Short communication: Occurrence and persistence of Prototheca zopfii in dairy herds of Korea.

Bovine mastitis caused by Prototheca has been reported globally, and its incidence is increasing in dairy herds. The present study aimed to investigate the occurrence of Prototheca and persistence of Prototheca zopfii strains in Korean dairy herds. A total of 187 (7.5%) P. zopfii strains were isolated from 2,508 quarter milk samples collected from 50 dairy farms throughout Korea from 2015 to 2017. Prototheca zopfii was isolated from one farm among the 50 farms over the 3-yr period. The P. zopfii isolates belonged to genotype 2. Overall, Prototheca-positive quarter milk samples showed high somatic cell counts with an average value of log 6.48 ± 6.54 cells/mL. Prototheca zopfii was found to be persistent in an infected farm over a 2-yr period. To the best of our knowledge, this is the first report on the presence and persistence of protothecal mastitis caused by P. zopfii genotype 2 in a Korean dairy herd. This disease leads to a significant increase in somatic cell counts in milk, which persists for more than 1 yr in the affected cow udder. These results suggest that P. zopfii could pose a serious risk to dairy herds. Thus, strict surveillance for protothecal mastitis is urgently needed and sanitary conditions regarding the environment and milk collection are essential because of the lack of effective treatment options.

Molecular Characterization of Prototheca strains isolated in China revealed the first cases of protothecosis associated with Prototheca zopfii genotype 1.

In this study, six strains of Prototheca isolated in China from human patients diagnosed as protothecosis and cows with mastitis were characterized by polymerase chain reaction (PCR) and nucleotide sequencing of the ribosomal RNA gene (rDNA) and by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). The results indicated that three strains isolated from the human patients were P. zopfii genotype 1, revealing the first cases of human protothecosis associated with P. zopfii genotype 1. The remaining three strains were shown to be P. zopfii genotype 2. Interestingly, one strain isolated from the cerebrospinal fluid of the human patient appeared to have both of the genotype 1- and 2-specific alleles in the small subunit (SSU) rDNA although it was classified by MALDI-TOF MS as genotype 2. For genotyping of certain strains of P. zopfii, it may be necessary to comprehensively evaluate the diversity in the SSU rDNA sequences and the MALDI-TOF MS results.

Prototheca zopfii Induced Ultrastructural Features Associated with Apoptosis in Bovine Mammary Epithelial Cells.

Prototheca zopfii infections are becoming global concerns in humans and animals. Bovine protothecal mastitis is characterized by deteriorating milk quality and quantity, thus imparting huge economic losses to dairy industry. Previous published studies mostly focused on the prevalence and characterization of P. zopfii from mastitis. However, the ultrastructural pathomorphological changes associated with apoptosis in bovine mammary epithelial cells (bMECs) are not studied yet. Therefore, in this study we aimed to evaluate the in vitro comparative apoptotic potential of P. zopfii genotype-I and -II on bMECs using flow cytometry, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). The results showed fast growth rate and higher adhesion capability of genotype-II in bMECs as compared with genotype-I. The viability of bMECs infected with P. zopfii genotype-II was significantly decreased after 12 h (p < 0.05) and 24 h (p < 0.01) in comparison with control cells. Contrary, genotype-I couldn't show any significant effects on cell viability. Moreover, after infection of bMECs with genotype-II, the apoptosis increased significantly at 12 h (p < 0.05) and 24 h (p < 0.01) as compared with control group. Genotype-I couldn't display any significant effects on cell apoptosis. The host specificity of P. zopfii was also tested in mouse osteoblast cells, and the results suggest that genotype-I and -II could not cause any significant apoptosis in these cell lines. SEM interpreted the pathomorphological alterations in bMECs after infection. Adhesion of P. zopfii with cells and further disruption of cytomembrane validated the apoptosis caused by genotype-II under SEM. While genotype-1 couldn't cause any significant apoptosis in bMECs. Furthermore, genotype-II induced apoptotic manifested specific ultrastructure features, like cytoplasmic cavitation, swollen mitochondria, pyknosis, cytomembrane disruption, and appearance of apoptotic bodies under TEM. The findings of the current study revealed that genotype-II has the capability to invade and survive within the bMECs, thus imparting significant damages to the mammary cells which result in apoptosis. This study represents the first insights into the pathomorphological and ultrastructure features of apoptosis in bMECs induced by P. zopfii genotype-II.

Occurrence of Prototheca spp. in cow milk samples.

Protothecosis is a potential zoonotic disease associated with bovine mastitis which can be transmitted to humans through contaminated milk. Considering the increasing prevalence of bovine mastitis due to Prototheca species, individual cow milk samples were analyzed using microbiological examination and biomolecular assay. Aspects related to health requirements for milk production, clinical and histological bovine mastitis were also described. The results showed 24/257 (9.3%) culture-positive samples and 42/257 (16.3%) PCR-positive samples. Moreover in 5 cows with somatic cell count over 106/mL presented histological features of mastitis. This study reveals that the presence of Prototheca species in dairy herds was related to the hygienic conditions of the milking equipment, showing an emerging public health issue.

Characterization of Prototheca zopfii associated with outbreak of bovine clinical mastitis in herd of Beijing, China.

Prototheca zopfii (P. zopfii) has become an important cause of bovine mastitis in many countries. In the present study, to better understand the occurrence of one clinical mastitis (CM) outbreak due to P. zopfii, the molecular characterization and resistance patterns of the microalga were described. P. zopfii strains were isolated from 17 of 23 quarters, which suffered CM in the outbreak, and 7 of 46 CM recovered quarters before the outbreak, as well as 2 of 75 environmental samples in the dairy farm. All strains were identified as genotype 2 by genotype-specific PCR analysis. Results of in vitro antimicrobial and antifungal susceptibility tests indicated that these strains were resistant to majority of tested drugs, with the only exception of amphotericin B, nystatin, streptomycin, gentamicin, and amikacin. This is the first report about CM outbreak caused by P. zopfii in China. These data suggest that P. zopfii may represent a serious risk in the studied herd, and this microalga could be an important potential pathogen causing mastitis in dairy herds of Beijing.

[Genotypical differentiation of Prototheca isolates of milk samples from mastitis affected cattle in Germany]. / Genotypische Differenzierung von Prototheca-Isolaten aus Milchproben mastitiskranker Rinder in Deutschland.

To elucidate the spectra of Prototheca species and P. zopfii genotypes in milk samples from mastitis affected cattle, 200 Prototheca-isolates of 57 German dairy cattle barns were analyzed by species- and genotype- specific PCR and RFLP analysis. The investigations showed that 177 (88.5%) of all isolates represented P. zopfii genotype 2, 21 (10.5%) isolates represented P. blaschkeae and two (1.0%) P. zopfii genotype 1. Therefore mainly P. zopfii genotype 2 but also P. blaschkeae act in the epidemiology of mastitis in cattle, whereas the role of P. blaschkeae in development of bovine protothecal mastitis should be further explored in epidemiological studies. In this study P. blaschkeae was demonstrated for the first time in association with bovine mastitis in Germany.

Host finding and feeding in Hydrotaea irritans (Diptera, Muscidae): the role of chemical senses.

Hydrotaea irritans is commonly considered to be the primary vector for the bacteria which cause summer mastitis in cattle. A behavioural physiology approach was used to investigate potential host odours (kairomones) that may be used by the fly in finding its host and to determine which substrates or sites on the host may be utilised in feeding. Attractant odours include CO2 and butyric, propionic and acetic acids: the latter three are also produced by the bacteria causing summer mastitis. When milk, slaver, nasal secretion, mastitis secretion and blood were offered to flies as feeding substrates only the last three produced significant increases in feeding duration in comparison to controls offered distilled water.