Embedded Scribd iPaper - Requires Javascript and Flash Player

Israel Journal of Veterinary Medicine  Vol. 69 (3)  September 2014 Kaufman, N. 136
INTRODUCTION
Paratuberculosis ( Johne’s disease), caused by Mycobacterium
avium subsp. paratuberculosis (MAP), is predominantly a dis-
ease of ruminants but may afect other animals as well (1).
MAP causes signifcant economic damages due to the neces-
sity to cull shedding or clinically ill animals and by the infection’s
indirect actions, such as reduced fertility and production (2).
Te disease has a worldwide distribution, increasing in
time, with only 26 countries reported free of the disease (3).
In Israel, out of 189 farms examined, about 66% tested posi-
tive (KO, unpublished data).
Following the isolation of MAP from human patients
sufering from Crohn’s Disease (CD), the microorganisms
involvement in the syndrome as causative agent or secondary
invader aggravating its symptoms has been suggested, origi-
nating an ongoing controversy (for a recent review see 4).
Young animals aged up to six months are at the highest
risk (5) and typically only about 10% of those infected will
develop clinical signs. Te most frequent mode of infection
is fecal-oral, although other means of transmission have
been described (6). Te microorganism enters the enteric
mucosa mostly but not exclusively through the lymphatic tis-
Assessing the Fecal Shedding Consistency of Mycobacterium Avium
Subsp. Paratuberculosis by Dairy Cows by qPCR: A Preliminary Study
Kaufman, N.,
1§
Koren, O.,
2
Shwimmer, A.,
3
Baider, Z.,
1
Mor, F.,
3
Grinberg, K.
1
and Elad, D.
1
*
1
Dept. of Clinical Bacteriology and Mycology, Te Kimron Veterinary Institute, P.O. Box 12, Bet Dagan 50250, Israel.
2
Hachaklait Veterinary Services Ltd., P.O. Box 3039, Caesarea Ind. Park, Israel.
3
Te National Service for Udder Health and Milk Quality - Israeli Dairy Board, 4 Derech Ha’Horesh, Yehud 56470, Israel.
§
In partial fulfllment of the requirements for the degree of Doctor in Veterinary Medicine at the Koret School of Veterinary
Medicine, Te Hebrew University in Jerusalem.
* Corresponding Author: Prof. Daniel Elad, DVM, PhD, Dept. of Clinical Bacteriology and Mycology, Te Kimron Veterinary Institute, P.O.Box 12,
Bet Dagan 50250, Israel. Tel: +972-3-9681688, Fax: +972-3-9601578, Email: daniel.elad@gmail.com
ABSTRACT
Paratuberculosis, caused by Mycobacterium avium subsp. paratuberculosis (MAP), is predominantly a disease
of ruminants. Te microorganism is shed principally in the feces of afected animals, even without clinical
signs. Young animals, up to the age of 6 months are at the highest risk of infection by oral exposure to
contaminated feces. Tere is no treatment and vaccination is not permitted in Israel. Te standard method
of reducing farm infection rates is by improving management hygiene and culling shedding cows, especially
super-shedders (more than 10
4
copies of the hspX gene). Consequently the prompt identifcation of animals
shedding MAP is of cardinal importance in eradicating the infection. Te aim of this preliminary study
was to devise a fecal sampling protocol that would maximize the likelihood of detecting MAP shedding
cows and assessing the infuence of parturition induced stress on shedding. Sixteen cows and 15 heifers,
raised on a commercial dairy farm of about 350 lactating cows, with 10% milk MAP ELISA positive
cows were included in the survey. Rectal feces, sampled for 5-7 consecutive days, before and after calving,
were examined by quantitative PCR (qPCR). Results indicate that the microorganism was shed by positive
animals regularly and quantitative variations were minimal. Consequently one sample is a good indication
of the animal’s shedding status. MAP shedding was found not be infuenced by parturition associated stress.
Keywords: Feces, Mycobacterium avium subsp. paratuberculosis, quantitative PCR, shedding.
Israel Journal of Veterinary Medicine  Vol. 69 (3)  September 2014 137 Mycobacterium Avium Subsp. Paratuberculosis by Dairy Cows
sue (Payer’s patches) (7). Foci of infection are formed, some
of which are cleared from the bacteria whereas others act as
source of its further dissemination and the formation of new
foci (8). At sites of permanent infection, a granulomatous
reaction ensues but does not limit the microorganisms’ spread
in the surrounding tissues (9). Te initial cellular immune
reaction is replaced after 2 or more years by an inefcient, hu-
moral one. Concomitantly, fecal shedding may increase (6).
Te intestinal lesions cause the thickening of the intestine’s
wall and consequently malabsorption.
Clinically 4 stages of the infection have been described
(10): a) latent: without clinical signs, shedding or antibodies,
b) subclinical: with a cellular immune response and some shed-
ding, c) clinical: characterized by weight decrease (in spite of
normal feeding), declining cellular and rising humoral immune
response and MAP shedding and d) advanced clinical: with
intensive diarrhea, development of edema and massive shed-
ding of MAP. About 10% of infected animals reach this stage.
Recently the classifcation of cows by shedding intensity
has been proposed (11):
a. Negative
b. Passive shedders – cows that shed a few bacteria follow-
ing their exposure to a contaminated environment with-
out the colonization of the gastrointestinal tract (GIT)
c. Active shedders with colonized GIT
d. High shedders (“super-shedders”) – cows that shed
more than 10000 bacteria/g feces. Tis group poses by
far the highest risk to act as source of infection of other
animals and thus have to be identifed and eliminated
from the herd.
Laboratory identifcation of animals infected by MAP
is either by methods assessing the presence of an immune
reaction or by showing the presence of the bacteria or their
subcellular components in feces. Each of these methods
sufers from shortcomings: the former have unsatisfactory
specifcity (for detecting the cellular response) or sensitivity
(due to the relative advanced stages of the disease in which
antibodies are produced) (12) whereas the latter’s sensitivity
has been reported to be hampered by the irregularity of the
fecal shedding of MAP (6).
In this preliminary study we aimed at identifying an
eventual short term pattern of MAP shedding so as to be
able to devise the optimal number and timing of fecal sam-
pling to improve the likelihood of identifying shedding cows.
Moreover, the two parameters that may infuence MAP
shedding were assessed: age and parturition induced stress.
A qPCR technique aimed at detecting the hspX gene, shown
to be specifc to MAP (13), was used. Te accuracy of this
technique was found to be comparable to culturing the mi-
croorganism, the latter being signifcantly longer (6-8 weeks
vs. 1-2 days) (14). To the best of our knowledge, this is the
frst publication of a MAP shedding study, using qPCR.
MATERIALS AND METHODS
Milk or serum antibodies, in cows and heifers respectively,
were assessed by ELISA (ID Screen, IDvet, Grabels, France).
Te survey was conducted in a commercial dairy farm of
about 350 lactating cows, 10% of which were MAP milk an-
tibody positive. Fifteen heifers, two of them seropositive, were
sampled for 5-7 consecutive days during the last trimester
before and during the frst two weeks following parturition.
Sixteen cows, 15 of which were positive for MAP milk anti-
bodies, were sampled under a similar protocol. None of the
animals showed clinical signs of paratuberculosis. Te samples
were frozen at -18°C and all the samples of a given series were
examined at the same time with a commercial quantitative
polymerase chain reaction (qPCR) kit, based on detection
of the hspX gene (Vetalert, Tetracore, Rockville MD, US).
Means of the samples before and after parturition were
statistically compared by the two tailed dependent paired
t test (https://statistics.laerd.com/calculators/dependent-
t-test-paired-samples-calculator.php) and the resulting t
value’s signifcance was evaluated (http://www.danielsoper.
com/statcalc3/calc.aspx?id=8).
Antibody levels and qPCR values before and after calving
were compared by the Correl() function of Microsoft Excel
@
.
To assess whether the observed variability in the qPCR results
for the same series resulted from irregular distribution of the
microorganism in the sample or diferences in shedding levels,
six samples from three cows were examined 5-6 times each.
RESULTS
Results showed that MAP shedding variability was limited
(Table 1 for cows and Table 2 for heifers). Te comparison
of pre- and post-parturient animals showed that three cows
each were positive, low shedders or negative respectively, be-
fore and after calving. One cow was positive before calving
but became negative 3 months later. Five cows, all nega-
tive, were available for only one series of testing. Among the
Reserch Articles
Israel Journal of Veterinary Medicine  Vol. 69 (3)  September 2014 Kaufman, N. 138
heifers, two were seropositive for MAP. All the heifers were
found to be negative or low shedders before calving. Nine
months later, one of the seropositive heifers became a high
shedder and was removed from the herd.
No signifcant statistical diference was found between
pre- and post-parturient shedding levels (p=0.3374 for heif-
ers and p=0.1482 for cows). Correlation coefcients were low
(0.20-0.27).
Ten samples from a historically negative (serologically
and clinically) paratuberculosis free herd were examined and
resulted in completely negative reading.
DISCUSSION
Results of the qPCR repeat tests (Table 3) showed that
sample variability was minimal and consequently the dis-
crepancies probably result from diferences in shedding lev-
Table 1: Anti-MAP milk antibody titers and qPCR results (number of hspX gene copies) - cows
No ELISA Last trimester of pregnancy Post-partum
1 Pos NE 0.39 1.16 7.61 4.37 0.14
2 Pos 165.21 534.11 151.24 715.00 173.34 130.85 56.01 86.51 257.09 793.22
3 Pos 9431.19 9747.90 4176.75 6965.82 2733.37 0.76 1742.49 23391.85 167.14 6.61
4 Pos 7.47 4.40 37.97 27.80 NE
5 Pos 24.13 14.97 12.08 11.58 77.15 22.89 28.04 26.13 18.38 14.96 10.04
6 Pos 7.96 58.46 29.95 69.40 21.38 51.26 59.43 337.47 46.38 31.03 348.26 25.82
7 Pos 1.94 1.53 30.22 54.85 13.41 15.13 0.69* 239.81 76.22 52.80 50.86 84.63
8 Pos 3.04 213.55 0.92 1360.49 1546.96 207.40 0.03 3.18 4.90 5.43 2.76
9 Pos NE 29.91 50.99 100.36 1.55 5.52
10 Neg 321.52 5.20 1.73 0.01* 1.53 0.04* 2.48 2.98 1.93 0.68* 2.00 90.21
11 Pos 2.99 8.61 10.61 7.53 42.66 2.71 7.47 5.97 0.06 0.13
12 Pos NE 25.08 1.18 2.82 0.23 0.62
13 Pos 273.71 0.40 0.18 3.60 4.79 16.16 99.88 7.13 130.63 13.30
14 Pos 5.39 4.99 2.79* 4.94 4.21* NE
15 Pos 3.24 2.73 29.69 7.86 0.34 1.63 0.95 7.83 12.70
16 Pos 1025.74 10042.3 2405.10 3885.55 1227.91 69.22 797.25 1493.02 827.61 1340.66
* Samples reexamined. See table 3. Low positive Positive High positive (supershedders)
Table 2: Anti MAP serum antibody titers and qPCR results (number of hspX gene copies) - heifers
No ELISA Last trimester of pregnancy Post-partum
1 Neg 7.11 34.86 1.95 2.15 2.00 4.70 4.41 7.50 7.56 4.18
2 Neg 1.10 1.48 4.41 0.09 1.53 187.12 65.35 20.18 3.09 6.10
3 Pos 39.35 3.35 18.43 28.99 1739.29 39461.14 30402.11 28933.78 34780.57 14401.21
4 Pos 0.10 3.61 65.65 0.47 1.50 2.38 1.64 14.86 105.52 1.80
5 Neg 1.62 0.14 0.10 16.52 1.92 1.27 5.51 10.91 7.67 2.42
6 Neg 0.35 0.01 0.00 0.01 1.27 17.32 5.24 8.34 3.46 33.69
7 Neg 2.84 0.01 0.62 607.64 18.76 4.78 31.56 12.15 7.23 3.69
8 Neg 32.39 3.17 0.82 2.34 0.29 0.87 1.24 16.07 6.34 5.92
9 Neg 11.30 1.47 2.01 6.08 10.91 0.33 20.03 1.94 2.31 0.51
10 Neg 0.50 0.01 0.00 0.88 510.25 19.33 9.46 1.63 25.65 6.08
11 Neg 0.07 0.54 0.04 0.93 1.67 16.74 3.07 86.50 1.32
12 Neg 6.14 4.60 2.74 3.71 3.97 9.09 32.60 13.83 11.04 14.02
13 Neg 20.82 41.63 73.91 89.12 1.89 19.74 2.70 4.01 3.16 38.41
14 Neg 5.11 3.70 2.86 1.97 0.45 7.57 14.41 10.50 11.42 24.31
15 Neg 5.65 4.66 7.90 14.77 1.39 2.71 0.00 2.04 0.00 5.28
Low positive Positive High positive (supershedders)
Reserch Articles
Israel Journal of Veterinary Medicine  Vol. 69 (3)  September 2014 139 Mycobacterium Avium Subsp. Paratuberculosis by Dairy Cows
els. Te conclusions of this preliminary study are that, un-
like previously reported (3) fecal MAP shedding levels are,
with a few exceptions, constant enough to classify animals
as negative, low shedder, positive and high shedder (super-
shedder), by a single sample. Tis may be the result of the
use of qPCR whereas, to the best of our knowledge, fecal
MAP shedding uniformity was thus far assessed by cultur-
ing the microorganism. In addition, the results indicate that
calving associated stress does not seems to have infuenced
fecal MAP shedding levels and that the microorganism’s
distribution in the fecal sample is homogenous enough to
allow a single qPCR test to determine fecal MAP shed-
ding levels.
Forty fve percent (64/142) and 67% (100/149) (Tables
1 and 2) of the samples from cows and heifers, respectively,
resulted in low qPCR results (<10 gene copies). Tis may
be the result of true shedding, passive or incipient active,
or due to the inaccuracy of the kit at low reading values.
Since the kit correctly identifed fecal samples from a his-
torically negative farm, we assume the former assumption
to be true.
It is our opinion that low qPCR values should be in-
terpreted in light of the status of the herd: in high preva-
lence herds these results are more likely to indicate passive
shedding due to heavy environmental exposure whereas
in low prevalence herds such results may indicate animals
in initial stages of shedding that should be tested periodi-
cally to identify eventual evolvement into more massive
shedders.
Tis study was conducted in one dairy herd in a lim-
ited number of animals. Consequently we recommend
that our conclusions should be substantiated further in
the future by expanding the number of the examined
population.
CONCLUSIONS
Our results indicate that:
a. MAP shedding level variations during 5-7 consecu-
tive days are small and thus one sample is likely to
represent the animals shedding status.
b. Parturition stress does not infuence shedding levels,
neither in pluriparous cows nor in heifers.
c. Low qPCR results indicate low shedding levels and
not kit inaccuracies. Tese results may indicate passive
shedding or infection.
Table 3: Results of qPCR test replicates of the same sample
7 10 10 10 14 14 Cow no.
0.69 0.01 0.04 0.68 2.79 4.21 Original result
2.32 2.23 34.49 1.64 4.94 3.51
Replicate results
2.10 1.48 3.30 3.79 2.37 4.58
4.23 1.40 9.29 2.48 3.02 0.17
3.62 1.49 1.56 2.66 2.05 64.90
2.67 0.28 4.58 5.08 0.36 2.36
1.53
Table 4: Two tailed, dependent, paired ttest of pre and postpartum qPCR results (animals sampled during only one period excluded)
Cow no. 3
rd
pregnancy trimester mean Postpartum mean Heifer no. 3
rd
pregnancy trimester mean Postpartum mean
2 306.26 264.74 1 5.67 9.61
3 5836.39 5061.77 2 56.37 1.72
4 15.70 4.95 3 29595.76 365.88
5 22.32 17.38 4 25.24 14.27
6 116.67 190.79 5 5.56 4.06
7 58.84 100.86 6 13.61 0.33
8 279.33 3.26 7 11.88 125.97
10 33.53 19.56 8 6.09 7.80
11 8.87 3.27 9 5.02 6.35
13 54.98 53.42 10 12.43 102.33
15 7.28 5.78 11 26.91 0.65
16 2311.47 905.55 12 16.06 4.23
13 13.60 45.47
14 13.64 2.82
15 2.01 6.87
p=0.1482 p=0.3374
Reserch Articles
Israel Journal of Veterinary Medicine  Vol. 69 (3)  September 2014 Kaufman, N. 140
ACKNOWLEDGEMENTS
Tis study was partially funded by the Israeli Dairy Board, grant
no. 845 - 0277 - 11.
We are grateful to Mr. Shamai Zur and Mr. Sagi Marcovics for
their technical help.
DECLARATI ON OF CONFLI CTI NG I NTERESTS
Te authors declare no potential conficts of interest with respect to
the research, authorship, and/or publication of this article.
REFERENCES
1. Beard, P.M., Daniels, M.J., Henderson, D., Pirie, A., Rudge,
K., Buxton, D., Rhind, S., Greig, A., Hutchings, M.R., McK-
endrick, I., Stevenson, K. and Sharp, J.M.: Paratuberculosis in-
fection of nonruminant wildlife in Scotland. J. Clin. Microbiol.
39:1517-1521,2001.
2. Pillars, R.B., Grooms, D.L., Wolf, C.A. and Kaneene, J.B.: Eco-
nomic evaluation of Johne’s disease control programs imple-
mented on six Michigan dairy farms. Prev. Vet. Med. 90:223-
232, 2009.
3. Kennedy, D.: International eforts at paratuberculosis control.
Vet. Clin. North Am. Food Anim. Pract. 27:647-654, 2011.
4. Chiodini, R.J. Chamberlin, W.M., Sarosiek, J. and McCallum,
R.W.: Crohn’s disease and the mycobacterioses: a quarter cen-
tury later. Causation or simple association? Crit. Rev. Microbi-
ol. 38:52-93, 2012.
5. Larsen, A.B., Merkal, R.S. and Cutlip, R.C.: Age of cattle as re-
lated to resistance to infection with Mycobacterium paratuberculo-
sis. Am. J. Vet. Res. 36:255-257, 1975.
6. Olsen, I., Sigurğardóttir, G. and Djønne, B.: Paratuberculosis
with special reference to cattle. A review. Vet. Q 24:12-28, 2002.
7. Sigurğardóttir, G., Press, C.M. and Evensen, O.: Uptake of My-
cobacterium avium subsp. paratuberculosis through the distal small
intestinal mucosa in goats: an ultrastructural study. Vet. Pathol.
38:184-189, 2001.
8. Cocito, C., Gilot, P., Coene, M., de Kesel, M., Poupart, P. and
Vannufel, P.: Paratuberculosis. Clin. Microbiol. Rev. 7:328-345,
1994.
9. Lei, L., Plattner, B.L. and Hostetter, J.M.: Live Mycobacterium
avium subsp. paratuberculosis and a killed-bacterium vaccine in-
duce distinct subcutaneous granulomas, with unique cellular and
cytokine profles. Clin. Vaccine Immunol. 15:783-793, 2008.
10. Whitlock, R.H. and Buergelt, C.: Preclinical and clinical man-
ifestations of paratuberculosis (including pathology). Vet. Clin.
North. Am. Food Anim. Pract. 12:345-356, 1996.
11. Pradhan, A.K., Mitchell, R.M., Kramer, A.J., Zurakowski, M.J.,
Fyock, T.L., Whitlock, R.H., Smith, J.M., Hovingh, E., Van
Kessel, J.A., Karns, J.S. and Schukken, Y.H.: Molecular epide-
miology of Mycobacterium avium subsp. paratuberculosis in a lon-
gitudinal study of three dairy herds. J. Clin. Microbiol. 49:893-
901, 2011.
12. Collins, M.T.: Diagnosis of paratuberculosis. Vet. Clin. North
Am. Food. Anim. Pract. 12:357-371, 1996.
13. Harris, N.B. and Barletta, R.G.: Mycobacterium avium sub-
sp. paratuberculosis in Veterinary Medicine. Clin Microbiol Rev.
14:489-512, 2001.
14. Alinovi, C.A., Ward, M.P., Lin, T.L., Moore, G.E. and Wu, C.C.:
Real time PCR, compared to liquid and solid culture media and
ELISA, for the detection of Mycobacterium avium ssp. paratuber-
culosis. Vet. Microbiol. 136:177-179, 2009.
Reserch Articles