In vitro algicidal effect of guanidine on Prototheca zopfii genotype 2 strains isolated from clinical and subclinical bovine mastitis.

Prototheca species have increasingly been reported to be opportunistic pathogens that cause mastitis in dairy herds, and it poses an emergent problem because at present, there are no effective therapies for the treatment of protothecal mastitis. This study investigated the in vitro algicidal effect of guanidine on 75 Prototheca zopfii genotype 2 strains isolated from 75 cases of clinical and subclinical bovine mastitis. All strains were susceptible to guanidine in vitro with minimal algaecide concentrations ranging from 0·001 to 0·035%. Guanidine is known to have a high microbicidal effect and is considered to be a new generation microbicidal compound. It is not toxic to human mucous membranes and conjunctivas at low concentrations and has been used as a disinfectant in swimming pools and as an antiseptic for human wounds. The algicidal action of guanidine at low concentrations indicates that it could be an alternative disinfectant or antiseptic for cleaning of the dairy environment and milking equipment, in pre- and postdipping solutions, in the chemical dry therapy of bovine teats and even in the intramammary therapy of P. zopfii infections. This is the first report of the in vitro algicidal effect of guanidine on P. zopfii strains of animal origin. SIGNIFICANCE AND IMPACT OF THE STUDY: Prototheca zopfii genotype 2 is an opportunistic pathogen of bovine mastitis. To date, no effective therapies against protothecal mastitis have been developed. The in vitro algicidal effect of guanidine on 75 P. zopfii genotype 2 strains isolated from cows revealed that all of the isolates were susceptible to the compound at low concentrations, which indicates that guanidine may be used as an antiseptic/disinfectant for dairy milking equipment, in pre- and postdipping solutions, and as a chemical dry therapy or an intramammary therapy. This study describes the in vitro algicidal effect of guanidine on P. zopfii for the first time.

Molecular typing and differences in biofilm formation and antibiotic susceptibilities among Prototheca strains isolated in Italy and Brazil.

Bovine mastitis caused by Prototheca is a serious and complex problem that accounts for high economic losses in the dairy industry. The main objective of this study was to identify and characterize at genetic level different Prototheca strains and provide the most complete data about protothecal antibiotic resistance. The study involves 46 isolates from Italian (13 strains) and Brazilian (33 strains) mastitic milk. These strains were identified by multiplex PCR and single strand conformation polymorphism analysis and characterized by randomly amplified polymorphic DNA (RAPD)-PCR. Moreover, biofilm production and antibiotic susceptibility were evaluated. Forty-two strains resulted as Prototheca zopfii genotype 2, whereas 4 isolates could belong to a potential new Prototheca species. The RAPD-PCR, performed with 3 primers (M13, OPA-4, and OPA-18), showed a notable heterogeneity among isolates and grouped the strains according to the species and geographical origin. Biofilm production was species-dependent and P. zopfii genotype 2 strains were classified as strong biofilm producers. In vitro antibiotic susceptibility tests indicated that Prototheca strains were susceptible to antibacterial drugs belonging to aminoglycosides group; the highest activity against Prototheca strains was observed in the case of colistin sulfate, gentamicin, and netilmicin (100% of susceptible strains). It is interesting to note that all the Italian P. zopfii genotype 2 strains showed lower minimum inhibitory concentration values than the Brazilian ones. Nisin showed more efficacy than lysozyme and potassium sorbate, inhibiting 31% of the strains. Results obtained in this study confirmed that RAPD-PCR is a rapid, inexpensive, and highly discriminating tool for Prototheca strains characterization and could give a good scientific contribution for better understanding the protothecal mastitis in dairy herd.