The 16th International Pig Veterinary Society Congress, Melbourne, Australia, 17-20 2000 Sept.

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STUDY OF PREVALENCE OF LAWSONIA INTRACELLULARIS IN PIG FARMS FROM VENEZUELA

E.J. Kwiecien
1
, S. McOrist
2
, and V. Bermudez
3

1
Escuela de Educacin Agrcola. Universidad Simn Rodrguez. Ncleo Canobo. Canoabo. Carabobo 2043. Venezuela.
(ekwiecie@telcel.net.ve).
2
Veterinary Pathology Services, 33 Flemington St, Glenside. SA 5065, Australia. (stevemcorist@vps.com.au)

3
Catedra de Anatoma Patolgica. Facultad de Ciencias Veterinarias. Universidad Central de Venezuela. Maracay. Aptdo. Postal 4563.
Aragua. Venezuela. (vbermud@looksmart.com)

Introduction

Prolifertive Enteritis (PE) is an important enteric diseases caused by
the obligate intracellular bacterium Lawsonia intracellularis wich it
can not be grown in cell free media, thereby only limited information
has hitherto been available regarding prevalence of this germ in pig
herds. Recently, the PCR method has shown to be an effective
technique to detect the bacterium from faeces
(1,2,3)
. The objective of this
study was to investigate the presence of L. Intracellularis among
different age groups in infected or not pig herds by using PCR and IFA
test.

Materials and Methods

Pigfarms:
The study was conducted on farrow to finish (F-F) and wean to finish
(W-F) pig farms where a diagnosis of acute or chronic PE had been
suspected by clinical signs, necropsy or histopthalogy results obtained
from affected pigs within the past year (Table 1).
Faeces samples from postweaning pigs obtained in 5 farms were
recoleted by rectal stimulus and put them into 50 ml cap plastic vial.
Then faecal samples were transfered to swaps (CULTURETTE) and
chilled until their proccesing in the laboratory. Aditionally, three
samples of ileum, colon and cecum tissues were taken from pig with
clinical signs of diarrhea in two different farms to perform the IFA test.
To obtain a representative sample, we used a 20% of prevalence at
95% of conficence so a sample from 14 pigs per farm was need to
detect a least one positive animal
4
.

Table 1. Distribution of samples collected.

Farms No. Pigs/Herd System No. Samples
A 8.000 F-F 52
B 10.000 F.F 2
C 11.000 F-F 14
D 3.500 W-F 15
E 9.000 F-F 16
Total 41.500 99

PCR:
Extraction of DNA from faecal samples was performed as decribed
previously
5
. Standar PCR assay incorporating primers specif for L.
Intracellularis DNA was used as recommended
1
. First reaction
incorporated primers for eubacterial DNA (P11-E, rP1) to confirm
bacterial DNA suitable for Lawsonia testing. Each PCR amplification
was performed by using 2 l of the boiled DNA sample, 0.2 M of
each DNA nucleotide, 3.0 mM MgCl and 1-0 unit Taq polimerase at a
total volume of 50 l in PCR buffer. After denaturation of the samples
at 94 for 3 minutes, 35 cycles were performed at respective optimal
temperture starting at 94 C, then 55 and finally 72, for 1 minute
each one.
The PCR products were analysed by 2% agarose gel electrophoresis
added with 1l of ethidium bromide stain for each 100 ml of gel.
Then the gels were photographed under UV light to read the results.

Histopathology:
Sample tissues were fixed in 10% buffered formalin and prepared by
routine mathods as paraffin emmbedded blocks, then stained by
hematoxilin-eosin and Warthin.Starry stain.

IFA Assay:
We used a standard IFA assay on fresh sections incorporating
monoclonal antibody IG4 specific for L. Intracellularis. Control
negative and positive sections were incorporated into the assays.

Results

Three farms (60%) were positive to L. intracellularis by PCR and the
overall mean of positive samples was 6.66%, ranged from 0.0% to
100% (Table 2). The mean of positivity per farm was 22.67%. Previous
studies of prevalence have reported a low prevalence like this
preliminary study in our country
6,7
.

Table 2. Results of PCR and IFA tests.

Farms Positive Samples Percentage
PCR
A
*
2/28 7.14
B 2/2 100.00
C 0/14 0.00
D 0/15 0.00
E 1/16 6.25
Total 5/75 6.66
IFA 2/3 66.66
*52 samples collected, 24 samples were excluded.

Since annecdotical reports of Ileitis in our country come from early
1990, this is the first data generated by PCR and IFA test where the
prevalence of the bacterium predicts a high enzootic disease in
Venezuela.

References

1. Jones, G.F., Ward, C.E., Murtaugh, M.P. and Gebhart, C.J. (1993).
Enhanced detection of intracellular organism of swine proliferative
enteritis, ileal symbiont intracellularis, in feces by polimerase chain
reaction. J. Clin. Microbiol. 31, 2611-2615.

2. McOrist, S., Gebhart, C.J. and Lawson, H.K. (1994). Polimerase
Chain Reaction fro diagnosis of porcine prolifeartive enteropathy. Vet.
Microbiol. 41, 205-212.

3. McOrist S, Gebhart CJ, Boid R, et al.. 1995. Characterization of L.
intracellularis, gen. nov., sp. nov., the obligately intracellular
bacterium of porcine proliferative enteropathy. Int J Syst Bacteriol. 45:
820-825.

4. Cannon, M.R. and Roe, R.T. 1982. Livestock Disease Survey: A field
Manual for Veterinarians. Camberra: Australian Bureau of Animal
Health.

5. Knitell, J.P.; Roof, M.; Schwartz, K.J. et al. 1997. Diagnosis of
Porcine proliferative enteritis. Comp. Cont. Educ.. 19: S26-S35.

6. Baccaro, M.R.; Moreno; A.M. and Coutinho, L.L. 1998. Porcine
Proliferative Enteritis: Histopathological Aspects and Diagnosis using
Polimerase Chain Reaction. Proc. 15
th
IPVS Congress. Birminghan,
England. p108.

7. Takahashi K.; Kishimoto, Y et al.. 1998. Porcine Prolifearive
Enteropathy caused by Lawsonia intracellularis in Japan. Proc. 15
th

IPVS Congress, Birminghan, England. p109.




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