Extracellular vesicles in infectious diseases caused by protozoan parasites in buffaloes

Extracellular vesicles (EVs) are small membrane-bound vesicles of growing interest in vetetinary parasitology. The aim of the present report was to provide the first isolation, quantification and protein characterization of EVs from buffalo (Bubalus bubalis) sera infected with Theileria spp. Methods: Infected animals were identified through optical microscopy and PCR. EVs were isolated from buffalo sera by size-exclusion chromatography and characterized using western blotting analysis, nanoparticle tracking analysis and transmission electron microscopy. Subsequently, the proteins from isolated vesicles were characterized by mass spectrometry. Results: EVs from buffalo sera have shown sizes in the 124-140 nm range and 306 proteins were characterized. The protein-protein interaction analysis has evidenced biological processes and molecular function associated with signal transduction, binding, regulation of metabolic processes, transport, catalytic activity and response to acute stress. Five proteins have been shown to be differentially expressed between the control group and that infected with Theileria spp., all acting in the oxidative stress pathway. Conclusions: EVs from buffaloes infected with Theileria spp. were successfully isolated and characterized. This is an advance in the knowledge of host-parasite relationship that contributes to the understanding of host immune response and theileriosis evasion mechanisms. These findings may pave the way for searching new EVs candidate-markers for a better production of safe biological products derived from buffaloes.(AU)

Extracellular vesicles in infectious diseases caused by protozoan parasites in buffaloes

Background: Extracellular vesicles (EVs) are small membrane-bound vesicles of growing interest in vetetinary parasitology. The aim of the present report was to provide the first isolation, quantification and protein characterization of EVs from buffalo (Bubalus bubalis) sera infected with Theileria spp. Methods: Infected animals were identified through optical microscopy and PCR. EVs were isolated from buffalo sera by size-exclusion chromatography and characterized using western blotting analysis, nanoparticle tracking analysis and transmission electron microscopy. Subsequently, the proteins from isolated vesicles were characterized by mass spectrometry. Results: EVs from buffalo sera have shown sizes in the 124-140 nm range and 306 proteins were characterized. The protein-protein interaction analysis has evidenced biological processes and molecular function associated with signal transduction, binding, regulation of metabolic processes, transport, catalytic activity and response to acute stress. Five proteins have been shown to be differentially expressed between the control group and that infected with Theileria spp., all acting in the oxidative stress pathway. Conclusions: EVs from buffaloes infected with Theileria spp. were successfully isolated and characterized. This is an advance in the knowledge of host-parasite relationship that contributes to the understanding of host immune response and theileriosis evasion mechanisms. These findings may pave the way for searching new EVs candidate-markers for a better production of safe biological products derived from buffaloes.(AU)

Transmissão congênita em cabras reinfectadas com Toxoplasma gondii / Congenital transmission in reinfected goats with Toxoplasma gondii

Avaliou-se o potencial de transmissão congênita em cabras experimentalmente reinfectadas com Toxoplasma gondii, em três estágios gestacionais (inicial, intermediário e final). Das 25 fêmeas não gestantes negativas para T. gondii, 20 foram inoculadas oralmente com 2,5x103 oocistos de T. gondii cepa ME49. Destas, 15 fêmeas gestantes cronicamente infectadas foram reinoculadas, via oral, com 2,5x103 oocistos T. gondii cepa VEG. Cinco grupos experimentais foram formados (n=5): I, II e III (reinoculações nos estágios gestacionais inicial, intermediário e final, respectivamente), IV (inoculação) e V (não inoculação). Exames clínicos e sorológicos (IgG RIFI [reação de imunofluorescência indireta]) em diferentes dias de avaliação, e bioensaio e PCR foram realizados em todos os animais. Nas cabras infectadas com T. gondii foram observados um pico de 40,2°C (IV) aos nove, soroconversão (IgG ≥64) aos 21 e estabilização (IgG <1024) aos 119 dias pós inoculação. Nas cabras reinfectadas com T. gondii ocorreu um aumento nos títulos de IgG (≥1024) aos 28 (I) , 7 (II) e 3 (III) dias pós-reinoculação. Durante o parto foram observados apenas nos grupos reinfectados: distocia, deformidades corporais, natimortalidade e fraqueza, e anticorpos IgG antiToxoplasma foram detectados em todas e em algumas crias das cabras reinfectadas e infectadas, respectivamente. Parasitismo tissular por T. gondii foi diagnosticado por bioensaio e PCR em cabras infectadas e reinfectadas e em sua prole. A toxoplasmose congênita foi possível em caprinos cronicamente infectados e reinfectados com T. gondii. A infecção primária com T. gondii não protegeu as cabras prenhes contra a doença congênita resultante de reinfecção toxoplásmica, em diferentes estágios de gestação (inicial, intermediário e final). (AU)

This study evaluated the potential of congenital transmission in goats experimentally infected and reinfected with Toxoplasma gondii, in three gestational stages (initial, intermediate and final). Of the 25 non-pregnant females negative for T. gondii, 20 were orally inoculated with 2.5 x 103 T. gondii ME49 oocysts. Of these, 15 pregnant females chronically infected were reinoculated, via oral, with 2.5 x 103 T. gondii VEG oocysts. Five experimental groups were formed (n=5): I, II and III (reinoculations in the initial, intermediate and final gestational stage, respectively), IV (inoculation) and V (no inoculation). Clinical and serological exams (IgG IFAT [indirect immunofluorescence antibody test]) in different days of evaluation, and bioassay and PCR were performed in all goats. In the infected goats with T. gondii a peak of 40.2°C (IV) at nine, seroconversion (IgG≥64) at 21 and stabilization (IgG<1024) at 119 days postinoculation were observed. In the reinfected goats with T. gondii occurred an increase in IgG titers (≥1,024) at 28 (I), 7 (II) and 3 (III) days post-reinoculation. During kidding were observed only in the reinfected groups: dystocia, malformation body, stillbirth and weakness, and IgG anti-Toxoplasma were detected in all and in some offsprings of the reinfected and infected goats, respectively. Tissue parasitism by T. gondii was diagnosed by bioassay and PCR in infected and reinfected goats and in their offspring. The congenital toxoplasmosis was possible in goats chronically infected and reinfected with T. gondii. The primary infection with T. gondii did not protect the pregnant goats against congenital disease resulting from toxoplasmic reinfection, in different gestational stages (initial, intermediate and final). (AU)

Transmissão congênita em cabras reinfectadas com Toxoplasma gondii / Congenital transmission in reinfected goats with Toxoplasma gondii

Avaliou-se o potencial de transmissão congênita em cabras experimentalmente reinfectadas com Toxoplasma gondii, em três estágios gestacionais (inicial, intermediário e final). Das 25 fêmeas não gestantes negativas para T. gondii, 20 foram inoculadas oralmente com 2,5x103 oocistos de T. gondii cepa ME49. Destas, 15 fêmeas gestantes cronicamente infectadas foram reinoculadas, via oral, com 2,5x103 oocistos T. gondii cepa VEG. Cinco grupos experimentais foram formados (n=5): I, II e III (reinoculações nos estágios gestacionais inicial, intermediário e final, respectivamente), IV (inoculação) e V (não inoculação). Exames clínicos e sorológicos (IgG RIFI [reação de imunofluorescência indireta]) em diferentes dias de avaliação, e bioensaio e PCR foram realizados em todos os animais. Nas cabras infectadas com T. gondii foram observados um pico de 40,2°C (IV) aos nove, soroconversão (IgG ≥64) aos 21 e estabilização (IgG <1024) aos 119 dias pós inoculação. Nas cabras reinfectadas com T. gondii ocorreu um aumento nos títulos de IgG (≥1024) aos 28 (I) , 7 (II) e 3 (III) dias pós-reinoculação. Durante o parto foram observados apenas nos grupos reinfectados: distocia, deformidades corporais, natimortalidade e fraqueza, e anticorpos IgG antiToxoplasma foram detectados em todas e em algumas crias das cabras reinfectadas e infectadas, respectivamente. Parasitismo tissular por T. gondii foi diagnosticado por bioensaio e PCR em cabras infectadas e reinfectadas e em sua prole. A toxoplasmose congênita foi possível em caprinos cronicamente infectados e reinfectados com T. gondii. A infecção primária com T. gondii não protegeu as cabras prenhes contra a doença congênita resultante de reinfecção toxoplásmica, em diferentes estágios de gestação (inicial, intermediário e final).

This study evaluated the potential of congenital transmission in goats experimentally infected and reinfected with Toxoplasma gondii, in three gestational stages (initial, intermediate and final). Of the 25 non-pregnant females negative for T. gondii, 20 were orally inoculated with 2.5 x 103 T. gondii ME49 oocysts. Of these, 15 pregnant females chronically infected were reinoculated, via oral, with 2.5 x 103 T. gondii VEG oocysts. Five experimental groups were formed (n=5): I, II and III (reinoculations in the initial, intermediate and final gestational stage, respectively), IV (inoculation) and V (no inoculation). Clinical and serological exams (IgG IFAT [indirect immunofluorescence antibody test]) in different days of evaluation, and bioassay and PCR were performed in all goats. In the infected goats with T. gondii a peak of 40.2°C (IV) at nine, seroconversion (IgG≥64) at 21 and stabilization (IgG<1024) at 119 days postinoculation were observed. In the reinfected goats with T. gondii occurred an increase in IgG titers (≥1,024) at 28 (I), 7 (II) and 3 (III) days post-reinoculation. During kidding were observed only in the reinfected groups: dystocia, malformation body, stillbirth and weakness, and IgG anti-Toxoplasma were detected in all and in some offsprings of the reinfected and infected goats, respectively. Tissue parasitism by T. gondii was diagnosed by bioassay and PCR in infected and reinfected goats and in their offspring. The congenital toxoplasmosis was possible in goats chronically infected and reinfected with T. gondii. The primary infection with T. gondii did not protect the pregnant goats against congenital disease resulting from toxoplasmic reinfection, in different gestational stages (initial, intermediate and final).

Candida parapsilosis como contaminante de cultivos celulares vero en la producción de neospora caninum antigen / Candida parapsilosis as vero cell culture contaminant in neospora caninum antigen production / Candida parapsilosis como contaminante de cultura de células vero na produção de antígeno de neospora caninum

Neospora caninum ha sido identificado como un importante agente causador de infecciones en bovinos y caninos. Este parásito es mantenido con éxito en cultivos celulares VERO. La contaminación de los cultivos celulares puede ocurrir de dos formas principales: la contaminación con otras líneas celulares o con microorganismos. Este trabajo relata un cultivo de células VERO para la producción de antígeno de Neospora caninum contaminada con Candida parapsilosis. La secuenciación de los amplicones de la región ITS presentó 99% de similitud con las secuencias de Candida parapsilosis depositadas en GenBank. Se destaca en esta comunicación la posibilidad de contaminación de los cultivos celulares por Candida parapsilosis y, por lo tanto, la importancia de redoblar los cuidados en la manipulación de estos.

Neospora caninum has been identified as an important cause of infectious in cattle and dogs. The parasite is successfully established in continuous passage in VERO cell cultures. Cell culture contamination may present itself in two mainly forms: contamination with other cell line or with microorganisms. In this paper we report a VERO cell culture of Neospora caninum antigen production contaminated with Candida parapsilosis. Sequencing of the ITS amplicons showed 99% similarity with deposited sequences of Candida parapsilosis at GenBank. It should be noted through this report the possibility of contamination by Candida parapsilosis in cell cultures, thus care in handling cultures should be redoubled.

Neospora caninum tem sido identificado como um importante agente causador de infecções em bovinos e caninos. O parasita é mantido com sucesso em passagens contínuas em culturas de células VERO. A contaminação de culturas células pode ocorrer de duas formas principais: contaminação com outras linhagens celulares ou com micro-organismos. Neste trabalho, relata-se uma cultura de células VERO para produção de antígeno de Neospora caninum contaminada com Candida parapsilosis. O sequenciamento dos amplicons da região ITS apresentou 99% de similaridade com Candida parapsilosis nas sequências depositadas no GenBank. Ressalta-se através deste relato a possibilidade de contaminação por Candida parapsilosis em cultivos celulares, devendo-se redobrar os cuidados na manipulação.

Accuracy of a multiplex pcr protocol for Staphylococcus aureus, Streptococcus agalactiae and Escherichia coli detection in bulk tanks

The objective of the present study was to evaluate the accuracy of a multiplex PCR (mPCR) protocol for Staphylococcus aureus, Streptococcus agalactiae and Escherichia coli detection in bovine milk samples collected from bulk tanks. Twenty dairy farms in mid-west of Sao Paulo State, Brazil were enrolled. Milk samples collected from each bulk tank were evaluated by mPCR and microbiological culture (gold standard test). S. aureus, S. agalactiae and E. coli were isolated by culture in 30%, 10% and 40% respectively, and detected by mPCR in 0%, 10% and 35% bulk tank milk samples respectively. mPCR has demonstrated 93.2% specificity and 37.5% sensitivity values, with 78.3% accuracy. It was possible to conclude that this mPCR protocol was efficient for S. agalactiae and E. coli detection in the evaluated bulk tank milk samples, but failed to detect S. aureus. Despite the high specificity values obtained by mPCR, the sensitivity levels were low, indicating that this molecular method should only be recommended as a complementary diagnosis test for milk quality monitoring in bulk tanks.