, 12 herds (11%, 95%CI: 5-17%) were positive for MAP infection based on IS900 nested PCR. The prevalence of positive milk samples in the three districts of Fars province was different ranging from 8.6% to 23% which was not statistically significant (P=0.19). It is recommended to conduct further epidemiologic studies to determine cow-level prevalence and risk factors for infection, and to evaluate the economic consequences of the MAP infection in the region.     

奶牛血清和乳中β-羟丁酸的高效液相色谱法的建立

酮体是由3种物质组成：即β-羟丁酸（Beta—hydroxybutyric acid，BHBA）是酮体的主要成分，约占酮体的70％，在肝脏内合成，经肝外组织利用。由于β-羟丁酸在酮体中所占比例最大，可以代表酮体的含量。因此β-羟丁酸被认为是人糖尿病和奶牛酮病的重要诊断指标之一。     

Risk factors for clinical ketosis and association with milk production and reproduction variables in dairy cows in a hot environment

Tropical Animal Health and Production - The aims of the present study were to investigate (1) the risk factors that influence the occurrence of clinical ketosis (CK; blood β-hydroxybutyrate...