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.

In vitro resistance of Aspergillus fumigatus to azole farm fungicide.

Azole resistance in Aspergillus fumigatus is mainly due to a point mutation in the 14α-sterol demethylase (CYP51A) gene, which encodes the target of azole fungicides. Moreover, overexpression of CYP51B or multidrug resistance (MDR) gene is supposedly related to the mechanism of azole resistance in A. fumigatus. In this study, we tried to induce resistance to tetraconazole, an azole fungicide, in strains of A. fumigatus from a farm and then investigated mutation and expression of their CYP51A, CYP51B, and multidrug resistance (MDR) genes. Three tetraconazole resistant strains were induced and their minimum inhibitory concentration (MIC) for tetraconazole was 145 mg/L. However, the MICs of itraconazole (ITZ), posaconazole (POS), and voriconazole (VRZ) obtained by an E-test of the three tetraconazole resistant strains were 0.064-0.19 mg/L for ITZ, 0.023-0.32 mg/L for POS, and 0.047-0.064 mg/L for VRZ. No gene mutations were detected in the CYP 51A sequence amplified in these strains. RT-PCR of cyp51A and cyp51B indicated that the tetraconazole resistant strains more highly expressed these genes than the susceptible strain in tetraconazole containing medium.

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.

Rapid molecular typing of Prototheca zopfii by high resolution melting real-time PCR (PCR-HRM).

Prototheca zopfii is an achlorophyllic alga that is ubiquitous around cow sheds. The alga is associated with bovine mastitis, which causes a reduction in milk production and secretion of thin watery milk containing white flakes. Isolates of P. zopfii from bovine mastitis were almost all identified as P. zopfii genotype 2, suggesting that it is the main causative agent of bovine protothecal mastitis. The ability to differentiate between genotype 1 and genotype 2 is therefore very important for preventing bovine mastitis. In this study, high resolution melting real-time PCR (PCR-HRM) analysis of the protothecal 18S rDNA domain successfully differentiated between genotypes of P. zopfii in less than 3 hours, while conventional sequence analysis requires more than 48 hours to differentiate between genotypes. PCR-HRM analysis clustered P. zopfii genotype 1 isolates separately from P. zopfii genotype 2 isolates, indicating that this molecular typing method is an effective tool for rapidly diagnosing bovine protothecal mastitis.

In vitro algaecide effect of disinfectants on Prototheca zopfii genotypes 1 and 2.

Bovine mastitis due to Prototheca zopfii leads to reduced milk production and is difficult to cure. Therefore, prevention is the best approach and this is best achieved through the use of effective disinfectants. The aim of this study was to evaluate the in vitro algaecide efficacy of conventional disinfectants against strains of P. zopfii genotype 1 and 2. The minimal algaecide concentration (MAC) of alkyldiaminoethylglycine hydrochloride, chlorhexidine, dioxide chlorine, povidone iodine and sodium hypochlorous acid against 10 isolates and the type strain (SAG2063(T)) of P. zopfii genotype 1 as well as 10 isolates and the type strain (SAG2021(T)) of P. zopfii genotype 2 were examined using the micro dilution method. This in vitro study indicated that alkyldiaminoethylglycine hydrochloride, chlorhexidine, povidone iodine and sodium hypochlorous acid, but not dioxide chlorine, are effective against both genotypes of P. zopfii.

In vitro susceptibility of Prototheca zopfii genotypes 1 and 2.

Prototheca zopfii causes bovine mastitis that leads to reduced milk production. Since P. zopfii isolates from mastitis have been assigned P. zopfii genotype 2, it suggests that this genotype is the etiologic agent of the infection. However, isolates of P. zopfii have not been investigated with regard to their in vitro drug susceptibility. In this study, we examine the susceptibility of genotype 2 strains from bovine mastitis and genotype 1 isolates recovered from cow-barn surroundings. The in vitro susceptibility of ten isolates and the type strain (SAG2063(T)) of P. zopfii genotype 1, and equal number of genotype 2 isolates and the type strain (SAG2021(T)) were assessed by E-test against amphotericin B (AMB), gentamicin (GM), kanamycin (KM) and itraconazole (ITZ). Results showed that P. zopfii genotype 1 isolates are more susceptible in vitro to AMB, GM and KM than those of genotype 2. Moreover, genotype 2 isolates and seven isolates of genotype 1, including the type strain, are not susceptible to ITZ (>10 µg/ml). Thus, drug susceptibility of P. zopfii differs between these two genotypes.

Experimental infection of bovine mammary gland with prototheca zopfii genotype 1.

Prototheca zopfii is divided into three genotypes, one of which, P. zopfii genotype 2, appears to be the main causative agent of bovine protothecal mastitis. However, the difference in pathogenicity between genotypes 1 and 2 has not been well investigated. In the present study, we experimentally infected normal bovine mammary gland with P. zopfii genotype 1 to investigate its pathogenicity. The mammary gland infected with P. zopfii genotype 1 showed no clinical signs. However, the histopathologic features of the infected mammary gland consisted of interstitial infiltrates of macrophages, plasma cells, lymphocytes, and fibroblasts with neutrophils in acinar lumens. Algae were present in macrophages and free in the alveolar lumens and the interstitium. Histopathology of the resultant tissue samples revealed that genotype 1 also induced a granulomatous lesion in the cow teat, similar to the mastitis lesion due to genotype 2.