An Investigation of the Innate Immune Response in Bovine Mammary Epithelial Cells Challenged by Prototheca zopfii.

Prototheca zopfii is an important bovine mastitis pathogen, which could result in severe mammary infection. However, the innate immune response in bovine mastitis associated with P. zopfii was not clear. Therefore, the aim of this study is to investigate in vitro innate immune responses implicated by P. zopfii. Bovine mammary epithelial cells (bMECs) were infected with 5.0 × 104 cells/ml P. zopfii genotypes I and II independently, and the mRNA expression of TLR-2, TLR-4, TNF-α, IL-1ß, IL-8, NOD-1, NOD-2 and ß-defensin-5 was determined by real-time polymerase chain reaction (RT-PCR) over a time course of 1, 3, 6, 12 and 24 h. The detection of the NF-κB p65 protein in nucleus and cytoplasm of infected bMECs over the same time course was evaluated. Results showed that P. zopfii genotype II has ability to up-regulate the expression of TLR-2, TLR-4, TNF-α, IL-1ß, IL-8, NOD-1, NOD-2 and ß-defensin-5 'more strongly than genotype I. Western blot results showed that when bMECs were challenged by P. zopfii genotype II, the expression of NF-κB p65 protein in the nucleus was up-regulated, while in cytoplasm it appeared to be repressed, which indicated that bMECs partly regulate the innate immune responses and inflammation by the NF-κB signaling pathway while being infected by P. zopfii genotype II. It was concluded that adhesion of genotype II was stronger than genotype I, and therefore the genotype II regulatory ability is more robust than that of the genotype I, which causes inflammation of bovine mammary tissue.

Short communication: ELISA system for screening of bovine mastitis caused by Prototheca zopfii.

Prototheca zopfii is an achlorophyllic alga that causes bovine mastitis, resulting in a reduction in milk production and the secretion of thin, watery milk with white flakes. This study evaluated the use of an ELISA system for distinguishing cows with mastitis due to P. zopfii genotype 2 from healthy cows and cows with chronic candidal mastitis. We also investigated the transitional changes of specific antibody titers in healthy cows injected with inactivated P. zopfii genotype 2 cells. The ELISA system exhibited the highest sensitivity (94%) and specificity (100%) for chronic protothecal mastitis when the positive cutoff value was set at 43.4 ELISA units. Anti-protothecal IgG titers were positive in all cows after they were inoculated with inactivated P. zopfii genotype 2 cells. These results indicated that ELISA detection of anti-protothecal IgG in serum provided specificity and sensitivity sufficient for diagnosing protothecal mastitis. Thus, an ELISA system incorporating this specific antiserum is expected to be valuable for definitive field-based diagnosis of bovine mastitis due to P. zopfii genotype 2.

Well-known surface and extracellular antigens of pathogenic microorganisms among the immunodominant proteins of the infectious microalgae Prototheca zopfii.

Microalgae of the genus Prototheca (P.) are associated with rare but severe infections (protothecosis) and represent a potential zoonotic risk. Genotype (GT) 2 of P. zopfii has been established as pathogenic agent for humans, dogs, and cattle, whereas GT1 is considered to be non-pathogenic. Since pathogenesis is poorly understood, the aim of this study was to determine immunogenic proteins and potential virulence factors of P. zopfii GT2. Therefore, 2D western blot analyses with sera and isolates of two dogs naturally infected with P. zopfii GT2 have been performed. Cross-reactivity was determined by including the type strains of P. zopfii GT2, P. zopfii GT1, and P. blaschkeae, a close relative of P. zopfii, which is known to cause subclinical forms of bovine mastitis. The sera showed a high strain-, genotype-, and species-cross-reactivity. A total of 198 immunogenic proteins have been analyzed via MALDI-TOF MS. The majority of the 86 identified proteins are intracellularly located (e.g., malate dehydrogenase, oxidoreductase, 3-dehydroquinate synthase) but some antigens and potential virulence factors, known from other pathogens, have been found (e.g., phosphomannomutase, triosephosphate isomerase). One genotype-specific antigen could be identified as heat shock protein 70 (Hsp70), a well-known antigen of eukaryotic pathogens with immunological importance when located extracellularly. Both sera were reactive to glyceraldehyde-3-phosphate-dehydrogenase of all investigated strains. This house-keeping enzyme is found to be located on the surface of several pathogens as virulence factor. Flow-cytometric analysis revealed its presence on the surface of P. blaschkeae.

Immunohistopathology of Prototheca wickerhamii in cutaneous lesions of protothecosis.

Protothecosis is a rare infection caused by pathogenic algae of the genus Prototheca. Prototheca wickerhamii causes cutaneous/subcutaneous opportunistic infections in humans and small animals. The diagnosis of protothecosis is based on histopathological examination of this organism, which can be confused with other fungi and inflammatory cells in infected tissues. In this study, immunohistopathological investigation was made of infected cutaneous human and animal tissues exhibiting protothecosis using rabbit antiserum against P. wickerhamii. Serum detected P. wickerhamii in human and feline protothecosis tissues, and did not react with Candida albicans in the human kidney tissues showing candidiasis. This antiserum can therefore differentiate P. wickerhamii cells from the yeast-like cells of C. albicans and Prototheca zopfii in target tissues.

Galactomannan enzymatic immunoassay cross-reactivity caused by Prototheca species.

We report a reactive Aspergillus galactomannan enzymatic immunoassay against the serum of a patient with invasive Prototheca zopfii infection. Analysis of the supernatants of suspensions of P. zopfii and other Prototheca isolates revealed positive results as well. These data suggest cross-reactivity with the serum Aspergillus galactomannan assay in invasive protothecosis.

Effect of Prototheca zopfii on neutrophil function from bovine milk.

This study was carried to investigate neutrophil function in the presence of Prototheca zopfii. For this purpose, bovine milk neutrophils were incubated in the absence (control) of and presence of P. zopfii, and then they were examined hydrogen peroxide (H(2)O(2)) production, antioxidant enzyme activities, and phagocytic capacity. Milk was collected from negative "California Mastitis Test" (CMT) quarter from three lactating Holstein cows after induction of leukocytosis with an intramammary infusion of oyster glycogen. H(2)O(2) production was measured using the phenol red method. Catalase activity was measured following H(2)O(2) reduction at 240 nm and the activity of glutathione reductase was determined by measuring the rate of NADPH oxidation at 340 nm. P. zopfii death was assessed by fluorescent microscopy using acridine orange assay and by colony forming units (CFUs). Comparisons between the groups were initially performed by analysis of variance (ANOVA). Significant differences were then compared using Tukey's test with a significance coefficient of 0.05. Hydrogen peroxide production, catalase and glutathione reductase activities by neutrophils incubated in presence of P. zopfii were stimulated five times, 21% and 27% respectively, compared to the unstimulated-neutrophils. Neutrophils did not affect P. zopfii death as shown by microscopy and CFUs. These observations led to the conclusion that the P. zopfii promote a high increase of H(2)O(2) production by neutrophils from bovine milk during algae exposition accompanied by increase of antioxidant enzyme activities; however, this process did not affect P. zopfii death.

[Eradication of Prototheca zopfii infection in a dairy cattle herd]. / Sanierung eines Prototheca zopfii-infizierten Milchviehbestandes.

Protothecosis is a severe form of mastitis in dairy cows caused by colorless algae of the genus Prototheca. Since P. zopfii is highly resistant to all known chemotherapeutics, infected cows must be removed from the herd. Eradication measures are difficult since many chronically infected cows may become intermittent shedders. Therefore, cultural methods are insufficient for control measures. In order to eradicate Prototheca zopfii-mastitis in dairy cattle herds, two isotype specific indirect ELISA for detection of IgA and IgG1 in whey were used in a dairy herd highly affected with protothecal mastitis. All cows (n = 313) were tested four times in intervals of six months. Milk specimens were examined in parallel by cultivation and serologically using two indirect ELISA systems for specific IgA and IgG1 in whey. Cows tested Prototheca positive were consequently separated from the herd and slaughtered. At the first examination, 15.6% of the animals were found positive by culture, and 23.3% were positive in at least one of the ELISA systems. Within two years, protothecal prevalence and incidence decreased to zero indicating that the eradication strategy used was successful. In summary, serological identification of P. zopfii-infected lactating cows is an useful tool to eradicate protothecal bovine mastitis in infected herds.

Emended phenotypic characterization of Prototheca zopfii: a proposal for three biotypes and standards for their identification.

A representative selection of Prototheca zopfii strains isolated from different environmental habitats or clinical cases was characterized in a polyphasic approach in order to assess their intraspecies taxonomic position. Recently, the recognition of distinct phenotypic clusters has been reported as the assignment of 'variants'. In the present study, 11 strains were compared by a number of phenotypic and genetic criteria, including growth characteristics, biochemical reactions and serotyping results. Based on emended standards for biotype identification, P. zopfii strains showed auxanographic differences and distinct assimilation patterns with respect to utilization of amino acids and glycerol. Serotyping by means of immunoblotting revealed that all isolates of variant II obtained from clinical cases, i.e. isolates from bovine mastitis or from human enteropathia, showed specific antigen patterns. They were found to be different from strains assigned to the other two variants with respect to their immunogenic antigens. Furthermore, comparison of partial 18S rDNA sequences confirmed distinct differences between the former variants. Based on these results, it is proposed that P. zopfii merits classification as a species comprising three biotypes.

Longitudinal analysis of Prototheca zopfii-specific immune responses: correlation with disease progression and carriage in dairy cows.

In order to characterize the humoral and cellular immune responses to bovine mammary protothecosis, serum and whey samples obtained from 72 dairy cows assigned to four different clinical stages of infection were examined for specific antibodies by indirect enzyme-linked immunosorbent assay techniques. Milk samples were analyzed for the total numbers of excreted algal cells and somatic cells. After characterization of the course of immune induction in bovine protothecal mastitis, a long-term sentinel study was performed in an affected herd in order to investigate disease progression. A total of 61 dairy cows with protothecal mastitis were examined for shedding of algae cells and for local immune responses three times in 6-month intervals. During acute and chronic stages of protothecosis, significantly elevated specific antibody activities in sera were detected. A strong correlation of whey immunoglobulin A (IgA) and whey IgG1 antibody activity with the total counts of somatic cells in milk was observed, whereas only a weak correlation of whey IgA and whey IgG1 concentrations to the number of algal cells excreted with the milk was seen. Our results from the sentinel long-term study of infected cows revealed that 70.5% of the persistently infected animals were continuously shedding the pathogen. About 4.9% of the animals showed an intermittent shedding, whereas 18% of the cows were tested culturally negative throughout the study. It can be assumed that Prototheca zopfii mastitis in dairy cows is maintained on the herd level by subclinically infected alga-shedding cows.

Serologic survey of rotavirus, Norwalk agent and Prototheca wickerhamii in wastewater workers.

Analysis of paired sera from 48 wastewater workers and controls who reported gastrointestinal illness did not reveal any excess of seroconversions to Norwalk agent or to rotavirus. Inexperienced wastewater-exposed workers had higher levels of antibody to Norwalk agent than did experienced and control workers and those with high and medium aerosol exposure had higher titres than those in the low aerosol category. Analysis for Prototheca antibody titres was essentially negative.

Phagocytosis and algicidal activity of human polymorphonuclear neutrophils against Prototheca wickerhamii.

Protothecosis is an uncommon infection due to Prototheca wickerhamii. Recently, a patient with progressive cutaneous protothecosis and an apparent defect in the ability of her polymorphonuclear neutrophils (PMNs) to kill P. wickerhamii was successfully treated. Therefore, the role of PMNs and serum opsonins in host defense against tissue invasion by this organism was evaluated. The results show that human PMNs ingested and kill P. wickerhamii. Ultrastructural studies revealed digestion of the organism by PMNs 60 min after ingestion. Optimal phagocytosis and killing of P. wickerhamii by PMNs required the presence of both specific IgG antibody and heat-labile opsonins.

Disseminated protothecosis with central nervous system involvement in a dog.

A 4-year-old mixed-breed spayed bitch was euthanatized after 2 1/2 months of progressive neurologic disease. Prototheca organisms were identified by histopathology, culture, and electron microscopy. Specific fluorescent antibody procedures revealed two species--Prototheca wickerhamii and Prototheca zopfii. Organisms and pyogranulomatous lesions were found in the brain, spinal cord, right eye, kidneys, and heart.