1. EVALUATION OF A NESTED PCR ASSAY FOR IDENTIFICATION OF VIRULENT Rhodococcus equi
M.L. Marenzoni1, F. Passamonti1, K. Cappelli1, S. Capomaccio 2, F. Cittadini1, M. Catanossi1, G. Coppola1, M. Coletti1.
Centro di Studio del Cavallo Sportivo
1Facoltà di Medicina Veterinaria, Perugia
2Facoltà di Agraria, Perugia

2. INTRODUCTION
Rhodococcus equi is a Gram-positive bacteria and an opportunistic pathogen of foals under 6 months of age
R equi is an inhabitant of both soil and intestinal tracts of animals
Suppurative bronchopneumonia of foals is the major disease caused
Pigs, cats and cattle can occasionally be infected
Pneumonia caused by R. equi has been reported in patients with human immunodeficiency virus infection
Only virulent strains of R. equi harbouring a virulence plasmid of 85 to 90 kb (VapA)
The disease tends to be insidious and lesions can be well advanced before the animal exhibits coughing, dyspnoea,and characteristic loud, moist rales on auscultation of the lung
Attempted culture of R. equi is often unrewarding

3. OBJECTIVE
To develop a new nested PCR protocol with the aim to increase sensitivity and specificity to detect R. equi and to differentiate strains that contain the virulence-associated gene (VapA) from strains that do not

4. Materials and Methods: PCR primers for 16S rRNA gene of Rhodococcus equi
Primer 16S-forward
5’-TCGTCCGTGAAAACTTGGG-3’
Primer 16S-reverse
5’-ACCACAAGGGGGCCGT-3’
5’-GAGGAGCGAAAGCGTGGGTA-3’
Primer 16S N-reverse
Primer 16S N-forward
5’-TTAGCCTTGCGGCCGTACTC-3’
Access Genbank X82052
*Sellon D.C., et al., 2001
Accuprime Taq DNA polymerase System è provvista di una proteina termostabile che aumenta la specificità con il stampo DNA permettendo l’attivazione della polimerasi solo se i primer sono completamente ibridati al loro sito specifico.

5. Materials and Methods: PCR primers for plasmid VapA of Rhodococcus equi
Primer VP N-forward
5’-TCGGAACTGCCCGAGAACAT-3’
Primer VP N-reverse
5’-GCTCCCAGAACCGACAATGC-3’
5’-GAGGGATCCGGTTCTCGTAACGCTACAATC-3’
Primer VP-reverse
Primer VP-forward
5’-GGTTCGTCTTTCTGAAGGTT-3’
Access Genbank AF116907
*Sellon D.C., et al., 2001
Accuprime Taq DNA polymerase System è provvista di una proteina termostabile che aumenta la specificità con il stampo DNA permettendo l’attivazione della polimerasi solo se i primer sono completamente ibridati al loro sito specifico.

6. MATERIALS AND METHODS THERMAL CYCLE: FIRST ROUND30 ‘‘ ’ 1
35 cicli
94°C
66°C
68°C 30 ‘‘ ’ 1
35 cicli
MATERIALS AND METHODS
THERMAL CYCLE: FIRST ROUND
THERMAL CYCLE: NESTED ROUND
Accuprime Taq DNA polymerase System è provvista di una proteina termostabile che aumenta la specificità con il stampo DNA permettendo l’attivazione della polimerasi solo se i primer sono completamente ibridati al loro sito specifico.
*Sellon D.C., et al., 2001

7. MATERIALS AND METHODS: specimens
To evaluate sensitivity serial tenfold diluitions from 20 ng to 0,2 fg.
of DNA from R. equi reference strain (ATCC 33701) were carried out
Twenty-four R. equi isolates (obtained from colonies on blood agar and identified by biochemical test -Api Coryne, Biomérieux- and CAMP test)
Ten biological specimens from horses with clinical disease (ie, tracheal wash, abscesses, lung tissues from foals with pneumonia)
To evaluate specificity Corynebacterium pseudotuberculosis, Escherichia coli, Klebsiella pneumoniae, Nocardia asteroides, Pasteurella spp., Staphylococcus aureus, Streptococcus equi, Mycobacterium spp. were tested with nested-PCR

8. MATERIALS AND METHODS: DNA EXTRACTION
R. equi isolates: 95 °C for 5’, centrifuged at rpm for 5’  2 µl for PCR.
Biological specimens: extraction with the QIAamp DNA Blood Mini Kit  200 ng or 5 µl for PCR.
DNA concentration and purity were measured with spectrophotometer and electrophoresis on agarose gel

9. MATERIALS and METHODS: PCR
FIRST ROUND PCR:
R. equi isolates: 2 µl supernatant
BIOLOGICAL SPECIMENS: 200 ng or 5 µl
NESTED CYCLE:
1 µl di of the first round product

10. RESULTS: sensitivity and specificity
Sensitivity: 0,2 ng for the first round and 2 pg for the nested round, for both genes (equivalent to 100 bacilli). Sensitivity is 100 fold higher than the first cycle
Specificity: no amplification occurred when different bacteria from R. equi were used
16S first round
VP first round
16S nested
M 0,2ng 0.02ng 2pg 0.2pg N BC+DNA R.equi N LINF+DNA R.equi
VP nested
M 0,2ng 0.02ng
BC+DNA R.equi N LINF+DNA R.equi
N: negative control;
BC: DNA buffy coat;
LINF:DNA lymph node.

11. RESULTS: specimens PCR R.equi isolates (n = 24):
All the isolates resulted positive for 16S and VapA genes in the first and in the nested cycles; however the DNA in 6 specimens, extracted by boiling and stored at -20°C for 3-4 years, resulted positive for the VapA gene only in the nested cycle
PCR biological specimens
16S gene: all 10 specimens resulted positive both in the first and in the nested cycle, with products of amplification more evident after the nested cycle
VapA : 8 resulted positive both in the first and in the nested cycle, whereas 2 (1 tracheal wash and 1 pulmonary nodul) only after the nested cycle

12. CONCLUSIONS
Rapid identification of species and virulence plasmid, both in contaminated samples and directly from specimens
Increase in analytical sensitivity and specificity, especially in clinical samples and in the identification of the virulence plasmid
Possible use in retrospective studies or environmental investigation

13. Grazie per l’attenzione!