- Email: [email protected]
Evaluation of indigenous milk ELISA with m-culture and m-PCR for the diagnosis of Bovine Johne’s disease (BJD) in lactating Indian dairy cattle
Available online at www.sciencedirect.com
Research in Veterinary Science 84 (2008) 30–37 www.elsevier.com/locate/rvsc
Evaluation of indigenous milk ELISA with m-culture and m-PCR for the diagnosis of Bovine Johne’s disease (BJD) in lactating Indian dairy cattle G. Sharma a, S.V. Singh a,*, I. Sevilla a,b, A.V. Singh a, R.J. Whittington a,c, R.A. Juste S. Kumar a, V.K. Gupta a, P.K. Singh a, J.S. Sohal a, V.S. Vihan a b
a,b
,
a Central Institute for Research on Goats, Makhdoom, 281 122 Farah, Mathura District, UP, India Animal Health, Instituto Vasco de Investigacion y Desarrollo Agrario (NEIKER), Berreaga, J, 48160 Derio, Bizkaia, Spain c NSW Agriculture, Elizabeth Macarthur Agricultural Institute PMB 8 Camden, NSW 2570, Australia
Accepted 30 March 2007
Abstract Present study is the first attempt to evaluate an indigenous milk ELISA with milk culture, standardize milk PCR, estimate lacto-prevalence of Map and genotype Map DNA from milk samples in few Indian dairy herds. In all 115 cows were sampled from 669 lactating cows in six dairy herds from three districts of North India. Fifty milk samples (four herds) were screened by three tests (milk culture, mELISA and m-PCR). Lacto-prevalence of Map in four dairy herds was 84.0% (50.0% in fat and 62.0% in sediment). Screening of both fat and sediment increased the sensitivity of culture. Colonies appeared between 45 and 120 DPI. In indigenous m-ELISA, protoplasmic antigen derived from native Map ‘Bison type’ strain of goat origin was used. Screening of 115 lactating cows by m-ELISA (‘herd screening test’) detected 32.1% positive lactating cows (lacto-prevalence). Sensitivity of ELISA was 28.5% and 42.8% in single point cutoff and S/P ratio, respectively. Lacto-prevalence of JD was high in dairy herds (66.6–100.0% by culture and 20.0–50.0% by m-ELISA). DDD farm, Mathura had very high (95.8%) and moderate prevalence of Map and lacto-antibodies, respectively. All cows were clinically suffering from JD. Specific IS 900 PCR was standardized in decontaminated fat and sediment of milk samples. DNA isolated from decontaminated pellets was amplified and characteristic 229 bp band was confirmatory for Map. Of the 50 milk samples, 6.0% were positive in m-PCR. The test needs further standardization. Map DNA were genotyped as Map ‘Bison type’ by IS 1311 PCR–REA. Of the three tests, milk culture was most sensitive followed by m-ELISA and m-PCR. Map DNA isolated from milk samples of dairy cattle were first time genotyped as Map, ‘Bison type’ in India. High prevalence of Map in milk of dairy herds, posed major health hazard for calves and human beings. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Dairy cattle; Lactating cows; Johne’s disease; Mycobacterium avium subsp. paratuberculosis; Milk culture; Milk ELISA test; Milk PCR; IS 1311 PCR–REA
1. Introduction Mycobacterium avium subspecies paratuberculosis (Map) is the cause of Johne’s disease (JD) in ruminants character* Corresponding author. Tel.: +91 565 2763260x269 (O), +91 565 2763262 (R); fax: +91 565 2763246; Mobile: +91 9719072856. E-mail addresses: [email protected], shoorvir_singh@rediffmail.com (S.V. Singh).
0034-5288/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2007.03.014
ized by weight loss and diarrhea leading to emaciation (Figs 1 and 2). Estimated annual losses to US dairy are between 200 and 250 million US$ (Ott et al., 1999; Chi et al., 2002) and nearly 40% of the US dairy herds reported to be infected (Stabel, 1998). Disease has been mostly reported from the cattle herds of developed nations (Jackobson et al., 2000; Muskens et al., 2000). Though majority of 185.5 million Indian cattle are low or un-productive, still never screened for JD. The lack of information on preva-
G. Sharma et al. / Research in Veterinary Science 84 (2008) 30–37
31
dairy cattle. This was the first attempt to evaluate indigenous milk ELISA (m-ELISA) with milk culture and standardize milk PCR (m-PCR) for the diagnosis of JD in lactating cows. Lacto-prevalence of antibodies and Map infecting few dairy herds in North India was also determined. Map isolates from milk of lactating cows were characterized and genotyped by IS 900 m-PCR and IS 1311 PCR–REA. 2. Materials and methods Fig. 1. Calf with clinical Johne’s disease.
2.1. Milk samples
Fig. 2. Cow with clinical Johne’s disease.
lence of JD is mainly due to in-attention and non-availability of indigenous diagnostic kits and reagents (Johnin) in the country. In developed countries commercial ELISA kits are popular for the diagnosis of JD (Collins et al., 2005). However, sensitive and cost effective diagnostic tests are essential for the control and eradication of JD. Milk and colostrum are primary source of infection in young calves (Clarke, 1997) and may also potentially infect human beings (Chiodini and Hermon-Taylor, 1993). Value of milk as clinical material for screening of dairy herds has been documented (Sweeney et al., 1994) and crucial for the control of disease in dairy animals. Map isolates from goats in India have been genotyped as ‘Bison type’ (Whittington, 2002 and Sevilla et al., 2005), therefore, it was important to know the genotype of Map infecting Indian
Fifty cows (non-descript local, Haryana and Jersey crosses) from four dairy cattle herds (three private: Balkeshwar dairy, Gokulpura Dairy, Pvt. dairy, and Government dairy: DDD farm) from Agra, Mathura and Etah districts (North India) were screened by milk culture, mELISA and m-IS 900 PCR (Table 1). The 10, 4, 12, and 24 milk samples were randomly sampled from 55 (Balkeshwar dairy, Agra), 82 (Gokulpura dairy, Agra) 68 (Soron dairy, Etah), and 214 (DDF, Mathura) lactating cows, respectively. More cows (12 each) were sampled from Gokulpura dairy (Agra) and DDF (Mathura). Samples were collected first time from 28 and 15 cows out of 100 and 50 lactating cows, respectively from herds in Agra and Farah (Table 3). The 115 cows were sampled (randomly), from 669 lactating cows in six dairy herds from three districts of North India. Milk samples from 14 weak and clinically suspected cows (not screened earlier) from four dairy herds, were processed for DNA isolation. DNA were genotyped by IS1311 and IS900 PCR–REA, in Spain. One milk sample represented one cow. From each cow 15–20 ml of milk was collected aseptically from four quarters after discarding first few streaks. Milk samples were centrifuged (3000 rpm for 45 min) to separate three layers (fat, whey and sediment). From each milk sample, fat and sediment parts (50 each) were screened by culture and IS 900 PCR, and whey part (50) by m-ELISA. Milk was cultured as per Singh and Vihan (2004). Milk/whey (10 ml) were treated with 3.0% citric acid, centrifuged to collect clear whey and stored at 4 °C.
Table 1 Screening of milk sample by culture and milk ELISA Place
Milk samples
Positives Culture
Milk ELISA
Fat
Sediment
Fat and/or sediment
Balkeshwar dairy, Agraa Pvt. dairy, Soron, Etaha Gokulpura Dairy, Agraa DDD farm, Mathurab
10 12 4 24
4 7 3 14
6 3 3 19
7 (70.0%) 8 (66.6%) 4 (100.0%) 23 (95.8%)
Total
50
28
31
42 (84.0%)
a b
Private dairy. Government dairy.
2 2 2 8
(20.0%) (16.6%) (50.0%) (33.3%)
14 (28.0%)
32
G. Sharma et al. / Research in Veterinary Science 84 (2008) 30–37
Fat and sediment were decontaminated in 0.9% HPC (hexadecylpyridinium chloride, Sigma) for 18–24 h at room temperature, inoculated on HEYM (Herrold’s egg yolk medium) slants with and without mycobactin J (Allied Monitor, Inc., USA). Remaining inoculum (<1.0 ml) was centrifuged (10,000 rpm) and pelleted for DNA isolation. Pellets were washed twice with PBS and stored at 20 °C till further use. Slants were observed weekly. Map colonies were characterized on the basis of morphology, cultural characteristics, mycobactin J dependency, acid fastness, and slow growth. DNA from fat and sediment was characterized by IS 900 PCR. and genotyped by IS 1311 PCR– REA. A cow was considered positive for infection with Map/JD, if Map colonies were seen in the culture of either fat and/or sediment layer. 2.2. Milk ELISA test ELISA kit developed for goats (Singh et al., in press) using protoplasmic antigen (PPA) from prevalent native ‘Bison type’ strain of Map (Sevilla et al., 2005; Whittington, 2002) of goat origin was standardized in cows to detect lacto-antibodies against Map. However, concentration of antigen, whey and conjugate were re-optimized (Conjugate, 1:8000; Map ‘Bison type’ antigen, 0.5 lg per well; whey, 1:8; and incubation, 30 min) for cows. Negative serum control was derived from a healthy and culturally negative cow from a cow of private owner with JD negative status. Positive serum control was derived from weak and culturally positive cow from government dairy herd (DDF, Mathura). Cutoff OD was: mean OD of negatives ±2 SD = 0.191 + 0.62 = 0.253. Samples with >0.253 OD were treated positives and <0.253 negatives. Sensitivity was calculated as per Arizmendi and Grimes (1995). 2.3. IS 900 PCR (Map cultures and decontaminated pellets) M-PCR was developed for screening of milk samples of cows. Of the 50 fat and sediment samples each, visible pellets (from <1 ml aliquot left after inoculation on HEYM) obtained from 22 fat and 18 sediments, were further processed for DNA isolation as per van Embden et al. (1993) and van Soolingen et al. (1993) with some modifications. DNA was amplified by PCR using specific IS 900 primers (Vary et al., 1990). Briefly, in a volume of 58 ll of master mix (forward primer: 150 C 24 mer, 1 ll, reverse primer: 921, 25 mer, 1 ll, Taq PCR master mix: Qiagen, 30 ll and deionised water, 26 ll) and 2 ll of template DNA was added (total volume 60 ll). Total of 35 cycles were performed in a thermocycler for complete amplification reaction. Total time taken for 36 cycles was 1.20 h. Reaction conditions were; initial de-naturation at 94 °C for 3 min (one cycle), de-naturation at 94 °C for 10 s, annealing at 61 °C for 10 s, extension at 72 °C for 10 s (35 cycles) and final extension at 72 °C for 3 min. Presence and yield of specific PCR product (229 bp) was analyzed by 1.2% agarose ethidium bromide gel electrophoresis.
2.4. IS 1311 and IS 900 PCR–REA Of the 14 milk samples, good quality DNA was obtained from seven each decontaminated fat and sediment layers. Five micro-liters of the re-suspended DNA were used in a IS 1311 PCR mix containing 0.8 lM of primers M-56 and M-119 (Marsh et al., 1999), which was otherwise standard (Innis and Gelfand, 1990). Thermal profile was: one cycle of 3 min at 94 °C and 37 cycles of 30 s at 94 °C, 15 s at 62 °C, and 1 min at 72 °C. Samples processed by IS 1311 PCR, were repeated in IS 900 PCR, as per Garrido et al. (2000). DNA bands of 608 and 389 bp were considered positive results for IS 1311 and IS 900 PCR, respectively, after separation in a 2.0% agarose gel stained with ethidium bromide. IS 1311 REA digestion consisted of 8 ll of positive IS 1311 PCR solution that was digested in a 16 ll reaction containing two units of each endo-nucleases Hinf-I and Mse-I supplemented with buffers (New England biolabs Inc., Beverly MA, USA). Band patterns were interpreted according to Whittington et al. (2001). 3. Results 3.1. Lacto-prevalence of Map by culture – organized dairy herds Lacto-prevalence of Map was 84.0% by culture of 50 milk samples from four dairy herds. Of the 84.0% positive lactating cows, 56.0% and 62.0% were positive for Map in fat and sediment, respectively and 34.0% were detected both in fat and sediment cultures. The 22.0% and 28.0% cows were detected exclusively by fat and sediment cultures, respectively (Tables 1 and 2). Colonies started appearing from 45 DPI and continued to appear up to 150 days. Maximum colonies appeared around 90 DPI (28.5%, Fig. 6). The 50.0% cultures each from fat layer and 38.7% and 61.2% from sediment layer, were pauci and multibacillary, respectively (Figs. 3 and 4). 3.2. M-ELISA as ‘herd screening test’ in dairy cows On the basis of screening of 115 milk samples against Map lacto-antibodies by m-ELISA, 32.1% cows were positive in six dairy herds located in three districts (Agra, Etah and Mathura). Lacto-prevalence of Map antibodies varied from 15.3% to 50.0% in dairy herds screened. Single government dairy herds (DDD farm, Mathura) had 33.3% lacto-prevalence (Table 3). Table 2 Comparative isolation of Map in cow milk culture (fat and sediment) Culture
Combinations
Fat Sediment
+ +
Total 50
17
+ + 8
11
Positive in culture: 42; positive in fat 28; Positive in sediment 31.
14
G. Sharma et al. / Research in Veterinary Science 84 (2008) 30–37
33
Table 4 Comparative evolution of sediment and fat culture and milk-ELISA Test
Comparison
Fat culture Sediment culture Milk ELISA
+ + +
Total 50
4
1
2
3
4
5
+
6
7
+ +
+ + 6
11
6
2
13
8 +
+ +
+
8
0
Sensitivity – 28.5% (OD – 2SD – 0.253).
Fig. 3. Paucibacillary colony of Map on HEY medium.
3.4. IS 900 PCR in milk samples of dairy herds From 50 fat and sediment samples each decontaminated, visible pellet was present in 22 fat and 18 sediment samples. DNA was checked under UV illumination and DNA was isolated in nine fat and four sediments. Only good quality DNA (three fat and two sediment) processed and 3.0% DNA samples (two fat and one sediment) amplified or 6.0% cows were positive in IS 900 PCR giving 229 bp band characteristic for Map (Fig. 5).
Fig. 4. Typical multibacillary colonies of Map on HEY medium. Table 3 Screening of milk samples (dairy herds) by m-ELISA Place Balkeshwar dairy, Agra Pvt. dairy, Soron, Etah Gokulpura Dairy, Agra DDD farm, Mathura Pvt. dairy, Agra DDD dairy, Farah, Total
Milk samples 10 12 16 36 28 13 115
ELISA positive 2 2 8 12 11 2
(20.0%) (16.6%) (50.0%) (33.3%) (39.2%) (15.3%)
Fig. 5. Screening of milk samples for MAP by PCR.
37 (32.1%)
10 8 6 4 2 0 45
60
75
90
105
120
135
150
Days Post Inoculation Number of Colonies
Using three tests (m-ELISA, fat and sediment culture) for the screening of 50 milk samples each there was agreement in 20.0% cows. The three tests in combination detected 88.0% milk samples (cows) as positive. Considering culture (both fat and sediment) as ‘Gold standard test’, 84.0% milk samples (cows) were true positives. Of the 88.0% positive cows, only 4.0% were exclusively detected positive in m-ELISA (Table 4). Of these true positive cows, 28.5% were detected by m-ELISA. Using single point cutoff m-ELISA detected 28.0% cows as positive (sensitivity 28.5%). But on continuous scale (S/P ratio) using 0.25 cutoff, in m-ELISA 40.0% cows were positive and of these 90.0% were detected in culture (sensitivity – 42.8%). However, of 60.0% m-ELISA negative milk samples (cows), 80.0% were positive in culture and only 20.0% were negative in culture (true negatives).
Number of Colonies
12
3.3. Comparison of m-ELISA with milk fat and sediment culture
14 12 10 8 6 4 2 0 45
60
75
90
105
120
135
Days Post Inoculation
Fig. 6. Appearance of Map colonies from cow’s milk.
150
34
G. Sharma et al. / Research in Veterinary Science 84 (2008) 30–37
Fig. 7. IS 1311-PCR.
3.5. IS 1311 PCR–REA Of the 14 good quality DNA, seven were positive in PCR (three fat and four sediment) belonging to weak and suspected cows from four different herds. The seven PCR positive DNA, were subjected to IS 1311 PCR– REA and three sediment and three fat were genotyped as ‘Bison type’ and one sediment (CMS 32) was ‘Bovine’ (Table 6, Fig. 7). The Map ‘S 5’ strain (from clinical case of JD in goat) used for preparation of antigen was also genotyped as ‘Bison type’ (Whittington, 2002; Sevilla et al., 2005). 4. Discussion Screening of large ruminants is often met with difficulties in sampling in India. Due to absence of proper restraining facilities, there is often lot of struggling by animals in collection of blood samples. Many times it requires felling of animals to ground and typing with ropes, which is time consuming and not liked by owners. There is ban on cowslaughter in India, therefore, collection of tissues for culture was not possible. Milk was very convenient material for screening from lactating cows. Since milk is important source of contamination to calves; therefore critical for the screening of cows and control of JD. Disease has been widely reported from developed nations (Muskens et al., 2000), but information is not available in Indian cattle. First time in India, using three sensitive tests, milk samples were screened to estimate prevalence of Map. Fifty fat and sediment each was screened by culture and m-PCR and 50 whey by m-ELISA. Therefore, for screening of 50 milk samples, in total 250 samples (100 in culture, 100 in DNA isolation and 50 in ELISA) were handled to conclude the status of each of 50 cows sampled. M-ELISA was used
as ‘herd screening test’, since it helped in screening of 65 more cows with little efforts, in addition to 50, screened by three tests. High lacto-prevalence of Map was reported in lactating cows from four dairy herds in three districts of North India. There were 84.0% cows true positives (culture positives). Centrifugation of milk partitioned Map in to fat and sediment layers (Gao et al., 2005). Culture of both fat and sediment increased the efficiency of milk culture test used for screening of lactating cows. Using this protocol, similar sensitivity has been reported by Kumar (2004) in goat milk. Low lacto-incidence of Map reported by Singh and Vihan (2004) in goat milk may be due to culture of only sediment layer. Gao et al. (2005) reported preferential partitioning of Map into cream layer and enhanced recovery by centrifugation and culture. Millar et al. (1996) reported overall 7.0% of 312 milk samples positive for Map. High incidence has been reported in culture of fecal samples of lactating cows from US dairy herds (Stabel et al., 2002). Variable prevalence has been reported from clinically (Taylor et al., 1981; Giese and Ahrens, 2000) and sub-clinically (Sweeney et al., 1994; Jackobson et al., 2000) ill cows. Presence of paucibacillary cultures indicated sub-clinical infection and multibacillary, active disease (super shedders) in lactating cows. Giese and Ahrens (2000) cultivated few colonies from milk of fecal culture positive cattle. Colonoes appeared between 45 and 150 DPI (Figs. 3, 4 and 6). Milk ELISA was standardized using indigenous protoplasmic antigen from native isolate of Map ‘Bison type’ of goat origin and compared with culture of milk. At single point cutoff 28.0% cows were positive (sensitivity – 28.5%). As ‘herd screening test’ m-ELISA detected 32.1% cows. Variable prevalence of Map using ELISA (serum and milk) has been reported in different countries (Muskens et al., 2000). Sweeney et al. (1994) using LAM ELISA in cattle milk reported 50% sensitivity. Using different antigens variable sensitivity of ELISA has been reported (Vannuffel et al., 1994; Collins et al., 2005). Low sensitivity may be due to use of single point cutoff and was 42.8% when OD values were analyzed by S/P ratio. Using PPA, Ferreira et al. (2002) reported similar sensitivity. Commercial ELISA kits (Collins et al., 2005; Nielsen et al., 2000) were less sensitive, since based on Map strain not reported in India (unpublished data). In present study PPA was sensitive at 0.5 lg/well concentration with 1:8 dilution of whey. Salgado et al. (2005), screened lacto-antibodies at 1:2 dilutions. There was high correlation between animals positive in m-ELISA and milk culture. At single point cutoff, of Table 5 S/P ratio and status of JD in lactating cows in m-ELISA and milk culture S/P ratios
Disease status
Lactating cows
Culture positive
0.00–0.09 0.10–0.24 0.25–0.39 0.40–0.99 1.0–10.0
Negative Suspected Low positive Positive Strong positive
30 00 00 06 14
24 00 00 06 12
G. Sharma et al. / Research in Veterinary Science 84 (2008) 30–37
28.0% m-ELISA positive cows, 85.7% were positive in milk culture. In S/P ratio, of 40.0% ELISA positive milk samples, 90.0% were positive in culture (Table 5). Study showed that m-ELISA using ‘Bison type’ Map antigen of goat origin could be used as ‘Herd screening test’ in lactating cows. Stabel et al. (2002) reported low sensitivity of mELISA than milk culture. Collins et al. (2005) showed similar sensitivity of m-ELISA. M-ELISA missed 60.0%, true positives, may be due to anergy condition (Waters et al., 1999). Koets et al. (2001), reported that there was no uniform association with increased antibody responses during progression from asymptomatic stage to clinical stage of BJD. Using m-ELISA, Collins et al. (1991) detected 59.7% of animals that shed Map in their feces. Stabel et al. (2002), detected only 25.0% of cows that were fecal culture positive by m-ELISA. Of culture positive milk samples, 47.6% were positive (positive + strong positive) in m-ELISA (Table 5). Ferreira et al. (2002) reported that PPA antigen detected 52.4% false negatives and 13.1% false positives. PCR has been standardized using decontaminated tissues and fecal samples from goat kids (Kumar et al., in press). PCR used frequently in cattle (Juste et al., 2005) was standardized for the screening of cow milk samples. DNA was isolated from 1 ml of decontaminated fat or sediment. PCR could be standardized from small aliquots. Of the decontaminated milk samples, 6.0% were positive in IS900 PCR. In this study milk culture was highly sensitive, m-ELISA moderately sensitive and sensitivity of IS900 PCR was significantly low. In m-PCR also screening of both fat and sediment layers was essential. In this study three cows were detected in PCR alone (Table 6), thus giving 90% cumulative positives in m-PCR and milk culture. M-PCR missed 41.5% culture positive samples. Low sensitivity of m-PCR may be due to poor standardization of DNA isolation from decontaminated material and large
Table 6 IS 1311 PCR on decontaminated material and comparison with culture and m-ELISA Milk sample
IS 1311
Culture
m-ELISA
CMF-4 CMF-6 CMF-12 CMF-15 CMF-18 CMF-19 CMF-33 CMS-27 CMS-30 CMS-32 CMS-25 CMS-40 CMS-45 CMS-48
P N P P N N N P P Pa N P P N
N P P P N P N P N N N P P P
N P N N N N P P N N P N N N
Total positives
8
8
4
Map ‘Bison type, CMS – cow milk sediment, CMF – cow milk fat, N – negative, P – positive. a REA cattle type and extremely weak.
35
number of paucibacillary samples. In this study DNA was amplified without dilution. Diluted DNA samples have been used to offset inhibitors in fecal and milk samples (Chui et al., 2004). When DNA was diluted 1:4 times in milk IS 1311 PCR–REA, of 14 samples (seven fat and seven sediment), eight were positive. Giese and Ahrens (2000) reported that m-PCR in raw cows milk could detect Map, but cultivation was more sensitive. Stabel et al. (2002) reported m-PCR was highly sensitive and specific in cattle. Map was almost equally found in pellet or cream by culture or PCR, (Giese and Ahrens, 2000). PCR has been used in bulk-tank milk samples to estimate prevalence of Map (Stephan et al., 2002). Pillai and Jayarao (2002) reported IS 900 PCR was superior to milk culture. Buergelt and Williams (2004) reported both culture and IS900 PCR could detect 10–100 cfu/ml of Map. Both IS 900 and IS 1311 PCR techniques were suitable for Map characterization and genotyping (Whittington et al., 2001). Map strains in this study were mainly of ‘Bison type’ and one was Map ‘Bovine’. ‘Bison type’ Map was similar to Bison (Bison bison) from Montana, USA (Whittington et al., 2001) and has been reported from goats, sheep and buffaloes in India (Whittington, 2002; Sevilla et al., 2005). 5. Conclusion Using protoplasmic antigen from native Map ‘Bison type’ strain of goat origin, indigenous milk ELISA, was successfully used to screen lactating cows. In m-ELISA, prevalence lacto-antibodies was moderate (32.0% positive) in six herds in North India. The m-ELISA was simple, sensitive and cost effective ‘herd screening test’. Comparative evaluation of m-ELISA with milk culture showed 28.5% and 42.8% sensitivity on single point and S/P ratio, respectively. Milk culture was the most sensitive of the three tests and lacto-prevalence of Map was high (84.0%) in lactating Indian cows. Screening of both fat and sediment independently improved the sensitivity of in m-culture and mPCR. DNA from decontaminated fat and sediment layers of milk of lactating cows were characterized and genotyped as Map ‘Bison type’ first time in the country.
Acknowledgement Authors are thankful to Director, CIRG, Mathura, for the facilities. References Arizmendi, F., Grimes, J.E., 1995. Comparison of Gimenez staining method and antigen detection ELISA with culture for detecting Chlamydia in bird. Journal of Veterinary Diagnostic Investigation 7, 400–401. Buergelt, C.D., Williams, J.E., 2004. Nested PCR on blood and milk for the detection of Mycobacterium avium subsp. paratuberculosis DNA in clinical and subclinical bovine paratuberculosis. Australian Veterinary Journal 82, 497–503.
36
G. Sharma et al. / Research in Veterinary Science 84 (2008) 30–37
Chi, J., Van Leeuwen, J.A., Wentink, A., Keefe, G.P., 2002. Direct production losses and treatment costs from bovine viral diarrhoea virus, bovine leukosis virus, Mycobacterium avium subspecies paratuberculosis and Neospora caninum. Preventive Veterinary Medicine 55, 137–153. Chiodini, R.J., Hermon-Taylor, J., 1993. The thermal resistance of Mycobacterium paratuberculosis in raw milk under conditions simulating pasteurization. Journal of Veterinary Diagnostic Investigation 5, 629–631. Chui, L.W., King, R., Lu, P., Manninen, K., Sim, J., 2004. Evaluation of four DNA extraction methods for the detection of Mycobacterium avium subsp. paratuberculosis by polymerase chain reaction. Diagnostic microbiology and infectious disease 48, 39–45. Clarke, C.J., 1997. The pathology and pathogenesis of paratuberculosis in ruminants and other species. Journal of Comparative Pathology 116, 217–261. Collins, M.T., Sockett, D.C., Ridge, S., Cox, J.C., 1991. Evaluation of a commercial enzyme linked immunosorbent assay for Johne’s disease. Journal of Clinical Microbiology 29, 272–276. Collins, M.T., Wells, S.J., Petrini, K.R., Collins, J.E., Schultz, R.D., Whitlock, R.H., 2005. Evaluation of five-antibody detection tests for diagnosis of bovine paratuberculosis. Clinical and Diagnostic Laboratory Immunology 12, 685–692. Ferreira, R., Fonseca, L.S., Lilenbaum, W., 2002. Comparison between a commercial and an in-house ELISA for anti-M. avium paratuberculosis antibodies detection in dairy herds in Rio de Janeiro, Brazil. Revista Latinoamericana de Microbiologia 44, 129–132. Gao, A., Odumeru, J., Raymond, M., Mutharia, L., 2005. Development of improved method for isolation of Mycobacterium avium subsp. paratuberculosis from bulk tank milk: effect of age of milk, centrifugation, and decontamination. Canadian Journal of Veterinary Research 69, 81–87. Garrido, J.M., Cortabarria, N., Qguiza, J.A., Aduriz, G., Juste, R.A., 2000. Use of a PCR method on faecal samples for diagnosis of sheep paratuberculosis. Veterinary Microbiology 77, 3–4. Giese, S.B., Ahrens, P., 2000. Detection of Mycobacterium avium subsp. paratuberculosis in milk from clinically affected cows by PCR and culture. Veterinary Microbiology 20, 291–297. Innis, M.A., Gelfand, D.H., 1990. Optimization of PCRs. In: Innis, Gelfand, Sninsky, White (Eds.), PCR Protocols. Academic Press, New York, pp. 3–12. Jackobson, M.B., Alban, L., Nielsen, S.S., 2000. A cross sectional study of paratuberculosis in 1155 Danish dairy cows. Preventive Veterinary Medicine 46, 15–27. Juste, R.A., Garrido, J.M., Geijo, M., Elguezabal, N., Aduriz, G., Atxaerandio, R., Sevilla, I., 2005. Comparison of blood polymerase chain reaction and enzyme linked immunosorbent assay for detection of Mycobacterium avium subsp. paratuberculosis infection in cattle and sheep. Journal of Veterinary Diagnostic Investigation 17, 354–359. Koets, A.P., Victor, P., Rutten, M.G., de Boer, M., Bakker, D., ValentinWeigand, P., van Eden, W., 2001. Differential changes in heat shock protein-, lipoarabinomannan-, and purified protein derivative-specific immunoglobulin G1 and G2 isotype responses during bovine Mycobacterium avium subsp. paratuberculosis infection. Infection and Immunity 69, 1492–1498. Kumar, S., 2004. Prevalence and molecular characterization of Mycobacterium avium subsp. paratuberculosis, the cause of Johne’s disease (Paratuberculosis) from goat milk in Agra region. M.Sc. Dissertation, Bundelkhand University, Jhansi, UP, India. Kumar, P., Singh, S.V., Bhatiya, A.K., Sevilla, I., Singh, A.V., Whittington, R.J., Juste, R.A., Gupta, V.K., Singh, P.K., Sohal, J.S., Vihan, V.S., in press. Juvenile Capri-Paratuberculosis (JCP) in India: incidence and characterization by six diagnostic tests. Small Ruminant Research. doi:10.1016/j.smallrumres, 2006.10.023. Marsh, I., Whittington, R.J., Cousins, D.V., 1999. PCR-restriction endonuclease analysis for identification and strain typing of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium
subsp. avium based on polymorphisms in IS1311. Molecular and Cellular Probes 13, 115–126. Millar, D., Ford, J., Sanderson, J., Withey, S., Tizard, M., Doran, T., Hermon-Taylor, J., 1996. IS 900 PCR to detect Mycobacterium paratuberculosis in retail milk supplies of whole pasteurized cow’s milk in England and Wales. Applied Environmental Microbiology 62, 3446–3452. Muskens, J., Barkema, H.W., Russchen, E., van Maanen, K., Schukken, Y.H., Bakker, D., 2000. Prevalence and regional distribution of paratuberculosis in dairy herds in the Netherlands. Veterinary Microbiology 77, 253–261. Nielsen, S.S., Thamsborg, S.M., Houe, H., Bitsch, V., 2000. Bulk tank milk ELISA antibodies for estimating the prevalence of paratuberculosis in Danish dairy herds. Preventive Veterinary Medicine 44, 1–2. Ott, S.L., Wells, S.J., Wagner, B.A., 1999. Herd-level economic losses associated with Johne’s disease on US dairy operations. Preventive Veterinary Medicine 40, 179–192. Pillai, S.R., Jayarao, V.M., 2002. Application of IS 900 PCR for detection of Mycobacterium avium subsp. paratuberculosis directly from raw milk. Journal of Dairy Science 85, 1052–1057. Salgado, M., Manning, E.J.B., Collins, M.T., 2005. Performance of a Johne’s disease enzyme linked immunosorbent assay adapted for milk samples from goats. Journal of Veterinary Diagnostic Investigation 17, 350–354. Sevilla, I., Singh, S.V., Garrido, J.M., Aduriz, G., Rodriguez, S., Geijo, M.V., Whittington, R.J., Saunders, V., Whitlock, R.H., Juste, R.A., 2005. Molecular typing of Paratuberculosis strains from different hosts and regions. Revue Scientifique et Technique (International Office of Epizootics) 24, 1061–1066. Singh, S.V., Vihan, V.S., 2004. Detection of M. avium subsp. paratuberculosis in goat milk. Small Ruminant Research 34, 231–235. Singh, S.V., Singh, A.V., Singh, P.K., Gupta, V.K., Kumar, S., Vohra, J., in press. Sero-prevalence of paratuberculosis in young kids using ‘Bison type’, Mycobacterium avium subsp. paratuberculosis antigen in plate ELISA. Small Ruminant Reseacrh. doi:10.1016/j.smallrumres. 2005.11.019. Stabel, J.R., 1998. Johne’s disease: a hidden threat. Journal of Dairy Science 81, 283–288. Stabel, J.R., Wells, S.J., Wagner, B.A., 2002. Relationship between fecal culture, ELISA and bulk tank milk test results for Johne’s disease in US dairy herds. Journal of Dairy Science 85, 525–531. Stephan, R., Buhler, K., Corti, S., 2002. Incidence of Mycobacterium avium subsp.paratuberculosis in bulk-tank milk samples from different regions in Switzerland. Veterinary Record 150, 762–770. Sweeney, R.W., Whitlock, R.H., Buckley, C.L., Pam, B.S., Spencer, M.S., Rosenberger, A.E., Hutchinson, L.J., 1994. Diagnosis of paratuberculosis in dairy cattle using enzyme linked immuno-sorbent assay for detection of antibodies against Mycobacterium avium subspecies paratuberculosis in milk. American Journal of Veterinary Research 55, 905–909. Taylor, T.K., Wilks, C.R., Mc Queen, D.S., 1981. Isolation of Mycobacterium paratuberculosis from the milk of a cow with Johne’s disease. The Veterinary Record 109, 532–533. van Embden, J.D.A., Cave, D., Crawford, J.T., Dale, J.W., Eisenach, K.D., Gicquel, B., Hermans, P., Martin, C., Mc Adam, R., Shinnick, T.M., Small, P.M., 1993. Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardized methodology. Journal of Clinical Microbiology 31, 406–409. Vannuffel, P., Gilot, P., Limbourg, B., Naerhuyzen, B., Dieterich, C., Coene, M., Machtelinckx, L., Cocito, C., 1994. Development of species specific ELISA for diagnosis of Johne’s disease in cattle. Journal of Clinical Microbiology 32, 1211–1216. van Soolingen, D., de Hass, E.W., Hermans, P.W., Groenen, P.M.A., van Embden, J.D., 1993. Comparison of various repeatitive DNA elements as genetic markers for strain differentiation and epidemiology of
G. Sharma et al. / Research in Veterinary Science 84 (2008) 30–37 Mycobacterium tuberculosis. Journal of Clinical Microbiology 31, 1987–1995. Vary, P.H., Andersen, P.R., Green, E., Hermon-Talyor, J., Mc Fadden, J.J., 1990. Use of highly specific DNA probes and the polymerase chain reaction to detect Mycobacterium paratuberculosis in Johne’s disease. Journal of Clinical Microbiology 28, 933–937. Waters, W.R., Stabel, J.R., Sacco, R.E., Harp, J.A., Pesch, A., Wannemuehler, M.J., 1999. Antigen-Specific B-Cell Unresponsiveness Induced by Chronic Mycobacterium avium subsp. paratuber-
37
culosis Infection of Cattle. Infection and Immunity 67, 1593– 1598. Whittington, R.J., 2002. Personal communication. Whittington, R.J., Marsh, I.B., Whitlock, R.H., 2001. Typing of IS1311 polymorphisms confirms that bison (Bison bison) with paratuberculosis in Montana are infected with a strain of Mycobacterium avium subsp. paratuberculosis distinct from that occurring in cattle and other domesticated livestock. Molecular and Cellular Probes 15, 139–145.
Research in Veterinary Science 84 (2008) 30–37 www.elsevier.com/locate/rvsc
Evaluation of indigenous milk ELISA with m-culture and m-PCR for the diagnosis of Bovine Johne’s disease (BJD) in lactating Indian dairy cattle G. Sharma a, S.V. Singh a,*, I. Sevilla a,b, A.V. Singh a, R.J. Whittington a,c, R.A. Juste S. Kumar a, V.K. Gupta a, P.K. Singh a, J.S. Sohal a, V.S. Vihan a b
a,b
,
a Central Institute for Research on Goats, Makhdoom, 281 122 Farah, Mathura District, UP, India Animal Health, Instituto Vasco de Investigacion y Desarrollo Agrario (NEIKER), Berreaga, J, 48160 Derio, Bizkaia, Spain c NSW Agriculture, Elizabeth Macarthur Agricultural Institute PMB 8 Camden, NSW 2570, Australia
Accepted 30 March 2007
Abstract Present study is the first attempt to evaluate an indigenous milk ELISA with milk culture, standardize milk PCR, estimate lacto-prevalence of Map and genotype Map DNA from milk samples in few Indian dairy herds. In all 115 cows were sampled from 669 lactating cows in six dairy herds from three districts of North India. Fifty milk samples (four herds) were screened by three tests (milk culture, mELISA and m-PCR). Lacto-prevalence of Map in four dairy herds was 84.0% (50.0% in fat and 62.0% in sediment). Screening of both fat and sediment increased the sensitivity of culture. Colonies appeared between 45 and 120 DPI. In indigenous m-ELISA, protoplasmic antigen derived from native Map ‘Bison type’ strain of goat origin was used. Screening of 115 lactating cows by m-ELISA (‘herd screening test’) detected 32.1% positive lactating cows (lacto-prevalence). Sensitivity of ELISA was 28.5% and 42.8% in single point cutoff and S/P ratio, respectively. Lacto-prevalence of JD was high in dairy herds (66.6–100.0% by culture and 20.0–50.0% by m-ELISA). DDD farm, Mathura had very high (95.8%) and moderate prevalence of Map and lacto-antibodies, respectively. All cows were clinically suffering from JD. Specific IS 900 PCR was standardized in decontaminated fat and sediment of milk samples. DNA isolated from decontaminated pellets was amplified and characteristic 229 bp band was confirmatory for Map. Of the 50 milk samples, 6.0% were positive in m-PCR. The test needs further standardization. Map DNA were genotyped as Map ‘Bison type’ by IS 1311 PCR–REA. Of the three tests, milk culture was most sensitive followed by m-ELISA and m-PCR. Map DNA isolated from milk samples of dairy cattle were first time genotyped as Map, ‘Bison type’ in India. High prevalence of Map in milk of dairy herds, posed major health hazard for calves and human beings. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Dairy cattle; Lactating cows; Johne’s disease; Mycobacterium avium subsp. paratuberculosis; Milk culture; Milk ELISA test; Milk PCR; IS 1311 PCR–REA
1. Introduction Mycobacterium avium subspecies paratuberculosis (Map) is the cause of Johne’s disease (JD) in ruminants character* Corresponding author. Tel.: +91 565 2763260x269 (O), +91 565 2763262 (R); fax: +91 565 2763246; Mobile: +91 9719072856. E-mail addresses: [email protected], shoorvir_singh@rediffmail.com (S.V. Singh).
0034-5288/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.rvsc.2007.03.014
ized by weight loss and diarrhea leading to emaciation (Figs 1 and 2). Estimated annual losses to US dairy are between 200 and 250 million US$ (Ott et al., 1999; Chi et al., 2002) and nearly 40% of the US dairy herds reported to be infected (Stabel, 1998). Disease has been mostly reported from the cattle herds of developed nations (Jackobson et al., 2000; Muskens et al., 2000). Though majority of 185.5 million Indian cattle are low or un-productive, still never screened for JD. The lack of information on preva-
G. Sharma et al. / Research in Veterinary Science 84 (2008) 30–37
31
dairy cattle. This was the first attempt to evaluate indigenous milk ELISA (m-ELISA) with milk culture and standardize milk PCR (m-PCR) for the diagnosis of JD in lactating cows. Lacto-prevalence of antibodies and Map infecting few dairy herds in North India was also determined. Map isolates from milk of lactating cows were characterized and genotyped by IS 900 m-PCR and IS 1311 PCR–REA. 2. Materials and methods Fig. 1. Calf with clinical Johne’s disease.
2.1. Milk samples
Fig. 2. Cow with clinical Johne’s disease.
lence of JD is mainly due to in-attention and non-availability of indigenous diagnostic kits and reagents (Johnin) in the country. In developed countries commercial ELISA kits are popular for the diagnosis of JD (Collins et al., 2005). However, sensitive and cost effective diagnostic tests are essential for the control and eradication of JD. Milk and colostrum are primary source of infection in young calves (Clarke, 1997) and may also potentially infect human beings (Chiodini and Hermon-Taylor, 1993). Value of milk as clinical material for screening of dairy herds has been documented (Sweeney et al., 1994) and crucial for the control of disease in dairy animals. Map isolates from goats in India have been genotyped as ‘Bison type’ (Whittington, 2002 and Sevilla et al., 2005), therefore, it was important to know the genotype of Map infecting Indian
Fifty cows (non-descript local, Haryana and Jersey crosses) from four dairy cattle herds (three private: Balkeshwar dairy, Gokulpura Dairy, Pvt. dairy, and Government dairy: DDD farm) from Agra, Mathura and Etah districts (North India) were screened by milk culture, mELISA and m-IS 900 PCR (Table 1). The 10, 4, 12, and 24 milk samples were randomly sampled from 55 (Balkeshwar dairy, Agra), 82 (Gokulpura dairy, Agra) 68 (Soron dairy, Etah), and 214 (DDF, Mathura) lactating cows, respectively. More cows (12 each) were sampled from Gokulpura dairy (Agra) and DDF (Mathura). Samples were collected first time from 28 and 15 cows out of 100 and 50 lactating cows, respectively from herds in Agra and Farah (Table 3). The 115 cows were sampled (randomly), from 669 lactating cows in six dairy herds from three districts of North India. Milk samples from 14 weak and clinically suspected cows (not screened earlier) from four dairy herds, were processed for DNA isolation. DNA were genotyped by IS1311 and IS900 PCR–REA, in Spain. One milk sample represented one cow. From each cow 15–20 ml of milk was collected aseptically from four quarters after discarding first few streaks. Milk samples were centrifuged (3000 rpm for 45 min) to separate three layers (fat, whey and sediment). From each milk sample, fat and sediment parts (50 each) were screened by culture and IS 900 PCR, and whey part (50) by m-ELISA. Milk was cultured as per Singh and Vihan (2004). Milk/whey (10 ml) were treated with 3.0% citric acid, centrifuged to collect clear whey and stored at 4 °C.
Table 1 Screening of milk sample by culture and milk ELISA Place
Milk samples
Positives Culture
Milk ELISA
Fat
Sediment
Fat and/or sediment
Balkeshwar dairy, Agraa Pvt. dairy, Soron, Etaha Gokulpura Dairy, Agraa DDD farm, Mathurab
10 12 4 24
4 7 3 14
6 3 3 19
7 (70.0%) 8 (66.6%) 4 (100.0%) 23 (95.8%)
Total
50
28
31
42 (84.0%)
a b
Private dairy. Government dairy.
2 2 2 8
(20.0%) (16.6%) (50.0%) (33.3%)
14 (28.0%)
32
G. Sharma et al. / Research in Veterinary Science 84 (2008) 30–37
Fat and sediment were decontaminated in 0.9% HPC (hexadecylpyridinium chloride, Sigma) for 18–24 h at room temperature, inoculated on HEYM (Herrold’s egg yolk medium) slants with and without mycobactin J (Allied Monitor, Inc., USA). Remaining inoculum (<1.0 ml) was centrifuged (10,000 rpm) and pelleted for DNA isolation. Pellets were washed twice with PBS and stored at 20 °C till further use. Slants were observed weekly. Map colonies were characterized on the basis of morphology, cultural characteristics, mycobactin J dependency, acid fastness, and slow growth. DNA from fat and sediment was characterized by IS 900 PCR. and genotyped by IS 1311 PCR– REA. A cow was considered positive for infection with Map/JD, if Map colonies were seen in the culture of either fat and/or sediment layer. 2.2. Milk ELISA test ELISA kit developed for goats (Singh et al., in press) using protoplasmic antigen (PPA) from prevalent native ‘Bison type’ strain of Map (Sevilla et al., 2005; Whittington, 2002) of goat origin was standardized in cows to detect lacto-antibodies against Map. However, concentration of antigen, whey and conjugate were re-optimized (Conjugate, 1:8000; Map ‘Bison type’ antigen, 0.5 lg per well; whey, 1:8; and incubation, 30 min) for cows. Negative serum control was derived from a healthy and culturally negative cow from a cow of private owner with JD negative status. Positive serum control was derived from weak and culturally positive cow from government dairy herd (DDF, Mathura). Cutoff OD was: mean OD of negatives ±2 SD = 0.191 + 0.62 = 0.253. Samples with >0.253 OD were treated positives and <0.253 negatives. Sensitivity was calculated as per Arizmendi and Grimes (1995). 2.3. IS 900 PCR (Map cultures and decontaminated pellets) M-PCR was developed for screening of milk samples of cows. Of the 50 fat and sediment samples each, visible pellets (from <1 ml aliquot left after inoculation on HEYM) obtained from 22 fat and 18 sediments, were further processed for DNA isolation as per van Embden et al. (1993) and van Soolingen et al. (1993) with some modifications. DNA was amplified by PCR using specific IS 900 primers (Vary et al., 1990). Briefly, in a volume of 58 ll of master mix (forward primer: 150 C 24 mer, 1 ll, reverse primer: 921, 25 mer, 1 ll, Taq PCR master mix: Qiagen, 30 ll and deionised water, 26 ll) and 2 ll of template DNA was added (total volume 60 ll). Total of 35 cycles were performed in a thermocycler for complete amplification reaction. Total time taken for 36 cycles was 1.20 h. Reaction conditions were; initial de-naturation at 94 °C for 3 min (one cycle), de-naturation at 94 °C for 10 s, annealing at 61 °C for 10 s, extension at 72 °C for 10 s (35 cycles) and final extension at 72 °C for 3 min. Presence and yield of specific PCR product (229 bp) was analyzed by 1.2% agarose ethidium bromide gel electrophoresis.
2.4. IS 1311 and IS 900 PCR–REA Of the 14 milk samples, good quality DNA was obtained from seven each decontaminated fat and sediment layers. Five micro-liters of the re-suspended DNA were used in a IS 1311 PCR mix containing 0.8 lM of primers M-56 and M-119 (Marsh et al., 1999), which was otherwise standard (Innis and Gelfand, 1990). Thermal profile was: one cycle of 3 min at 94 °C and 37 cycles of 30 s at 94 °C, 15 s at 62 °C, and 1 min at 72 °C. Samples processed by IS 1311 PCR, were repeated in IS 900 PCR, as per Garrido et al. (2000). DNA bands of 608 and 389 bp were considered positive results for IS 1311 and IS 900 PCR, respectively, after separation in a 2.0% agarose gel stained with ethidium bromide. IS 1311 REA digestion consisted of 8 ll of positive IS 1311 PCR solution that was digested in a 16 ll reaction containing two units of each endo-nucleases Hinf-I and Mse-I supplemented with buffers (New England biolabs Inc., Beverly MA, USA). Band patterns were interpreted according to Whittington et al. (2001). 3. Results 3.1. Lacto-prevalence of Map by culture – organized dairy herds Lacto-prevalence of Map was 84.0% by culture of 50 milk samples from four dairy herds. Of the 84.0% positive lactating cows, 56.0% and 62.0% were positive for Map in fat and sediment, respectively and 34.0% were detected both in fat and sediment cultures. The 22.0% and 28.0% cows were detected exclusively by fat and sediment cultures, respectively (Tables 1 and 2). Colonies started appearing from 45 DPI and continued to appear up to 150 days. Maximum colonies appeared around 90 DPI (28.5%, Fig. 6). The 50.0% cultures each from fat layer and 38.7% and 61.2% from sediment layer, were pauci and multibacillary, respectively (Figs. 3 and 4). 3.2. M-ELISA as ‘herd screening test’ in dairy cows On the basis of screening of 115 milk samples against Map lacto-antibodies by m-ELISA, 32.1% cows were positive in six dairy herds located in three districts (Agra, Etah and Mathura). Lacto-prevalence of Map antibodies varied from 15.3% to 50.0% in dairy herds screened. Single government dairy herds (DDD farm, Mathura) had 33.3% lacto-prevalence (Table 3). Table 2 Comparative isolation of Map in cow milk culture (fat and sediment) Culture
Combinations
Fat Sediment
+ +
Total 50
17
+ + 8
11
Positive in culture: 42; positive in fat 28; Positive in sediment 31.
14
G. Sharma et al. / Research in Veterinary Science 84 (2008) 30–37
33
Table 4 Comparative evolution of sediment and fat culture and milk-ELISA Test
Comparison
Fat culture Sediment culture Milk ELISA
+ + +
Total 50
4
1
2
3
4
5
+
6
7
+ +
+ + 6
11
6
2
13
8 +
+ +
+
8
0
Sensitivity – 28.5% (OD – 2SD – 0.253).
Fig. 3. Paucibacillary colony of Map on HEY medium.
3.4. IS 900 PCR in milk samples of dairy herds From 50 fat and sediment samples each decontaminated, visible pellet was present in 22 fat and 18 sediment samples. DNA was checked under UV illumination and DNA was isolated in nine fat and four sediments. Only good quality DNA (three fat and two sediment) processed and 3.0% DNA samples (two fat and one sediment) amplified or 6.0% cows were positive in IS 900 PCR giving 229 bp band characteristic for Map (Fig. 5).
Fig. 4. Typical multibacillary colonies of Map on HEY medium. Table 3 Screening of milk samples (dairy herds) by m-ELISA Place Balkeshwar dairy, Agra Pvt. dairy, Soron, Etah Gokulpura Dairy, Agra DDD farm, Mathura Pvt. dairy, Agra DDD dairy, Farah, Total
Milk samples 10 12 16 36 28 13 115
ELISA positive 2 2 8 12 11 2
(20.0%) (16.6%) (50.0%) (33.3%) (39.2%) (15.3%)
Fig. 5. Screening of milk samples for MAP by PCR.
37 (32.1%)
10 8 6 4 2 0 45
60
75
90
105
120
135
150
Days Post Inoculation Number of Colonies
Using three tests (m-ELISA, fat and sediment culture) for the screening of 50 milk samples each there was agreement in 20.0% cows. The three tests in combination detected 88.0% milk samples (cows) as positive. Considering culture (both fat and sediment) as ‘Gold standard test’, 84.0% milk samples (cows) were true positives. Of the 88.0% positive cows, only 4.0% were exclusively detected positive in m-ELISA (Table 4). Of these true positive cows, 28.5% were detected by m-ELISA. Using single point cutoff m-ELISA detected 28.0% cows as positive (sensitivity 28.5%). But on continuous scale (S/P ratio) using 0.25 cutoff, in m-ELISA 40.0% cows were positive and of these 90.0% were detected in culture (sensitivity – 42.8%). However, of 60.0% m-ELISA negative milk samples (cows), 80.0% were positive in culture and only 20.0% were negative in culture (true negatives).
Number of Colonies
12
3.3. Comparison of m-ELISA with milk fat and sediment culture
14 12 10 8 6 4 2 0 45
60
75
90
105
120
135
Days Post Inoculation
Fig. 6. Appearance of Map colonies from cow’s milk.
150
34
G. Sharma et al. / Research in Veterinary Science 84 (2008) 30–37
Fig. 7. IS 1311-PCR.
3.5. IS 1311 PCR–REA Of the 14 good quality DNA, seven were positive in PCR (three fat and four sediment) belonging to weak and suspected cows from four different herds. The seven PCR positive DNA, were subjected to IS 1311 PCR– REA and three sediment and three fat were genotyped as ‘Bison type’ and one sediment (CMS 32) was ‘Bovine’ (Table 6, Fig. 7). The Map ‘S 5’ strain (from clinical case of JD in goat) used for preparation of antigen was also genotyped as ‘Bison type’ (Whittington, 2002; Sevilla et al., 2005). 4. Discussion Screening of large ruminants is often met with difficulties in sampling in India. Due to absence of proper restraining facilities, there is often lot of struggling by animals in collection of blood samples. Many times it requires felling of animals to ground and typing with ropes, which is time consuming and not liked by owners. There is ban on cowslaughter in India, therefore, collection of tissues for culture was not possible. Milk was very convenient material for screening from lactating cows. Since milk is important source of contamination to calves; therefore critical for the screening of cows and control of JD. Disease has been widely reported from developed nations (Muskens et al., 2000), but information is not available in Indian cattle. First time in India, using three sensitive tests, milk samples were screened to estimate prevalence of Map. Fifty fat and sediment each was screened by culture and m-PCR and 50 whey by m-ELISA. Therefore, for screening of 50 milk samples, in total 250 samples (100 in culture, 100 in DNA isolation and 50 in ELISA) were handled to conclude the status of each of 50 cows sampled. M-ELISA was used
as ‘herd screening test’, since it helped in screening of 65 more cows with little efforts, in addition to 50, screened by three tests. High lacto-prevalence of Map was reported in lactating cows from four dairy herds in three districts of North India. There were 84.0% cows true positives (culture positives). Centrifugation of milk partitioned Map in to fat and sediment layers (Gao et al., 2005). Culture of both fat and sediment increased the efficiency of milk culture test used for screening of lactating cows. Using this protocol, similar sensitivity has been reported by Kumar (2004) in goat milk. Low lacto-incidence of Map reported by Singh and Vihan (2004) in goat milk may be due to culture of only sediment layer. Gao et al. (2005) reported preferential partitioning of Map into cream layer and enhanced recovery by centrifugation and culture. Millar et al. (1996) reported overall 7.0% of 312 milk samples positive for Map. High incidence has been reported in culture of fecal samples of lactating cows from US dairy herds (Stabel et al., 2002). Variable prevalence has been reported from clinically (Taylor et al., 1981; Giese and Ahrens, 2000) and sub-clinically (Sweeney et al., 1994; Jackobson et al., 2000) ill cows. Presence of paucibacillary cultures indicated sub-clinical infection and multibacillary, active disease (super shedders) in lactating cows. Giese and Ahrens (2000) cultivated few colonies from milk of fecal culture positive cattle. Colonoes appeared between 45 and 150 DPI (Figs. 3, 4 and 6). Milk ELISA was standardized using indigenous protoplasmic antigen from native isolate of Map ‘Bison type’ of goat origin and compared with culture of milk. At single point cutoff 28.0% cows were positive (sensitivity – 28.5%). As ‘herd screening test’ m-ELISA detected 32.1% cows. Variable prevalence of Map using ELISA (serum and milk) has been reported in different countries (Muskens et al., 2000). Sweeney et al. (1994) using LAM ELISA in cattle milk reported 50% sensitivity. Using different antigens variable sensitivity of ELISA has been reported (Vannuffel et al., 1994; Collins et al., 2005). Low sensitivity may be due to use of single point cutoff and was 42.8% when OD values were analyzed by S/P ratio. Using PPA, Ferreira et al. (2002) reported similar sensitivity. Commercial ELISA kits (Collins et al., 2005; Nielsen et al., 2000) were less sensitive, since based on Map strain not reported in India (unpublished data). In present study PPA was sensitive at 0.5 lg/well concentration with 1:8 dilution of whey. Salgado et al. (2005), screened lacto-antibodies at 1:2 dilutions. There was high correlation between animals positive in m-ELISA and milk culture. At single point cutoff, of Table 5 S/P ratio and status of JD in lactating cows in m-ELISA and milk culture S/P ratios
Disease status
Lactating cows
Culture positive
0.00–0.09 0.10–0.24 0.25–0.39 0.40–0.99 1.0–10.0
Negative Suspected Low positive Positive Strong positive
30 00 00 06 14
24 00 00 06 12
G. Sharma et al. / Research in Veterinary Science 84 (2008) 30–37
28.0% m-ELISA positive cows, 85.7% were positive in milk culture. In S/P ratio, of 40.0% ELISA positive milk samples, 90.0% were positive in culture (Table 5). Study showed that m-ELISA using ‘Bison type’ Map antigen of goat origin could be used as ‘Herd screening test’ in lactating cows. Stabel et al. (2002) reported low sensitivity of mELISA than milk culture. Collins et al. (2005) showed similar sensitivity of m-ELISA. M-ELISA missed 60.0%, true positives, may be due to anergy condition (Waters et al., 1999). Koets et al. (2001), reported that there was no uniform association with increased antibody responses during progression from asymptomatic stage to clinical stage of BJD. Using m-ELISA, Collins et al. (1991) detected 59.7% of animals that shed Map in their feces. Stabel et al. (2002), detected only 25.0% of cows that were fecal culture positive by m-ELISA. Of culture positive milk samples, 47.6% were positive (positive + strong positive) in m-ELISA (Table 5). Ferreira et al. (2002) reported that PPA antigen detected 52.4% false negatives and 13.1% false positives. PCR has been standardized using decontaminated tissues and fecal samples from goat kids (Kumar et al., in press). PCR used frequently in cattle (Juste et al., 2005) was standardized for the screening of cow milk samples. DNA was isolated from 1 ml of decontaminated fat or sediment. PCR could be standardized from small aliquots. Of the decontaminated milk samples, 6.0% were positive in IS900 PCR. In this study milk culture was highly sensitive, m-ELISA moderately sensitive and sensitivity of IS900 PCR was significantly low. In m-PCR also screening of both fat and sediment layers was essential. In this study three cows were detected in PCR alone (Table 6), thus giving 90% cumulative positives in m-PCR and milk culture. M-PCR missed 41.5% culture positive samples. Low sensitivity of m-PCR may be due to poor standardization of DNA isolation from decontaminated material and large
Table 6 IS 1311 PCR on decontaminated material and comparison with culture and m-ELISA Milk sample
IS 1311
Culture
m-ELISA
CMF-4 CMF-6 CMF-12 CMF-15 CMF-18 CMF-19 CMF-33 CMS-27 CMS-30 CMS-32 CMS-25 CMS-40 CMS-45 CMS-48
P N P P N N N P P Pa N P P N
N P P P N P N P N N N P P P
N P N N N N P P N N P N N N
Total positives
8
8
4
Map ‘Bison type, CMS – cow milk sediment, CMF – cow milk fat, N – negative, P – positive. a REA cattle type and extremely weak.
35
number of paucibacillary samples. In this study DNA was amplified without dilution. Diluted DNA samples have been used to offset inhibitors in fecal and milk samples (Chui et al., 2004). When DNA was diluted 1:4 times in milk IS 1311 PCR–REA, of 14 samples (seven fat and seven sediment), eight were positive. Giese and Ahrens (2000) reported that m-PCR in raw cows milk could detect Map, but cultivation was more sensitive. Stabel et al. (2002) reported m-PCR was highly sensitive and specific in cattle. Map was almost equally found in pellet or cream by culture or PCR, (Giese and Ahrens, 2000). PCR has been used in bulk-tank milk samples to estimate prevalence of Map (Stephan et al., 2002). Pillai and Jayarao (2002) reported IS 900 PCR was superior to milk culture. Buergelt and Williams (2004) reported both culture and IS900 PCR could detect 10–100 cfu/ml of Map. Both IS 900 and IS 1311 PCR techniques were suitable for Map characterization and genotyping (Whittington et al., 2001). Map strains in this study were mainly of ‘Bison type’ and one was Map ‘Bovine’. ‘Bison type’ Map was similar to Bison (Bison bison) from Montana, USA (Whittington et al., 2001) and has been reported from goats, sheep and buffaloes in India (Whittington, 2002; Sevilla et al., 2005). 5. Conclusion Using protoplasmic antigen from native Map ‘Bison type’ strain of goat origin, indigenous milk ELISA, was successfully used to screen lactating cows. In m-ELISA, prevalence lacto-antibodies was moderate (32.0% positive) in six herds in North India. The m-ELISA was simple, sensitive and cost effective ‘herd screening test’. Comparative evaluation of m-ELISA with milk culture showed 28.5% and 42.8% sensitivity on single point and S/P ratio, respectively. Milk culture was the most sensitive of the three tests and lacto-prevalence of Map was high (84.0%) in lactating Indian cows. Screening of both fat and sediment independently improved the sensitivity of in m-culture and mPCR. DNA from decontaminated fat and sediment layers of milk of lactating cows were characterized and genotyped as Map ‘Bison type’ first time in the country.
Acknowledgement Authors are thankful to Director, CIRG, Mathura, for the facilities. References Arizmendi, F., Grimes, J.E., 1995. Comparison of Gimenez staining method and antigen detection ELISA with culture for detecting Chlamydia in bird. Journal of Veterinary Diagnostic Investigation 7, 400–401. Buergelt, C.D., Williams, J.E., 2004. Nested PCR on blood and milk for the detection of Mycobacterium avium subsp. paratuberculosis DNA in clinical and subclinical bovine paratuberculosis. Australian Veterinary Journal 82, 497–503.
36
G. Sharma et al. / Research in Veterinary Science 84 (2008) 30–37
Chi, J., Van Leeuwen, J.A., Wentink, A., Keefe, G.P., 2002. Direct production losses and treatment costs from bovine viral diarrhoea virus, bovine leukosis virus, Mycobacterium avium subspecies paratuberculosis and Neospora caninum. Preventive Veterinary Medicine 55, 137–153. Chiodini, R.J., Hermon-Taylor, J., 1993. The thermal resistance of Mycobacterium paratuberculosis in raw milk under conditions simulating pasteurization. Journal of Veterinary Diagnostic Investigation 5, 629–631. Chui, L.W., King, R., Lu, P., Manninen, K., Sim, J., 2004. Evaluation of four DNA extraction methods for the detection of Mycobacterium avium subsp. paratuberculosis by polymerase chain reaction. Diagnostic microbiology and infectious disease 48, 39–45. Clarke, C.J., 1997. The pathology and pathogenesis of paratuberculosis in ruminants and other species. Journal of Comparative Pathology 116, 217–261. Collins, M.T., Sockett, D.C., Ridge, S., Cox, J.C., 1991. Evaluation of a commercial enzyme linked immunosorbent assay for Johne’s disease. Journal of Clinical Microbiology 29, 272–276. Collins, M.T., Wells, S.J., Petrini, K.R., Collins, J.E., Schultz, R.D., Whitlock, R.H., 2005. Evaluation of five-antibody detection tests for diagnosis of bovine paratuberculosis. Clinical and Diagnostic Laboratory Immunology 12, 685–692. Ferreira, R., Fonseca, L.S., Lilenbaum, W., 2002. Comparison between a commercial and an in-house ELISA for anti-M. avium paratuberculosis antibodies detection in dairy herds in Rio de Janeiro, Brazil. Revista Latinoamericana de Microbiologia 44, 129–132. Gao, A., Odumeru, J., Raymond, M., Mutharia, L., 2005. Development of improved method for isolation of Mycobacterium avium subsp. paratuberculosis from bulk tank milk: effect of age of milk, centrifugation, and decontamination. Canadian Journal of Veterinary Research 69, 81–87. Garrido, J.M., Cortabarria, N., Qguiza, J.A., Aduriz, G., Juste, R.A., 2000. Use of a PCR method on faecal samples for diagnosis of sheep paratuberculosis. Veterinary Microbiology 77, 3–4. Giese, S.B., Ahrens, P., 2000. Detection of Mycobacterium avium subsp. paratuberculosis in milk from clinically affected cows by PCR and culture. Veterinary Microbiology 20, 291–297. Innis, M.A., Gelfand, D.H., 1990. Optimization of PCRs. In: Innis, Gelfand, Sninsky, White (Eds.), PCR Protocols. Academic Press, New York, pp. 3–12. Jackobson, M.B., Alban, L., Nielsen, S.S., 2000. A cross sectional study of paratuberculosis in 1155 Danish dairy cows. Preventive Veterinary Medicine 46, 15–27. Juste, R.A., Garrido, J.M., Geijo, M., Elguezabal, N., Aduriz, G., Atxaerandio, R., Sevilla, I., 2005. Comparison of blood polymerase chain reaction and enzyme linked immunosorbent assay for detection of Mycobacterium avium subsp. paratuberculosis infection in cattle and sheep. Journal of Veterinary Diagnostic Investigation 17, 354–359. Koets, A.P., Victor, P., Rutten, M.G., de Boer, M., Bakker, D., ValentinWeigand, P., van Eden, W., 2001. Differential changes in heat shock protein-, lipoarabinomannan-, and purified protein derivative-specific immunoglobulin G1 and G2 isotype responses during bovine Mycobacterium avium subsp. paratuberculosis infection. Infection and Immunity 69, 1492–1498. Kumar, S., 2004. Prevalence and molecular characterization of Mycobacterium avium subsp. paratuberculosis, the cause of Johne’s disease (Paratuberculosis) from goat milk in Agra region. M.Sc. Dissertation, Bundelkhand University, Jhansi, UP, India. Kumar, P., Singh, S.V., Bhatiya, A.K., Sevilla, I., Singh, A.V., Whittington, R.J., Juste, R.A., Gupta, V.K., Singh, P.K., Sohal, J.S., Vihan, V.S., in press. Juvenile Capri-Paratuberculosis (JCP) in India: incidence and characterization by six diagnostic tests. Small Ruminant Research. doi:10.1016/j.smallrumres, 2006.10.023. Marsh, I., Whittington, R.J., Cousins, D.V., 1999. PCR-restriction endonuclease analysis for identification and strain typing of Mycobacterium avium subsp. paratuberculosis and Mycobacterium avium
subsp. avium based on polymorphisms in IS1311. Molecular and Cellular Probes 13, 115–126. Millar, D., Ford, J., Sanderson, J., Withey, S., Tizard, M., Doran, T., Hermon-Taylor, J., 1996. IS 900 PCR to detect Mycobacterium paratuberculosis in retail milk supplies of whole pasteurized cow’s milk in England and Wales. Applied Environmental Microbiology 62, 3446–3452. Muskens, J., Barkema, H.W., Russchen, E., van Maanen, K., Schukken, Y.H., Bakker, D., 2000. Prevalence and regional distribution of paratuberculosis in dairy herds in the Netherlands. Veterinary Microbiology 77, 253–261. Nielsen, S.S., Thamsborg, S.M., Houe, H., Bitsch, V., 2000. Bulk tank milk ELISA antibodies for estimating the prevalence of paratuberculosis in Danish dairy herds. Preventive Veterinary Medicine 44, 1–2. Ott, S.L., Wells, S.J., Wagner, B.A., 1999. Herd-level economic losses associated with Johne’s disease on US dairy operations. Preventive Veterinary Medicine 40, 179–192. Pillai, S.R., Jayarao, V.M., 2002. Application of IS 900 PCR for detection of Mycobacterium avium subsp. paratuberculosis directly from raw milk. Journal of Dairy Science 85, 1052–1057. Salgado, M., Manning, E.J.B., Collins, M.T., 2005. Performance of a Johne’s disease enzyme linked immunosorbent assay adapted for milk samples from goats. Journal of Veterinary Diagnostic Investigation 17, 350–354. Sevilla, I., Singh, S.V., Garrido, J.M., Aduriz, G., Rodriguez, S., Geijo, M.V., Whittington, R.J., Saunders, V., Whitlock, R.H., Juste, R.A., 2005. Molecular typing of Paratuberculosis strains from different hosts and regions. Revue Scientifique et Technique (International Office of Epizootics) 24, 1061–1066. Singh, S.V., Vihan, V.S., 2004. Detection of M. avium subsp. paratuberculosis in goat milk. Small Ruminant Research 34, 231–235. Singh, S.V., Singh, A.V., Singh, P.K., Gupta, V.K., Kumar, S., Vohra, J., in press. Sero-prevalence of paratuberculosis in young kids using ‘Bison type’, Mycobacterium avium subsp. paratuberculosis antigen in plate ELISA. Small Ruminant Reseacrh. doi:10.1016/j.smallrumres. 2005.11.019. Stabel, J.R., 1998. Johne’s disease: a hidden threat. Journal of Dairy Science 81, 283–288. Stabel, J.R., Wells, S.J., Wagner, B.A., 2002. Relationship between fecal culture, ELISA and bulk tank milk test results for Johne’s disease in US dairy herds. Journal of Dairy Science 85, 525–531. Stephan, R., Buhler, K., Corti, S., 2002. Incidence of Mycobacterium avium subsp.paratuberculosis in bulk-tank milk samples from different regions in Switzerland. Veterinary Record 150, 762–770. Sweeney, R.W., Whitlock, R.H., Buckley, C.L., Pam, B.S., Spencer, M.S., Rosenberger, A.E., Hutchinson, L.J., 1994. Diagnosis of paratuberculosis in dairy cattle using enzyme linked immuno-sorbent assay for detection of antibodies against Mycobacterium avium subspecies paratuberculosis in milk. American Journal of Veterinary Research 55, 905–909. Taylor, T.K., Wilks, C.R., Mc Queen, D.S., 1981. Isolation of Mycobacterium paratuberculosis from the milk of a cow with Johne’s disease. The Veterinary Record 109, 532–533. van Embden, J.D.A., Cave, D., Crawford, J.T., Dale, J.W., Eisenach, K.D., Gicquel, B., Hermans, P., Martin, C., Mc Adam, R., Shinnick, T.M., Small, P.M., 1993. Strain identification of Mycobacterium tuberculosis by DNA fingerprinting: recommendations for a standardized methodology. Journal of Clinical Microbiology 31, 406–409. Vannuffel, P., Gilot, P., Limbourg, B., Naerhuyzen, B., Dieterich, C., Coene, M., Machtelinckx, L., Cocito, C., 1994. Development of species specific ELISA for diagnosis of Johne’s disease in cattle. Journal of Clinical Microbiology 32, 1211–1216. van Soolingen, D., de Hass, E.W., Hermans, P.W., Groenen, P.M.A., van Embden, J.D., 1993. Comparison of various repeatitive DNA elements as genetic markers for strain differentiation and epidemiology of
G. Sharma et al. / Research in Veterinary Science 84 (2008) 30–37 Mycobacterium tuberculosis. Journal of Clinical Microbiology 31, 1987–1995. Vary, P.H., Andersen, P.R., Green, E., Hermon-Talyor, J., Mc Fadden, J.J., 1990. Use of highly specific DNA probes and the polymerase chain reaction to detect Mycobacterium paratuberculosis in Johne’s disease. Journal of Clinical Microbiology 28, 933–937. Waters, W.R., Stabel, J.R., Sacco, R.E., Harp, J.A., Pesch, A., Wannemuehler, M.J., 1999. Antigen-Specific B-Cell Unresponsiveness Induced by Chronic Mycobacterium avium subsp. paratuber-
37
culosis Infection of Cattle. Infection and Immunity 67, 1593– 1598. Whittington, R.J., 2002. Personal communication. Whittington, R.J., Marsh, I.B., Whitlock, R.H., 2001. Typing of IS1311 polymorphisms confirms that bison (Bison bison) with paratuberculosis in Montana are infected with a strain of Mycobacterium avium subsp. paratuberculosis distinct from that occurring in cattle and other domesticated livestock. Molecular and Cellular Probes 15, 139–145.