Multilocus Short Sequence Repeat technique to
identify Mycobacterium avium subspecies
paratuberculosis strains in supershedders and
contemporary Pass through or Active passiveshedders in three Northeastern US dairy herds
Drs. A.J. Kramer
Studentnr. 0352179
Supervisors:
Dr. R. Jorritsma
Dr. Y.H. Schukken
Cornell University, Quality Milk Production Service
August – November 2008
CONTENTS
1. Abstract
2
2. Introduction
3
2.1 Johnes disease general
3
2.2 Supershedders, Pass through cows and Active passive shedders
3
2.3 MAP Multilocus Short Sequence Repeats
4
3. Materials and Methods
6
3.1. Dairy farm and sample collection
6
3.2. Bacterial analysis
7
3.3. DNA extraction
8
3.4. PCR and Gel
8
3.5. Purification, Quantification, and Sequencing
10
4. Results
10
4.1. SS cows and cows that are shedding on the same time
10
4.2. Collecting date
14
4.3. Longitudinal follow-up
16
5. Discussion
17
5.1. MLSSR strain typing
17
5.2. Pass through and Active passive-shedding
18
6. Conclusion
20
7. Acknowledgements
20
8. Thank you
21
9. References
22
1
1. ABSTRACT
Trefwoorden: Mycobacterium avium subsp. Paratuberculosis, Supershedders, Multilocus
Short Sequence Repeats.
Paratuberculosis in cows is caused by Mycobacterium avium subsp. paratuberculosis. (MAP)
Infected cows spread MAP in the environment. Recently Supershedder (SS) cows were
recognized. These cows are spreading extremely high numbers of MAP in the environment.
These MAP in the environment can be ingested by herd-mates causing positive fecal samples
in these herd-mates. We hypothesized that these fecal positive cows become Pass through
cows, ingesting the MAP and shedding MAP in the feces without becoming positive on tissue
rather than Active passive-shedders, ingesting the MAP, shedding MAP in the feces and
becoming positive on tissue.
By using the Multilocus Short Sequence Repeats (MLSSR) method for 3 different loci (1, 2
and 8) on SS cows and cows that were fecal positive at the same time as the SS, in three
different herds in the North East of the US, we aimed to evaluate if low shedders should be
considered Pass through cows or Active passive-shedders and if Active passive-shedders were
infected by the SS.
We observed that a large percentage of the cows that were positive on fecal samples at the
same date as the SS, were shedding the same strain as the SS. When cows were found positive
on fecal culture, the majority also were eventually positive in tissue at slaughter. Of these
tissue positive cows several were found having the same strain in their tissue as the SS. We
2
therefore conclude can be that a large number of cows become Active passive-shedders rather
than being Pass through cows.
2. INTRODUCTION
2.1 Johne’s disease general
Paratuberculosis in cows, also known as Johne’s disease, is caused by Mycobacterium avium
subspecies paratuberculosis (MAP) and causes large economic losses on dairy farms (13).
Clinical signs of the disease appear after a long incubation period of several years and include
a decline in milk production, a loss of body weight despite normal appetite, and diarrhea. The
disease is transmitted to young animals by the ingestion of feed, milk, and water contaminated
by feces of infected animals excreting the pathogen (13).
Shedding of MAP can occur without showing clinical signs. The majority of the animals are
infected within the age of one year (16). Daughters from infected dams have a high risk of
becoming infected. Calves from non-infected dams that were born shortly after a calving of an
infected dam and calves growing up with future high shedders are more likely to get infected
(3). Although the subclinical shedders MAP are seen as the main factor in keeping the herds
infected, farms still appear to remain infected with MAP even after participating in
eradication programs that focus on culling of known shedding animals (9).
2.2 Supershedders, Pass through cows and Active passive-shedders
The quantity of bacteria detected in the feces from infected animals varies from very low (<5
CFU/tube), low (<10 CFU/tube) to moderate (10-50 CFU/tube) and high (>50 CFU/tube) (6)
3
(17). Recently, supershedders (SS) were identified in dairy herds (17), which were defined as
animals that are shedding more than 10,000 CFU of MAP/g of feces, the number of CFU/tube
in those cows exceeds usually the 300 or is to numerous to count (TNTC). (17). SS are
shedding extremely high numbers of MAP into the environment leading to the increased MAP
bio-burden in the dairy herd. It has been indicated that SS may have a significant role in
creating Pass through and Active passive-shedders in dairy herds. The high concentration of
MAP in the feces of SS may contaminate the environment of uninfected herd mates, including
feed materials such as total mixed ration (TMR). When the contaminated TMR is consumed
by previously uninfected herd-mates, some of these animals will become test positive for
MAP in their fecal samples. This makes the SS the original source of MAP and the herdmates
with a positive fecal culture are considered pass through cows, acting merely as a passthrough of infectious material.(17). In our study, Pass through of MAP is defined as detection
of MAP in fecal samples of an adult cow with subsequent fecal samples testing culturenegative, negative Johne’s serological tests, and culture negative tissues obtained at slaughter.
However, our preliminary data and bacterial genetic analyses suggested that many of these
tentative Pass through cows, (shedding only once in the presence of a SS and all subsequent
fecal tests negative) would be truly infected when the same strain of MAP was identified in
their intestinal tissues at slaughter. These cows are considered Active passive-shedders (1).
Initially, SS were considered as unique biological events that responded to some trigger.
Thereby stimulating Johne’s infected cattle to shed increasing quantities of MAP. However,
more recently it was recognized that all active-shedders have the potential to become SS and
if they remain in the herd (17). Active shedders are defined as the cattle infected with MAP
and have MAP in manure from gastro-intestine (GI) mucosal cell turnover and cell lysis
releasing MAP into the gut contents-manure.
4
2.3 Molecular techniques for strain typing
Methods for differentiation or subtyping of bacterial strains provide important information for
molecular epidemiologic analysis. DNA-based molecular sub typing techniques such as
multiplex PCR for IS900 integrations loci (MPIL) (4) (12), restriction fragment length
polymorphism (RFLP) analysis (5, 5, 7, 14), and amplified fragment length polymorphism
(AFLP) (12) have been used to investigate genetic variation in Map (2).
Amonsin et al. (2004) recently described DNA fingerprinting techniques for subtyping of
MAP isolates by using multilocus short sequence repeats (MLSSRs). Short sequence repeats
(SSRs) have also been used in subtyping other bacteria such as Yersinia pestis, Salmonella
subsp., Escherichia coli and Bacillus anthracis (2, 8). The SSRs consist of simple
homopolymeric tracts of a single nucleotide (mononucleotide repeats) or multimeric tracts
(homogeneous or heterogeneous repeats), such as di- or tri- nucleotide repeats, which can be
identified as variable-number tandem repeats (VNTRs) in the genome of the organism (18).
Amonsin et al. (2004) identified 11 polymorphic SSR loci for MAP and locus 1, locus 2, and
locus 8 were identified as the loci associated with the highest Simpson’s diversity indices (D
value) of 0.700, 0.616 and 0.668, respectively. The D value is also known as genetic diversity
or discriminatory power. Amonsin et al. (2004) calculated D value on the basis of the allele
frequency and the number of alleles. This revealed an average number of alleles per locus of
3.20, with an average D value of 0.393 (a range of 0.1 to 0.7). Harris et al. (2006) also
suggested that these loci have the highest D values. While some cross-sectional studies have
used these loci to track MAP infection, it has been recognized that the use of well-designed
longitudinal studies using several herds in multiple states is essential to apply these loci in
understanding the epidemiology of Johne’s disease (8). The use of SSR strain-typing provided
5
the opportunity to get a better insight of herd infection and transmission dynamics of MAP.
(8, 11).
By using this SSR typing method on MAP isolates from cows identified as SS and cows
shedding at the same collection time when SS were identified, we aim to evaluate (i) if low
shedders should be considered pass through cows or active passive shedders, and (ii) if active
passive shedders were potentially infected (as adults) by the SS.
3. MATERIALS AND METHODS
3.1 Dairy farms and sample collection
For this study longitudinal data from the Regional Dairy Quality Management Alliance
(RDQMA) project was used. This longitudinal research project is a multi state research
program conducted under a cooperative research agreement between the United States
Department of Agriculture Research Service (USDA-ARS) and four universities (University
of Vermont, Pennsylvania State University, University of Pennsylvania, and Cornell
University. In this project 3 different herds in the Northeast were followed for approximately
5 years. For a more complete description see Pradhan et al. (2008).
Briefly, each of the three enrolled farms (Farms A, B, and C) received quarterly farm visits
from the project team in their state. At each visit, an online management survey was
completed, environmental samples were collected, and blood samples were taken from all
lactating cows. Individual fecal samples were collected from all cows every six months.
Additionally, all culled cows were tracked from the farm to the slaughter house using Radio
Frequency Identification (RFID) tags. At slaughter, in addition to fecal sample, tissues
6
samples were collected from four locations (lymph nodes and ileum valves) and carcass data
was obtained.
Fecal samples were collected from the rectum with disposable gloves and transferred into
sterile vials (50 mL plastic tube, BD FalconTM propylene screw top, BD Biosciences, San
Jose, CA). All samples where labelled with the corresponding sample numbers and placed
into a large Ziploc bag, which where than placed into coolers with ice packs and submitted for
bacterial analysis to the Johne’s research laboratory at the University of Pennsylvania (15).
3.2 Bacterial analysis
Two grams of fecal samples was put into a 50 mL plastic tube containing 35 mL of water
(fecal-water tube). This sample was then shaken for 30 min in a mechanical shaker. Tubes
were then incubated for 30 min at room temperature. Five mL was taken from the top portion
of the fecal-water tube and transferred into a second 50 mL plastic centrifuge tube containing
25 mL of 0,9% hexodecylpyridiniumchloride (HPC) in ½ strength brain heart infusion (BHI)
broth solution (final concentration of HPC 0,75%). In the next step (decontamination or
germination step) tubes where incubated at 35 to 37˚C for 18 to 24 h. After this, tubes were
centrifuged for 30 min at 900 x g. The supernatant was discarded and the pellet was resuspended by adding 1 mL antibiotic brew (1L of ½ strength BHI 18,5 g/L, Amphorericin B
50 mg/L, Nalidixic acid 100 mg/L, vancomicin 100 mg/L); this was vortexed inside a class II
biological safety cabinet. The re-suspended pellets were incubated overnight up to a
maximum of 3 d at 35 to 37˚C. After this, 4 tubes of Herold’s Egg Yolk Media (HEYM; 2 inhouse and 2 commercial (BD Diagnostics)) were inoculated with 0.2 mL per tube and then
incubated in a slanted position at 37˚C. The tubes were read every 2 wk and finally read at 16
wk. Slightly raised white-yellow colonies were evaluated for typical acid-fastness and
7
morphological appearance of MAP. Each culture with colony growth was sub cultured for
mycobactin dependency prior to reporting the culture positive for MAP (15).
3.3 DNA extraction
The extraction of MAP DNA was done by using a Qiamp® DNA mini kit, (Qiagen Inc.,
Valencia, Calif.) using previously described methods with a few modifications (12). Colonies
were scraped from the flask by a 10 μL loop and put in a 2 mL tube containing 1 mL ultrapure
distilled, DNAse-, RNAse- free water (Invitrogen Corporation Carisbad, CA) in 0.25 mL
zirconia/silia beads (Biospec, OK, USA). To the tubes 0.65 mL of AL buffer (Qiagen) was
added. The content in the tubes was homogenized with the bead-beater (Mini-BeadBeater8™, Biospec Products) for 5 min and then incubated for 30 min at 70˚C. In the next step, 600
µL of the incubated sample was transferred into a new 2 mL tube containing 600 µL buffer
AL (Qiagen) and 60µL proteinase K (Qiagen). The content was vortexed briefly and
incubated for 30 min at 70˚C. Following the incubation, 600 µL of 100% ethanol was added
and vortexed. The mixed samples were then centrifuged through a spin column at 6000 x g for
1 min. Next 500 µL of buffer AW1 (Qiagen) was added and centrifuged through the spin
column at 6000 x g for 1 min and the flow-through was discarded, the same procedure was
done for buffer AW2 (Qiagen). After 3 min of centrifugation at 13000 g x, membranes of the
spin columns were placed in a new 1.5 mL tube and successively 100 µL and 50 µL of
distilled, DNAse-, RNAse- free water (Invitrogen Corporation Carisbad, CA) was added to
the membrane and centrifuged. The 1.5 mL tubes containing DNA were stored at -20 ºC. An
extraction blank was included in each batch, which was used as a negative control for
molecular analyses.
8
3.4 PCR and Gel run
PCR was done using three different loci (locus 1, locus 2, and locus 8) as described by
Amonsin et al. (2004). The loci were identified as the ones with the highest D values. A
mastermix of 20 µL containing 12.5 μL GoTaq green [10 µM], 0.625 μL [10 μM] forward
primer (Table 1), 0.625 μL [10 µM] reverse primer (see Table 1), and 6.25 μL of distilled,
DNAse, RNAse- free water (Invitrogen Corporation Carisbad, CA) and 5 µl DNA template
was used per sample in the PCR reaction. For those samples with an unsuccessful PCR
amplification for locus 1, an adjusted volume for mastermix (24 μL) and 1 μL of DNA
template was used. The 24 µL mastermix contained 12.5 µL GoTaq green [10 µM], 0.625 µL
[10 µM] Forward primer (table 1), 0.625 µL [10 µM] Reverse primer (Table 1) and 10.25 µL
of distilled, DNAse-, RNAse- free water (Invitrogen Corporation). A set of modified primers
were also used for locus 1 for samples for which PCR amplification was unsuccessful with the
conventional primer (Table 1).
TABLE 1: Primers (F-forward and R-reverse) for 3 loci (1, 2, and 8) used for MLSSR
analysis.
MLSSR-1F
5’ TCA GAC TGT GCG GTA TGA AA 3’
MLSSR-1R
5’ GTG TTC GGC AAA GTC GTT GT 3’
MLSSR-1F
modified
MLSSR-1R
modified
MLSSR-2F
5’ GTG TTC GGC AAA GTC GTT GT 3’
MLSSR-2R
5’ TGC ACT TGC ACG ACT CTA GG 3’
MLSSR-8F
5’ AGA TGT CGA CCA TCC TGA CC 3’
MLSSR-8R
5’ AAG TAG GCG TAA CCC CGT TC 3’
5’ GCG GTA CAC CTG CAA G 3’
5’ GTG ACC AGT GTT TCC GTG TG 3’
9
The amplification conditions consisted of an initial denaturation at 94˚C for 2 min, followed
by 35 cycles of denaturation at 94˚C for 30 s, annealing at 60˚C for 1 min, and extension at
72˚C for 1 min, with a final extension step at 72˚C for 7 min.
After completion of the PCR reaction, gel electrophoresis (at 104 V and 55 mA) was
performed for the amplified products. For electrophoresis, 5 μL of PCR product was loaded
onto a 1.5% agarose gel in a 0.5 TBE buffer for 45 min. Gel’s were stained in ethidium
bromide for 2 min and after de-staining for about 1 hr, gel’s were photographed under UV
light (Biorad Hercalus, CA) (10).
3.5 Purification, quantification, and sequencing
PCR products were purified by using an Invitrogen Purelink™ PCR purification kit
(Invitrogen Carisbad, CA). DNA quantification was performed using a nanodrop (ND-1000
spectrophotometer V3.1.1., PEQLAB Biotechnologie GmbH Erlangen, Germany) device.
PCR products were sent for sequencing to Cornell University Life Sciences Core Laboratories
Center where DNA sequencing was performed using the Applied Biosystems Automated
3730 DNA Analyzer. Sequence data was analysed using DNASTAR (DNASTAR Inc.,
Madison, Wis). The number of tandem repeats was determined for each locus to reflect the
number of copies represented in the SSR sequence. (8)(2).
4. RESULTS
4.1 Supershedders and cows shedding on the same time
10
Supershedders on 3 herds were identified based on the shedding level (>10,000 cfu of MAP/g
feces were considered supershedders). Six, 2, and 4 animals were identified as supershedders
on Farms A, B, and C, respectively. The fecal samples from SS cows (n = 12 cows), from
other cows that were fecal culture-positive at the same time that a SS was on the farm (n = 44
cows), as well as available tissue samples at slaughter (n= 15 cows) at slaughter were
analyzed (n= 137 tissue samples) for the 3 different loci using MLSSR strain typing
technique. The PCR products showing strong bands at gel electrophorese with good DNA
yield were sent for subsequent sequencing (Fig. 2).
FIGURE 1: An example of sequence results with G repeats at 208-216 bp.
Sequence results were obtained from a total of 373 MAP culture-positive PCR products (see
Fig. 1 for an example).
11
With the number of tandem repeats per locus known at this point we were able to indentify
different strains of MAP within and between the herds (Tables 2, 3, and 4). Based on the data,
a total of 14 different strains were found with certainty. Based on preliminary analysis, an
additional 3 strains maybe identified when genetic information are completed for the
incomplete samples.
1
2 3
4 5
6 7 8
9 10 11 12 13 14 15 16 17 18 19 20
21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40
FIGURE 2: PCR for MLSSR 2 and MLSSR 8
Upper gel: MLSSR 2, lane 16 extraction blank, lane 18 PCR blank
Lower gel: MLSSR 8, lane 37 extraction blank, lane 39 PCR blank
For most of the isolates the number of repeats for the 3 different loci (loci 1, 2, and 8) were
evaluated, whereas for others the number of repeats that was evaluated was only at two
different loci (loci 2 and 8). This was due to unsuccessful amplification of DNA template for
a number of isolates during PCR of locus 1. When the number of repeats for the strains with
only two known loci (2 and 8) were the same as those strains where three different loci (loci
1, 2, and 8) were known, these strains were defined as the same strain. This means that if a
12
given strain had the combination 7–10–6, a strain with the combination 10–6 was defined for
this analysis as the same strain.
TABLE 2: Repeats for loci 1, 2, and 8 in tissue and faeces on subsequent days for
supershedders and cows that are shedding at the same time as that of supershedders on
Herd A
Herd A
COWS
SS
693
1085
1099
1160
1180
1182
Other
1078
1127
1148
1149
1161
1167
1171
**1179
1181
1200
1255
1280
1288
1294
1416
1645
2005
2044
2653
2669
DATES
CULLING
Birthdate 17-2-2004 5-10-2004 12-4-2005 3-10-2005 1-5-2006 9-10-2006 16-4-2007 15-10-2007 13-4-2008 Cull date LN 1
31-8-1997 7 - 10 - 6
7-27-2004 n/a
20-10-1999 7 -10 - 5 17 - 10 - 5
3-22-2005 n/a
11-12-1999 neg
7-9-5
3-29-2005 n/a
19-8-2000 neg
7 - 10 - 6
7 - 10 - 6
8-16-2005 n/a
17-11-2000 7 - 10 - 6 7 - 10 - 6
7 - 10 - 6 lost by contamination
17-1-2006 x - 10 - 6
24-11-2000 7 - 10 - 6
9-8-2004 n/a
LN 2
n/a
n/a
n/a
n/a
7 - 10 - 6
n/a
7-10-1999 neg
1-1-1998 7 - 10 - 6
15-7-2000 neg
23-7-2000 neg
28-8-2000 7 - 10 - 6
17-9-2000 7 - 10 - 6
12-10-2000 neg
11-11-2000 neg
22-11-2000 neg
25-12-2000 neg
10-3-2001 7 - 10 - 5
23-5-2001 neg
10-6-2001 7 - 11 - 6
1-7-2001 neg
1-12-1999 neg
25-10-2003 neg
10-8-1998 neg
12-9-1998 neg
26-12-1998 neg
5-2-1999 7 - 10 - 5
7-9-6
7-9-6
neg
7 - 11 - 6
neg
7 - 10 - 6
neg
x - 10 - 5
neg
7- 10 - 6
7 - 10 - 6 x - 10 - 6 x - 10 - 6 x - 10 - 6
neg
7 - 10 - 6
7 - 11 - 6
neg
neg
neg
neg
neg
neg
neg
7 - 10/11 - 5
neg
neg
7 - 10 - 5
neg
7 - 10 - 6
neg
neg
neg
neg
neg
7 - 14 - 4
neg
neg
neg
7 - 10/11 -5
7 - 10 -5
7- 10 - 6
7 - 10 - 6
7 - 11 - 6
x - 10 - 6
neg
x-9-6
neg
x - 11 - 6
x - 11 - 6
neg
neg
neg
x-9-5
neg
7 - 11/12- 4
x - 10 - 6
neg
7 - 10 - 6
neg
neg
neg
neg
neg
neg
neg
neg
n/a
neg
28-9-2006 7 - 10 - 6
18-1-2005 7 - 9 - 6
5-3-2008
7-12-2004 n/a
28-9-2004 n/a
24-8-2004 7 - 9 - 6
19-10-2006 7- 9 - 6
31-7-2006 x - 10 - 6
29-8-2006 x - 11 - 6
IL-IC valveIleum
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
x - 10 - 6 x - 10 - 6
n/a
n/a
7 - 10 - 6
7-9-5
7 - 10 - 6
n/a
n/a
n/a
n/a
7 - 10 - 6 7 - 10 - 6
x-9-6 7-9-6
7 - 10 - 6 7 - 10 - 6
7 - 11 - 6 7 - 10 - 6
x - 10 - 6
n/a
n/a
x-9-6
x- 10 - 6
7 - 11 - 6
Fec slaug
n/a
n/a
n/a
n/a
7 - 10 - 6
n/a
x - 10 - 6
n/a
n/a
7 - 10 - 6
x - 11 - 6
28-6-2005
7 - 10 - 6 x - 10 - 6 x - 11 - 6
10-7-2006 n/a
16-4-2007
19-9-2006 n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
n/a
22-2-2005 n/a
n/a
n/a
n/a
n/a
neg
x: PCR failed
**: actively infected cows
n/a: no data on tissues available
TABLE 3: Supershedders and cows that are shedding at the same time as the supershedders,
with number of repeats for loci 1, 2, and 8 Herd B
13
Herd B
COWS
DATES
SS
51
52
CULLING
Birthdate
22-3-2004 12-9-2004 15-12-2005
1-12-1999 7 - 11/12 - 57 - 10/11 - 5
20-1-1999 16 - 10 - 5
6-6-2005 5-12-2005 Cull date LN 1
10-27-2004n/a
n/a
LN 2
n/a
n/a
IL-IC ValveIleum
n/a
n/a
n/a
n/a
Fec slaug
n/a
n/a
n/a
neg
n/a
n/a
n/a
n/a
n/a
neg
n/a
n/a
n/a
n/a
n/a
neg
n/a
n/a
n/a
n/a
Other
30 2-11-1998 13 - 10 - 5
73 15-10-1999 12 - 11 - 5
74 5-11-1999 7 - 10 - 5
87 15-1-2000 14 -12 - 5
214
2-8-1999 7 - 12 - 5
Tulip
15-9-1999 neg
neg
neg
neg
neg
neg
7 - 10 - 5
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
1-13-2006 n/a
1-9-2006 neg
10-18-2004n/a
march 2005n/a
3-10-2006 n/a
9-19-2004 n/a
n/a
neg
n/a
n/a
n/a
n/a
n/a: no data on tissues available
TABLE 4: Supershedders and cows that are shedding at the same time as that of
supershedders, with number of repeats for loci 1, 2, and 8 Herd C
Herd C
COWS
SS
66
102
106
152
Other
*40
**107
114
124
**133
*136
164
170
**171
178
184
189
193
208
270
**491
493
495
Birthdate
21-6-1999
27-4-2000
22-6-2000
10-7-2001
15-11-2004 9-5-2005
7-9-6
7-9-6
neg
8-9-6
7-9-6
7-9-6
x-9-6
16-10-1998
27-6-2000
26-7-2000
11-10-2000
17-1-2001
20-2-2001
1-10-2001
13-11-2001
17-11-2001
30-1-2002
9-4-2002
26-4-2002
22-5-2002
13-8-2002
4-10-2002
29-9-2001
1-1-2000
1-1-2000
7-9-6
neg
7-9-6
7-9-6
7-9-6
7-9-6
7-9-6
7-9-6
7-9-6
neg
7-9-6
7-9-6
7-9-6
not test
no test
7-9-6
7-9-6
7-9-6
DATES
CULLING
14-11-2005 15-5-2006 13-11-2006 Cull date LN 1
x-9-6
x-9-6
15-6-2006 x - 9 - 6
x-9-6
15-12-2005 n/a
7-9-6
2-2-2006 n/a
9-2-2005 x - 8 - 6
LN 2
x-9-6
n/a
n/a
7-8-6
IL-IC Valve
x-9-6
n/a
n/a
7-8-6
Ileum
x-9-6
n/a
n/a
7-8-6
Fec Slaug
neg
x-9-5
n/a
pos
neg
neg
neg
n/a
pos
neg
neg
neg
7-9-5
7-9-6
pos
neg
neg
neg
x-9-6
7-9-6
pos
7 - 11/12 - 5
neg
13-11-2006 n/a
29-12-2005 7 - 9 - 6
n/a
neg
n/a
neg
n/a
neg
n/a
neg
8-8-2005 x - 9 - 6
15-8-2005 n/a
26-6-2006 n/a
neg
n/a
n/a
neg
n/a
n/a
neg
n/a
n/a
neg
n/a
n/a
14-4-2005
6-7-2006
10-10-2006
29-8-2005
6-7-2006
14-4-2005
7-9-6
7-9-6
7 - 10 - 6
neg
neg
7-9-6
neg
x-9-6
neg
neg
neg
neg
neg
7 - 11 - 6
neg
neg
neg
neg
no test
neg
neg
neg
neg
neg
neg
neg
neg
neg
neg
7-9-6
no test
neg
neg
neg
neg
neg
neg
neg
neg
neg
no sample
neg
neg
neg
neg
neg
neg
x-9-6
7-9-6
pos
x-9-6
neg
n/a
n/a
7-9-6
neg
neg
neg
x: PCR failed
*: pass through cows
**: actively infected cows
n/a: no data on tissues available
4.2 Percentage of cows shedding same strain as that of SS at multiple collection dates
Data was evaluated according to collection date, where the percentage of cows that were
shedding the same strain as that of SS was calculated (Table 5).
14
For herd A, there were 4 different collection dates where a SS was on the farm, with
percentages of cows shedding the same strain being 83%, 33%, and 25%. From the fourth
collection date onwards nothing can be concluded with certainty because the fecal sample of
the SS cow that was lost due culture contamination, after this sample date the cow left the
farm. But if we assume that this SS keeps shedding the same strain at the fourth time, 60% of
the cows that were positive on that collection date are shedding the same strain as the SS
(Table 5).
For herd B there were 2 different collection dates where a SS was on the farm. At these dates,
25% and 0% of the cows were shedding the same strain. When assuming that the number of
alleles for loci 2 was 10 instead of 11 , this last percentage was 100% (Table 3). Locus 2 of
this MAP isolate will be sequenced again to resolve this inconsistency. On the second
collection, only 1 other cow appeared positive in addition to the SS, leading to result of
identical shedding as the SS as either 0% or 100%.
Herd C had 4 different collection dates with a SS on the farm. On the first collection date,
100% of all the positive cows in addition to the SS were shedding the same strain as that of
SS, the second time this was 50%, and the last 2 times was 100% (Table 5). It has to be noted
that only 1 or 2 other cows were culture-positive for MAP. Still, the percentage of cows
shedding the same strain as the SS was the highest for this farm.
Of all the cows that were shedding at the same time as the SS, 66% were shedding the same
strain once or more as that of SS. This percentage includes the fourth collection time on farm
A as a valid one and assuming that cow 1180 of herd A was shedding the same strain at the
time of culture contamination as identified before. Also this analysis assumed that cow 51
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(SS) in herd B was shedding MLSSR type 7-12-5 at the first collection and MLSSR type 710-5 at the second collection. Fifty-two percent of the cows were found to be shedding the
same strain as one of the SS once or more, when these cows (1180, 51) were excluded.
TABLE 5: Percentage of cows shedding the same strain as SS at a collection date.
Herd
Collection
A
B
C
1
83%
25%
100%
2
33%
0% or 100%*
50%
3
25%
100%
4
60%**
100%
*: 100% is valid if cow 51 (SS) is shedding 7-12-5 the first collection and 7-10-5 the second time (Table 3)
**: 100% is valid if cow1180 is shedding same strain as before on fourth collection (Table 2)
4.3 Longitudinal follow-up
When evaluating cows within their lifetime, several trends were seen. Some cows appeared
culture positive during life time only once or twice and were not seen positive again, not even
in tissues at slaughter. Those cows that were positive with the same strain as the SS were
therefore defined as Pass through cows. This was only seen for 2 cows out of all the cows we
studied. These two cows were in herd C (Table 3).
Other cows were identified as culture positive at 1 or 2 collection dates and negative on other
collection dates but were then positive in tissues at slaughter for the same strain. When strains
16
were the same as the strains seen at the SS, these cows were defined as cows that became
Active passive-shedders caused by the SS. For herd A, this was observed for 1 of the 20 cows
(Table 2). Assuming that cow 1180 was shedding the same strain at the fourth collection date
this can be defined for 3 of the 20 cows.
Herd B had no data on tissue at slaughter except for 1 cow that was negative. In herd C, 5 out
of the 18 cows were found positive showing this pattern (Table 4).
Because tissue samples at slaughter were not collected for all cows, there are cows that were
not identified as Pass through or Active passive-shedders cows but still showed negative fecal
samples during their life. In herd A and C, respectively, 1 and 8 cows with this phenomenon
were identified. For herd A, an additional 2 cows were found assuming that cow 1180 was
shedding the same strain at the fourth collection.
Another group of cows were found to shed the same strains in life while these strains and
different strains were found at slaughter. Also variation within the samples during their lifetime and tissues collected at slaughter were found. (Tables 2, 3, and 4)
5. DISCUSSION
Because of the economical losses on dairy farms caused by Johne’s disease (13) control of
this disease is important. Several eradication programs are set up to control the disease. But
even with the control programs herds still appear to remain infected with MAP (9). Although
Johne’s disease is known to transmit particularly in young animal, animals are only found to
become positive on fecal tests later on in their life (17). The observation of SS brought new
17
information to this aspect. It was suggested that SS spread MAP in such a high amounts that
the environment gets contaminated with MAP. This contamination may cause the so called
pass-through cows or may even infect cows (17).
5.1 MLSSR strain typing
The MLSSR approach provided a better insight into the epidemiology of MAP and the
concept of Pass-through and Active passive shedders within the herds. MLSSR has been
described as a useful discrimination technique between MAP strains within the same species
(11) and even within the same herds (8). By using 3 different loci 14 (or 17) different strains
were identified in these 3 herds. The number of repeats found in these herds was within the
same variation as observed by previous investigators (2, 8, 10). The biggest variation was
found in Herd B with 4 or 5 different strains in a total of 9 positive samples. Herd C had the
least variation with 6 or 8 different strains in a total of 51 positive samples. In Herd A and C a
predominant strain was recognized. Harris et al., (2006) also found that there is shared
genotype among MAP isolates within herds and between geographically distinct sites..
Because some of the samples had unsuccessful PCR amplification for locus 1, strain typing
for locus 1 was not complete yet at the time of writing this report. A different, modified, set of
primers and different optimum PCR conditions are currently being tested to get more
successful amplifications for locus 1 PCR products. Our preliminary data indicated that these
modifications were working reasonably well for successful amplification. Previous
researchers had also identified unsuccessful amplification or sequencing for locus 1 (10) It is
noted that for a proportion of the MAP isolates, PCR was not completed for locus 1. It is
expected that the amplification and sequencing of this locus will be completed soon. For those
PCR products where the number of nucleotides repeats could not be identified with certainty,
18
further sequencing in both directions (forward and reverse) as well as duplicate sample testing
were done.
5.2 Pass through and Active passive-shedding
The data collected during this study is not conclusive with regard to the moment time at
which cows are actually getting infected. Cows could be either infected within the first year of
life by the dam or the environment or as an adult by the SS. Cows that were seen only positive
once or twice at fecal culture with a low CFU and are negative at all other collections are
likely adult Pass through infections.
Two cows appeared positive in their lifetime with the same strain as that of SS and were
found negative in tissues at slaughter. These cows can be seen as Pass through cows, who
ingested MAP from their environment and were shedding it in their feces. Experiments
conducted by Sweeney et al. (1992) in heifers revealed that 100,000 CFU was sufficient
enough to cause Pass through shedding.
This data also includes several cows that are culture-positive during all collections and in
tissues and in fecal sample obtained during slaughter, even with the same strain observed for
the SS. These cows might be infected during their early life. Further analyses in combination
with the amount of CFU cows are shedding needs to be done.
A total of 5 or 7 cows were identified as cows that became Active passive-shedders because
they were positive on fecal culture once or more, but were also negative on fecal culture and
were again culture positive in tissue at slaughter. One of these cows also shed a different
strain during its lifetime when there were no SS on the farm anymore. Also 2 cows shedding
the same strain as the SS only once during their life, were found culture positive in fecal
samples with this strain and another strain in their tissue at the time of slaughter.
19
Several cows appeared positive with multiple strains during life and in tissues obtained at
slaughter. This was also seen for some of the SS. This might indicate that cows that got
infected with multiple strains got infected at different times during their life.
From all the fecal culture-positive cows where tissue samples were obtained during slaughter
(n = 20) only 3 appear negative. This suggests that if cows were positive in fecal culture, this
most likely indicates an active infection. Data presented by Whitlock. (2008) suggests that
approximately 50% fecal cultures are passive shedders. Data on this research suggests that a
high percentage of these cows become Active passive-shedders.
6. CONCLUSION
It can be concluded that more cows were Active passive-shedders rather than being Pass
through cows. Of all cows for which data on tissue obtained at slaughter was available, 83%
was positive. Several actively infected low shedding cows (41%) have the same strain as a
contemporary SS, suggesting adult active infection may have taken place.
7. ACKNOWLEDGEMENTS
We express our appreciation to the farm owners and personnel that participated in the study
both at the farms and in the laboratories. This project was supported in part by the USDAAgricultural Research Service (Agreements. 58-1265-3-155, 58-1265-3-156,58-1265-3-158,
20
and 58-1265-4-020) for the Regional Dairy Quality Management Alliance (RDQMA) and the
Johne’s Disease Integrated Program (JDIP, USDA contract 45105).
8. THANK YOU
After these months of research it’s time to say “thank you” to some people. First of all I
would like to thank Quality Milk Production Service and Cornell University for the
opportunity to do this research with them. Especially I would like to thank Ynte Schukken for
supervising me through these months, his enthusiasm, all the possibilities and of course his
bike, which provided me the best way of transportation to QMPS until the snow came. I also
would like to thank Abani Pradhan who introduced me in the world of the RDQMA project,
MAP and Multilocus Short Sequence Repeats. Abani Pradhan answered all my questions and
proved to be a good troubleshooter for PCR problems. All the other people at QMPS: “thanks
for everything you have done for me!”. Sharinne for having and helping me in the lab, Vicky
and Tully for their company at the front office and their help with all the forms and questions.
All others, thank you for giving me such a good time.
Ruurd Jorritsma thank you for being my supervisor in Utrecht and the flexibility you gave me
doing this project at Cornell.
21
Katrien van den Brink for taking care of all the paper work in Netherlands so I can start my
clinical rotations at the end of January and still spend some more time in the USA. Thank you
you’re a great friend!
And than at the last but certainly not the least I would like to thank two people who basically
kidnapped me the first two weeks. By doing that, they gave me the best time ever in Ithaca.
Laura and Rick thank you for all the good times! We’ll meet again in Wisconsin and of course
in Holland for your honeymoon!
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