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Title: Phenotypic and genotypic characterization of Haemophilus parasuis isolates involved in a multifarm o


1
Phenotypic and genotypic characterization of
Haemophilus parasuis isolates involved in a
multi-farm outbreak S. R. Oliveira, A. Ruiz, C.
PijoanDepartment of Clinical and Population
Sciences, College of Veterinary Medicine,
University of Minnesota, Minnesota, USA 55108.
Introduction Haemophilus parasuis is the
causative agent of the Glassers Disease, a
syndrome that affects swine of all ages,
characterized by fibrinous meningitis and
polyserositis. Outbreaks involving H. parasuis
occur mainly in high health or specific pathogen
free herds, where most pigs are naïve to this
agent. To date, 15 serotypes of H. parasuis are
recognized. A high percentage (26,2) of H.
parasuis isolates is nontypable. Serotypes 1, 5,
10, 12, 13 and 14 are believed to be the most
virulent ones, while serotypes 2, 4 and 15 are
classified as moderately virulent (1).
Correlation between serovar and virulence has not
achieved consensus in the literature. Some
studies have shown that different H. parasuis
strains from the same serovar can have different
virulence (2). Phenotypic and genotypic
characterization of H. parasuis isolates can be
used for identification of virulent strains
isolated from an outbreak. This technique may
yield better information than serotyping,
especially for autogenous vaccine production. In
this report we used rep-PCR and found that only
two strains of H. parasuis were involved in a
large farm outbreak.
Materials and methods Thirty-one isolates of H.
parasuis obtained from a multi-farm outbreak were
characterized by their outer membrane protein
(OMP) and DNA profiles. The farms belonged to a
large swine company and were obtained from
different site 2 nurseries derived from different
sow herds. Twenty isolates from respiratory site
(lung) and 11 isolates from systemic sites
(joint, brain, heart, and spinal fluid) were
evaluated. OMP isolation was conducted as
described previously (4). Determination of
protein concentration in each sample was assessed
using a microplate assay (DC Protein Assay
BIO-RAD). Protein concentration was adjusted to
50?g/ 25?l/ sample. OMP samples were run in a
discontinuous SDS-PAGE gel, with a 4 stacking
gel and a 10 separating gel and the two-buffer
system of Laemmli (5). Samples were loaded at a
concentration of 16?g of protein per lane and run
at constant current of 30mA for approximately 2
hours. Bands were visualized by staining SDS-gel
with Coomassie blue R250. Molecular weight from
OMP bands was calculated by comparison with
protein standards. Genomic fingerprints from H.
parasuis isolates were determined using a
repetitive element-based polymerase chain
reaction (rep-PCR) with ERIC1R and ERIC2 primers
(6). Bands from fingerprints were assigned by
hand. Strains with similar fingerprints and
similar OMP were clustered separately.
Figure 1. Genetic patterns (A and B) of 6 H.
parasuis isolates recovered
from diseased pigs (lanes 1 to 6). ERIC-PCR
technique. (M) Molecular weight
marker.
Results The thirty-one H. parasuis isolates were
clustered into two genotypes (A and B) and two
phenotypes (1 and 2), based on ERIC-PCR and OMP
profiles respectively (Figures 1 and 2).
Fourteen isolates were classified as genotype A
and 17 isolates as genotype B. Most of the
systemic isolates (9/11) were classified as
genotype A and most of the lung isolates (15/20)
were classified as genotype B. Nine isolates were
classified as OMP pattern 1 and 22 as OMP pattern
2. There was no clear relationship between OMP
profiles and site of isolation or between
ERIC-PCR and OMP patterns, since isolates showing
similar fingerprints had slightly different OMP
profiles.
Figure 2. OMP patterns (1 and 2) from 12 H.
parasuis isolates recovered
from diseased pigs. (M) Molecular weight marker.
(CR) Clonally related.
Discussion Thirty-one H. parasuis isolates
recovered from clinical cases from a farm
outbreak were compared based on DNA and OMP
profiles. Both techniques could be successfully
used to characterize virulent strains. However,
the ERIC-PCR technique was more discriminative
than OMP profiles regarding strain
differentiation. Our results showed that even in
large farm outbreaks few H. parasuis strains are
involved, in this case, only two. Evaluation of
genomic fingerprints showed that systemic and
respiratory isolates tended to be clustered in
distinct genotypic groups, indicating that
virulent strains can be genetically related.
Similar results were described in a study that
evaluated strains isolated from several outbreaks
(3). Definite conclusions regarding association
between virulence and genotype cannot be made
based on our results, since we did not evaluate
all organs from the diseased pigs. Further
studies regarding the characterization of H.
parasuis strains isolated within and between
farms have to be done in order to better define
associations between genotype patterns and
virulence. Construction of dendograms based on
genotypes of H. parasuis strains with known
epidemiological and clinical information can be
useful in the identification of clonally related
virulent strains.
Implications H. parasuis infections in high
health herds are associated mainly with lack of
immunity against the farms virulent strain.
Control of the disease can be made by vaccination
of naïve pigs. However, commercial vaccines lack
effectiveness due to poor cross-reactivity
between H. parasuis serovars and strains. We have
showed in this study that genotypic
characterization of H. parasuis strains by the
ERIC-PCR technique is a valid tool in defining
the virulent strains involved in a farm outbreak.
Characterization of virulent H. parasuis strains
present in a farm is potentially useful to define
which of these should be included in an
autogenous vaccine.
References (1) Kielstein, P. and
Rapp-gabrielson, V. 1992. J. Clin. Microb.
30862-865. (2) Rapp-Gabrielson, V.J., Kocur,
G.J., Clark, J.T., Muir, S.K. 1997.Vet.
Med._83-90. (3) Ruiz, A., Torremorell, M., and
Pijoan C. 1998. Proc. A. Leman Swine Conference.
Minneapolis, p.3. (4) Carlone, G.M., Thomas,
M.L., Rumschlag, H.S., Sottnek, F.O. 1986. J.
Clin. Microb. 24330-332. (5) Laemmli, U.K. 1970.
Nature, 227680-685. (6) Versalovic J., Koeuth,
T., Lupski, J.R.. (1994) Meth. Mol. C. Biol.
525-40
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