Paratuberculosis, commonly known as Johne’s disease, is a production-limiting disease of dairy cattle caused by Mycobacterium avium
(MAP) and has a substantial financial effect on the dairy industry [1
]. The disease was first reported in Korea in 1984 and its prevalence was subsequently reported to be 18.7% and 11.7% in dairy and beef cattle, respectively [2
]. In Korea, prevalences of 6.1% and 1.2% were recorded in dairy and beef cattle, respectively, in Gyeongnam province in 2009 [3
], and a 5.2% prevalence was observed in Jeju province in 2013 [4
]. Previous studies have reported outbreaks of MAP in other countries. A cattle herd MAP prevalence of 16.7% was reported in Canada, and a 68% MAP prevalence was recorded in US dairy farms in 2008 [5
]. Johne’s disease results in decreased milk production and increased cow replacement costs, which has resulted in economic losses in the US dairy industry estimated at US$
200 to US$
250 million annually, or US$
22 to US$
27 per cow [6
The main clinical signs of paratuberculosis are persistent diarrhea, reduced milk production, weight loss, and progressive emaciation. This infectious disease develops slowly and is characterized by chronic degenerative granulomatous enteritis that affects the distal part of the small intestine, as well as the colon and associated lymphoid tissue. Young animals have a greater possibility of getting an MAP infection when their age is below six months. Following infection by MAP, seroconversion usually occurs around two years of age [7
]. Not all infected animals develop clinical signs, but the disease can be detected by diagnostic testing a few years after the initial infection [8
Therefore, adequate diagnostics are essential to reduce the prevalence of MAP, which is a key component of the control of paratuberculosis in a farm [9
]. Fecal culture and PCR are the diagnostic tools for detection of the MAP antigen, whereas the ELISA methods are commonly used for detection of MAP antibodies. However, fecal samples sometimes show negative results in true MAP-positive animals due to the intermittent shedding of bacteria, resulting in differing sensitivity of each test depending on the stage of infection [10
]. Although the serum/milk ELISA has low sensitivity and high specificity, it is commonly used in dairy herds for detection of MAP, due to its acceptable diagnostic performance, as well its time efficiency and cost effectiveness.
Dairy farms participating in the Johne’s disease eradication program in Korea perform MAP screening tests by using antibody detection ELISA regularly, once or twice per year, with irregular fecal antigen detection PCR, and removing MAP-positive animals from the herd. However, annually new MAP-positive animals have been coming out of these participating farms for several years. We presumed that MAP-positive animals were being missed by single time screening, indicating that the evaluation of MAP antibody status is needed throughout the animal’s lifetime. Therefore, the objective of this study was to evaluate MAP antibody kinetics in serum and milk samples throughout the lactation period and to assess the diagnostic performance of the milk ELISA test.
2. Materials and Methods
2.1. Ethical Statement
All animal procedures and the study design were approved by the Institutional Animal Care and Use Committee (IACUC) at the National Institute of Animal Science (NIAS), Republic of Korea. The Reference number was NIAS (2013-046).
2.2. Selection of Animals
A longitudinal study was performed on three commercial dairy farms that were enrolled in a Johne’s disease control program in Korea. The cows on these farms underwent one- or two-time screenings per month for Johne’s disease by using MAP antibody detection ELISA (IDEXX Laboratories, Inc. Westbrook, ME, USA). More than a hundred cows were tested from three dairy farms and among them eight cows were diagnosed as MAP-positive during the screening period. The cows had no apparent clinical signs of Johne’s disease and showed normal reproductive and milk yield performance. Two healthy MAP-negative dairy cows were used as a negative control. The control group animals were negative in both fecal PCR and serum/milk ELISA results. Cow-specific information about age, calving date, and milk production was recorded for all tested cows. The ages of all animals ranged from 4 to 8 years. The animals were housed in free-stall housing systems and their average daily production of milk was 27–36 kg/cow.
2.3. Sampling and MAP Antibody Testing
Blood and milk samples were collected simultaneously for MAP antibody ELISA testing from eight MAP-positive and two healthy MAP-negative cows throughout the year. We collected blood and milk samples one to two times per month; however, a few samples were missed due to inappropriate sample handling or recording. Only blood samples were collected during the dry period. Approximately 5 mL of blood was collected from the jugular vein of each animal and transferred to the laboratory for further processing. For serum preparation, blood samples were centrifuged at 1800× g for 15 min. The serum was then transferred into a screw-cap tube and stored at −20 °C until analysis. Serum samples were tested by MAP-specific antibody ELISA (IDEXX Laboratories, Inc. Westbrook, ME, USA), following the manufacturer’s recommendations. Samples with a sample-to-positive (S/P) ratio of 55 or higher were considered seropositive for MAP antibody detection, as determined by IDEXX kit cutoffs.
Milk samples were collected aseptically from all four teats after discarding the first few streaks. The milk was taken in a vial, transported to the laboratory, and stored at 4 °C until processed. Samples were tested using a commercially available MAP antibody detection ELISA (IDEXX Laboratories, Inc. Westbrook, ME, USA) as per the procedure recommended by the manufacturer. To determine the number of animals that were MAP-antibody-positive by milk ELISA, an S/P ratio of 30 was used as the cut-off value for a MAP-positive test result, as recommended by IDEXX.
2.4. Statistical Analysis
Serum and milk ELISA S/P ratios were compared by using correlation analysis and controlling for repeated measures. Pearson’s correlation coefficient (R2
) was calculated to determine the linear relationship between test results, and the differences were considered significant at p
< 0.05. Interpretation of the R2
values was done as previously reported [12
]; that is, strong agreement (1 > R2
> 0.7), moderate agreement (0.7 > R2
> 0.5), weak agreement (0.5 > R2
> 0.3), and none or very weak agreement (0.3 > R2
The eight MAP positive cows (denoted as cows A to H) were enrolled as Johne’s disease cows and their MAP antibody levels were observed in serum and milk ELISA throughout the year (Figure 1
). Two healthy MAP-negative cows were used as negative control animals (cows N1 and N2) and their MAP ELISA testing was performed for six months (Figure 1
). All animals had different age records and normal calving history, except one animal that had a history of abortion (cow B).
3.1. MAP Antibody Fluctuation
The antibody level of all MAP-positive animals fluctuated throughout the experimental period. (Figure 1
). In cow A, the serum and milk MAP antibody titers fluctuated between the cut-off values. The serum MAP antibody titers were above the cut-off level in the first to fourth observations, then it decreased and again increased for a single-time observation and continued to maintain its fluctuating nature. The serum MAP antibody titer of cow A was above the cut-off level at ten different times throughout the study period and was below the cut-off level at five other times. Most of the serum MAP antibody levels were higher than the milk MAP antibody levels over the observational period; although, at two observations, the milk MAP antibody titer was higher than the serum titer. Cow B aborted twin calves in the fourth month of pregnancy, and its serum MAP antibody titer fluctuated within the borderline of the cut-off value over the study period. The serum MAP antibody titer of cow H also fluctuated between the cut-off points followed by decreased antibody titer after calving. In general, the MAP antibody titer of cows A, B, and H fluctuated between the borderline of cut-off value. Due to the fluctuating nature of the MAP antibody and depending on the sampling time, these MAP-positive animals would be diagnosed as MAP-negative.
3.2. Dynamics of MAP Antibody Changes After Parturition
: The changing pattern of MAP antibody kinetics in the serum and milk samples (cows A to H) observed after parturition (Figure 1
). The antibody titer of cows C, D, E, and F showed an increased trend in MAP titer after parturition. Based on our field experience, we also categorized our animals into high (>200) and low (<100) MAP antibody titer groups. The serum and milk MAP antibody kinetics of cows C and E fluctuated at a low titer level (<100) until mid-lactation and then the fluctuation continued at a high titer level (>200) throughout the remaining period. The serum MAP antibody kinetic pattern did not follow the milk MAP antibody kinetic pattern before mid-lactation, but afterward, both the serum and milk MAP kinetics followed similar patterns.
Cows D and F showed a rapidly increasing trend of MAP antibody titers after parturition, and their serum MAP antibody titer was higher than that of milk except for one or two unusual observations. The patterns of MAP antibody changes were similar in serum and milk over the entire observational period, with the antibody titers being high in both serum and milk. Cow F had clinical symptoms of Johne’s disease, including watery diarrhea, weight loss, and dehydration, after its fourth calving, and samples of its serum and milk were collected more frequently for one month after it developed clinical symptoms. In cow F, its milk MAP antibody titers were approximately 85% of the serum levels.
Steady-state/decreased trend: After parturition, the MAP antibody kinetics of cow G maintained a steady-state trend in MAP antibody titers, whereas cows A, B, and H showed a decreasing trend. In the case of cow G, the serum MAP antibody levels were higher than those in milk, and both the serum and milk MAP antibody titers maintained a steady-state pattern after parturition. The milk MAP antibody titer remained near the cut-off value over the entire study period. The MAP antibody titers of serum and milk for cow G were 100.5 and 48.4, respectively, at the first observation after calving, both of which were higher than the standard cut-off values. The MAP antibody titers of cow A showed a decreased trend and maintained their fluctuating nature after parturition. Cow B also showed a decreasing and fluctuating trend after abortion. In cow H, the serum MAP antibody titer fluctuated until parturition and then sharply increased followed by a gradual decrease to below the cut-off value at one month after parturition.
For a comparison of MAP-negative and MAP-positive cows, two healthy cows (N1 and N2) were selected as a MAP-negative controls and their MAP antibody levels in both serum and milk were observed throughout the observational period (Figure 1
). Cows N1 and N2 maintained low S/P ratios due to the non-specific reaction of the ELISA test kits. The average ELISA S/P ratios were <10 in serum samples and <5 in milk samples in both MAP-negative animals.
3.3. Correlation between Serum and Milk MAP Antibodies
The repeated serum and milk ELISA measurements underwent correlation-based analysis (Figure 2
and Figure 3
). Approximately 180 serum and milk samples were collected from the MAP-positive cows and analyzed by ELISA. The serum and milk ELISA results showed a moderate agreement (R2
= 0.5358) between the serum and milk MAP antibody levels of the MAP-positive cows (Figure 2
). For further analysis, the MAP antibody titers were categorized as high (>200) or low (<100) level (Figure 3
). A strong agreement (R2
= 0.7335) was observed for the relationship between the serum and milk sample titers in the high MAP antibody titer group (>200), which indicates that serum and milk MAP antibody titer patterns were similar (Figure 3
A). However, the low MAP antibody titer group (<100) showed a very weak correlation between serum and milk samples (R2
= 0.0198), indicating that the milk MAP antibody pattern did not closely follow the serum MAP antibody pattern (Figure 3
B), indicating that the milk ELISA test would provide low diagnostic performance in low MAP antibody titer cows.
MAP antibody titers fluctuated in both serum and milk samples over the year, with the fluctuations occurring near the MAP-positive and MAP-negative cut-off borderline. These fluctuations make it difficult to diagnose a MAP-positive cow by only a single time measurement. The study result indicates that periodic MAP screening in a dairy herd is needed due to the fluctuating trend in MAP antibody level. In addition, the serum MAP antibody levels were gradually increased in high MAP antibody titer (>200) cows after parturition. However, some cows showed steady-state or decreasing trends in MAP antibody levels in low titer (<100) cows after parturition. There was a significant relationship between serum and milk sample results in cows with high MAP antibody titer (>200), but a weak relationship in cows with low (<100) MAP antibody titer. This weak agreement between serum and milk samples in low MAP antibody titer cows is indicative of low diagnostic performance of the milk MAP ELISA. Finally, the results of this study suggest that those farms applying Johne’s disease eradication programs should list cows as potentially MAP positive if their antibody titer lies near the cut-off value and periodic MAP ELISA testing is recommended due to the fluctuating nature of MAP antibody kinetics in dairy cattle.