Proceedings of the 10th International Colloquium on Paratuberculosis

#107
Multilocus Short Sequence Repeat Analysis <strong>of</strong> Mycobacterium avium subsp.
paratuberculosis Isolates from Dairy Herds in Nor<strong>the</strong>astern United States <strong>of</strong> a
Longitudinal Study Indicates Low Shedders are Truly Infected
Pradhan AK, 1* Kramer AJ, 2 Mitchell RM, 1 Whitlock RH, 3 Smith JM, 4 Hovingh E, 5 Van Kessel JS, 6
Karns JS, 6 and Schukken YH 1
1Quality Milk Production Services, Department <strong>of</strong> Population Medicine and Diagnostic Sciences,
College <strong>of</strong> Veterinary Medicine, Cornell University, Ithaca, NY, USA; 2 Faculty <strong>of</strong> Veterinary
Medicine, Utrecht University, The Ne<strong>the</strong>rlands; 3 New Bolton Center, Department <strong>of</strong> Clinical
Studies, School <strong>of</strong> Veterinary Medicine, University <strong>of</strong> Pennsylvania, Kennett Square, PA, USA;
4 Department <strong>of</strong> Animal Science, University <strong>of</strong> Vermont, Burlington, VT, USA; 5 Department <strong>of</strong>
Veterinary and Biomedical Science, Pennsylvania State University, University Park, PA, USA;
6 Environmental Microbial and Food Safety Laboratory, ANRI, USDA-ARS, Beltsville, MD, USA
ABSTRACT
The objectives <strong>of</strong> this study were to evaluate whe<strong>the</strong>r low shedders <strong>of</strong> Mycobacterium avium
subsp. paratuberculosis (MAP) were pass-through animals or whe<strong>the</strong>r <strong>the</strong>y were truly infected.
We also evaluated whe<strong>the</strong>r <strong>the</strong>se animals were possibly infected by <strong>the</strong> super-shedders. The
MAP isolates were obtained from a longitudinal study that involved three different herds in <strong>the</strong>
nor<strong>the</strong>astern US. The shedding levels <strong>of</strong> animals at each culture-positive occasion were
determined. Selected isolates were collected from all animals that were culture-positive at <strong>the</strong>
same time super-shedders were present in <strong>the</strong> herds and from super-shedders. Using a
multilocus short sequence repeat (MLSSR) approach we found 15 different strains from a total
<strong>of</strong> 142 isolates analyzed. The results indicated herd-specific infections; a clonal infection in herd
C with 89% <strong>of</strong> animals sharing <strong>the</strong> same strain, different strains in herds A and B. In herd C,
100% and in herd A, 17 to 70% <strong>of</strong> cows shed <strong>the</strong> same strain as that <strong>of</strong> contemporary supershedders
at a given collection date. About 82% <strong>of</strong> available tissue samples were culture-positive
indicating a true infection. Taken toge<strong>the</strong>r <strong>the</strong> results <strong>of</strong> MAP strain-typing and shedding levels,
we conclude that at least 50% <strong>of</strong> low shedders have same strain as that <strong>of</strong> a contemporary
super-shedder. The results <strong>of</strong> this study indicate that very few cows had characteristics <strong>of</strong> a
possible pass-through animal; many more cows were actively infected. The sharing <strong>of</strong> same
strain <strong>of</strong> low shedders with <strong>the</strong> contemporary super-shedders suggests that low shedders may
be infected as adults by <strong>the</strong> super-shedders.
Key words: Mycobacterium avium subsp. paratuberculosis, MLSSR analysis, longitudinal
study, low shedders, truly infected
INTRODUCTION
Mycobacterium avium subsp. paratuberculosis is <strong>the</strong> causative agent <strong>of</strong> paratuberculosis (or
Johne’s disease), a debilitating chronic gastroenteritis in ruminants (Möbius et al., 2008) and is
characterized by a very long incubation period (up to several years). Shedding <strong>of</strong> MAP can
occur without showing clinical signs. Recently, using fur<strong>the</strong>r serial dilutions to determining <strong>the</strong>
range <strong>of</strong> shedding in animals, super-shedders were identified and defined as cattle shedding
more than 10,000 MAP/g <strong>of</strong> manure (Whitlock et al., 2005). It has been indicated that supershedders
represent <strong>the</strong> greatest risk to spread <strong>of</strong> Johne’s disease (Whitlock et al., 2005).
Although several molecular subtyping techniques have been previously applied to investigate
genetic variation in MAP, MLSSR approach has recently gained much attention and been
applied in different studies (Amonsin et al., 2004; Harris et al., 2006). While only a limited
number <strong>of</strong> cross-sectional studies have used this method with a restricted set <strong>of</strong> isolates, it has
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