Technical note: Identification of Prototheca species from bovine milk samples by PCR-single strand conformation polymorphism

Abstract

We report the development of a PCR-single strand conformation polymorphism (SSCP) method to identify Prototheca spp. responsible for bovine mastitis: P. zopfii and P. blaschkeae. The method was set up using reference strains belonging to P. zopfii genotype 1, P. zopfii genotype 2, and P. blaschkeae as target species and P. stagnora, and P. ulmea as negative controls. The assay was applied on 50 isolates of Prototheca spp. isolated from bovine mastitic milk or bulk-tank milk samples, and all isolates were identified as P. zopfii genotype 2. We conclude that the described PCR-SSCP approach is accurate, inexpensive, and highly suitable for the identification of P. zopfii genotype 2 on field isolates but also directly on milk, if preceded by a specific DNA extraction method.

Simultaneous identification by multiplex PCR of major Prototheca spp. isolated from bovine and buffalo intramammary infection and bulk tank

“…The identification of all isolates was also confirmed by the PCR–single strain conformational polymorphism SSCP typing methodology (Cremonesi et al . ) (data not shown).…”

“…(reviewed in Cremonesi et al . ); however, no one has been designed for the identification of Prototheca spp. associated with IMI in a single PCR.…”

Mastitis associated with Prototheca zopfii - an emerging health and economic problem on dairy farms

“…In cows infected with P. zopfii, the SCC in quarter and composite milk samples can be lower than 100,000 cells/mL (24). The infection is commonly subclinical and is discovered only after significant increase of the SCC (8). If the composite milk sample is tested for the SCC, the infection of individual quarters can remain unnoticed.…”

“…The main drawback of the standard method of isolation and identification of P. zopfii is its duration -it usually takes more than three days. Thus, molecular techniques (PCR) for prompt identification of Prototheca zopfii genotypes 2 directly from milk samples are available today (8,9). It has been proven that PCR-and RFLPassays may distinguish between pathogenic and apathogenic strains of P. zopfii and between these and P. blaschkeae, a putative milk contaminant (22).…”

“…The genus Prototheca encompasses six generally accepted species: P. stagnora, P. ulmea, P. blaschkeae, P. wickerhamii, P. zopfii, and P. cutis sp. nov. (8,12,30). The species P. blaschkeae, P. wickerhamii, P. zopfii, and P. cutis are considered causal agents of local human and/or animal infections (18,21,28,30,31).…”