# Molecular and Phenotypic Characterization of Prototheca Species Isolates Associated with Bovine Mastitis Cases in Chile

## TL;DR

This study characterizes Prototheca species causing mastitis in Chilean dairy cows, revealing high genetic diversity and antibiotic resistance patterns.

## Contribution

The first comprehensive study of Prototheca in Chile and the third globally to assess P. bovis genetic diversity.

## Key findings

- 65 out of 66 isolates were identified as P. bovis, and one as P. ciferrii, the first report of the latter in bovine mastitis in Chile.
- All isolates showed resistance to most antibiotics except aminoglycosides, with gentamicin showing potential as a treatment.
- P. bovis isolates exhibited high genetic diversity and moderate biofilm formation, which may aid infection persistence.

## Abstract

Bovine mastitis is a significant disease impacting dairy cows worldwide, primarily due to the substantial economic losses it causes. Among the pathogens involved, Prototheca spp. has gained increasing attention due to the rising number of protothecal mastitis cases and the associated treatment challenges, often resulting in the culling of affected animals. In Chile, only two outdated studies from 2011 have reported protothecal infections in cattle, underscoring the need for updated data to understand the epidemiology of this infection. This study aimed to characterize Prototheca isolates from Chilean dairy cows through molecular identification at the species level and assessment of their genetic diversity, antimicrobial susceptibility, and biofilm formation capacity. A total of 66 Prototheca spp. isolates were analyzed, with 65 identified as P. bovis and one as P. ciferrii. Genetic analysis revealed 18 distinct P. bovis genotypes without clustering by farm origin, indicating high genetic heterogeneity. All isolates exhibited resistance to most antibiotics tested except aminoglycosides. Additionally, the isolates demonstrated weak to moderate biofilm production, which may contribute to the persistence of infections. This work represents the first comprehensive study of Prototheca in Chile and the third worldwide to assess P. bovis genetic diversity, providing valuable insights for the development of alternative therapeutic strategies.

Background: Bovine mastitis caused by Prototheca spp. is the most significant animal disease of algal origin, with an increasing number of cases reported worldwide. Currently, there is no effective treatment, so control requires the culling of infected animals. In Chile, information is limited, and a discrepancy remains in the literature regarding the Prototheca species involved in bovine mastitis. Methods: This study aimed to molecularly type and phenotypically characterize Prototheca isolates associated with bovine mastitis in Chile. Sixty-six Prototheca isolates obtained from individual bovine mastitis milk samples and bulk tank milk samples were analyzed through cytochrome b gene (cytb) sequencing, Random Amplified Polymorphic DNA–Polymerase Chain Reaction (RAPD-PCR) analysis, and phenotypic evaluation (morphology, antimicrobial susceptibility, and biofilm formation). Results: Sixty-five isolates were identified as P. bovis and one as P. ciferrii, marking the first report of the latter in bovine mastitis in Chile. RAPD analysis revealed a high genetic diversity in P. bovis. All strains exhibited resistance to the antibiotics tested from the Fluoroquinolone, β-lactam, and sulfonamide groups; however, 100% of the strains showed susceptibility to aminoglycosides, with gentamicin standing out as a potential therapeutic option. Most P. bovis strains formed weak (81.5%, 53/65) or moderate (15.4%, 10/65) biofilms, which could favor the persistence of infection. Conclusions: These findings provide novel insights into the molecular and phenotypic characteristics of Prototheca spp. in Chile, highlighting the predominance of P. bovis, the emergence of P. ciferri, and the implications for antimicrobial management and disease control.