ABSTRACT
Bovine campylobacteriosis caused by Campylobacter fetus is associated with reproductive losses. The knowledge about the mechanisms of bacterial pathogenesis is limited, then a murine experimental model is proposed. BALB/c females and males were used. Two-cell embryos were cultured in Ham-F10 as control group (CG). Treatment groups were constituted by the addition of Cfv 1 and 3, or Cff 2 and 5. Morulae were placed in Ham-F10 (CG); treatment groups were constituted by the addition of Cfv27, CFF (cell-free filtrate) and Brucella broth (BB). Blastocysts were cultured in MEM (CG); challenge group were constituted by the addition of Cfv 27. Differentiation, hatching, hatched, adhesion and expansion were evaluated. Results were analyzed by Chi2 test. In two-cell embryo, the differentiation rate was not modified when the study strains were added (p > 0.05). The differentiation rate at 24 h for embryos at the morula stage was lower for BB, Cfv, and CFF, compared with CG (p < 0.05). After 48 h culture, no differences were observed in blastocyst formation for Cfv and BB, compared to CG (p > 0.05). However, the differentiation rate for the CFF group was lower than for CG (p < 0.05). At 48 and 72 h, the hatching rate was higher in CFF and Cfv groups than in CG (p < 0.05). Differences were not detected in blastocyst cultures. In conclusion, under these experimental conditions, Cf was not detrimental to the development of murine embryos. Efforts will be intensified to establish in vitro infection models that reproduce their pathogenicity.
La campilobacteriosis bovina caudada por Campylobacter fetus produce pérdidas reproductivas existiendo poca información de los mecanismos de patogenicidad de dicha bacteria, por lo cual se propone un modelo utilizando ratones BALC/c. Embriones de dos células fueron cultivados en Ham-F10: grupo control (GC), los grupos experimentales fueron adicionados con las cepas Cfv 1, Cfv 3, Cff 2 y Cff 5. Mórulas fueron cultivadas en Ham-F10 (GC); los grupos tratados recibieron Cfv27, CFF (filtrado libre de células) y caldo Brucella (BB). Blastocistos fueron cultivados en MEM (GC) y MEM más Cfv 27 (grupo desafiado). Se evaluó: diferenciación, "hatching", "hatched", adhesión y expansión. Los resultados fueron analizados por Chi2. En embriones de dos células, la diferenciación no fue modificada por acción de las cepas evaluadas (p > 0,05). Para embriones en estadío de mórula, la diferenciación a las 24 h de cultivo fue menor para BB, Cfv, y CFF, comparado con el GC (p < 0,05). Luego de 48 h de cultivo, no hubo diferencias entre Cfv, BB, y CG (p > 0,05), no obstante para el grupo CFF la diferenciación fue menor al CG (p < 0,05). El porcentaje de "hatching" (48 y 72 h de cultivo), fue mayor en los grupos CFF y Cfv comparado con el GC (p < 0,05). La adición de Cfv 27 no modificó el desarrollo de blastocistos. En el modelo propuesto, Cf no afectó negativamente el desarrollo embrionario. Futuros trabajos serán necesarios para establecer un modelo de infección in vitro en pos de reproducir su patogenicidad.
Subject(s)
Animals , Mice , Blastocyst/microbiology , Campylobacter Infections , Campylobacter fetus/physiology , Embryo, Mammalian/microbiology , Morula/microbiology , Culture Techniques , Mice, Inbred BALB CABSTRACT
A bovine viral diarrhea virus (BVDV) amplification method combined with an enzyme immunoassay was developed to detect BVDV antigens in seropositive cattle. Reconstitution assays conducted by adding decreasing amounts of BVDV (Singer strain) to Madin-Darby bovine kidney (MDBK) cells showed that the sensitivity threshold of the combined assay was 10-7 TCID50. BVDV amplification was carried out in polycation (DEAE-Dextran and polybrene)- treated MDBK cells. Treated cells were able to replicate both ether-treated virus and neutralizing antibody-coated virus. Ammonium chloride decreased virus replication in polycation-treated cells, suggesting viral penetration by endocytosis. BVDV detection was tested in leukocytes from 104 seropositive cattle from 2 unvaccinated commercial closed dairy herds with high seroprevalence. Lysates and co-cultures of peripheral blood leukocytes (PBL) were tested, directly or after up to 6 blind passages in normal or polycation-treated cells. BVDV was detected in 10/104 cattle after only one co-culture of PBL in treated cells. No virus was detected in whole blood or plasma samples. BVDV positive and negative cattle were retested three times, achieving consistent results. The finding of immune carriers supports the possibility that these animals may constitute an epidemiological risk.
Se desarrolló un método de detección de antígenos del virus de la diarrea viral bovina (BVDV) combinando amplificación viral con enzimoinmunoensayo. El método combinado presentó una sensibilidad de 10-7 TCID50 en ensayos con diluciones decrecientes de BVDV cepa Singer sobre la línea celular MDBK. La amplificación del título viral se efectuó sobre células MDBK tratadas con policationes Estas células replicaron tanto el BVDV tratado con éter como el unido a anticuerpos. La replicación viral en las células tratadas disminuyó ante la presencia de cloruro de amonio, lo que sugiere la penetración viral por endocitosis. El BVDV se determinó en leucocitos de 104 bovinos seropositivos de dos rodeos en producción, cerrados y con alta seroprevalencia. Los leucocitos de sangre periférica (LSP) fueron lisados y analizados directamente o luego de hasta 6 pasajes ciegos sobre células normales o tratadas con policationes. El BVDV se detectó en 10 de los 104 animales después de solamente un cultivo de LSP en células tratadas. No se pudo detectar presencia viral en las muestras de sangre o plasma. Los estudios se repitieron tres veces en animales BVDV positivos y negativos, con resultados consistentes. El hallazgo de bovinos seropositivos portadores del virus indica la posibilidad de que estos animales puedan significar un riesgo epidemiológico.
Subject(s)
Animals , Cattle , Female , Bovine Virus Diarrhea-Mucosal Disease/virology , Diarrhea Viruses, Bovine Viral/isolation & purification , Enzyme-Linked Immunosorbent Assay , Virus Cultivation/methods , Blood/virology , Bovine Virus Diarrhea-Mucosal Disease/diagnosis , Cell Line/drug effects , Cell Line/virology , DEAE-Dextran/pharmacology , Hexadimethrine Bromide/pharmacology , Kidney , Plasma/virology , Reproducibility of Results , Reverse Transcriptase Polymerase Chain Reaction , Sensitivity and SpecificityABSTRACT
Se presenta dos casos de carcinoma mucoepidermoide central de la mandíbula y los resultados de la revisión bibliográfica. En ellos se cumplieron los criterios que definen esta afección: 1) corticales intactas; 2) evidencia radiológica de obstrucción ósea; 3) confirmación histopatológica con tinción de mucicarmín. El caso N§ 1 fue tratado con resección parcial de la mandíbula y reconstrucción con injerto de cresta ilíaca; seguido por 14 años sin recidiva local, regional o a distancia. El caso N§ 2 fue tratado con resección marginal mandibular y radioterapia post-operatoria, caso reciente. Se discute aspectos clínicos, patológicos, radiológicos y terapéuticos, los que están en corcondancia con las comunicaciones extranjeras
Subject(s)
Male , Female , Adult , Aged , Carcinoma, Mucoepidermoid/diagnosis , Mandibular Neoplasms/diagnosis , Biopsy/statistics & numerical data , Carcinoma, Mucoepidermoid , Carcinoma, Mucoepidermoid/pathology , Surgical Procedures, OperativeABSTRACT
To test whether the requirements for GPI-attachment are the same in mammalian cells and parasitic protozoa, we expressed the GPI-linked variant surface glycoprotein (VSG) of Trypanosoma brucei (T. brucei) in COS cells. Although large amounts of VSG were produced, only a small fraction became GPI-linked. This impaired processing is not due to the VSG ectodomain since replacement of the VSG GPI-signal with that of decay accelerating factor (DAF) produced GPI-linked VSG. Further, whereas fusion of the DAF GPI-signal to the COOH-terminus of human growth hormone (hGH) produces GPI-linked hGH, an analogous fusion using the VSG GPI-signal does not, indicating that the VSG GPI-signal functions poorly in mammalian cells. By constructing chimeric VSG-DAF GPI-signals and fusing them to the COOH-terminus of hGH, we show that of the two critical elements that comprise the GPI-signal - the cleavage/attachment site and the hydrophobic domain - the former is responsible for the impaired activity of the VSG GPI-signal in COS cells. To confirm this, we show that the VSG GPI-signal can be converted to a viable signal for mammalian cells by altering the amino acid configuration at the cleavage/attachment site. We also show that when fused to hGH, the putative GPI-signal from the malaria circumsporozoite (CS) protein produces low levels of GPI-anchored hGH, suggesting that the CS protein is indeed GPI-linked, but that the CS protein-signal, like the VSG-signal, functions poorly in COS cells