Abstract Book of EAVLD2012 - eavld congress 2012

S1 - P - 25
GENOTYPIC VARIABILITY DETERMINATION OF PATHOGENIC PROTOTHECA
Sara Marques 1 , Eliane Silva 1 , Gertrude Thompson 1 , Volker Huss 2
1
University of Porto, ICBAS – CIBIO, Porto, Portugal
2
University of Erlangen-Nuremberg, Biology Department – MPP, Erlangen, Germany
Prototheca spp.; bovine mastitis; internal transcribed spacer (ITS); 16S rDNA; intergenic spacer (IGS)
Introduction
Members of the genus Prototheca are ubiquitous nonphotosynthetic
green algae that can be associated with
pathologies in humans and animals (1). The known pathogenic
species in the genus are P. zopfii, P. wickerhamii and P.
blaschkeae (2). Both P. zopfii and P. blaschkeae have been
associated with bovine mastitis (1, 2). Presently this pathology is
recognized as endemic worldwide and is considered a public
health problem (3). Prototheca identification is generally achieved
by phenotypic and molecular characterization, the latter being
used to easily identify Prototheca species. Phylogenetic
relationships of Prototheca can be inferred from 18S and 28S
rDNA sequence comparisons (1, 2, 4). The ribosomal internal
transcribed spacer (ITS) regions and plastid ribosomal RNA
operon are less conserved than 18S and 28S rRNA genes and
provide greater interspecific differences that are useful for the
differentiation of strains (5). Thus, the aim of the present study
was to determine the genotypic variability within P. zopfii and P.
blaschkeae strains by amplification and sequencing of these
regions.
Materials & methods
Prototheca isolates used in this study belong to a major collection
of milk pathogens obtained from different dairy herds from the
Northwest of Portugal. Thirty seven isolates were previously
identified as P. zopfii genotype 2 and five as P. blaschkeae (1).
These isolates were spread and grown on Sabouraud Dextrose
Agar medium (Merck Laboratories, Darmstadt, Germany). In this
study PCR amplification of the nuclear ITS region and plastid 16S
rDNA, intergenic spacer (IGS) and partial 23S rDNA were
performed. Briefly, genomic DNA preparation was performed as
previously described (1). For amplification of the ITS region
several sets of primers were applied and some additives were
used to improve the reaction. The amplification products were
analysed on a 0.8% (wt/vol) agarose gel after staining with
ethidium bromide. Since some of the amplifications produced two
bands, these were purified with the QIAquick ® PCR purification kit
(Qiagen, Crawley, UK) and cloned into Topo ® XL PCR Cloning
Kit (Invitrogen, Carlsbad, USA). As some sequences displayed
similarities to the Prototheca plastid IGS region between 16S and
23S rDNA, several other primers for the ITS region were tested
and amplification of the 16S rDNA, IGS and part of the 23S rDNA
were also performed.
Results
ITS amplification of Prototheca isolates retrieved bands of the
expected size only in few cases (Fig. 1). Sequencing of the
amplified products retrieved nuclear ITS of only 3 P. zopfii strains
with some differences between them. No ITS sequences were
obtained for P. blaschkeae.
zopfii isolates had identical sequences, and the 5 P. blaschkeae
isolates sequences could be assigned to 2 groups which differed
in two positions within the 16S rRNA gene and one position in the
tRNA-alanine gene.
Figure 2: 16S rDNA amplification of some P. zopfii (Pz1-2) and P.
blaschkeae (Pb1-2) isolates with 16S universal primers. All PCR
fragments have a size close to the expected size of about 1,500
bp. Lane EcoRI + HindIII – molecular weight standard, -C,
negative control without DNA.
Discussion & conclusions
The variability of nuclear ITS and plastid rDNA sequences
between and within P. zopfii and P. blaschkeae strains is shown
for the first time. Within the ITS sequences of the P. zopfii
isolates, similarities between 92 and 96% were found. The plastid
sequences obtained for P. zopfii and P. blaschkeae isolates
showed only 82.7% similarity between each other. Similarity of
the complete 16S rDNA alone was 85.3%, which was lower than
that of the respective 18S rDNA (98.0%) (1). Solving the
amplification problem and sequencing of the ITS region together
with the plastid rRNA operon from all isolates will be of great
value for Prototheca population genetics and epidemiology.
Acknowledgements
This work was supported by Fundação para a Ciência e
Tecnologia, Portugal, grant SFRH/BD/28892/2006.
References
1. Marques, S, Silva, E, Kraft, C, Carvalheira, J, Videira, A, Huss, V,
Thompson, G (2008). Bovine mastitis associated with Prototheca
blaschkeae. J Clin Microbiol; 46, 1941-1945.
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F, Huszenicza, G (2001). Review of the microbiological, pathological, and
clinical aspects of bovine mastitis caused by the alga Prototheca zopfii. Vet
Q, 23, 58-61.
3. Malinowski, E, Lassa, H, Klossowska, A (2002). Isolation of Prototheca
zopfii from inflamed secretion of udders. Bull Vet Inst Pulawy, 46, 295-299.
4. Moller, A, Truyen, U, Roesler, U (2007). Prototheca zopfii genotype 2:
the causative agent of bovine protothecal mastitis? Vet Microbiol, 120,
370-374.
5. Pröschold, T, Darienko, T, Silva, PC, Reisser, W, Krienitz, L (2011). The
systematics of Zoochlorella revisited employing an integrative approach.
Environ Microbiol, 13, 350-364.
Figure 1: ITS amplification of some P. zopfii (Pz1-6) and P.
blaschkeae (Pb) isolates with ITS primers. All PCR fragments
should have a size of approximately 1,300 bp, however different
sizes were found and in some isolates two bands were amplified.
Lane ClaI – molecular weight standard, -C, negative control
without DNA.
The complete plastid 16S rDNA of most of the isolates was
amplified (Fig. 2). Almost complete sequences of the plastid
rRNA operon were obtained for 8 Prototheca isolates. Three P.