Prototheca Zopfii Genotype Research Articles

Herd characteristics and cow-level factors associated with Prototheca mastitis on dairy farms in Ontario, Canada

Prototheca spp. are algae that cause incurable acute or chronic mastitis in dairy cows. The aim of this case-control study was the identification of cow- and herd-level risk factors for this unusual mastitis pathogen. Aseptically collected composite milk samples from 2,428 milking cows in 23 case and 23 control herds were collected between January and May 2011. A questionnaire was administered to the producers, and cow-level production and demographic data were gathered. In 58 of 64 isolates, Prototheca spp. and Prototheca zopfii genotypes were differentiated using PCR and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. All isolates were identified as Prototheca zopfii genotype 2. The mean within-herd prevalence for Prototheca spp. was 5.1% (range 0.0–12.5%). Case herds had a significantly lower herd-level prevalence of Staphylococcus aureus and a higher prevalence of yeasts than did control herds. The final logistic regression model for herd-level risk factors included use of intramammary injections of a non-intramammary drug [odds ratio (OR) = 136.8], the number of different injectable antibiotic products being used (OR = 2.82), the use of any dry cow teat sealant (external OR = 80.0; internal OR = 34.2), and having treated 3 or more displaced abomasums in the last 12 mo OR = 44.7). The final logistic regression model for cow-level risk factors included second or greater lactation (OR = 4.40) and the logarithm of the lactation-average somatic cell count (OR = 2.99). Unsanitary or repeated intramammary infusions, antibiotic treatment, and off-label use of injectable drugs in the udder might promote Prototheca udder infection.

Disseminated protothecosis diagnosed by evaluation of CSF in a dog

A 5-year-old female spayed Shetland Sheepdog Mix dog was evaluated for a history of recent seizure activity, progressive hind limb ataxia, polyuria, and polydipsia and no history of gastrointestinal signs. Physical examination findings included conscious proprioceptive deficits, ataxia, and anterior uveitis along with a hypermature cataract in the right eye. Results of a CBC, serum biochemical profile, urinalysis, and computed tomography scan of the brain were unremarkable. Cerebrospinal fluid (CSF) analysis revealed marked eosinophilic pleocytosis and rare organisms consistent with Prototheca spp within neutrophils and macrophages. On postmortem histologic examination, mononuclear inflammation and numerous intralesional algal organisms, similar to those seen on the cytologic preparation of CSF, were found in the brain, eyes, kidneys, and heart. Abnormalities were not detected on gross and histologic examination of the gastrointestinal tract. Cultures of CSF and subdural/olfactory bulb, but not intestinal tract, yielded growth of Prototheca spp, and PCR analysis and DNA sequencing confirmed the organism as Prototheca zopfii genotype 2. We have reported a rare case of disseminated protothecosis that was diagnosed by evaluation of CSF in a dog presented with neurologic signs and no overt enteric disease. Protothecosis should be considered as a rare cause of seizures, even in the absence of obvious enteric signs, and should be included in the differential diagnosis of eosinophilic pleocytosis.

Epidemiological analysis of worldwide bovine, canine and human clinicalProtothecaisolates by PCR genotyping and MALDI-TOF mass spectrometry proteomic phenotyping

This study presents information on the phenotypic and genotypic characterization of clinical Prototheca spp. isolates obtained from different geographic regions. Of 350 isolates studied, 342 came from cattle, six from canines and two from humans. Phenotypic characterization was carried out by a matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) proteomic analysis. The peptide extraction that was used for this analysis included the additional steps of washing and sonication to increase the yield of peptide. Genotypic analysis was conducted using species- and genotype-specific primers. The study revealed that among the cattle isolates, 310 (90.6%) belonged to Prototheca zopfii genotype 2, 30 (8.8%) to P. blaschkeae, and two (0.6%) to P. zopfii genotype 1. P. zopfii genotype 2 is the principal etiological agent of protothecal mastitis in cattle regardless of the geographic region. Similarly, all canine and human isolates also belonged to the P. zopfii genotype 2, suggesting that this is probably the most virulent species of the genus. The role of P. blaschkeae needs further epidemiologic studies to ascertain its etiologic role in bovine mastitis. To the best of our knowledge, this is the first comprehensive study on phenotypic and genotypic characterization of P. zopfii and P. blaschkeae isolates originating from diverse clinical specimens from different countries.

A novel DNA extraction and duplex polymerase chain reaction assay for the rapid detection of Prototheca zopfii genotype 2 in milk

To develop a PCR-based assay to detect Prototheca zopfii (P. zopfii) and its mastitis-related subtype (genotype 2) directly from milk samples. The DNA extraction method herein is based on the lysing properties of chemical agents, mechanical grinding and DNA-binding properties of silica particles; this method was developed to rapidly extract DNA directly from P. zopfii in bovine milk. Two pairs of primers specific for P. zopfii and genotype 2 were used in the duplex PCR, and a sensitivity test showed that the detection level was 5 × 10(2) colony-forming units (CFU) ml(-1) for P. zopfii and 5 × 10(3) CFU ml(-1) for genotype 2. Furthermore, a practical survey of 23 milk samples showed that the assay produced results that were in accordance with those obtained by the conventional microbiology method. The DNA extraction method is effective in isolating sufficient quantities of DNA from P. zopfii in milk for PCR analysis. The PCR assay is economical, sensitive and more rapid than the conventional culture method. The assay could be used as an alternative method for the rapid the detection of bovine mastitis resulting from P. zopfii genotype 2.

Molecular characterization of Prototheca strains isolated from Italian dairy herds

One hundred sixty-one Prototheca spp. strains isolated from composite milk and barn-surrounding environmental samples (bedding, feces, drinking, or washing water, surface swabs) of 24 Italian dairy herds were characterized by genotype-specific PCR analysis. Overall, 97.2% of strains isolated from composite milk samples were characterized as Prototheca zopfii genotype 2, confirming its role as the main mastitis pathogen, whereas Prototheca blaschkeae was only sporadically isolated (2.8%). Regarding environmental sampling, 84.9% of isolates belonged to P. zopfii genotype 2, 13.2% to P. blaschkeae, and 1.9% to P. zopfii genotype 1. The data herein contradict previous hypotheses about the supposed exclusive role of P. zopfii genotype 2 as the causative agent of protothecal mastitis and, on the contrary, confirm the hypothesis that such pathology could be caused by P. blaschkeae in a few instances.

Prototheca zopfii genotypes isolated from cow barns and bovine mastitis in Japan

This study is the first investigation on Japanese isolates of Prototheca zopfii from bovine mastitis and the cow-barn surroundings by molecular characterization to clarify routes of infection for bovine protothecal mastitis. We performed isolation of Prototheca from cow-barn surroundings (drinking water, sewage and feces) and milk samples from cases of bovine mastitis. Genotypes of the 32 isolates of P. zopfii from cow-barn surroundings and 67 isolates from mastitis were analyzed by genotype-specific PCR assays and restriction fragment length polymorphism (RFLP) assays. All mastitis isolates were identified as P. zopfii genotype 2. Conversely, 29 isolates from cow-barn surroundings were identified as P. zopfii genotypes 1 and 3 isolates as genotype 2, respectively. Given these results, both genotypes of P. zopfii could exist in cow-barn surroundings, but no sites were identified as frequent sources of P. zopfii genotype 2. P. zopfii isolates should thus be further explored with regard to genotype to clarify the reservoir of etiological agents in bovine Prototheca mastitis.