EVALUATION OF A NESTED PCR ASSAY FOR

IDENTIFICATION OF VIRULENT Rhodococcus equi

M.L. MarenzoniM.L. Marenzoni11, F. Passamonti, F. Passamonti11, K. Cappelli, K. Cappelli11, S. , S. Capomaccio Capomaccio 22, F. Cittadini, F. Cittadini11, M. Catanossi, M. Catanossi11, G. , G.

CoppolaCoppola11, M. Coletti, M. Coletti11..Centro di Studio del Cavallo Sportivo Centro di Studio del Cavallo Sportivo

11Facoltà di Medicina Veterinaria, PerugiaFacoltà di Medicina Veterinaria, Perugia 22Facoltà di Agraria, PerugiaFacoltà di Agraria, Perugia

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

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

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-reversePrimer 16S N-forward

5’-TTAGCCTTGCGGCCGTACTC-3’

*Sellon D.C., et al., 2001

Access Genbank X82052

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-reversePrimer VP-forward

5’-GGTTCGTCTTTCTGAAGGTT-3’*Sellon D.C., et al., 2001

Access Genbank AF116907

94°C

66°C

68°C30 ‘‘

30 ’

1 ’

35 cicli94°C

1 ’

*Sellon D.C., et al., 2001

94°C

62°C

68°C30 ‘‘

30 ’

1 ’

35 cicli94°C

1 ’

THERMAL CYCLE: FIRST ROUND

THERMAL CYCLE: NESTED ROUND

MATERIALS AND METHODS

• 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

MATERIALS AND METHODS: specimens

MATERIALS AND METHODS: DNA EXTRACTION

• R. equi isolates: 95 °C for 5’, centrifuged at 14.000rpm 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

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

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

N: negative control;BC: DNA buffy coat; LINF:DNA lymph node.

VP nested

M 0,2ng 0.02ng BC+DNA R.equi N LINF+DNA R.equi

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

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

Grazie per l’attenzione!