Culture-attenuated pathogenic Leptospira lose the ability to survive to complement-mediated-killing due to lower expression of factor H binding proteins

Several pathogens including Gram-negative bacteria hijack complement regulators to escape host's innate response. Pathogenic Leptospira species bind Factor H, C4b binding protein and vitronectin from the complement system. We evaluated the ability of low passage (LP) and culture-attenuated (CA) pathogenic strains of Leptospira, to bind Factor H. We used LOCaS46 (Leptospira interrogans sv Canicola), LOVe30 (L. interrogans sv Icterohaemorrhagiae) and MOCA45 (L. santarosai sv Tarassovi), and ten high passage strains of Leptospira [used in the microscopic agglutination test (MAT)]. Afterwards, we assessed their survival in normal human serum (NHS). Interestingly, the ability in binding Factor H was higher for LOCaS46 and LOVe30 LP strains, than for the respective CA strains suggesting that the ability of evading the alternative complement pathway is lost after culture attenuation. Accordingly, the level of mRNA expression of the Factor H binding proteins, LigA, LigB and Lsa23 was higher in these LP strains than in the corresponding CA strains. Unexpectedly, no difference in Factor H binding and surviving was observed between LP and CA MOCA45 strains. The high passage MAT-reference strains showed variation in Factor H binding ability, but, in most cases, the ability for capturing Factor H by Leptospira strains correlated with their survival in NHS.

Culture-attenuated pathogenic Leptospira lose the ability to survive to complement-mediated-killing due to lower expression of factor H binding proteins

Several pathogens including Gram-negative bacteria hijack complement regulators to escape host's innate response. Pathogenic Leptospira species bind Factor H, C4b binding protein and vitronectin from the complement system. We evaluated the ability of low passage (LP) and culture-attenuated (CA) pathogenic strains of Leptospira, to bind Factor H. We used LOCaS46 (Leptospira interrogans sv Canicola), LOVe30 (L. interrogans sv Icterohaemorrhagiae) and MOCA45 (L. santarosai sv Tarassovi), and ten high passage strains of Leptospira [used in the microscopic agglutination test (MAT)]. Afterwards, we assessed their survival in normal human serum (NHS). Interestingly, the ability in binding Factor H was higher for LOCaS46 and LOVe30 LP strains, than for the respective CA strains suggesting that the ability of evading the alternative complement pathway is lost after culture attenuation. Accordingly, the level of mRNA expression of the Factor H binding proteins, LigA, LigB and Lsa23 was higher in these LP strains than in the corresponding CA strains. Unexpectedly, no difference in Factor H binding and surviving was observed between LP and CA MOCA45 strains. The high passage MAT-reference strains showed variation in Factor H binding ability, but, in most cases, the ability for capturing Factor H by Leptospira strains correlated with their survival in NHS.

International regulatory requirements for Leptospira vaccine potency testing. Roundtable: current requirements and opportunity for harmonization.

Progress continues to be made in the ongoing efforts to replace, reduce, or refine the use of laboratory animals for Leptospira vaccine potency testing in certain markets/regions. Leptospira-containing vaccines, as with many veterinary vaccines, are manufactured and distributed both on a regional basis by local manufacturers and internationally by large multinational firms. Three general scenarios exist for the international testing and distribution of veterinary vaccines including: 1) the importing country recognizes the country of origin's testing and batch release data with no additional testing; 2) the importing country requires the manufacturer to conduct a specific potency assay based on the current importing market's regulations for the importing country or 3) the importing country requires retesting of the product in country prior to distribution. Scenarios 2 and 3 both have the potential to significantly increase the usage of laboratory animals for what may be considered redundant testing. Specific requirements for the importation of Leptospira vaccines in the United States, Europe, and Mexico were presented as well as efforts to reduce the use of laboratory animal testing through the availability of internationally recognized tests.

Detección de Leptospira santarosai y L. kirschneri en bovinos: nuevos aislados con potencial impacto en producción bovina y salud pública / Detection of Leptospira santarosai and L. kirschneri in cattle: new isolates with potential impact in bovine production and public health

Bovine leptospirosis causes high economic losses in cattle mainly due to reproductive failure, as well as representing public health risk. Since the last century, antibody titers against several Leptospira serovars have been detected by the microscopic agglutination test (MAT) in Mexico. With the exception of very few cases, the presence of serovars causing leptospirosis in cattle and other animal species has not been demonstrated by isolation in Mexico, and in such cases characterization had to be done abroad by complex and slow immunological approaches, by comparison with a number of reference strains. The present study was conducted to perform the molecular characterization of Leptospira isolates by multiple locus sequencing typing (MLST). A hundred and ninety seven sera and kidneys samples were collected immediately after slaughter, from grazing cattle coming from the south-eastern states of Mexico. Anti-Leptospira antibodies were detected by the MAT and kidneys were inoculated into EMJH and Fletcher's specific medium. A seropositivity of 60.4% (119 out of 197), with titers from 1:100 up to 1:3 200 was detected. Four isolates (2.03%), referred as CAL4, CAL6, CAL7 and MOCA45, were characterized by serology, ribotyping and MLST as L. kirschneri serovar Grippotyphosa; L. interrogans serovar Hardjo; L. santarosai serovar Mini and L. santarosai serovar Tarassovi, respectively. With the exception of serovar Hardjo, the three other isolates belong to serovars and species not previously isolated in Mexico. These findings make it necessary to evaluate the potential distribution of such serovars among cattle and their role on animal production and public health.

La leptospirosis bovina causa grandes pérdidas económicas a la ganadería por problemas reproductivos y también es un riesgo de salud pública. En México, desde el siglo pasado se ha registrado la presencia de anticuerpos contra serovariedades de Leptospira por la técnica de aglutinación microscópica (AM), en bovinos y otras especies animales. En muy pocos casos, la enfermedad fue demostrada por el aislamiento de Leptospira, y en tales casos, su caracterización se basó en métodos inmunológicos lentos que requirieron la comparación con cepas de referencia y que fueron realizados fuera de México. En el presente trabajo se realizó la caracterización molecular mediante la secuenciación de locus múltiples (MLST), de aislados de Leptospira obtenidos de riñones de bovinos recolectados en rastro, procedentes de las zonas Golfo y sur de México. Se obtuvieron muestras de suero y riñones de 197 bovinos para realizar la AM, y el cultivo en medios específicos EMJH y Fletcher. Se detectó una seropositividad del 60.4% (119 de 197), con títulos desde 1:100 hasta 1:3,200 y se obtuvieron cuatro aislados de Leptospira (2.03%), denominados CAL4, CAL6, CAL7 y MOCA45. Los aislados fueron caracterizados por serología, ribotipificación y MLST, como L. kirschneri serovariedad Grippotyphposa; L. interrogans serovariedad Hardjo; L. santarosai serovariedad Mini y L. santarosai serovariedad Tarassovi, respectivamente. A excepción de la serovariedad Hardjo, los aislados pertenecen a especies y serovariedades no aisladas anteriormente en la República Mexicana, esto sugiere la necesidad de evaluar su diseminación entre bovinos y su potencial efecto en la producción animal y en la salud pública.

Aislamiento de levaduras de glándulas mamarias con diferentes presentaciones de mastitis en el Altiplano Mexicano / Yeasts isolation from bovine mammary glands under different mastitis status in the Mexican High Plateu

Antecedentes. Las mastitis subclínicas y clínicas en bovinos por hongos se han incrementado principalmente por levaduras del género Candida. Objetivo. Conocer las levaduras presentes en la leche de glándulas mamarias de bovinos clínicamente sanos, y de aquellos con mastitis subclínica y clínica. Método. Se evaluó la presencia de levaduras en 1.095 muestras de leche de 342 glándulas mamarias sanas, 383 con mastitis subclínica y 370 con mastitis clínica, de los estados de Querétaro, Hidalgo, Puebla y la ciudad de México, Distrito Federal, que forman parte del Altiplano Mexicano. El estado sanitario de las glándulas mamarias se determinó por examen clínico y la prueba de California. La identificación de levaduras fue realizada por métodos morfológicos y bioquímicos. Resultados. Se identificaron 20 especies diferentes del género Candida a partir de 282 (25,75%) de las muestras de leche. Las especies encontradas con mayor frecuencia en los bovinos sanos y con mastitis clínica fueron Candida glabrata y Candida krusei. El grupo de las muestras con mastitis subclínica mostró una diversidad de especies de Candida, incluidas Candida zeylanoides, Candida norvegica, Candida viswanathii, Candida guilliermondii y Candida tropicalis. Candida albicans fue aislada solo en 11 (3,9%) de las muestras de mastitis clínica (6) y subclínica (5). Conclusiones. Estos resultados sugieren el posible papel de otras especies de Candida diferentes a C. albicans como causantes de mastitis micótica(AU)

Background. The mastitis subclinical and clinical in cows caused by fungi has been increased specially by yeast of the genus Candida. Objective. To identify what yeasts were present in milk samples obtained from mammary glands of healthy cows, and others suffering subclinical or clinical mastitis. Methods. From a total of 1,095 milk samples 342 were from mammary glands of healthy dairy cows, 383 with subclinical mastitis, and 370 with clinical mastitis, were taken, in the states of Querétaro, Hidalgo, Puebla and Mexico City (Distrito Federal) in the so called Mexican High Plateu. The clinical status of the mammary glands was determined by clinical examination and the California Mastitis Test. Yeasts identification was carried out by morphology and biochemical methods. Results. Twenty different species of Candida were identified out of 282 (25.75%) milk samples. The most frequently identified species in the healthy cows and cows with clinical mastitis groups were Candida glabrata and Candida krusei. On the other hand, samples from the subclinical mastitis group showed a diversity of Candida species, including Candida zeylanoides, Candida norvegica, Candida viswanathii, Candida guilliermondii, and Candida tropicalis. Candida albicans was isolated only in 11 (3.9%) samples from the clinical and subclinical mastitis groups. Conclusions. These results suggest the possible role that Candida species other than C. albicans may play in mycotic mastitis in cows(AU)