Felipe Masiero Salvarani | UFPA - Federal University of Pará (original) (raw)
Papers by Felipe Masiero Salvarani
Arquivo Brasileiro De Medicina Veterinaria E Zootecnia, 2008
Multiplex PCR was optimized to detect Clostridium chauvoei and Clostridium septicum in pure cultu... more Multiplex PCR was optimized to detect Clostridium chauvoei and Clostridium septicum in pure cultures. In each reaction, a pair of primers for a specific segment of the flagellin gene of C. chauvoei and a pair of primers for a specific segment of alpha toxin gene of C. septicum were employed. Reference strains of both microorganisms were used as control. The multiplex PCR was evaluated by testing 16 clinical isolates of C. chauvoei from ruminants, 15 clinical isolates of C. septicum from ruminants and, four vaccine strains of each one of these agents. Reference strains of both microorganisms were used as control. To evaluate the specificity, genomic DNA of the following microorganisms was used: C. sordellii, C. novyi type A, C. novyi type B, C. perfringens type A, C. haemolyticum, C. botulinum type D, Pseudomonas aeruginosa, Staphylococcus aureus, Enterobacter aerogenes, Escherichia coli, and Salmonella typhimurium. All the isolates and vaccine strains of C. chauvoei and C. septicum were positive by PCR assay and cross reactions were not observed with the other species of clostridia, the other bacterial species or amongst both investigated agents. The smallest concentrations of DNA detected from C. chauvoei and C. septicum were 45pg/µl and 30pg/µl, respectively. The multiplex PCR was useful for the specific identification of C. chauvoei and C. septicum in pure cultures.
Ciencia Rural, 2009
Clostridium perfringens type D is the aetiological agent of enterotoxemia in ruminants. The disea... more Clostridium perfringens type D is the aetiological agent of enterotoxemia in ruminants. The disease is caused by epsilon toxin characterized by cardiac, pulmonary, kidney and brain edema. Monoclonal antibodies were produced by using myeloma cell line P3-X63-Ag8 653 fused with spleen cells from Balb/c mice, immunized with epsilon toxoid of C. perfringens type D. Six hybrids were established secreting monoclonal antibodies of the IgM class and IgG3 subclass.
... Felipe Masiero Salvarani I ; Flávia Ferreira Pinto I ; Francisco Carlos Faria Lobato I ; Ronn... more ... Felipe Masiero Salvarani I ; Flávia Ferreira Pinto I ; Francisco Carlos Faria Lobato I ; Ronnie Antunes de Assis II ; Luciana Aramuni Gonçalves I ; Nelson Éder Martins I ; Inácio José Clementino III ; Adrienny Trindade Reis Costa IV. ... [ Links ]. 17 PRIETO, C. et al. ...
Ciencia Rural, 2008
Botulism is a poisoning caused by the ingestion of toxins produced by Clostridium botulinum, that... more Botulism is a poisoning caused by the ingestion of toxins produced by Clostridium botulinum, that infects mammals and birds. This article reports an outbreak of botulism in two different flocks of ruminants at Paraíba, Brazil. In one, 85 out of 88 (96.6%) cattles died. In the other, 145 (96.7%) sheeps, 233 (57.8%) goats and 30 (96.8%) cattles died. Clinical signs were progressive paralysis, difficulties in moving, sialorrhoe and dyspnoe. Death occurred 24 to 48 hours after the beginning of clinic signs and at post-mortem examination no noteable changes were observed. Type C and D toxins were demonstrated in the intestinal contents and poultry litter by neutralization test in mice.
Arquivo Brasileiro De Medicina Veterinaria E Zootecnia, 2006
This report describes a case of bovine enterotoxaemia in Morro da Garça, Minas Gerais, Brazil. Cl... more This report describes a case of bovine enterotoxaemia in Morro da Garça, Minas Gerais, Brazil. Clostridium perfringens type D was isolated in pure culture and was characterized by biochemical reactions and PCR. By the mouse neutralization test, the presence of epsilon toxin from intestinal content was detected.
Brazilian Journal of Microbiology, 2009
Clostridium perfringens is a normal inhabitant of the intestinal tract of chickens as well as a p... more Clostridium perfringens is a normal inhabitant of the intestinal tract of chickens as well as a potential pathogen that causes necrotic enteritis and colangio hepatitis. The minimum inhibitory concentration (MIC) of seven different compounds used for therapy, growth promotion or prevention of coccidiosis was determined by agar dilution method for 55 C. perfringens strains isolated from the intestines of broiler chickens. All strains showed high susceptibility to penicillin, avilamycin, monensin and narasin. Only 7.3% of the strains showed an intermediated sensitivity to lincomycin, and 49 (89.1%) were considered susceptible. For tetracycline and bacitracin, 41.8% and 47.3% of strains, respectively, were considered resistant.
Ciencia Rural, 2009
Botulism is an intoxication caused by the ingestion of toxins produced by Clostridium botulinum. ... more Botulism is an intoxication caused by the ingestion of toxins produced by Clostridium botulinum. It affects mammals and birds, and is characterized by flaccid paralysis of the limbs. This report describes an outbreak of botulism in turkeys of various ages in the city of Santa Luzia, state of Minas Gerais, Brazil. The animals showed incoordination followed by flaccid paralysis involving the muscles of the legs, wings and neck. Within 24 hours, all 29 (100%) turkeys died. The post-mortem examination revealed the presence of fly larvae in the crop and the C. botulinum type C toxin was demonstrated in the sera of two affected animals by serum neutralization test.
Ciencia Rural, 2008
Botulism is an intoxication caused by the ingestion of toxins produced by Clostridium botulinum, ... more Botulism is an intoxication caused by the ingestion of toxins produced by Clostridium botulinum, that affects mammals and birds, characterized by a flaceid paralysis. This report describes a case of botulism in a goose in Santa Luzia, Minas Gerais State, Brazil. Clinical examinations showed dropping feathers and flaccid paralysis involving the muscles of the wings, legs and neck. post-mortem examination showed no significant gross or macroscopic lesions C. botulinum type C toxin was demonstrated in the serum of the affected animal through serum neutralization test in mice.
Ciencia Rural, 2010
Enterotoxemia (also called pulpy kidney disease) is an enteric disease, that affect ruminants, pr... more Enterotoxemia (also called pulpy kidney disease) is an enteric disease, that affect ruminants, produced by epsilon toxin from Clostridium perfringens type D, an anaerobic commonly isolated from soil and feces of healthy animals. The diagnostic is based on detection of this exotoxin in the intestinal content by soroneutralization in mice. Therefore, this study aimed to standardize a test for detection and titration of the toxin in vitro, and compare it with the phenomenon in vivo. A volume of epsilon toxin was titrated in mice and in some cell lines. After concluding the most sensitive cell line, were held in vitro titrations of dilutions from a toxin wich one had in vivo titer known. The results were grouped and a mathematical equation was developed. MDCK cell line showed that the phenomenon observed in vitro can be expressed by a mathematical equation wich shows a correlation of 98.33% with a minimum lethal dose determined in vivo. Therefore, the soroneutralization using MDCK allows a specific, sensitive, practical, fast, and doesn't requere use of animal titration of epsilon toxin.
Arquivo Brasileiro De Medicina Veterinaria E Zootecnia, 2007
Descreve-se a aplicabilidade de uma técnica de imunofluorescência direta, para o diagnóstico de m... more Descreve-se a aplicabilidade de uma técnica de imunofluorescência direta, para o diagnóstico de mionecroses causadas por clostrídios, a partir de tecidos fixados em formol e incluídos em parafina. Essa técnica pode auxiliar no diagnóstico do carbúnculo sintomático e da gangrena gasosa, contribuindo para determinar a real prevalência dessas doenças no país.
Pesquisa Veterinaria Brasileira, 2009
This paper describes the clinical, pathological, and microbiologic aspects of paratuberculosis (J... more This paper describes the clinical, pathological, and microbiologic aspects of paratuberculosis (Johne's disease) in a dairy Gyr herd in the State of Paraíba, northeastern Brazil. An eight years old cow with chronic unresponsive diarrhea was clinically examined and euthanized for pathological evaluation. Fecal samples from all 160 animals over 12 months of age from the herd were collected for isolation of Mycobacterium avium subsp. paratuberculosis. Clinically, the index case cow was severely dehydrated, cachectic, with profuse mucous diarrhea. The main post-mortem findings were emaciation and thickened intestinal wall. Microscopically, the intestinal lamina propria and submucosa were infiltrated by macrophages, epithelioid cells, and Langhans giant cells with numerous alcohol-acid resistant bacilli in the cytoplasm. Two fecal samples displayed growth in slants of Herrold's egg-yolk agar supplemented with mycobactin J, 150 days after incubation. No growth was noticed in slants without mycobactin J. Microscopic examination of the isolated microorganisms stained by Ziehl-Neelsen revealed considerable amounts of alcohol-acid resistant bacilli, morphologically compatible with Mycobacterium spp. Based on the clinical signs, gross and histological lesions, growth time, bacterial morphology in Ziehl-Neelsen staining, and dependence of mycobactin J, the first diagnosis of paratuberculosis in Zebu cattle was made.
Genetics and Molecular Research, 2010
Epsilon toxin produced by Clostridium perfringens types B and D causes enterotoxemia in sheep, go... more Epsilon toxin produced by Clostridium perfringens types B and D causes enterotoxemia in sheep, goats and calves. Enterotoxemia can cause acute or superacute disease, with sudden death of the affected animal. It provokes huge economic losses when large numbers of livestock are affected. Therapeutic intervention is challenging, because the disease progresses very rapidly. However, it can be prevented by immunization with specific immunogenic vaccines. We cloned the etx gene, encoding epsilon toxin, into vector pET-11a; recombinant epsilon toxin (rec-ε) was expressed in inclusion bodies and was used for animal immunization. Serum protection was evaluated and cross-serum neutralization tests were used to characterize the recombinant toxin. To analyze the potency of the ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 9 (1): 266-276 (2010) Immunization using recombinant epsilon toxin from C. perfringens toxin (as an antigen), rabbits were immunized with 50, 100 or 200 µg recombinant toxin, using aluminum hydroxide gel as an adjuvant. Titers of 10, 30 and 40 IU/mL were obtained, respectively. These titers were higher than the minimum level required by the European Pharmacopoeia (5 IU/mL) and by the USA Code of Federal Regulation (2 IU/mL). This rec-ε is a good candidate for vaccine production against enterotoxemia caused by epsilon toxin of C. perfringens type D.
Archives of Microbiology, 2009
Clostridium perfringens type D produces enterotoxemia, an enteric disease in ruminants, also know... more Clostridium perfringens type D produces enterotoxemia, an enteric disease in ruminants, also known as pulpy kidney disease. Caused by epsilon toxin, enterotoxemia is a major exotoxin produced by this microorganism. Epsilon toxin is also the main component of vaccines against this enteric disorder. In this study, a standardized dot-blot was used to choose strains of C. perfringens type D that are producers of epsilon toxin. Clones producing epsilon toxin were chosen by limiting dilution; after three passages, lethal minimum dose titers were determined by soroneutralization test in mice. These clones produced epsilon toxin 240 times more concentrated than the original strain. The presence of the epsilon toxin gene (etx) was verified by polymerase chain reaction. All clones were positive, including those determined to be negative by dot-blot tests, suggesting that mechanisms in addition to the presence of the etx gene can influence toxin production. The dot-blot test was efficient for the selection of toxigenic colonies of C. perfringens type D and demonstrated that homogeneous populations selected from toxigenic cultures produce higher titers of epsilon toxin.
Arquivo Brasileiro De Medicina Veterinaria E Zootecnia, 2008
Multiplex PCR was optimized to detect Clostridium chauvoei and Clostridium septicum in pure cultu... more Multiplex PCR was optimized to detect Clostridium chauvoei and Clostridium septicum in pure cultures. In each reaction, a pair of primers for a specific segment of the flagellin gene of C. chauvoei and a pair of primers for a specific segment of alpha toxin gene of C. septicum were employed. Reference strains of both microorganisms were used as control. The multiplex PCR was evaluated by testing 16 clinical isolates of C. chauvoei from ruminants, 15 clinical isolates of C. septicum from ruminants and, four vaccine strains of each one of these agents. Reference strains of both microorganisms were used as control. To evaluate the specificity, genomic DNA of the following microorganisms was used: C. sordellii, C. novyi type A, C. novyi type B, C. perfringens type A, C. haemolyticum, C. botulinum type D, Pseudomonas aeruginosa, Staphylococcus aureus, Enterobacter aerogenes, Escherichia coli, and Salmonella typhimurium. All the isolates and vaccine strains of C. chauvoei and C. septicum were positive by PCR assay and cross reactions were not observed with the other species of clostridia, the other bacterial species or amongst both investigated agents. The smallest concentrations of DNA detected from C. chauvoei and C. septicum were 45pg/µl and 30pg/µl, respectively. The multiplex PCR was useful for the specific identification of C. chauvoei and C. septicum in pure cultures.
Ciencia Rural, 2009
Clostridium perfringens type D is the aetiological agent of enterotoxemia in ruminants. The disea... more Clostridium perfringens type D is the aetiological agent of enterotoxemia in ruminants. The disease is caused by epsilon toxin characterized by cardiac, pulmonary, kidney and brain edema. Monoclonal antibodies were produced by using myeloma cell line P3-X63-Ag8 653 fused with spleen cells from Balb/c mice, immunized with epsilon toxoid of C. perfringens type D. Six hybrids were established secreting monoclonal antibodies of the IgM class and IgG3 subclass.
... Felipe Masiero Salvarani I ; Flávia Ferreira Pinto I ; Francisco Carlos Faria Lobato I ; Ronn... more ... Felipe Masiero Salvarani I ; Flávia Ferreira Pinto I ; Francisco Carlos Faria Lobato I ; Ronnie Antunes de Assis II ; Luciana Aramuni Gonçalves I ; Nelson Éder Martins I ; Inácio José Clementino III ; Adrienny Trindade Reis Costa IV. ... [ Links ]. 17 PRIETO, C. et al. ...
Ciencia Rural, 2008
Botulism is a poisoning caused by the ingestion of toxins produced by Clostridium botulinum, that... more Botulism is a poisoning caused by the ingestion of toxins produced by Clostridium botulinum, that infects mammals and birds. This article reports an outbreak of botulism in two different flocks of ruminants at Paraíba, Brazil. In one, 85 out of 88 (96.6%) cattles died. In the other, 145 (96.7%) sheeps, 233 (57.8%) goats and 30 (96.8%) cattles died. Clinical signs were progressive paralysis, difficulties in moving, sialorrhoe and dyspnoe. Death occurred 24 to 48 hours after the beginning of clinic signs and at post-mortem examination no noteable changes were observed. Type C and D toxins were demonstrated in the intestinal contents and poultry litter by neutralization test in mice.
Arquivo Brasileiro De Medicina Veterinaria E Zootecnia, 2006
This report describes a case of bovine enterotoxaemia in Morro da Garça, Minas Gerais, Brazil. Cl... more This report describes a case of bovine enterotoxaemia in Morro da Garça, Minas Gerais, Brazil. Clostridium perfringens type D was isolated in pure culture and was characterized by biochemical reactions and PCR. By the mouse neutralization test, the presence of epsilon toxin from intestinal content was detected.
Brazilian Journal of Microbiology, 2009
Clostridium perfringens is a normal inhabitant of the intestinal tract of chickens as well as a p... more Clostridium perfringens is a normal inhabitant of the intestinal tract of chickens as well as a potential pathogen that causes necrotic enteritis and colangio hepatitis. The minimum inhibitory concentration (MIC) of seven different compounds used for therapy, growth promotion or prevention of coccidiosis was determined by agar dilution method for 55 C. perfringens strains isolated from the intestines of broiler chickens. All strains showed high susceptibility to penicillin, avilamycin, monensin and narasin. Only 7.3% of the strains showed an intermediated sensitivity to lincomycin, and 49 (89.1%) were considered susceptible. For tetracycline and bacitracin, 41.8% and 47.3% of strains, respectively, were considered resistant.
Ciencia Rural, 2009
Botulism is an intoxication caused by the ingestion of toxins produced by Clostridium botulinum. ... more Botulism is an intoxication caused by the ingestion of toxins produced by Clostridium botulinum. It affects mammals and birds, and is characterized by flaccid paralysis of the limbs. This report describes an outbreak of botulism in turkeys of various ages in the city of Santa Luzia, state of Minas Gerais, Brazil. The animals showed incoordination followed by flaccid paralysis involving the muscles of the legs, wings and neck. Within 24 hours, all 29 (100%) turkeys died. The post-mortem examination revealed the presence of fly larvae in the crop and the C. botulinum type C toxin was demonstrated in the sera of two affected animals by serum neutralization test.
Ciencia Rural, 2008
Botulism is an intoxication caused by the ingestion of toxins produced by Clostridium botulinum, ... more Botulism is an intoxication caused by the ingestion of toxins produced by Clostridium botulinum, that affects mammals and birds, characterized by a flaceid paralysis. This report describes a case of botulism in a goose in Santa Luzia, Minas Gerais State, Brazil. Clinical examinations showed dropping feathers and flaccid paralysis involving the muscles of the wings, legs and neck. post-mortem examination showed no significant gross or macroscopic lesions C. botulinum type C toxin was demonstrated in the serum of the affected animal through serum neutralization test in mice.
Ciencia Rural, 2010
Enterotoxemia (also called pulpy kidney disease) is an enteric disease, that affect ruminants, pr... more Enterotoxemia (also called pulpy kidney disease) is an enteric disease, that affect ruminants, produced by epsilon toxin from Clostridium perfringens type D, an anaerobic commonly isolated from soil and feces of healthy animals. The diagnostic is based on detection of this exotoxin in the intestinal content by soroneutralization in mice. Therefore, this study aimed to standardize a test for detection and titration of the toxin in vitro, and compare it with the phenomenon in vivo. A volume of epsilon toxin was titrated in mice and in some cell lines. After concluding the most sensitive cell line, were held in vitro titrations of dilutions from a toxin wich one had in vivo titer known. The results were grouped and a mathematical equation was developed. MDCK cell line showed that the phenomenon observed in vitro can be expressed by a mathematical equation wich shows a correlation of 98.33% with a minimum lethal dose determined in vivo. Therefore, the soroneutralization using MDCK allows a specific, sensitive, practical, fast, and doesn't requere use of animal titration of epsilon toxin.
Arquivo Brasileiro De Medicina Veterinaria E Zootecnia, 2007
Descreve-se a aplicabilidade de uma técnica de imunofluorescência direta, para o diagnóstico de m... more Descreve-se a aplicabilidade de uma técnica de imunofluorescência direta, para o diagnóstico de mionecroses causadas por clostrídios, a partir de tecidos fixados em formol e incluídos em parafina. Essa técnica pode auxiliar no diagnóstico do carbúnculo sintomático e da gangrena gasosa, contribuindo para determinar a real prevalência dessas doenças no país.
Pesquisa Veterinaria Brasileira, 2009
This paper describes the clinical, pathological, and microbiologic aspects of paratuberculosis (J... more This paper describes the clinical, pathological, and microbiologic aspects of paratuberculosis (Johne's disease) in a dairy Gyr herd in the State of Paraíba, northeastern Brazil. An eight years old cow with chronic unresponsive diarrhea was clinically examined and euthanized for pathological evaluation. Fecal samples from all 160 animals over 12 months of age from the herd were collected for isolation of Mycobacterium avium subsp. paratuberculosis. Clinically, the index case cow was severely dehydrated, cachectic, with profuse mucous diarrhea. The main post-mortem findings were emaciation and thickened intestinal wall. Microscopically, the intestinal lamina propria and submucosa were infiltrated by macrophages, epithelioid cells, and Langhans giant cells with numerous alcohol-acid resistant bacilli in the cytoplasm. Two fecal samples displayed growth in slants of Herrold's egg-yolk agar supplemented with mycobactin J, 150 days after incubation. No growth was noticed in slants without mycobactin J. Microscopic examination of the isolated microorganisms stained by Ziehl-Neelsen revealed considerable amounts of alcohol-acid resistant bacilli, morphologically compatible with Mycobacterium spp. Based on the clinical signs, gross and histological lesions, growth time, bacterial morphology in Ziehl-Neelsen staining, and dependence of mycobactin J, the first diagnosis of paratuberculosis in Zebu cattle was made.
Genetics and Molecular Research, 2010
Epsilon toxin produced by Clostridium perfringens types B and D causes enterotoxemia in sheep, go... more Epsilon toxin produced by Clostridium perfringens types B and D causes enterotoxemia in sheep, goats and calves. Enterotoxemia can cause acute or superacute disease, with sudden death of the affected animal. It provokes huge economic losses when large numbers of livestock are affected. Therapeutic intervention is challenging, because the disease progresses very rapidly. However, it can be prevented by immunization with specific immunogenic vaccines. We cloned the etx gene, encoding epsilon toxin, into vector pET-11a; recombinant epsilon toxin (rec-ε) was expressed in inclusion bodies and was used for animal immunization. Serum protection was evaluated and cross-serum neutralization tests were used to characterize the recombinant toxin. To analyze the potency of the ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 9 (1): 266-276 (2010) Immunization using recombinant epsilon toxin from C. perfringens toxin (as an antigen), rabbits were immunized with 50, 100 or 200 µg recombinant toxin, using aluminum hydroxide gel as an adjuvant. Titers of 10, 30 and 40 IU/mL were obtained, respectively. These titers were higher than the minimum level required by the European Pharmacopoeia (5 IU/mL) and by the USA Code of Federal Regulation (2 IU/mL). This rec-ε is a good candidate for vaccine production against enterotoxemia caused by epsilon toxin of C. perfringens type D.
Archives of Microbiology, 2009
Clostridium perfringens type D produces enterotoxemia, an enteric disease in ruminants, also know... more Clostridium perfringens type D produces enterotoxemia, an enteric disease in ruminants, also known as pulpy kidney disease. Caused by epsilon toxin, enterotoxemia is a major exotoxin produced by this microorganism. Epsilon toxin is also the main component of vaccines against this enteric disorder. In this study, a standardized dot-blot was used to choose strains of C. perfringens type D that are producers of epsilon toxin. Clones producing epsilon toxin were chosen by limiting dilution; after three passages, lethal minimum dose titers were determined by soroneutralization test in mice. These clones produced epsilon toxin 240 times more concentrated than the original strain. The presence of the epsilon toxin gene (etx) was verified by polymerase chain reaction. All clones were positive, including those determined to be negative by dot-blot tests, suggesting that mechanisms in addition to the presence of the etx gene can influence toxin production. The dot-blot test was efficient for the selection of toxigenic colonies of C. perfringens type D and demonstrated that homogeneous populations selected from toxigenic cultures produce higher titers of epsilon toxin.