Multiple-locus Variable-number Analysis Research Articles

Multiple-Locus Variable-Number Tandem Repeat Analysis Genotypes of Listeria monocytogenes Isolated from Farms, Abattoirs, and Retail in Gauteng Province, South Africa

The use of multiple-locus variable-number analysis (MLVA) of tandem repeats (TRs) for subtyping Listeria monocytogenes has proven to be reliable and fast. This study determined the MLVA genotypes of 60 isolates of L. monocytogenes recovered from cattle farms, abattoirs, and retail outlets in Gauteng province, South Africa. The distribution of the 60 L. monocytogenes isolates analyzed by type of sample was as follows: raw beef (28, 46.7%), ready-to-eat beef products (9, 15.0%), beef carcass swabs (9, 15.0%), cattle environment (6, 10.0%), and cattle feces (8, 13.3%). The serogroups of the isolates were determined using PCR and the MLVA genotypes based on six selected loci. The frequency of the 60 serogroups detected was as follows: 1/2a-3a (IIa) (27, 45.0%); 4b-4d-4e (1Vb) (24, 40.0%); 1/2c-3c (IIc) (8, 13.3%); and 1/2b-3b (IIb) (1, 1.7%). MLVA successfully clustered genetically related isolates and differentiated nonrelated isolates, irrespective of their sources, sample types, and serogroups, as demonstrated by 16 MLVA pattern types detected. For serogroup 4b-4d-4e (IVb), there was no variation in TRs LM-TR2, LM-TR4, and LM-TR6, which each contained only one allele (02, 00, and 93, respectively). However, across the sources and sample types of isolates, there was variation in serogroup 4b-4d-4e (IVb): LM-TR1 contained 00, 03, and 05; LM-TR3 contained 14, 20, and 22; and LM-TR5 contained 14, 21, and 25. Similar patterns of variation in the TRs were detected in the other serogroups (1/2a-3a, 1/2b-3b, and 1/2c-3c). BioNumeric data analysis identified at least five types in Gauteng province. MLVA epidemiologically clustered the related isolates and differentiated unrelated isolates.

MOLECULAR TYPING OF DUTCH ISOLATES OF XANTHOMONAS ARBORICOLA pv. PRUNI ISOLATED FROM ORNAMENTAL CHERRY LAUREL

Xanthomonas arboricola pv. pruni (Xap) has been found in several cherry laurel (Prunus laurocerasus) nurseries in the Netherlands, causing leaf spot. As no information is available yet about the epidemiology of this quarantine bacterium in cherry laurel, molecular typing of Xap isolates can considerably improve our understanding of pathogen spread between various cherry laurel production systems in different regions of the Netherlands and pathogen relatedness among different disease outbreaks. In this study, the genotypic diversity within a population of 25 Xap isolates isolated from different cherry laurel cultivars grown in different locations in the Netherlands between 2008-2010, was assessed using Multiple-locus variable-number analysis (MLVA). The identity of these Xap isolates was initially determined based on the EPPO standard PM 7/64. Confirmation of the identity of these Xap isolates was additionally achieved with diverse methodologies, including gyrase subunit B (gyrB) sequence typing, BOXand ERIC-PCR, AFLP, and Xap-specific PCR’s: one based on the previously described Pagani primers (2004) (conventional-PCR and its TaqMan-PCR variant) and one based on the recently described Pothier primers (2011c). Based on the results of the MLVA analysis, the Dutch population of Xap isolates could be divided into two groups; however no correlation with the geographical origin or any other character of these isolates could be established. Additionally, based on colony morphology, a panel of 5 look-a-likes were isolated from symptomatic leaves of P. laurocerasus which reacted in the Xap-specific PCR described by Pagani (2004) but that did not react in the Xap-specific PCR described by Pothier et al. (2011c). Further characterisation of these look-a-like isolates with AFLP, BOXand ERIC-PCR, and gyrB sequencing showed that the Xap-specific PCR described by Pagani does not discriminate between Xap and the look-a-like isolates. Similarly to Pagani PCR, the performance of a pathogenicity test with a pure culture of the isolate was not always discriminative between Xap and the look-a-like isolates, unraveling a complexity in Xanthomonas pathogenicity. Therefore, in routine screening based on the EPPO standard PM 7/64, complementary techniques such as BOX- ERIC-PCR, gyrB sequencing, Xap-specific PCR described by Pothier (2011c), MLVA and AFLP should be used to obtain a reliable diagnosis of Xap and avoid false positive results.