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Paolicchi, F1; Morsella C1; Verna,
A1; Spath E.1,
Martinis D.3;
Zumarraga, M2; Giofree, A2; Cataldi, A2 and Romano,
M2
1Grupo de Sanidad Animal, Departamento de Producción Animal
EEA INTA– Facultad Ciencias Agrarias.
Universidad Nacional Mar del Plata - CC 276, Balcarce (7620), Argentina. 2Instituto de Biotecnología, CNIA INTA Castelar, Argentina.
3Facultad Ciencias Veterinarias, UNNE, Argentina.
7mo. International Colloquium on Paratuberculosis. Bilbao, España. 11 al 14
de junio de 2002.
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Introduction
Paratuberculosis (Ptbc) is an infectious disease characterized by chronic
enteritis that causes a progressive deterioration of the affected animals. The
etiological agent of Ptbc is Mycobacterium avium subsp. paratuberculosis (M.
paratuberulosis) that affects domestic and wild species. The disease causes
important economic losses in infected herds, production losses between 6 to 19%
of either meat or milk or both were reported. Losses occurs not only in animals
with clinical symptoms but also in the so-called sub clinical carriers of Ptbc.
M. paratuberculosis is antigenically and genetically closely related to other
Mycobacteria but the most evident genetic difference is the presence of the
specific insertion sequence IS900 which is used as target of primers by the PCR.
M. paratuberculosis is a slow growing microorganism requiring mycobactin to
grow. The use of immunologic methods such as ELISA and gIFN tests are important
for the rapid diagnosis of Ptbc. By the other hand, IS900 RFLP typing can be
applied to M. paratuberculosis isolates to compare local strains with isolates
from other regions and countries.
Ptbc activities at the Laboratory of the Animal Health Group
Research and diagnosis of Ptbc in ruminants started in our Lab in 1985,
through serological, bacteriological, and immunopathological methods.
Serological studies:
Between 1992 and 2002 the serological technique used has been the absorbed
indirect ELISA test with a diagnostic sensitivity of 66% and a specificity of
98% (ROC analysis MedCalc Program). This ELISA uses a protoplasmatic antigen
PPA-3 (Allied Monitor, USA), sera are preabsorbed with Mycobacterium phlei to
avoid unspecific reactions.
During this period a total of 68,335 sera (cattle: n=61,525, cervidae:
n=6,670 and ovine: n=140) were processed, while sera belonged to a total of
5,197 samples from reports of 4-year-old or older cattle of the Provinces of:
Buenos Aires (BA:n=3,160), La Pampa (LP:n=716), Corrientes (C:n=761), La Rioja
(LR:n=101), Neuquén (N:n=74), and Río Negro (RN:n=385) has been studied.
Apparent seroprevalences were adjusted toobtain the real seroprevalences which
were in BA:
26.5% (beef) and 56% (dairy), LP: 2.4%, C: 1%, LR: 0.2%, N: 0%, RN: 7%.
Recently other techniques were introduced in our Laboratory and applied to sera
from cattle with M.paratuberculosis isolation (Paracheck ELISA Kit, CSL,
Australia) or in dairy heifers belonging to herds with clinical Ptbc (Bovigam
Gamma Interferon Kit, CSL, Australia). More information is required about the
use of these new techniques in our country.
Bacteriological and Molecular Analyses:
Since 1985 faeces (F), organs (OR) lymph nodes and intestine, or milk (M)
samples have been processed. The medium used was Herrold´d medium with
mycobactin, piruvate
and antibiotics. A total of 136 Strains of M. paratuberculosis were isolated
from: F (n=100: 66 beef cattle {mc}, 28 dairy cattle {dc}, 6 Deer {de}), OR
(n=34: 12 mc, 3 dc, 19 de), and M (n=2 dc). All the isolated strains were stored
in liquid nitrogen, and later typed by Polymerase Chain
Reaction (P.C.R.) to confirm IS900 insertion sequence, and 61 strains of them
have also been typed by Restriction Fragment Length Polymorphism (RFLP) in 4 different
patterns designated "A" (75%), "B" (10%), "C"
(6,1%), and "E" (13%), and were compared with isolates from Europe.
The more prevalent pattern in Argentina has been identical to the less frequent
in Europe, R9(C17), while the other patterns were not found in Europe. Isolates
of deer only have pattern "A".
Histological Analysis:
Histopathologic methods are routinely used in our Laboratory to confirm
compatible Ptbc lesions in cattle, sheep and deer that died with clinical
symptoms. Routine staining procedures like Haematoxylin and Eosin and Ziehl
Neelsen are used. Immunohistochemical streptovidine - biotine labelled methods
are also used to increase the specificity of the microscopic lesions found in
the specimens.
Teaching and Training Activities:
These activities are carried out in our Animal Health Group (INTA) jointly
with the National University of Mar del Plata. The Graduate Research and
Training Programs are aimed to a) Veterinary Residents, b) Magister or Doctoral
Students, and c) Short Trainings Courses and Workshops for Veterinary Laboratory
Diagnosticians from the private and official sectors. Figure.
Conclusions and future prospects in our Region
The results of our studies demonstrated the widespread distribution of Ptbc
in the Pampas region from Argentina, and the need to identify infected herds to
organize first a national control and afterwards an eradication program.
Standardized diagnostic methods should be used for individual diagnosis and
especially for epidemiological
surveillance to determine the real prevalence of Ptbc in beef and dairy cattle,
deer and sheep.
To start a national control program it is necessary to organize a Network of
Laboratories with the capacity to diagnose Ptbc, using standardized
bacteriological and serological techniques to isolate M. paratuberculosis and
detect infected herds.
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