A Comparative Study on the Efficiency of Two Mycobacterium avium subsp. paratuberculosis (MAP)-Derived Lipopeptides of L3P and L5P as Capture Antigens in an In-House Milk ELISA Test

Mycobacterium avium subsp. paratuberculosis (MAP) surface-exposed lipopeptides could be specific capture-antigen molecules targeting antibodies against MAP, in milk, through ELISA. Previous studies have revealed that MAP strains, isolated from sheep (S) or cow (C), could produce specific lipopeptides, L3P or L5P, respectively. In this study, we used L3P and L5P as capture antigens in an in-house milk ELISA (H-MELISA) to assess how these antigens perform, in comparison with other ELISA tests, on well-defined milk samples from MAP-infected sheep. The overall positivity rates of H-MELISA via L3P and L5P varied by the source of milk samples, in which, at bulk-tank-milk (BTM) level, the majority of positive cases (63.83%) reacted more against L5P, whereas a predominant number (69.14%) of milk samples were more responsive against L3P at the individual level. To clarify whether the positivity status of milk samples in H-MELISA L3P/L5P were predictive of MAP strain-types (S/C), strain-typing was carried out using PCR IS1311-restriction enzyme analysis. Although the presence of three MAP strains (S/C/bison types) was detected among the milk samples, the C-type (46.67%) and S-type (75%) MAP strains were detected with higher incidence among BTMs and individual milk samples, respectively. However, further examination on the H-MELISA L3P/L5P-positivity pattern of each C/S-type-MAP sample revealed that some samples had a reverse reactivity against both L3P and L5P. These results could be the consequence of either cross-reactivity between L3P and L5P (due to the similarity in the structures of the two epitopes) or simply a within-herd mixed infection with MAP strains of C and S types. These findings suggest that lipopeptide antigens could contribute a diagnostic test with optimal performance, considering the diversity of MAP strains.

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

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.

Validation of I’screen AFLA M1 Milk for Detection of Aflatoxin M1 in Raw Bovine Milk and Powdered Milk: AOAC Performance Tested MethodSM 072002

Background Aflatoxin M1 (AFM1) is the major metabolite of Aflatoxin B1 (AFB1) and can be found in the milk of animals fed with feed containing AFB1. The frequency of occurrence of AFM1 in milk has led to the development of specific quantitative methods of analysis to mitigate the risk of adversely affect human health. Objective: The objective was to demonstrate that I’screen AFLA M1 Milk ELISA kit can quantify AFM1 in raw bovine milk and in powdered milk. Methods Assay performance was evaluated studying lot-to-lot consistency, assay stability, robustness and possible interferences of related molecules. Raw bovine milk samples spiked at 0, 5.0, 20, 50, 100, 200 ng/L of AFM1 and powdered milk reference materials and spiked samples at 100 and 200 ng/L were tested to determine recovery, repeatability and bias. LOD and LOQ were also determined for both matrixes. Results: High selectivity for AFM1 was demonstrated and performances were consistent, robust and stable. The LOQ was validated at 5 ng/L for raw milk and 50 ng/L for powdered milk. Recoveries for spiked raw and powdered milk were 97–122%, with RSDr < 10%, and 106–111% for reference materials, with RSDr < 5%. Conclusions: The data collected validate the method as a selective, specific, sensitive, accurate and precise tool for the analysis of AFM1 in raw bovine milk and powdered milk. Highlights: We demonstrated that I’screen AFLA M1 is a reliable kit and a proper screening tool suitable for high analytical throughputs.

From six to zero per cent Mycobacterium avium subsp. paratuberculosis milk ELISA positivity in three years – probably induced by Mycobacterium vaccae

This research communication reports the results of a study aimed at investigating the effects of introducing Mycobacterium vaccae on paratuberculosis carriage in a dairy herd. M. vaccae is a non-pathogenic member of the Mycobacteriaceae, with immunomodulatory and immunotherapeutic capabilities, acting by stimulating the cellular immune system, important in protection against paratuberculosis. Starting in 2014 we administered, by gavage, 1010 live M. vaccae bacteria to all new-born heifers on a dairy farm, first within 24 h of birth and again 2 weeks later. Paratuberculosis carriage was monitored yearly by milk ELISA. Faecal samples of 50% of cows, aged 3 years, born 1, 2 or 3 years before the experiment's onset, were tested by qPCR for MAP shedding and compared to 100% treated cows of the same age. Within 3 years, milk ELISA positivity was reduced from 6 to 0% and remained unchanged for the subsequent 2 years. One qPCR positive control cow was found each year for a total of 3 animals (2.46%). One positive cow (1%) was found among the treated cows. Two of the 3 positive control animals, still present on the farm at the end of 2019, tested negative whereas the positive test cow continued shedding MAP. M. vaccae shedding heifers mixing with adult cows were the probable means of the microorganism's propagation. The results of this investigation indicate that the introduction of live M. vaccae may be an inexpensive and fast alternative to current paratuberculosis control practices, justifying further exploration of the topic.