Cryoprotection effectiveness of low concentrations of natural and lyophilized LDL (low density lipoproteins) on canine spermatozoa

The aim of this study was to evaluate the use of low concentrations of natural and lyophilized low density lipoprotein (LDL) from hen's egg yolk for cryopreservation of canine semen. Different ammonium sulphate concentrations were tested to extract LDL from egg yolk. The yolk was centrifuged, and LDL was isolated using 10, 20, 40, 45, or 50% ammonium sulphate solution (ASS). The LDL-rich floating fraction was collected for chemical characterization. Dry matter content was lowest (P<0.05) in the LDL extracted with the 50% ASS. The purification of LDL increased in association with increasing ammonium sulphate concentrations. SDS-PAGE showed that the 50% ASS solution yielded a purer fraction of LDL from egg yolk. For semen cryopreservation, TRIS extender was used replacing 20% egg yolk (control) by natural or lyophilized LDL using 1, 2, and 3% (w/v). Semen was centrifuged (755Xg for 7 min), diluted with one of the extenders, packed into 0.5mL straws (100x106 sperm/mL), and placed in a programmable cryopreservation machine. Thawed semen (37°C/ 30s) was analyzed for sperm motility, morphology, and by the hypoosmotic and epifluorescence tests (CFDA/ PI). Natural LDL extracted with 50% ASS was as effective as whole egg yolk to preserve canine frozen sperm when using low concentrations. The lyophilized LDL, mainly in the two higher concentrations tested (2 and 3%), was unsuitable to maintain the effectiveness of the LDL cryoprotective effect on dog sperm

Cryoprotection effectiveness of low concentrations of natural and lyophilized LDL (low density lipoproteins) on canine spermatozoa

The aim of this study was to evaluate the use of low concentrations of natural and lyophilized low density lipoprotein (LDL) from hen's egg yolk for cryopreservation of canine semen. Different ammonium sulphate concentrations were tested to extract LDL from egg yolk. The yolk was centrifuged, and LDL was isolated using 10, 20, 40, 45, or 50% ammonium sulphate solution (ASS). The LDL-rich floating fraction was collected for chemical characterization. Dry matter content was lowest (P<0.05) in the LDL extracted with the 50% ASS. The purification of LDL increased in association with increasing ammonium sulphate concentrations. SDS-PAGE showed that the 50% ASS solution yielded a purer fraction of LDL from egg yolk. For semen cryopreservation, TRIS extender was used replacing 20% egg yolk (control) by natural or lyophilized LDL using 1, 2, and 3% (w/v). Semen was centrifuged (755Xg for 7 min), diluted with one of the extenders, packed into 0.5mL straws (100x106 sperm/mL), and placed in a programmable cryopreservation machine. Thawed semen (37°C/ 30s) was analyzed for sperm motility, morphology, and by the hypoosmotic and epifluorescence tests (CFDA/ PI). Natural LDL extracted with 50% ASS was as effective as whole egg yolk to preserve canine frozen sperm when using low concentrations. The lyophilized LDL, mainly in the two higher concentrations tested (2 and 3%), was unsuitable to maintain the effectiveness of the LDL cryoprotective effect on dog sperm

Stability and toxicity of Clostridium perfringens type D epsilon prototoxin treated by iodine Estabilidade e toxicidade da protoxina epsilon de Clostridium perfringens tipo D

Purified epsilon prototoxin of Clostridium perfringens type D was produced, purified, and detoxified by the stoiechiometric method of non-radioactive iodine incorporation. Different degrees of iodination were perfomed and the toxicity of the derivatives were analysed by in vivo studies. Toxicity decreased inversely to the iodine incorporation. Eletrophoretic analysis showed different levels of stability of samples kept under different temperatures 4ºC, - 20ºC, and -80 ºC. The iodinated prototoxins were stocked for a period of four months.A prototoxina epsílon de Clostridium perfringens tipo D foi produzida, purificada e destoxificada por estoiquiometria. Diferentes quantidades de iodo foram incorporadas à estrutura protéica da prototoxina e a toxicidade dos derivados foi analisada em estudos in vivo. Verificou-se que a toxicidade diminuiu à medida que os átomos de iodo foram incorporados à prototoxina. A análise eletroforética demonstrou a estabilidade das amostras quando armazenadas a 4ºC, -20ºC e -80ºC. A prototoxina iodada foi estocada por um período de quatro meses

Prevention Of Nonspecific Reactions On Reversed Passive Latex Agglutination Assay (rpla) For Detecting Low Amounts Of Staphylococcal Enterotoxins

The SET-RPLA, from Denka Seiken Co. Ltd., Tokio, a commercial reversed passive latex agglutination test kit, has been recommended to establish the enterotoxicity capacity of some staphylococcal strains, implicated in food poisoning outbreaks that produce low levels of enterotoxins (SE). Despite the RPLA specificity, the occurrence of nonspecific reactions when testing low- SE-producing is common. In order to control these nonspecific reactions the addition of purified normal rabbit IgG purified was applied on approximately 350 staphylococcal isolates from human milk and anatomic sites of healthy dental student carriers. The results indicated that addition of 5% (v/v) of purified normal rabbit IgG (0.74 mg/mL) to the culture supernatant fluid is a simple and reliable tool for the controlling of nonspecific reactions in the RPLA assay.3901/02/155763Adesyin, A.A., Esbach, M., Lenz, W., Schall, K.P., Detection of enterotoxigenicity of Staphylococcus aureus strains: A comparative use of the modified Outcherlony precipitation test, reversed passive latex agglutination test, and avidin-biotin ELISA (1992) Can. J. Microbiol., 38, pp. 1097-1101Bergdoll, M.S., Staphylococcus aureus (1989) Foodborne Bacterial Pathogens., pp. 463-523. , Doyle, M.P. (ed.) INC, New YorkBergdoll, M.S., Importance of staphylococci that produce nanogram quantities of enterotoxin (1995) Zbl. Bakt., 282, pp. 1-6Ey, P.L., Prowsa, S.J., Jenken, C.R., Isolation of pure IgG1, IgG2a, and IgG2b immunoglobulins from mouse serum using protein A-Sepharose (1987) Biochem., 15, pp. 429-436Evenson, M.L., Hinds, M.W., Bernstein, R.S., Bergdoll, M.S., Estimation of human dose of staphylococcal enterotoxin A from a large outbreak involving chocolate milk (1988) Int. J. Food Microbiol., 7, pp. 311-316Foster, T.J., McDevitt, D., Surface associated proteins of Staphylococcus aureus: Their possible roles in virulence (1994) FEMS Microbiol. Letters, 118, pp. 199-206Halander, H., Production of large quantities of enterotoxin B and other staphylococcal toxins on solid media (1965) Acta. Pathol. Microbiol. Scand., 63, pp. 299-305Igasashi, H., Figikawa, H., Shingaki, M., Bergdoll, M.S., Latex agglutination test for staphylococcal toxic shock syndrome toxin-1 (1986) J. Clin. Microbiol., 23, pp. 509-512Jarvis, A.W., Lawrence, R.C., Production of high titers of enterotoxins for the routine testing of staphylococci (1970) Appl. Microbiol., 19, pp. 698-699Koper, J.W., Hagenaars, A.M., Notermans, S., Prevention of cross-reactions in the enzyme linked immunosorbent assay (ELISA) for the detection of Staphylococcus aureus enterotoxin type B in culture filtrates and foods (1980) J. Food Safety, 2, pp. 35-45Park, C.E., Szabo, R., Evaluation of the reversed passive latex agglutination (RPLA) test kit for detecting of staphylococcal enterotoxins A, B, C, and D in foods (1986) Can. J. Microbiol., 32, pp. 723-727Pereiera, J.P., Salsberg, S.P., Bergdoll, M.S., Production of staphylococcal enterotoxin D in foods by low-enterotoxin-producing staphylococci (1991) Int. J. Food Microbiol., 14, pp. 19-26Pereira, M.L., Carmo, L.S., Santos, E.J., Sellos, I.T., Bergdoll, M.S., Staphylococci in breast milk from women with and without mastitis (1995) Rev. Microbiol., 26, pp. 117-120Pereira, M.L., Carmo, L.S., Santos, E.J., Souki, M.Q., Carvalho, M.A.R., Bergdoll, M.S., Staphylococci from healthy dental student (1996) Braz. Dent. J., , in pressPereira, M.L., Heneine, L.G., Pereira, J.L., Bergdoll, M.S., Controlling of nonspecific reactions on reversed passive latex agglutination (RPLA) for detecting nanogram quantities of staphylococcal enterotoxins (1996) Arq. Bras. Med. Vet. Zootec., , in pressPereira, M.L., Carmo, L.S., Santos, E.J., Pereira, J.L., Bergdoll, M.S., Enterotoxin H in staphylococcal food poisoning (1995) J. Food Protect., 5, pp. 559-561Oda, T., Ohkubo, T., Nagai, M., Nishimoto, Y., Ohmaru, K., Detection of staphylococcal enterotoxins in foods by reversed passive agglutination test (1979) Ann. Rep. Fakuoka City Hyg. Lab., 4, pp. 33-37Robbins, R., Gould, S., Bergdoll, M.S., Detecting the enterotoxigenicity of Staphylococcus aureus strains (1974) Appl. Microbiol., 28, pp. 946-950Salomon, L.L., Tew, R.W., Assay of enterotoxin B by latex agglutination (1968) Proc. Soc. Exp. Biol. Med., 129, pp. 539-554Shingaki, M., Igarashi, H., Fugikawa, H., Ushioda, H., Tereyama, T., Sakai, Study on reversed passive agglutination for the detection of staphylococcal enterotoxins A, B, and C (1981) Ann. Rep. Fakuoka City Hyg. Lab., 32, pp. 128-131Valle, J., Gómez-Lucia, E., Piriz, S., Goyache, J., Orden, J.A., Vadillo, S., Enterotoxin production by staphylococci isolated from healthy goats (1990) Appl. Environ. Microbiol., 56, pp. 1323-1326Vanderzant, C., Spillitoesser, D.F., (1992) Compendium for the Microbiological Examination of Foods, , American Public Health Association. 3ed. Washington,DC., 1219

Diagnóstico do complexo teníase-cisticercose bovina em São João Evangelista, Minas Gerais, Brasil

Com o objetivo de diagnosticar a situação do complexo teníase-cisticercose bovina em Minas Gerais, Brasil, foi selecionado o município de São João Evangelista, onde foram coletadas amostras de sangue de 339 bovinos em 15 propriedades rurais, sorteadas aleatoriamente. Em cada propriedade, foi aplicado um questionário socioeconômico para a análise de fatores que favorecem a manutenção do complexo teníase-cisticercose bovina. Foi realizado também o diagnóstico de teníase humana por meio de exame coproparasitológico dos habitantes das propriedades. Encontrou-se a prevalência de 4,1% para cisticercose bovina e a frequência de 2,94% para teníase humana. Entre os fatores de risco para a manutenção do complexo teníase-cisticercose analisados, foi observada uma relação estatisticamente significativa (P=0,042) entre a ocorrência de cisticercose bovina e a ingestão de carne malpassada pelos entrevistados. Concluiu-se que a cisticercose bovina está presente no município de São João Evangelista, MG, em índices considerados endêmicos, sendo o consumo de carne malpassada e não inspecionada o principal fator de risco para a manutenção do complexo teníase-cisticercose, o que reforça a necessidade da adoção de medidas de controle com contínua vigilância epidemiológica e sanitária