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Hiroshi Kamata - One of the best experts on this subject based on the ideXlab platform.
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short communication elisa system for screening of bovine mastitis caused by Prototheca zopfii
Journal of Dairy Science, 2016Co-Authors: Rui Kano, Hideto Sobukawa, Kazuyuki Suzuki, Atsuhiko Hasegawa, Takaaki Ito, Ayano Sato, Yuko Sato, Hiroshi KamataAbstract: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.
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rapid molecular typing of Prototheca zopfii by high resolution melting real time pcr pcr hrm
Medical Mycology Journal, 2013Co-Authors: Hideto Sobukawa, Kazuyuki Suzuki, Hiroshi Kamata, Rui Kano, Takaaki Ito, Masato Ibaraki, Atsuhiko HasegawaAbstract: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.
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short communication molecular typing of Prototheca zopfii from bovine mastitis in japan
Journal of Dairy Science, 2012Co-Authors: Hideto Sobukawa, Masanobu Onozaki, Kazuyuki Suzuki, Atsuhiko Hasegawa, S. Yamaguchi, Hiroshi KamataAbstract:Abstract Prototheca zopfii causes bovine mastitis, resulting in reduced milk production and the secretion of thin watery milk with white flakes. Prototheca zopfii has been biochemically and serologically divided into at least 2 genotypes, P. zopfii genotype 1 and P. zopfii genotype 2. The latter is known to be the main causative agent of bovine Protothecal mastitis. Prototheca zopfii was later reclassified into 5 varieties: var. zopfii (genotypes 1 and 2), var. 1 (formerly Prototheca blaschkeae ), var. 3 (formerly P. moriformis ), and var. portoricensis . In this study, the 18S ribosomal DNA sequences of diverse clinical specimens from different areas in Japan were studied to clarify the pathogenicity of P. zopfii var. zopfii . The phylogenetic tree revealed that all genotype 2 isolates were grouped in a cluster of P. zopfii var. zopfii SAG 2021 T (type strain genotype 2), and were independent from the cluster of the genotype 1 isolates. Thus, all isolates from bovine mastitis in Japan were identified as P. zopfii genotype 2. Therefore, P. zopfii var. zopfii genotype 2 is associated with bovine mastitis.
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In vitro algaecide effect of disinfectants on Prototheca zopfii genotypes 1 and 2.
The Journal of veterinary medical science, 2011Co-Authors: Hideto Sobukawa, Masanobu Onozaki, Atsuhiko Hasegawa, Mitsuhiro Watanabe, Rui Kano, Takaaki Ito, Hiroshi KamataAbstract: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.
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in vitro susceptibility of Prototheca zopfii genotypes 1 and 2
Medical Mycology, 2011Co-Authors: Hideto Sobukawa, Masanobu Onozaki, Atsuhiko Hasegawa, Rui Kano, Koichi Makimura, Hiroshi KamataAbstract: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 (SAG2063T) of P. zopfii genotype 1, and equal number of genotype 2 isolates and the type strain (SAG2021T) 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 μ...
Atsuhiko Hasegawa - One of the best experts on this subject based on the ideXlab platform.
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short communication elisa system for screening of bovine mastitis caused by Prototheca zopfii
Journal of Dairy Science, 2016Co-Authors: Rui Kano, Hideto Sobukawa, Kazuyuki Suzuki, Atsuhiko Hasegawa, Takaaki Ito, Ayano Sato, Yuko Sato, Hiroshi KamataAbstract: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.
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rapid molecular typing of Prototheca zopfii by high resolution melting real time pcr pcr hrm
Medical Mycology Journal, 2013Co-Authors: Hideto Sobukawa, Kazuyuki Suzuki, Hiroshi Kamata, Rui Kano, Takaaki Ito, Masato Ibaraki, Atsuhiko HasegawaAbstract: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.
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detection and identification of genotypes of Prototheca zopfii in clinical samples by quantitative pcr analysis
Japanese Journal of Infectious Diseases, 2013Co-Authors: Masanobu Onozaki, Koichi Makimura, Kazuo Satoh, Atsuhiko HasegawaAbstract:: In this study, a specific quantitative PCR system for the detection and identification of Prototheca zopfii genotypes was developed using a TaqMan(®) MGB probe and ResoLight dye. The P. zopfii-specific primers 18PZF1 and 18PZR1 were generated on the basis of the alignment of the small subunit ribosomal DNA domain base sequences of the genera Chlorella and Prototheca obtained from DDBJ/EMBL/GenBank, and the TaqMan(®) MGB probe PZP1 was designed corresponding to this amplification region. Analysis of the melting curves of the amplicons using ResoLight dye was able to differentiate between P. zopfii genotypes 1 and 2. The specificity of this detection system was examined using strains from a culture collection (28 strains) and clinical isolates (140 strains). The TaqMan(®) MGB probe amplicon was detected only in reference strains of P. zopfii (n = 12) and clinical isolates (n = 135). Ninety-two clinical specimens from cows with mastitis (36 samples) and healthy controls (56 samples) were also tested. All isolates from milk samples (n = 92) and clinical isolates (n = 135) were identified as P. zopfii genotype 2.
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short communication molecular typing of Prototheca zopfii from bovine mastitis in japan
Journal of Dairy Science, 2012Co-Authors: Hideto Sobukawa, Masanobu Onozaki, Kazuyuki Suzuki, Atsuhiko Hasegawa, S. Yamaguchi, Hiroshi KamataAbstract:Abstract Prototheca zopfii causes bovine mastitis, resulting in reduced milk production and the secretion of thin watery milk with white flakes. Prototheca zopfii has been biochemically and serologically divided into at least 2 genotypes, P. zopfii genotype 1 and P. zopfii genotype 2. The latter is known to be the main causative agent of bovine Protothecal mastitis. Prototheca zopfii was later reclassified into 5 varieties: var. zopfii (genotypes 1 and 2), var. 1 (formerly Prototheca blaschkeae ), var. 3 (formerly P. moriformis ), and var. portoricensis . In this study, the 18S ribosomal DNA sequences of diverse clinical specimens from different areas in Japan were studied to clarify the pathogenicity of P. zopfii var. zopfii . The phylogenetic tree revealed that all genotype 2 isolates were grouped in a cluster of P. zopfii var. zopfii SAG 2021 T (type strain genotype 2), and were independent from the cluster of the genotype 1 isolates. Thus, all isolates from bovine mastitis in Japan were identified as P. zopfii genotype 2. Therefore, P. zopfii var. zopfii genotype 2 is associated with bovine mastitis.
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In vitro algaecide effect of disinfectants on Prototheca zopfii genotypes 1 and 2.
The Journal of veterinary medical science, 2011Co-Authors: Hideto Sobukawa, Masanobu Onozaki, Atsuhiko Hasegawa, Mitsuhiro Watanabe, Rui Kano, Takaaki Ito, Hiroshi KamataAbstract: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.
Hideto Sobukawa - One of the best experts on this subject based on the ideXlab platform.
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short communication elisa system for screening of bovine mastitis caused by Prototheca zopfii
Journal of Dairy Science, 2016Co-Authors: Rui Kano, Hideto Sobukawa, Kazuyuki Suzuki, Atsuhiko Hasegawa, Takaaki Ito, Ayano Sato, Yuko Sato, Hiroshi KamataAbstract: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.
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rapid molecular typing of Prototheca zopfii by high resolution melting real time pcr pcr hrm
Medical Mycology Journal, 2013Co-Authors: Hideto Sobukawa, Kazuyuki Suzuki, Hiroshi Kamata, Rui Kano, Takaaki Ito, Masato Ibaraki, Atsuhiko HasegawaAbstract: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.
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short communication molecular typing of Prototheca zopfii from bovine mastitis in japan
Journal of Dairy Science, 2012Co-Authors: Hideto Sobukawa, Masanobu Onozaki, Kazuyuki Suzuki, Atsuhiko Hasegawa, S. Yamaguchi, Hiroshi KamataAbstract:Abstract Prototheca zopfii causes bovine mastitis, resulting in reduced milk production and the secretion of thin watery milk with white flakes. Prototheca zopfii has been biochemically and serologically divided into at least 2 genotypes, P. zopfii genotype 1 and P. zopfii genotype 2. The latter is known to be the main causative agent of bovine Protothecal mastitis. Prototheca zopfii was later reclassified into 5 varieties: var. zopfii (genotypes 1 and 2), var. 1 (formerly Prototheca blaschkeae ), var. 3 (formerly P. moriformis ), and var. portoricensis . In this study, the 18S ribosomal DNA sequences of diverse clinical specimens from different areas in Japan were studied to clarify the pathogenicity of P. zopfii var. zopfii . The phylogenetic tree revealed that all genotype 2 isolates were grouped in a cluster of P. zopfii var. zopfii SAG 2021 T (type strain genotype 2), and were independent from the cluster of the genotype 1 isolates. Thus, all isolates from bovine mastitis in Japan were identified as P. zopfii genotype 2. Therefore, P. zopfii var. zopfii genotype 2 is associated with bovine mastitis.
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In vitro algaecide effect of disinfectants on Prototheca zopfii genotypes 1 and 2.
The Journal of veterinary medical science, 2011Co-Authors: Hideto Sobukawa, Masanobu Onozaki, Atsuhiko Hasegawa, Mitsuhiro Watanabe, Rui Kano, Takaaki Ito, Hiroshi KamataAbstract: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.
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in vitro susceptibility of Prototheca zopfii genotypes 1 and 2
Medical Mycology, 2011Co-Authors: Hideto Sobukawa, Masanobu Onozaki, Atsuhiko Hasegawa, Rui Kano, Koichi Makimura, Hiroshi KamataAbstract: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 (SAG2063T) of P. zopfii genotype 1, and equal number of genotype 2 isolates and the type strain (SAG2021T) 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 μ...
Masanobu Onozaki - One of the best experts on this subject based on the ideXlab platform.
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the first case of human protothecosis caused by Prototheca zopfii in japan
Journal of Infection and Chemotherapy, 2014Co-Authors: Mina Takano, Masanobu Onozaki, Shuichiro Hoshi, Kumiko Nagai, Haruka Ishidaira, Kazuo Satoh, Koichi MakimuraAbstract:Abstract This report describes a fatal case Prototheca zopfii genotype 2 infection in an immunosuppressed patient. The patient was a 62-year-old housewife who presented general malaise in April 2011. Hairy cell leukemia was highly suspected. Chemotherapy was started because the patient developed severe pancytopenia in October 2011. Itraconazole capsules (100 mg/day) and trimethoprim (320 mg/day) plus sulfamethoxazole (1600 mg/day) combinations were orally administered for prophylaxis of fungal infections. Of BacT/ALERT 3D FA aerobic culture bottles and FN anaerobic culture bottles, only FA aerobic blood culture bottles produced positive reactions when the patient developed fever in January 2012. Gram-staining of blood culture bottles revealed Gram-negative elliptical sporangia. Culturing on Sabouraud dextrose agar produced smooth and creamy white, yeast-like colonies. Partial DNA sequences of the nuclear 18S rDNA and 28S rDNA D1/D2 domains of the isolated strain were identical to those of P . zopfii genotype 2. The MICs and minimal lethal concentrations of antifungals revealed that it was susceptible to amphotericin B and itraconazole. The patient died, at which time plasma (1 → 3)-β-D-glucan was positive (131 pg/mL).
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the first case of human protothecosis caused by Prototheca zopfii in japan
Journal of Infection and Chemotherapy, 2014Co-Authors: Mina Takano, Masanobu Onozaki, Shuichiro Hoshi, Kumiko Nagai, Haruka Ishidaira, Kazuo Satoh, Koichi MakimuraAbstract:Abstract This report describes a fatal case Prototheca zopfii genotype 2 infection in an immunosuppressed patient. The patient was a 62-year-old housewife who presented general malaise in April 2011. Hairy cell leukemia was highly suspected. Chemotherapy was started because the patient developed severe pancytopenia in October 2011. Itraconazole capsules (100 mg/day) and trimethoprim (320 mg/day) plus sulfamethoxazole (1600 mg/day) combinations were orally administered for prophylaxis of fungal infections. Of BacT/ALERT 3D FA aerobic culture bottles and FN anaerobic culture bottles, only FA aerobic blood culture bottles produced positive reactions when the patient developed fever in January 2012. Gram-staining of blood culture bottles revealed Gram-negative elliptical sporangia. Culturing on Sabouraud dextrose agar produced smooth and creamy white, yeast-like colonies. Partial DNA sequences of the nuclear 18S rDNA and 28S rDNA D1/D2 domains of the isolated strain were identical to those of P . zopfii genotype 2. The MICs and minimal lethal concentrations of antifungals revealed that it was susceptible to amphotericin B and itraconazole. The patient died, at which time plasma (1 → 3)-β-D-glucan was positive (131 pg/mL).
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detection and identification of genotypes of Prototheca zopfii in clinical samples by quantitative pcr analysis
Japanese Journal of Infectious Diseases, 2013Co-Authors: Masanobu Onozaki, Koichi Makimura, Kazuo Satoh, Atsuhiko HasegawaAbstract:: In this study, a specific quantitative PCR system for the detection and identification of Prototheca zopfii genotypes was developed using a TaqMan(®) MGB probe and ResoLight dye. The P. zopfii-specific primers 18PZF1 and 18PZR1 were generated on the basis of the alignment of the small subunit ribosomal DNA domain base sequences of the genera Chlorella and Prototheca obtained from DDBJ/EMBL/GenBank, and the TaqMan(®) MGB probe PZP1 was designed corresponding to this amplification region. Analysis of the melting curves of the amplicons using ResoLight dye was able to differentiate between P. zopfii genotypes 1 and 2. The specificity of this detection system was examined using strains from a culture collection (28 strains) and clinical isolates (140 strains). The TaqMan(®) MGB probe amplicon was detected only in reference strains of P. zopfii (n = 12) and clinical isolates (n = 135). Ninety-two clinical specimens from cows with mastitis (36 samples) and healthy controls (56 samples) were also tested. All isolates from milk samples (n = 92) and clinical isolates (n = 135) were identified as P. zopfii genotype 2.
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short communication molecular typing of Prototheca zopfii from bovine mastitis in japan
Journal of Dairy Science, 2012Co-Authors: Hideto Sobukawa, Masanobu Onozaki, Kazuyuki Suzuki, Atsuhiko Hasegawa, S. Yamaguchi, Hiroshi KamataAbstract:Abstract Prototheca zopfii causes bovine mastitis, resulting in reduced milk production and the secretion of thin watery milk with white flakes. Prototheca zopfii has been biochemically and serologically divided into at least 2 genotypes, P. zopfii genotype 1 and P. zopfii genotype 2. The latter is known to be the main causative agent of bovine Protothecal mastitis. Prototheca zopfii was later reclassified into 5 varieties: var. zopfii (genotypes 1 and 2), var. 1 (formerly Prototheca blaschkeae ), var. 3 (formerly P. moriformis ), and var. portoricensis . In this study, the 18S ribosomal DNA sequences of diverse clinical specimens from different areas in Japan were studied to clarify the pathogenicity of P. zopfii var. zopfii . The phylogenetic tree revealed that all genotype 2 isolates were grouped in a cluster of P. zopfii var. zopfii SAG 2021 T (type strain genotype 2), and were independent from the cluster of the genotype 1 isolates. Thus, all isolates from bovine mastitis in Japan were identified as P. zopfii genotype 2. Therefore, P. zopfii var. zopfii genotype 2 is associated with bovine mastitis.
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In vitro algaecide effect of disinfectants on Prototheca zopfii genotypes 1 and 2.
The Journal of veterinary medical science, 2011Co-Authors: Hideto Sobukawa, Masanobu Onozaki, Atsuhiko Hasegawa, Mitsuhiro Watanabe, Rui Kano, Takaaki Ito, Hiroshi KamataAbstract: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.
Muhammad Qasim Shahid - One of the best experts on this subject based on the ideXlab platform.
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Prototheca zopfii genotype ii induces mitochondrial apoptosis in models of bovine mastitis
Scientific Reports, 2020Co-Authors: Muhammad Qasim Shahid, H W Barkema, Eduardo R Cobo, Liben Chen, Paloma Araujo Cavalcante, Siyu Xu, Cameron G Knight, John P KastelicAbstract:Prototheca zopfii is an alga increasingly isolated from bovine mastitis. Of the two genotypes of P. zopfii (genotype I and II (GT-I and -II)), P. zopfii GT-II is the genotype associated with acute mastitis and decreased milk production, although its pathogenesis is not well known. The objective was to determine inflammatory and apoptotic roles of P. zopfii GT-II in cultured mammary epithelial cells (from cattle and mice) and murine macrophages and using a murine model of mastitis. Prototheca zopfii GT-II (but not GT-I) invaded bovine and murine mammary epithelial cells (MECs) and induced apoptosis, as determined by the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling assay. This P. zopfii GT-II driven apoptosis corresponded to mitochondrial pathways; mitochondrial transmembrane resistance (ΔΨm) was altered and modulation of mitochondrion-mediated apoptosis regulating genes changed (increased transcriptional Bax, cytochrome-c and Apaf-1 and downregulated Bcl-2), whereas caspase-9 and -3 expression increased. Apoptotic effects by P. zopfii GT-II were more pronounced in macrophages compared to MECs. In a murine mammary infection model, P. zopfii GT-II replicated in the mammary gland and caused severe inflammation with infiltration of macrophages and neutrophils and upregulation of pro-inflammatory genes (TNF-α, IL-1β and Cxcl-1) and also apoptosis of epithelial cells. Thus, we concluded P. zopfii GT-II is a mastitis-causing pathogen that triggers severe inflammation and also mitochondrial apoptosis.
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Prototheca zopfii genotype ii induces mitochondrial apoptosis in models of bovine mastitis
bioRxiv, 2019Co-Authors: Muhammad Qasim Shahid, H W Barkema, Eduardo R Cobo, Liben Chen, Paloma Araujo Cavalcante, Siyu Xu, Cameron G Knight, John P KastelicAbstract:Abstract Prototheca zopfii is an alga increasingly isolated from bovine mastitis. Of the two genotypes of P. zopfii (genotype I and II (GT-I and II)), P. zopfii GT-II is the genotype associated with acute mastitis and decreased milk production by unknown mechanisms. The objective was to determine inflammatory and apoptotic roles of P. zopfii GT-II in cultured mammary epithelial cells (from cattle and mice) and murine macrophages and using a murine model of mastitis. Prototheca zopfii GT-II (but not GT-I) invaded bovine and murine mammary epithelial cells (MECs) and induced apoptosis, as determined by the terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end labeling assay. This P. zopfii GT-II driven apoptosis corresponded to mitochondrial pathways; mitochondrial transmembrane resistance (ΔΨm) was altered and modulation of mitochondrion-mediated apoptosis regulating genes changed (increased transcriptional Bax, cytochrome-c and Apaf-1 and downregulated Bcl-2), whereas caspase-9 and -3 expression increased. Apoptotic effects by P. zopfii GT-II were more pronounced in macrophages compared to MECs. In a murine mammary infection model, P. zopfii GT-II replicated in the mammary gland and caused severe inflammation with infiltration of macrophages and neutrophils and upregulation of pro-inflammatory genes (TNF-α, IL-1β and Cxcl-1) and also apoptosis of epithelial cells. Thus, we concluded P. zopfii GT-II is a mastitis-causing pathogen that triggers severe inflammation and also mitochondrial apoptosis. Author summary Bovine mastitis (inflammation of the udder) reduces milk production and quality, causing huge economic losses in the dairy industry worldwide. Although the alga Prototheca zopfii is a major cause of mastitis in dairy cows, mechanisms by which it damages mammary tissues are not well known. Here, we used cell cultures and a mouse model of mastitis to determine how Prototheca caused inflammation and cell death in mammary tissues. Prototheca invaded mammary gland cells, from cattle and mice, as well as macrophages (white cells that take up and kill pathogens) and caused cell death by interfering with mitochondria. Furthermore, Prototheca causes severe inflammation and tissue damage when injected into the mammary glands of mice. Although there are two genotypes of P. zopfii, only genotype II causes tissue damage, whereas gentotype I, common in farm environments, does not damage mammary tissues. Since P. zopfii is an alga and not a bacterium, antibiotic treatments, frequently used to treat mastitis in cattle, are not effective against this organism. Understanding how P. zopfii damages mammary tissue and causes mastitis is important new knowledge to promote future development of evidence-based approaches to prevent and treat mammary gland infections with this organism.
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Prototheca zopfii isolated from bovine mastitis induced oxidative stress and apoptosis in bovine mammary epithelial cells
Oncotarget, 2017Co-Authors: Muhammad Qasim Shahid, Youtian Deng, Naveed Sabir, Yanan Zhou, Jingliang SuAbstract:// Muhammad Shahid 1 , Jian Gao 1 , Yanan Zhou 1 , Gang Liu 1 , Tariq Ali 1 , Youtian Deng 1 , Naveed Sabir 1 , Jingliang Su 1 , Bo Han 1 1 College of Veterinary Medicine, China Agricultural University, Beijing 100193, P R China Correspondence to: Bo Han, email: hanbo@cau.edu.cn Keywords: bovine mastitis, Prototheca zopfii, bMECs, apoptosis, oxidative stress Received: February 17, 2017 Accepted: March 19, 2017 Published: March 29, 2017 ABSTRACT Bovine Protothecal mastitis results in considerable economic losses worldwide. However, Prototheca zopfii induced morphological alterations and oxidative stress in bovine mammary epithelial cells (bMECs) is not comprehensively studied yet. Therefore, the aim of this current study was to investigate the P. zopfii induced pathomorphological changes, oxidative stress and apoptosis in bMECs. Oxidative stress was assessed by evaluating catalase (CAT), superoxide dismutase (SOD), glutathione peroxidase (GPx), malondialdehyde (MDA) contents and lactate dehydrogenase (LDH) activity, while ROS generation and apoptosis was measured by confocal laser scanning microscopy. The results revealed that infection of P. zopfii genotype II (GTII) significantly changed bMECs morphology, increased apoptotic rate and MDA contents at 12 h ( p < 0.05) and 24 h ( p < 0.01) in comparison with control group, in time-dependent manner. LDH activity and ROS generation was also increased ( p < 0.01) at 12 h and 24 h. However, SOD and CAT contents in bMECs infected with GTII were decreased ( p < 0.05) at 12 h, while GPx ( p < 0.01), SOD ( p < 0.05) and CAT ( p < 0.01) levels were reduced at 24 h. In case of GTI, only CAT and GPx activities were significantly decreased when the duration prolonged to 24 h but lesser than GTII. This suggested that GTII has more devastating pathogenic effects in bMECs, and the findings of this study concluded that GTII induced apoptosis and oxidative stress in bMECs via the imbalance of oxidant and antioxidant defenses as well as the production of intracellular ROS.
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characterization of Prototheca zopfii genotypes isolated from cases of bovine mastitis and cow barns in china
Mycopathologia, 2016Co-Authors: Muhammad Qasim Shahid, Limei Zhang, Shiyao Zhang, Laidi Ding, Zhaoju Deng, Abdur RahmanAbstract:Protothecal mastitis, caused mostly by Prototheca zopfii (P. zopfii), is increasing in dairy herds and is being reported globally. The present study was aimed at studying the epidemiology of mastitis and at molecular characterization of P. zopfii isolates from dairy herds and their surroundings in three provinces of China using microbiological, biochemical and molecular methods, and antibiotic susceptibility tests. Samples from milk (n = 620) of mastitic cows and their barns sources (n = 410) including feces, feed, bedding materials and drinking water were analyzed. Among other pathogens recovered from mastitic milk, 84 (13.5 %) of the isolates were identified as P. zopfii. All of the P. zopfii isolates recovered from milk were recognized as genotype 2, whereas 58 (73.4 %) and 21 (26.6 %) isolates from environmental sources were found to be P. zopfii genotypes 1 and 2, respectively. The isolates were susceptible to some antibiotics and antifungal agents, including amikacin (78.1 %), streptomycin (58.5 %), gentamicin (17.8 %), amphotericin B (68.6 %) and nystatin (64.4 %). Additionally, the two genotypes displayed versatile patterns of susceptibility to different antimicrobials agents. Phylogeny of the genotypes on the basis of 18S SSU rDNA and 28S SSU rDNA was also investigated. The isolates of the two genotypes separated into different clades, and no interrelationship was observed among these as shown by phylogenetic analysis. The genotype 1 isolates from cow barn sources were non-pathogenic and may not present any risk of mastitis. We conclude that P. zopfii genotype 2 might play an important role in bovine mastitis in China.
