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

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-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.

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.

MATERIALS AND METHODS THERMAL CYCLE: FIRST ROUND 30 ‘‘ ’ 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

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

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 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.

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

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