Milk Enzyme-linked Immunosorbent Assay Research Articles

Evaluation of a Commercial Serum Competitive Enzyme-Linked Immunosorbent Assay for Detection of Neospora caninum-Specific Antibodies in Raw Milk of Ruminants

Bovine neosporosis is an infection caused by the protozoan parasite Neospora caninum and has substantial veterinary hazards. Neosporosis cannot be controlled by vaccination or chemotherapy. Thus, accurate diagnosis followed by isolation and culling of infected animals is regarded as the most efficient method of control. In vivo diagnosis often relies on serologic testing of the animals, and milk represents a non-invasive and easy-to-collect sample matrix. However, indirect enzyme-linked immunosorbent assay (ELISA) specifically designed for antibody detection in milk are sometimes not easily available and it is tempting to use ELISA kits that are originally designed for use in serum in milk samples instead. Herein, we evaluated a widely used commercial ELISA (ID Screen® Neospora caninum competition Multispecies ELISA (ID. Vet, Grabels, France)), developed for detection of N. caninum antibodies in serum samples, for its performance on milk samples. Milk samples from dairy ruminants (cows, buffaloes, sheep, and goats; n = 149) were tested in parallel with the serum ELISA and a commercial milk ELISA as a standard test (Neospora caninum Milk Competitive ELISA, ID. Vet, Grabels, France). The detected prevalence values were 28.2% (42/149), 17.4% (26/149), and 17.4% (26/149) using milk ELISA, serum ELISA, and both ELISAs, respectively. Sensitivity, specificity, positive predictive value, and negative predictive value for the serum ELISA used with milk samples were 61.9%, 100%, 100%, and 87%, respectively. The agreement and kappa value between the two ELISAs were 89.3% and 0.70, respectively, suggesting substantial agreement. High values of Pearson correlation coefficient (0.904, p ≥ 0.0001) and area under the receiver operating characteristic (ROC) curve (0.789, p ≥ 0.0001) demonstrated the high diagnostic performance of the serum ELISA in milk samples. Also, a Bland–Altman Plot and histogram describing the frequency of distribution of ELISA optical densities confirmed the high agreement of both serum and milk ELISAs. The current results revealed the high specificity but moderate sensitivity of the serum ELISA used for milk samples compared with the milk ELISA. However, the excellent positive predictive value of the serum ELISA makes it an alternative option in case of the unavailability of milk ELISAs. With this study, we provided additional evidence that a widely used serum ELISA test kit may also be used for the detection of N. caninum antibodies in milk samples.

Development of a predictive model for bovine leukemia virus proviral load.

BackgroundThere is currently no commercially available method in Canada to identify bovine leukemia virus (BLV)‐positive cows with high proviral load (PVL).ObjectivesFirst, develop a model to predict PVL using common, commercially available, cost‐effective diagnostic tests. Second, investigate the relationship between lymphocyte count and PVL in BLV‐positive cows.AnimalsA total of 339 BLV‐positive and 62 BLV‐seronegative cows on 15 dairy farms.MethodsCross‐sectional study. Blood and milk samples were collected from all lactating BLV‐positive cows on each farm and 5 to 10 BLV‐seronegative cows depending on herd size. Blood and milk samples were tested for anti‐BLV antibodies using enzyme‐linked immunosorbent assay (ELISA). Complete blood counts were performed on blood samples, and standard components analyses were obtained for milk samples. Proviral load was determined by quantitative polymerase chain reaction for each cow.ResultsThe inverse of lymphocyte count, the square of the inverse of lymphocyte count, and milk ELISA percent positivity were positively associated with increasing PVL in BLV‐positive cows. For BLV‐positive cows, lymphocyte count >5.2 × 109/L predicted a high PVL (BLV:Bovine DNA of >1 in blood) with a sensitivity of 92.4% and a specificity of 79.8%. For BLV‐positive cows, white blood cell count >10.8 × 109/L predicted a high PVL, with a sensitivity of 85.5% and a specificity of 83.6%.Conclusions and Clinical ImportanceBased on these results, producers can implement commonly available diagnostic tests to identify cows with high probability of having high PVL, which may help in designing effective disease control strategies for BLV‐positive herds.

The National Johne's Management Plan: using risk and data analysis to optimise Johne's control in dairy herds

The National Johne's Management Plan (NJMP) is now a compulsory element of the Red Tractor Farm assurance scheme. Over 95% of UK dairy farms will need to undertake surveillance, risk assessments, and have a written veterinary control plan, and 70% of participants are utilising milk enzyme-linked immunosorbent assay (ELISA) testing. To support the NJMP a new Johne's Progress Tracker has been developed that uses key outcome measures and drivers to provide new insights into Johne's disease (JD) development. Benchmark measures have been developed allowing for graphical comparison of all measures. The JD Tracker delivers a practical opportunity to help JD veterinary advisors to evaluate the reasons why their herds are succeeding or failing to control JD. The development of enhanced risk assessment tools is also essential to identify the expanded list of risks, and engaging the farmer with a successful JD control plan helps secure the farmer's prospects.

The Occurrence of Mycobacterium avium Subspecies paratuberculosis Positive Milk Antibody ELISA Results in Dairy Cattle under Varying Time Periods after Skin Testing for Bovine Tuberculosis.

Simple SummaryMilk from dairy cows is screened for antibodies to the bacterium causing Johne’s disease, which causes diarrhoea. The bacterium causing bovine tuberculosis has similarities in its structure with the bacterium causing Johne’s disease. Bovine tuberculosis is diagnosed by injecting avian and bovine tuberculin in the skin of the neck and comparing the reactions three days later. Potentially, tuberculosis testing may interfere with Johne’s disease testing. This study aimed to clarify the relationship between the time interval between tuberculosis and Johne’s disease testing and the number of Johne’s disease-positive cows. Data were collected from 51 herds, totalling 46,738 cow observations. Analysis showed that Johne’s disease testing in each 14 day interval increased the probability of detecting Johne’s disease-positive cows compared to Johne’s disease testing over 85 days after tuberculosis testing. The probability was 2.5-fold higher in the first 14 day interval after tuberculosis testing, increasing each two-week period to 4.0-fold higher at 57–70 days before dropping again. A previous history of tuberculosis within the herd increased the probability by 1.2 fold. This was less important compared to the timing of Johne’s disease testing after tuberculosis testing.Enzyme-linked immunosorbent assays (ELISA) are used to screen cows for Mycobacterium avium subspecies paratuberculosis (MAP) infections, informing Johne’s disease (JD) management practices in dairy herds. The causative agent of bovine tuberculosis (bTB), Mycobacterium bovis, and MAP share multiple antigens. Moreover, Mycobacterium avium subspecies avium is used in the single intradermal cervical comparative tests (SICCT) that are routinely used in early detection of cows infected with bTB. Although these are different types of immune responses, potentially the SICCT may interfere with the levels of MAP antibodies. This study aimed to clarify the relationship between the SICCT-MAP milk ELISA testing interval and apparent prevalence of JD risk statuses. Data from 51 herds were used, totalling 46,738 cow observations. The Poisson models showed that MAP milk ELISA testing at 14 day intervals post-SICCT statistically significantly increased the odds of detecting JD-positive cows compared to JD testing 85+ days post-SICCT. The odds ratio (OR) started at 2.5 in the first 14 day interval post-SICCT, increasing each two-week period to an OR of 4.0 at 57–70 days, to subsequently drop. Additionally, a herd history of bTB increased the odds of detecting JD-positive cows (OR = 1.2); this was relatively limited compared to the magnitude of the post-SICCT effect.

Detection of Brucella spp. in raw milk from various livestock species raised under pastoral production systems in Isiolo and Marsabit Counties, northern Kenya

IntroductionBrucellosis is an important zoonotic disease in Kenya, and identifying the bacteria in milk is important in assessing the risk of exposure in people.MethodsA cross-sectional study that involved 175 households was implemented in the pastoral counties of Marsabit and Isiolo in Kenya. Pooled milk samples (n = 164) were collected at the household level, and another 372 were collected from domesticated lactating animals (312 goats, 7 sheep, 50 cattle and 3 camels). Real-time polymerase chain reaction (qPCR) testing of the milk samples was performed to identify Brucella species. Brucella anti-LPS IgG antibodies were also detected in bovine milk samples using an indirect enzyme-linked immunosorbent assay (ELISA).ResultsBased on the qPCR, the prevalence of the pathogen at the animal level (considering samples from individual animals) was 2.4% (95% confidence interval (CI) 1.1–4.5) and 3.0% (CI: 1.0–7.0) in pooled samples. All 14 samples found positive by qPCR were from goats, with 10 contaminated with B. abortus and 4 with B. melitensis. The Brucella spp. antibody prevalence in bovine milk using the milk ELISA was 26.0% (95% CI: 14.6–40.3) in individual animal samples and 46.3% (95% CI: 30.7–62.6) in pooled samples.ConclusionThe study is the first in Kenya to test for Brucella spp. directly from milk using qPCR without culturing for the bacteria. It also detected B. abortus in goats, suggesting transmission of brucellosis between cattle and goats. The high prevalence of Brucella spp. is a significant public health risk, and there is a need for intervention strategies necessary in the study area.

Genomic prediction of bovine leukosis incidence in a US Holstein population

Bovine leukosis (BL) is a dairy cattle disease with a significant negative impact on several economically important traits such as milk yield, fertility and survival of animals. As there is no treatment or vaccine for this disease, finding a possible genetic solution to alleviate the problem would be extremely beneficial. Like other complex traits, the utilization of molecular marker information within a genomic selection approach might help in making selection decisions and potentially reduce the prevalence of the disease in dairy herds. However, the choice of the appropriate response variable and the statistical model are required for a successful genomic prediction program. As such, the objective of this study was to assess the prediction quality of genomic selection models for BL incidence. Milk Enzyme-Linked Immunosorbent Assay (ELISA) data obtained from a US Holstein population were analyzed using two modeling approaches: Bayes A and Bayes B, and three alternative response variables (i.e. pseudo-phenotypes): estimated breeding value (EBV), deregressed proofs free of parent average (DRP), and DRP with parent average added back after deregression (DRP_PA). The prediction ability for each combination of model and pseudo-phenotype was assessed based on the reliability, which was calculated as the square Pearson correlation coefficient between the response variable and the estimated genomic breeding values. Furthermore, to assess potential bias of predictions, response variables were regressed on their corresponding estimated genomic breeding values. Bayes A and Bayes B showed similar results across the three response variables analyzed in this study. Using DRP_PA yielded 6% and 10% higher prediction ability compared to EBV and DRP, respectively. In addition, DRP_PA revealed lower bias estimates. Genomic selection can be potentially applied for BL incidence to reduce the prevalence of the disease in dairy cattle herds. Adding back parents average to DRP may increase the reliability and reduce the bias of genomic selection for this trait.

Determination of Aflatoxin M1 and Ochratoxin A in Raw, Pasteurized and UHT Milk in Turkey

Background: Mycotoxins produced by yeast and fungi have toxic effects on human and animal health. Aflatoxin B1 (AFB1) is the most toxic hepatocarcinogen to mammals. Aflatoxin M1 (AFM1), which has been found in milk and dairy products, is the hydroxylated metabolite of AFB1. Aflatoxin M1 is formed by the cytochrome P450 enzyme in the liver. Ochratoxin A (OTA) is synthesized by Aspergillus and Penicillium species. Ochratoxin A is known to cause teratogenic, immunotoxic, nephrotoxic and carcinogenic effects. Due to the potential harmful effects on human and animal health, OTA has also been receiving increased attention globally; however, there is limited information on the presence of OTA in milk and dairy products. The aim of this study was to determine how mycotoxins impact the hygienic quality of raw and heat-processed milk.Materials, Methods & Results: In this study, a total of 105 milk samples were analyzed (35 raw, 35 pasteurized and 35 UHT) to identify AFM1 and OTA in raw, pasteurized and ultra-high temperature processing (UHT) milk. The levels of AFM1 were detected by using the enzyme-linked immunosorbent assay (ELISA). The milk samples were centrifugedin order to remove the fat content from the milk. After centrifugation, the upper cream layer was withdrawn with a pipette. The non-fat liquid portion was placed in wells at 100 μL for analysis. The concentration of AFM1 in the milk samples was analyzed by AFM1 test kit.The milk samples with AFM1 levels greater than 50 ng/L were confirmed by using High-Performance Liquid Chromatography (HPLC). An Ochratoxin A Serum / Milk ELISA test kit was used for the analyses of OTA. The analyses were made according to the manufacturer’s instructions, and samples were analyzed in duplicate. The absorbance value of milk samples was obtained from the ELISA plate reader at 450 nm. The mean value of AFM1 was found to be 19.54 ng/L in the milk samples. According to the European Commission (EC), the maximum limit for AFM1 in milk is 50 ng/L. In our study, eight (7.61%) of the 105 samples exceeded this limit. The mean value of OTA was found to be 119 ng/L in the milk samples. The relationship between milk type and levels of AFM1 was found to be significant at (P < 0.01). The mean value of AFM1 in pasteurized milk was found statistically significant and lower than raw milk (P < 0.05). The difference between levels of OTA and milk type was not statistically significant at (P > 0.05).Discussion: Milk is a great protein source especially for children in the age of growth. Yeasts such as Fusarium, Aspergillus and Penicillium produce mycotoxins that cause food, feed contamination. Owing to carcinogenic, mutagenic and teratogenic effects of AFM1, presence of AFM1 in milk samples may adversely affect human health. The presence of AFM1 in different contamination levels can be observed in milk and milk products. Factors such as ration type, climate conditions, feed storage conditions, feeding regime and health status of dairy animals may be effective in the occurrence of these contamination. It is necessary to establish legal limits by conducting effective research on the existence of OTA in animal-derived products. The existence of mycotoxins in milk and dairy products can be reduced by preventing the contamination of feed materials with yeast and molds used in the feeding of dairy cows. Milk is one of the most important protein source for the human, effective hygienic controls should be applied to prevent microbiological and chemical hazards. Our data suggest that heat-treated milk may also be dangerous to human health, mycotoxins contamination should be controled with monitoring programs routinely in milk and feed materials for food safety. Determination of Aflatoxin M1 and Ochratoxin A in Raw, Pasteurized and UHT Milk in Turkey

Bulk tank milk ELISA to detect IgG1 prevalence and clustering to determine spatial distribution and risk factors of Fasciola hepatica-infected herds in Mexico.

Fasciola hepatica is a helminth parasite that causes huge economic losses to the livestock industry worldwide. Fasciolosis is an emerging foodborne zoonotic disease that affects both humans and grazing animals. This study investigated the associations between climatic/environmental factors (derived from satellite data) and management factors affecting the spatial distribution of this liver fluke in cattle herds across different climate zones in three Mexican states. A bulk-tank milk (BTM) IgG1 enzyme-linked immunosorbent assay (ELISA) test was used to detect F. hepatica infection levels of 717 cattle herds between January and April 2015. Management data were collected from the farms by questionnaire. The parasite's overall herd prevalence and mean optical density ratio (ODR) were 62.76% and 0.67, respectively. The presence of clustered F. hepatica infections was studied using the spatial scan statistic. Three marked clusters in the spatial distribution of the parasite were observed. Logistic regression was used to test three models of potential statistical association from the ELISA results using climatic, environmental and management variables. The final model based on climatic/environmental and management variables included the following factors: rainfall, elevation, proportion of grazed grass in the diet, contact with other herds, herd size, parasite control use and education level as significant predictors. Geostatistical kriging was applied to generate a risk map for the presence of parasites in dairy herds in Mexico. In conclusion, the spatial distribution of F. hepatica in Mexican cattle herds is influenced by multifactorial effects and should be considered in developing regionally adapted control measures.

The Effect of Different Methods of Fermentation on the Detection of Milk Protein Residues in Retail Cheese by Enzyme‐Linked Immunosorbent Assay (ELISA)

Many commercially available enzyme-linked immunosorbent assays (ELISAs) are not validated for detection of allergenic residues in fermented or hydrolyzed products. This research seeks to determine if commercial milk ELISAs can detect milk residues in varieties of cheese that have undergone different styles of fermentation and different degrees of proteolysis. Only certain milk ELISA kits are capable of detecting residues in all varieties of cheese. However, commercial milk ELISA kits are capable of semiquantitative detection of cheese residues in foods, or in industry settings for the validation of allergen cleaning programs.

Commercial Milk Enzyme-Linked Immunosorbent Assay (ELISA) Kit Reactivities to Purified Milk Proteins and Milk-Derived Ingredients.

Numerous commercial enzyme-linked immunosorbent assay (ELISA) kits exist to quantitatively detect bovine milk residues in foods. Milk contains many proteins that can serve as ELISA targets including caseins (α-, β-, or κ-casein) and whey proteins (α-lactalbumin or β-lactoglobulin). Nine commercially-available milk ELISA kits were selected to compare the specificity and sensitivity with 5 purified milk proteins and 3 milk-derived ingredients. All of the milk kits were capable of quantifying nonfat dry milk (NFDM), but did not necessarily detect all individual protein fractions. While milk-derived ingredients were detected by the kits, their quantitation may be inaccurate due to the use of different calibrators, reference materials, and antibodies in kit development. The establishment of a standard reference material for the calibration of milk ELISA kits is increasingly important. The appropriate selection and understanding of milk ELISA kits for food analysis is critical to accurate quantification of milk residues and informed risk management decisions.

Sensitivity and specificity of repeated test results from a commercial milk enzyme-linked immunosorbent assay for detection of Mycobacterium avium subspecies paratuberculosis in dairy cattle.

To investigate the sensitivity and specificity of results of initial and repeated milk ELISAs (at 6- or 12-month intervals) to detect cows that were shedding Mycobacterium avium subsp paratuberculosis (ie, were infectious) and to evaluate factors influencing the probability that the results of a repeated milk ELISA would be positive for an infectious cow if the results of the initial milk ELISA were negative. Prospective cohort study. 3,145 dairy cows from 32 herds. Herds from the 3 Maritime provinces in Canada (Prince Edward Island, New Brunswick, and Nova Scotia), participating in a Dairy Herd Improvement program, and that had undergone a prior Mycobacterium avium subsp paratuberculosis awareness project were selected for the study. Sample collection occurred between April 2009 and March 2011 with milk and fecal samples collected from all lactating cows in study herds every 6 months. Herds completing < 3 herd visits with collection of individual cow fecal or milk samples, within this sampling timeframe, were excluded from analyses. Fecal samples were cultured in liquid medium and a cow was defined as infectious if ≥ 1 sample was culture positive (reference test). A milk ELISA (index test) was completed with a commercial kit, following manufacturer's instructions. For a 6-month test interval, sensitivities of the milk ELISA to detect infectious cows were 22.0% and 32.6% for initial and combined initial and repeated tests (parallel interpretation), respectively. Specificity of the initial ELISA was 99.6% and was 99.2% for combined tests. For a 12-month test interval, sensitivities of the milk ELISA to detect infectious cows were 25.6% and 45.3% for initial and combined initial and repeated tests (parallel interpretation), respectively. Specificity of the initial ELISA was 99.6% and was 98.9% for combined tests. In infectious cows, magnitude of the initial negative ELISA result was a positive predictor for a positive repeated ELISA result. Results of a repeated milk ELISA improved detection of Mycobacterium avium subsp paratuberculosis infectious cows, with minimal loss of specificity. A 12-month test interval provided a greater increase in sensitivity, relative to an initial test, than did a 6-month interval. Infectious cows with an initial negative milk ELISA result close to the cutoff for a positive test were more likely to have positive results on a repeated ELISA. Repeated testing improved detection of infectious cows and reduced risk of misclassification compared with a single ELISA result.

ELISA for Raw and Heat-Treated Cows and Buffalos Milk in the Milk of Other Species and Sources

Due to the lower prices of cow’s and buffalo’s milk, fraudulent mixing with higher priced milk of other species and sources is economically attractive but illegal and dangerous for allergic consumers. For the rapid detection of cow’s milk, several immunoassays are available but most of them lack the possibility to detect heat-treated cow’s milk due to denaturation of the target protein(s). In the present indirect enzyme-linked immunosorbent assay (ELISA), a mouse monoclonal antibody (Mab) raised against the bovine milk protein A¸-casein is labelled with the enzyme horseradish peroxidase and the binding to the bovine A¸-casein-coated wells of a 96-wells microtiter plate is inhibited by free bovine A¸-casein in the sample or standard. The Mab recognises a 5 amino acids-containing epitope on the glycomacropeptide (GMP) part of bovine A¸-casein which is absent in A¸-casein of goat, sheep, horse, donkey, camel, etc, but is present in A¸-casein of buffalo. Due to the indirect assay format, the cow’s and buffalo’s milk ELISA also recognises heat-treated milk containing denatured A¸-casein. The measurement range of the bovine A¸-casein calibration standards is from 0.1 to 2.5 μg/ml, the limit of detection is 0.05 μg/ml and the only milk sample preparation needed is dilution (100 or 1000 times). This new fast and easy-to-apply ELISA is well suited for the detection of the lower priced raw and heated milk of cows and buffalos in the higher priced milk of other species and sources within the range of 0.25 till 50%.

The Single Intradermal Cervical Comparative Test Interferes with Johne's Disease ELISA Diagnostics.

Enzyme-linked immunosorbent assays (ELISA) of milk and serum samples are a routinely used method of screening herds for Mycobacterium avium subspecies paratuberculosis (MAP). Infection with MAP causes granulomatous enteritis of ruminants known as Johne’s disease (JD). The sensitivity (Se) and specificity (Sp) of MAP ELISAs leads to difficulties in the identification of both infected and infectious animals. Interference with MAP ELISA Se and Sp has been reported in MAP seronegative cows following administration of purified protein derivative (PPD) as part of intradermal testing for bovine tuberculosis (bTB). The aim of this study is to examine the impact of the single intradermal cervical comparative test (SICCT) for bTB, on both serum and milk MAP ELISA tests, in a herd containing both seropositive and seronegative cows pre-SICCT. A secondary objective is to provide appropriate timing of JD ELISA tests in relation to the SICCT. A herd of 139 cows were serum and milk sampled pre- and post-SICCT administration. Prior to SICCT, 6% of the herd tested seropositive for MAP using milk ELISA, with 8% positive on serum. ID Screen Paratuberculosis Indirect Screening Test (ID Vet) was used to screen the herd. Within 14 days of PPD administration, a significant increase in the prevalence of seropositive cows was recorded. Identical prevalence’s were recorded with both test matrices (39%). ELISA values remained significantly higher until day 43 post-SICCT in milk (P = 0.850), and day 71 in serum (P = 0.602). If the “new” positives detected post-bTB testing are deemed false positives due to generation of cross-reacting antibodies by administration of PPD, milk would appear a more suitable sample for JD ELISA testing within 2 months of SICCT. In summary, sampling for JD utilizing milk ELISA should be avoided in the 43-day period following PPD administration, with serum ELISA sampling avoided for an additional 28 days.

Associations between Mycobacterium avium subsp. paratuberculosis antibodies in bulk tank milk, season of sampling and protocols for managing infected cows.

BackgroundThe objective of this study was to identify associations between the concentration of Mycobacterium avium subsp. paratuberculosis (MAP) antibodies in bulk milk and potential risk factors in herd management and herd characteristics, explaining high MAP antibody titers in milk. An extensive questionnaire was administered to 292 organic and conventional dairy farms from New York, Wisconsin and Oregon. Bulk milk samples were taken from each farm for MAP enzyme-linked immunosorbent assay (ELISA). A general linear model was constructed with MAP ELISA value as the outcome variable and the management factors and herd characteristics as independent variables, while at the same time controlling for the study design variables of state, herd size, and production system (organic or conventional). High bulk tank MAP ELISA value may be due to either a high prevalence of MAP in a herd with many cows contributing to the antibody titer or due to a few infected cows that produce large quantities of antibodies.ResultsResults of the regression models indicated that bulk milk ELISA value was associated with season of sampling and the presence or absence of protocols for managing MAP-positive cows. The concentration of MAP antibodies in bulk milk varied seasonally with a peak in the summer and low concentrations in the winter months. When compared to farms that had never observed clinical Johne’s disease, keeping MAP-positive cows or only culling them after a period of delay was associated with an increase in optical density.ConclusionsThe seasonal variation in MAP antibody titers, with a peak in the summer, may be due to a seasonal increase in MAP-bacterial load. Additionally, seasonal calving practices may contribute to seasonal fluctuations in MAP antibody titers in bulk tank milk. Keeping MAP-positive cows increases the antibody titer in bulk milk, likely due to direct antibody production in the infected cow and indirect triggering of antibody production in herdmates.

Bayesian estimation of sensitivity and specificity of Coxiella burnetii antibody ELISA tests in bovine blood and milk

Serological tests for Coxiella burnetii (the causative agent of Q fever) antibodies are usually based on enzyme linked immunosorbent assay (ELISA) although this method is not thoroughly evaluated. The objective of this study was to determine the sensitivity and specificity of an ELISA for detection of C. burnetii antibodies in milk and blood samples, using latent class models in a Bayesian analysis. Blood and milk samples of 568 lactating cows from 17 Danish dairy cattle herds collected in 2008 were used.The best combination of sensitivity and specificity estimates was revealed at a sample to positive (S/P) cut-off of 40 for both blood and milk ELISAs. At this cut-off, sensitivity of milk ELISA was 0.86 (95% posterior credibility interval [PCI] [0.76; 0.96]). This was slightly but insignificantly higher than sensitivity of blood ELISA (0.84; 95% PCI [0.75; 0.93]). The specificity estimates of the ELISA methods on milk and blood were equal at 0.99. No conditional dependence was observed between the specificity estimates of the two test methods. However, the sensitivity estimates of both tests were significantly reduced when conditional covariances ≥40 were used. Collection of milk samples from lactating cows is relatively easy, non-invasive and inexpensive and hence milk ELISA may be a better option for screening lactating cows. But, blood ELISA is an option for screening non-lactating cattle.

Effect of season and lactation stage on the diagnostic sensitivity of direct real-time PCR assay for detection of fecal shedding of Mycobacterium avium subsp paratuberculosis

Mycobacterium avium subsp paratuberculosis (MAP) is the causative organism of Johne's disease. Dairy Farmers of Canada lists this production-limiting disease as one of the top two animal health priorities for the Canadian dairy industry. Infection of newborn calves from ingestion of MAP-infected feces, colostrum, or milk is considered the main route of transmission and a major concern in a herd. Current diagnostics rely on identification of the bacterium in feces via culture and molecular tests or identification of MAP antibodies in milk or serum via enzyme-linked immunosorbent assays (ELISA). However, these tests are inadequate to meet industry needs for herd biosecurity and environmental-transmission control of MAP, especially when MAP-infected cows are in the subclinical stages of the disease. Milk ELISAs have poor predictive values due to imperfect sensitivity and specificity, especially when used in herds with a low prevalence of MAP-infected cows. Although culture of fecal samples for MAP is currently the gold standard diagnostic test for identification of MAP-infected cows, the long incubation times, costs, and intermittent shedding of MAP in feces hinder its use as an efficient screening tool. The goal of this study was to assess how shedding patterns of MAP in feces vary with lactation stage and season, as determined via direct real-time polymerase chain reaction (PCR) assay.

Cost-effectiveness of diagnostic strategies to identify Mycobacterium avium subspecies paratuberculosis super-shedder cows in a large dairy herd using antibody enzyme-linked immunosorbent assays, quantitative real-time polymerase chain reaction, and bacterial culture

Diagnostic strategies to detect Mycobacterium avium subsp. paratuberculosis (MAP) super-shedder cows in dairy herds have been minimally studied. The objective of the current study was to compare the cost-effectiveness of strategies for identification of MAP super-shedders on a California dairy herd of 3,577 cows housed in free-stall pens. Eleven strategies that included serum or milk enzyme-linked immunosorbent assay (ELISA), quantitative real-time polymerase chain reaction (qPCR) or culture of environmental samples, pooled or individual cow fecal samples, or combinations thereof were compared. Nineteen super-shedders (0.5%) were identified by qPCR and confirmed by culture as cows shedding ≥ 10,000 colony forming units (CFU)/g feces (median of 30,000 CFU/g feces). A stratified random sample of the study herd based on qPCR results of fecal pools was the most sensitive (74%) strategy and had the highest cost ($5,398/super-shedder). The reference strategy with the lowest cost ($1,230/super-shedder) and sensitivity (47%) included qPCR testing of fecal samples from ELISA-positive lactating (milk) and nonlactating (serum) cows housed in pens with the highest MAP bioburden. The most cost-effective alternative to the reference was to perform qPCR testing of fecal samples from ELISA-positive cows (milk and serum for milking and dry cows, respectively) for a sensitivity of 68% and cost of $2,226/super-shedder. In conclusion, diagnostic strategies varied in their cost-effectiveness depending on the tests, specimen type, and labor costs. Initial qPCR testing of environmental samples from free-stall pens to target cows in pens with the highest MAP bioburden for further testing can improve the cost-effectiveness of strategies for super-shedder identification.

Evaluation of combined high-efficiency DNA extraction and real-time PCR for detection of Mycobacterium avium subsp. paratuberculosis in subclinically infected dairy cattle: comparison with faecal culture, milk real-time PCR and milk ELISA

BackgroundJohne’s disease is caused by Mycobacterium avium subsp. paratuberculosis (Map) and it is one of the most important diseases in cattle worldwide. Several laboratory tests for Map detection are available; however, these are limited by inadequate sensitivity and specificity when used in subclinically infected populations. To identify Map shedders in subclinically infected cattle, we used a new, high-yield method for DNA-extraction from Map in faeces combined with quantitative real-time PCR (qPCR) for amplification of the insertion sequence IS900 of Map (HYDEqPCR). Evaluation of HYDEqPCR was carried out in comparison with faecal culture, milk qPCR, and milk enzyme-linked immunosorbent assay (ELISA), on 141 faecal and 91 milk samples, from 141 subclinically infected dairy cattle.ResultsThe qPCR proved to be highly sensitive, with a detection limit of 2 IS900 DNA copies/μl in 67 % of the reactions. It also showed 100 % specificity, as determined from 50 Map and non-Map strains, and by the sequencing of qPCR amplicons. The detection limit of HYDEqPCR was 90 Map/g Map-spiked faeces, which corresponds to 2.4 colony forming units/g Map-spiked faeces, with an estimated efficiency of 85 % (±21 %). When tested on the field samples, HYDEqPCR showed 89 % of the samples as positive for Map, whereas faecal culture, milk qPCR, and milk ELISA detected 19 %, 36 % and 1 %, respectively. Fisher’s exact tests only show statistical significance (p ≤0.05) for the correlation between HYDEqPCR and faecal culture. The agreement between HYDEqPCR and milk qPCR and milk ELISA was poor, slight, and non-significant.ConclusionsThis study highlights the advantages of HYDEqPCR for detection of Map in subclinically infected populations, in comparison with faecal culture, milk qPCR and milk ELISA. HYDEqPCR can detect low-level Map shedders that go undetected using these other methods, which will thus underestimate the proportions of Map-shedders in herds. Identification of these shedding animals is extremely important for prevention of the spread of Map infection in an animal population. Due to the relatively high sensitivity and specificity of HYDEqPCR, it can be applied to test for Map at the herd or individual level, regardless of animal age or production stage. HYDEqPCR will allow early detection and control of Map in any population at risk.

Bovine IFNGR2, IL12RB1, IL12RB2, and IL23R polymorphisms and MAP infection status

Mycobacterium avium ssp. paratuberculosis (MAP) infection causes a chronic granulomatous inflammatory condition of the bovine gut that is characterized by diarrhea, progressive weight loss, and emaciation, and ultimately leads to loss in productivity and profitability of dairy operations. The host cytokine machinery is known to play an important role in protecting against MAP infection. Therefore, the goal of the present study was to assess whether polymorphisms in candidate genes encoding important cytokines and cytokine receptors are associated with MAP infection status of dairy cattle. MAP infection status was evaluated based on serum and milk enzyme-linked immunosorbent assays (ELISAs) for MAP-specific antibodies. Twenty previously reported polymorphisms in genes encoding bovine interferon gamma (IFNG), IFNGR1, IFNGR2, IL22, IL22RA1, IL12RB1, IL12RB2, and IL23R were genotyped in a resource population of 446 dairy Holsteins with known MAP infection status, and logistic regression was used to assess the statistical association with a binomial MAP infection status phenotype. Four SNPs in IFNGR2, IL12RB1, IL12RB2, and IL23R were found to be associated with the MAP infection status of the resource population. These results underscore the importance of cytokines and their receptors in conferring protection against MAP infection and warrant further functional characterization of these associations.

Towards assessing fine-scale indicators for the spatial transmission risk of Fasciola hepatica in cattle

In order to improve the spatial resolution of current risk maps for fasciolosis in cattle, more knowledge is needed with respect to farm-level factors that determine infection risk. In this study, we visited 39 dairy farms within a predefined low- and high-risk area for fasciolosis in Belgium and assessed their infection status by an indirect bulk tank milk enzyme-linked immunosorbent assay (ELISA). Management factors were collected and all pastured lands of the farms were visited to identify and georeference potential snail habitats. The habitats were visually characterised, investigated for the presence of the intermediate host snails of Fasciola hepatica (i.e. Galba truncatula and Radix spp) and used in a geographical information system (GIS) to construct overlays including information on soil and hydrology. A linear regression model was used to evaluate associations between bulk tank milk ELISA results and farm level management and habitat factors. A logistic, mixed model was used to identify possible risk factors for the presence of intermediate host snails on a potential habitat. Potential snail habitats were found in 35 out of 39 farms. A total of 87 potential habitats were identified and on 29% of these, intermediate host snails were found. The number of potential habitats, the presence of snails, drainage of pastures, month of turnout of the cows, stocking rate, type of watering place and risk area were significantly associated with the bulk tank milk ELISA result and explained 85% of the observed variation. Intermediate host snails were more likely to be present with increasing surface of the potential habitat and on loamy soils. This study confirms the importance of farm management factors in the infection risk for F. hepatica in cattle and highlights that the combination of management factors with characterization of snail habitats is a powerful means to predict the infection risk with F. hepatica at the individual farm level. Further research is needed to investigate how this knowledge can be incorporated in nation-wide spatial distribution models of the parasite.