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1.
Reprod Domest Anim ; 49 Suppl 2: 28-32, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24947858

RESUMO

Pyometra is a reproductive disorder very common in bitches over 8 years of age in which physiological effects of progesterone on the uterus play a major role. The traditional therapy for pyometra is ovariohysterectomy. The main advantage of ovariohysterectomy over medical management is that it is both curative and preventive for recurrence of pyometra. However, surgery is associated with the risk of anaesthesia and renders the bitch sterile. During the last 10 years, numerous medical treatments have been proposed to treat both open and closed cervix pyometra. The most effective medical treatment with minor side effects seems to be the repeated administration of aglepristone with or without the additional treatment with low doses of prostaglandins.


Assuntos
Doenças do Cão/tratamento farmacológico , Estrenos/uso terapêutico , Piometra/veterinária , Receptores de Progesterona/antagonistas & inibidores , Animais , Cães , Estrenos/administração & dosagem , Feminino , Prostaglandinas/administração & dosagem , Prostaglandinas/uso terapêutico , Piometra/tratamento farmacológico
2.
Reprod Domest Anim ; 46(1): e1-6, 2011 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-20403133

RESUMO

Maedi Visna virus (MVV) causes progressive degenerative inflammatory disease in multiple organs including the lungs (pneumonia, 'maedi'), mammary gland, joints and nervous system (meningoencephalomyelitis, 'visna') in sheep. Maedi Visna Virus has been detected in macrophages of several tissues and epithelial cells in vivo: bone marrow, cells of the central nervous system, lung and bronchial tissues, milk epithelial cells recovered from milk samples and epithelial cells of mammary tissue. However, the presence of MVV in the genital tracts of naturally infected ewes has not previously been studied. The aim of this study was to use nested-PCR, targeting the gag gene, to determine whether genital tissues (ovaries, oviducts and uterus) from 83 ewes originating from various breeding herds in the South-East of France were positive for MVV-proviral DNA. Peripheral blood mononuclear cells (PBMC) tested positive for MVV-proviral DNA, using nested-PCR analysis, in 57.8% of ewes (48/83). The provirus was also identified in 47% (78/166) of the ovaries, 38.6% (64/166) of the oviducts and 45.8% (38/83) of the uteri sampled. These findings clearly demonstrate, for the first time, that tissue samples from the genital tract of ewes (ovary, oviduct and uterus) can be infected with MVV. This suggests that there is a risk of vertical and/or horizontal transmission of MVV during embryo transfer from embryos produced in vivo or in vitro.


Assuntos
DNA Viral/isolamento & purificação , Genitália Feminina/virologia , Infecções por Lentivirus/veterinária , Provírus/genética , Doenças dos Ovinos/virologia , Vírus Visna-Maedi/genética , Animais , Tubas Uterinas/virologia , Feminino , Infecções por Lentivirus/virologia , Ovário/virologia , Reação em Cadeia da Polimerase/veterinária , Ovinos , Útero/virologia
3.
Reprod Domest Anim ; 45(6): e461-4, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20088851

RESUMO

Pyometra in an inguinal hernia was diagnosed in a 10-year-old intact cross-bred bitch which had had dysorexia, depression and inguinal distension. The hernia contained caudal portions of the two uterine horns, uterine cervix and cranial part of the vagina. As the organs were enlarged and full of pus, manual attempt to push back the uterine horns and the vagina in the abdominal cavity through the inguinal canal was unsuccessful. Herniated uterine horns were ligated and cut in their median portion, so it became possible to remove the cervix and the caudal portion of the horns through the hernial orifice, and the ovaries and the cranial part of the horns through a peritoneal midline incision. This bitch was not intended for breeding purposes and, given the presence of a huge pyometra associated with an inguinal hernia, an ovario-hysterectomy was recommended. Uterine herniation should be considered as a differential diagnosis of a caudal lateral inguinal mass. When pushing the uterus back in the abdominal cavity is impossible, a surgical procedure should be performed to detect ischemia­reperfusion injury and/or a septic risk.


Assuntos
Hérnia Inguinal/veterinária , Piometra/veterinária , Animais , Cães , Feminino , Hérnia Inguinal/patologia , Hérnia Inguinal/cirurgia , Piometra/patologia , Piometra/cirurgia
4.
Reprod Domest Anim ; 44 Suppl 2: 167-9, 2009 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-19754560

RESUMO

To evaluate the efficacy and safety of aglepristone 15 mg kg(-1) for induction of parturition in bitches, 22 pregnant beagle bitches were injected subcutaneously on day 60 post-estimated LH surge, and again 24 h later with aglepristone and subsequently were given 0.15 IU kg(-1) oxytocin at hourly intervals until delivery of the last puppy. Six pregnant beagle bitches were used as a non-treated control group. In the control group, parturition occurred at 63.2 +/- 0.5 days, 29 pups were born and the average expulsion time per puppy was 1.0 +/- 0.6 h. In the treated group, parturition was obtained on average 29.7 +/- 5.6 h after aglepristone administration, 121 pups were born and average expulsion time per pup was 1.1 +/- 0.4 h. The percentage of live puppies, 7 weeks after birth, was 86.1% (25/29) and 86.8% (105/121) for the control and treated groups, respectively. No significant difference was observed between the control and treated groups for the average expulsion time per live puppy and for the percentage of live puppies at birth, 48 h, 7 days or 7 weeks after birth (p > 0.05). This study confirms previous results and demonstrates that the combination of aglépristone and oxytocin can be safely and reliably used to induce parturition in beagle bitches, at 60 days post-estimated LH surge.


Assuntos
Cães , Estrenos/farmacologia , Trabalho de Parto Induzido/veterinária , Ocitócicos/farmacologia , Ocitocina/farmacologia , Animais , Estrenos/administração & dosagem , Feminino , Ocitócicos/administração & dosagem , Ocitocina/administração & dosagem , Gravidez
5.
Theriogenology ; 69(4): 473-80, 2008 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-18082249

RESUMO

The aim of this study was to determine the infectious status of semen and genital tract tissues from male goat naturally infected with the caprine lentivirus. Firstly, polymerase chain reaction (PCR) was used to detect the presence of CAEV proviral-DNA in the circulating mononuclear cells, semen (spermatozoa and non-spermatic cells), and genital tract tissues (testis, epididymis, vas deferens, and vesicular gland) of nine bucks. RT-PCR was used to detect the presence of CAEV viral RNA in seminal plasma. Secondly, in situ hybridization was performed on PCR-positive samples from the head, body, and tail of the epididymis. CAEV proviral-DNA was identified by PCR in the blood cells of 7/9 bucks and in non-spermatic cells of the seminal plasma of 3/9 bucks. No CAEV proviral-DNA was identified in the spermatozoa fraction. The presence of CAEV proviral-DNA in non-spermatic cells and the presence of CAEV in the seminal plasma was significantly higher (p<0.01) in bucks with PCR-positive blood. Two of the three bucks with positive seminal plasma cells presented with at least one PCR-positive genital tract tissue. Proviral-DNA was found in the head (3/9), body (3/9), and tail (2/9) of the epididymis. In situ hybridization confirmed the presence of viral mRNA in at least one of each of these tissues, in the periphery of the epididymal epithelium. This study clearly demonstrates the presence of viral mRNA and proviral-DNA in naturally infected male goat semen and in various tissues of the male genital tract.


Assuntos
Vírus da Artrite-Encefalite Caprina/genética , DNA Viral/análise , Genitália Masculina/virologia , Cabras/virologia , Sêmen/virologia , Animais , Vírus da Artrite-Encefalite Caprina/isolamento & purificação , Hibridização In Situ , Masculino , Reação em Cadeia da Polimerase , Reação em Cadeia da Polimerase Via Transcriptase Reversa
6.
Theriogenology ; 69(4): 408-15, 2008 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-18036653

RESUMO

The aim of this study was to demonstrate that embryo transfer can be used to produce CAEV-free kids from CAEV-infected biological mothers when appropriate procedure is implemented. Twenty-eight goats that had tested positive for CAEV using PCR on vaginal secretions were used as embryo donors. Embryos with intact-ZP were selected and washed 10 times; they were then frozen and used for transfer into CAEV-free recipient goats. Nineteen of the 49 recipient goats gave birth, producing a total of 23 kids. Three blood samples were taken from each recipient goat, 10 days before, during, and 10 days after parturition; these were tested for CAEV antibodies using ELISA and for CAEV proviral DNA using PCR. The mothers were then euthanized. Tissue samples were taken from the lungs, udder, and retromammary and prescapular lymph nodes. The kids were separated from their mothers at birth. Seven of them died. At 4 months of age, 16 kids were subjected to drug-induced immunosuppression. Blood samples were taken every month from birth to 4 months of age; samples were then taken on days 15, 21, and 28 after the start of the immunosuppressive treatment. The kids were then euthanized and tissue samples taken from the carpal synovial membrane, lung tissue, prescapular lymph nodes, inguinal and retromammary lymph nodes, and uterus. All samples from the 19 recipient goats and 23 kids were found to be negative for CAEV antibodies and/or CAEV proviral DNA. Under acute conditions for infection this study clearly demonstrates that embryo transfer can be safely used to produce CAEV-free neonates from infected CAEV donors.


Assuntos
Vírus da Artrite-Encefalite Caprina , Transferência Embrionária/veterinária , Doenças das Cabras/transmissão , Doenças das Cabras/virologia , Infecções por Lentivirus/veterinária , Animais , Vírus da Artrite-Encefalite Caprina/genética , Criopreservação , DNA Viral/análise , Feminino , Doenças das Cabras/prevenção & controle , Cabras , Infecções por Lentivirus/prevenção & controle , Infecções por Lentivirus/transmissão , Reação em Cadeia da Polimerase , Gravidez , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Risco , Coleta de Tecidos e Órgãos/veterinária
7.
Reprod Domest Anim ; 43(4): 429-36, 2008 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-18179634

RESUMO

To improve the results obtained with a reference cryopreservation extender (control extender: Triladyl + 20% (v/v) egg yolk + 6.4% (v/v) glycerol) for freezing caprine semen, glutamine was added to 18 split ejaculates at concentrations of 0, 20, 40, 80 and 120 mM (experiment 1). In experiment 2, glutamine was added to 18 split ejaculates at concentrations of 20, 25, 30, 35 and 40 mM. In the third experiment, the egg yolk was replaced with the low-density lipoprotein (LDL) fraction of egg yolk. The quality of frozen then thawed spermatozoa in each extender was compared using computer-assisted semen analysis. In experiment 1, glutamine at concentrations of 20 mm and 40 mm significantly improved sperm motility compared with the control extender. However, at 120 mM, a significant decrease in motility and velocity was observed. In experiment 2, motility, curvilinear velocity and amplitude of lateral head displacement (ALH) were improved in glutamine at 25 mM compared with the control. In experiment 3, 8% LDL and 25 mM glutamine significantly improved sperm motility, straight line velocity and ALH. In the fourth experiment, the quality of the previously defined freezing extender (Triladyl + 8% (v/v) LDL + 25 mM glutamine + 6.4% (v/v) glycerol) was tested by comparing acrosome, tail membrane, plasma membrane and DNA integrity in 18 split ejaculates of frozen then thawed spermatozoa with spermatozoa that had been frozen then thawed in the control extender, and with spermatozoa from fresh, unfrozen sperm. The percentage of spermatozoa with intact acrosomes and tail membranes was significantly higher with the newly defined extender than that observed with the control extender. There was no significant difference in the percentage of spermatozoa with intact DNA between the frozen and fresh semen.


Assuntos
Criopreservação/veterinária , Glutamina/farmacologia , Cabras , Lipoproteínas LDL/farmacologia , Preservação do Sêmen/veterinária , Sêmen , Acrossomo/efeitos dos fármacos , Acrossomo/fisiologia , Reação Acrossômica/efeitos dos fármacos , Reação Acrossômica/fisiologia , Animais , Criopreservação/métodos , Crioprotetores/farmacologia , Relação Dose-Resposta a Droga , Gema de Ovo/química , Cabras/fisiologia , Masculino , Sêmen/citologia , Sêmen/efeitos dos fármacos , Sêmen/fisiologia , Preservação do Sêmen/métodos , Motilidade dos Espermatozoides/efeitos dos fármacos , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/efeitos dos fármacos , Espermatozoides/fisiologia
8.
Theriogenology ; 106: 259-264, 2018 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-29096274

RESUMO

Previous work demonstrated that after infection of in vivo derived caprine embryos, Coxiella burnetti (C. burnetii) showed a strong tendency to adhere to the zona pellicida (ZP). To investigate the risk of C. burnetii transmission via embryo transfer of in vitro-produced goat embryos the aim of this study was, (i) to evaluate the ability of C. burnetii to adhere to the intact zona pellicida of in vitro-produced goat embryos and to determine by confocal microscopy the location of the bacteria, (ii) to test the efficacy of IETS recommended rules for the washing of bovine embryos to eliminate C. burnetii. One hundred ZP-intact caprine embryos, produced in vitro, at the 8 to 16 cell stage, were randomly divided into 11 batches of eight to nine embryos. Nine batches were incubated for 18 h with 109Coxiella/ml of CbB1 strain (IASP, INRA Tours). The embryos then were recovered and washed in batches in 10 successive baths following the IETS guidelines. In parallel, two batches of embryos were subjected to similar procedures but without exposure to C. burnetii, to serve as the control group. One of the nine batches of infected embryos and one of the two non-infected control batches were separated to perform immunolabeling to locate the bacteria. C. burnetii DNA was detected by C-PCR in all eight batches of infected embryos after 10 successive washings. However, bacterial DNA was not detected in the embryo control batch. The first five washing media of the infected group were consistently found to be positive and Coxiella DNA was detected in the wash bath up to the 10th wash for two batches. After immunolabeling, the observation of embryos under confocal microscopy allowed C. burnetti to be found on the external part of the zona pellucida without deep penetration. This study clearly demonstrates that C. burnetii, after in vitro infection at 109Coxiella/ml, stick strongly to the external part of the zona pellucida of in vitro produced caprine embryos without deap penetration and that the 10 washings protocol recommended by IETS to eliminate the pathogenic agents of bovine embryos is unable to eliminate these bacteria from in vitro-produced goat embryo.


Assuntos
Aderência Bacteriana/fisiologia , Coxiella burnetii/fisiologia , Embrião de Mamíferos/microbiologia , Cabras/embriologia , Zona Pelúcida/microbiologia , Animais , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Microscopia Confocal
9.
Theriogenology ; 67(9): 1485-91, 2007 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-17459463

RESUMO

The objective of this study was to determine whether the 10 wash cycles proposed by the International Embryo Transfer Society (IETS) for bovine embryos efficiently decontaminated equine embryos exposed to equine herpes virus 1 (EHV-1) in vitro. Donor mares and stallions were individually screened and shown to be negative for the virus by PCR detection of EHV-1 DNA in blood leukocytes, semen, and uterine lavages in which embryos were recovered. Twenty embryos were recovered and randomly assigned to one of two groups: 10 embryos were exposed for 24h to infectious EHV-1 at 10(6)TCID(50)/ml, and 10 embryos were used as negative controls. Exposed embryos were washed in accordance with IETS recommendations for ruminant and porcine embryos, before being incubated for 24 h with semiconfluent rabbit kidney (RK13) cells to detect any cytopathic effects (CPE), and finally tested for the presence of EHV-1 viral DNA by PCR. The embryo washing media were also assayed for the virus on RK 13 cells and by PCR. Control embryos were neither exposed to the virus nor washed. EHV-1 was not found in the control embryos, or in the last five washes of the exposed embryos. However, the virus was detected in 7/10 of the embryos exposed to EHV-1 for 24h, as well as in the first five washes of the embryos. The gradual disappearance of EHV-1 from the 10 successive wash solutions from the exposed embryos and the detection of viral DNA in 7/10 washed embryos by PCR, demonstrated that the washing procedure was unable to remove EHV-1 and suggested that EHV-1 could be attached to the acellular layer surrounding embryos (zona pellucida or capsule) or had penetrated the embryo.


Assuntos
Transferência Embrionária/veterinária , Embrião de Mamíferos/virologia , Infecções por Herpesviridae/veterinária , Herpesvirus Equídeo 1/patogenicidade , Doenças dos Cavalos/transmissão , Animais , Feminino , Infecções por Herpesviridae/transmissão , Herpesvirus Equídeo 1/isolamento & purificação , Doenças dos Cavalos/virologia , Cavalos , Masculino , Reação em Cadeia da Polimerase/veterinária , Fatores de Risco
10.
Theriogenology ; 66(6-7): 1550-6, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16563489

RESUMO

The aim of the study was to evaluate the efficacy of aglepristone (10 mg/kg on days 1, 2 and 8) for the treatment of metritis or pyometra in bitches (n = 67) either alone for cases of metritis (n = 15), or in cases of pyometra (n = 52) with (n = 32) or without (n = 20) the addition of low doses (1 microg/kg) of cloprostenol for 5 days (days 3-7). Examinations performed on day 90, in addition to days 8, 14 and 28, determined that treatments had been curative in the long term in 54/67 bitches (80.6%). Bitches in whom pyometra did not resolve, were given additional aglepristone on day 14 (n = 38) and day 28 (n = 20). Aglepristone alone was curative in 15/15 bitches with metritis. In 17/17 bitches with closed pyometra, cervical opening occurred within 48 h of aglepristone administration. Amongst the 52 bitches with open (n = 35) or closed (n = 17) pyometra, the additional treatment with cloprostenol from days 3 to 7, significantly improved the overall success rate at day 90, which was 27/32 (84.4%), compared to 12/20 (60.0%) in bitches without cloprostenol (P < 0.05). The leucocyte count and plasma progesterone concentrations significantly decreased over the course of treatment. Thirteen of 15 bitches in whom plasma progesterone concentrations were initially low (< 3.18 nmol/L) were cured. The recurrence rate after 12 and 24 months was 13.0% (3/23) and 19.0% (4/21), respectively.


Assuntos
Cloprostenol/uso terapêutico , Doenças do Cão/tratamento farmacológico , Doenças do Cão/patologia , Hiperplasia Endometrial/tratamento farmacológico , Hiperplasia Endometrial/veterinária , Estrenos/uso terapêutico , Animais , Doenças do Cão/sangue , Cães , Quimioterapia Combinada , Hiperplasia Endometrial/sangue , Hiperplasia Endometrial/patologia , Endometrite/tratamento farmacológico , Endometrite/patologia , Endometrite/veterinária , Feminino , Contagem de Leucócitos/veterinária , Progesterona/sangue , Doenças Uterinas/tratamento farmacológico , Doenças Uterinas/patologia , Doenças Uterinas/veterinária
11.
Theriogenology ; 66(5): 1131-9, 2006 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-16620938

RESUMO

The aim of this study was to examine the Maedi-Visna virus (MVV) infection status of oocytes, cumulus cells, and follicular fluid taken from 140 ewes from breeding flocks. MVV proviral-DNA and MVV RNA were detected using nested-PCR and RT-PCR MVV gene amplification, respectively in the gag gene. Nested-PCR analysis for MVV proviral-DNA was positive in peripheral blood mononuclear cells in 37.1% (52/140) of ewes and in 44.6% (125/280) of ovarian cortex samples. The examination of samples taken from ovarian follicles demonstrated that 8/280 batches of cumulus cells contained MVV proviral-DNA, whereas none of the 280 batches of oocytes taken from the same ovaries and whose cumulus cells has been removed, was found to be PCR positive. This was confirmed by RT-PCR analysis showing no MVV-viral RNA detection in all batches of oocytes without cumulus cells (0/280) and follicular fluid samples taken from the last 88 ovaries (0/88). The purity of the oocyte fraction and the efficacy of cumulus cell removal from oocytes was proved by absence of granulosa cell-specific mRNA in all batches of oocytes lacking the cumulus cells, using RT-PCR. This is the first demonstration that ewe cumulus cells harbor MVV genome and despite being in contact with these infected-cumulus cells, the oocytes and follicular fluid remain free from infection. In addition, the enzymatic and mechanical procedures we used to remove infected-cumulus cells surrounding the oocytes, are effective to generate MVV free-oocytes from MVV-infected ewes.


Assuntos
Infecções por Lentivirus/veterinária , Lentivirus Ovinos-Caprinos/isolamento & purificação , Oócitos/citologia , RNA Viral/análise , Doenças dos Ovinos/transmissão , Animais , Sequência de Bases , Fragmentação do DNA , Feminino , Líquido Folicular/virologia , Infecções por Lentivirus/genética , Infecções por Lentivirus/transmissão , Oócitos/virologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/veterinária , Ovinos , Doenças dos Ovinos/genética , Visna/genética , Visna/transmissão , Vírus Visna-Maedi/isolamento & purificação
12.
Theriogenology ; 86(6): 1482-1488, 2016 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-27341774

RESUMO

The objectives of this study were to determine (i) whether Chlamydia abortus would adhere to or penetrate the intact zona pellucida (ZP-intact) of early in vivo-derived caprine embryos, after in vitro infection; and (ii) the efficacy of the International Embryo Transfer Society (IETS) washing protocol for bovine embryos. Fifty-two ZP-intact embryos (8-16 cells), obtained from 14 donors were used in this experiment. The embryos were randomly divided into 12 batches. Nine batches (ZP-intact) of five embryos were incubated in a medium containing 4 × 10(7)Chlamydia/mL of AB7 strain. After incubation for 18 hours at 37 °C in an atmosphere of 5% CO2, the embryos were washed in batches in 10 successive baths of a phosphate buffer saline and 5% fetal calf serum solution in accordance with IETS guidelines. In parallel, three batches of ZP-intact embryos were used as controls by being subjected to similar procedures but without exposure to C. abortus. The 10 wash baths were collected separately and centrifuged for 1 hour at 13,000 × g. The washed embryos and the pellets of the 10 centrifuged wash baths were frozen at -20 °C before examination for evidence of C. abortus using polymerase chain reaction. C. abortus DNA was found in all of the infected batches of ZP-intact embryos (9/9) after 10 successive washes. It was also detected in the 10th wash fluid for seven batches of embryos, whereas for the two other batches, the last positive wash bath was the eighth and the ninth, respectively. In contrast, none of the embryos or their washing fluids in the control batches were DNA positive. These results report that C. abortus adheres to and/or penetrates the ZP of in vivo caprine embryos after in vitro infection, and that the standard washing protocol recommended by the IETS for bovine embryos, failed to remove it. The persistence of these bacteria after washing makes the embryo a potential means of transmission of the bacterium during embryo transfer from infected donor goats to healthy recipients and/or their offspring. Nevertheless, the detection of C. abortus DNA by polymerase chain reaction does not prove that the bacteria found was infectious. Further studies are required to investigate whether enzymatic and/or antibiotic treatment of caprine embryos infected by C. abortus would eliminate the bacteria from the ZP.


Assuntos
Infecções por Chlamydia/veterinária , Chlamydia , Transferência Embrionária/veterinária , Doenças das Cabras/embriologia , Doenças das Cabras/microbiologia , Animais , Chlamydia/genética , Chlamydia/isolamento & purificação , Infecções por Chlamydia/transmissão , DNA Bacteriano/análise , Embrião de Mamíferos/microbiologia , Cabras , Reação em Cadeia da Polimerase/veterinária , Zona Pelúcida/microbiologia
13.
Theriogenology ; 64(7): 1656-66, 2005 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-15885763

RESUMO

The aim of this study was to determine whether oocytes taken from ovarian follicles in 123 naturally infected goats were carrying the proviral CAEV genome. Examination of DNA isolated from 190 batches of oocytes with intact cumulus cells and 190 batches of oocytes whose cumulus cells had been removed, taken from follicles of the same ovaries, demonstrated that 42/190 batches of oocytes with intact cumulus cells had the proviral CAEV genome, whereas none of the 190 batches of oocytes without cumulus cells were positive for the provirus. To confirm that the proviral genome was present in the cumulus cells and not in the oocyte cells, 586 oocytes from 56 different ovaries, were separated from their cumulus cells. The DNA was then extracted from each fraction and examined. The purity of the oocyte fraction was verified by searching for granulosa cell-specific mRNA, using RT-PCR; this was negative in all the batches of oocytes in which the cumulus cells had been removed. PCR analysis demonstrated that none of the oocytes without cumulus cells were positive, whereas 22/56 of the batches with cumulus cells were found to be positive. This study clearly demonstrates that despite being surrounded by infected cumulus cells, the oocytes are not infected, and that the enzymatic and mechanical technique for removing the cells surrounding the oocyte, as used in this study, is effective, thus enabling CAEV-free oocytes to be obtained from infected goats.


Assuntos
Vírus da Artrite-Encefalite Caprina/genética , DNA Viral/análise , Doenças das Cabras/virologia , Infecções por Lentivirus/veterinária , Oócitos/virologia , Folículo Ovariano/virologia , Animais , Feminino , Cabras , Infecções por Lentivirus/transmissão , Infecções por Lentivirus/virologia , Reação em Cadeia da Polimerase , Provírus/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa
14.
Arch Ital Biol ; 143(3-4): 199-206, 2005 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-16097496

RESUMO

Taste receptor cells (TRCs) represent an unique opportunity to study a dynamic population of excitable cells that undergoes two basic neurobiological processes: postnatal development and cell turnover. We have begun to investigate the functional properties of TRCs and how they mature over time by applying the patch-clamp technique to single cell in taste buds isolated from mouse vallate papilla during postnatal development. We have focussed our attention on a well-defined functional group of taste cells, called Na/OUT cells, and on their voltage-gated K+, and Cl- currents (I(K) and I(Cl), respectively). As in neurons, I(K) and I(Cl) underlie action potential waveform and firing properties in these cells. By analyzing the relative occurrence of I(K) and I(Cl) among cells, we found that in adult mice three different electrophysiological phenotypes of Na/OUT cells could be detected: cells with only I(K) (K cells); cells with both I(K) and I(Cl) (K + Cl cells); and cells with I(Cl) (Cl cells). On the contrary, at early developmental stages (2-4 postnatal day, PD) there were no Cl cells, which appeared at PD 8. The analysis of the changes in current amplitude (which continuously increased in developing cells) during postnatal development suggested that Cl cells and K + Cl cells likely represented a single functional line different from K cells. In addition, electrophysiological data were consistent with the interpretation that Cl cells derived from some K + Cl cells by suppression of I(K). The dynamics of the expression of I(K) and I(Cl) during postnatal development likely reflects a mechanism that could also operate during turnover.


Assuntos
Diferenciação Celular/fisiologia , Membrana Celular/fisiologia , Canais Iônicos/fisiologia , Papilas Gustativas/fisiologia , Potenciais de Ação/fisiologia , Envelhecimento/fisiologia , Animais , Diferenciação Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Canais de Cloreto/efeitos dos fármacos , Canais de Cloreto/fisiologia , Técnicas In Vitro , Canais Iônicos/efeitos dos fármacos , Camundongos , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/efeitos dos fármacos , Canais de Potássio/fisiologia , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Canais de Sódio/fisiologia , Papilas Gustativas/citologia , Papilas Gustativas/efeitos dos fármacos
15.
Virus Res ; 79(1-2): 165-72, 2001 Nov 05.
Artigo em Inglês | MEDLINE | ID: mdl-11551657

RESUMO

Recent reports demonstrated the susceptibility of epithelial cells from different organs to caprine arthritis-encephalitis virus (CAEV) both in vitro and in vivo. Since granulosa cells (GC) are of epithelial origin and currently used for in vitro oocyte maturation, we addressed the question whether these cells are susceptible or resistant to CAEV infection. GC were isolated from goats from certified CAEV-free herds. PCR analysis on GC DNA using CAEV specific primers confirmed the absence of CAEV infection and immunocytochemistry using specific K813 anti-cytokeratin monoclonal antibodies confirmed the epithelial nature of GC. These cells were then inoculated with CAEV using two strains: the CAEV-pBSCA molecular clone and the CAEV-3112 French field isolate. Cytopathic effects (CPE) were observed on cell culture monolayers inoculated with both CAEV strains. Expression of CAEV proteins was shown both by immunocytochemistry using anti-p24 gag specific antibodies and by immunoprecipitation using an hyperimmune serum. Supernatant of infected cells were shown to contain high titers (ranging 10(5) tissue culture infectious doses 50 per ml: TCID(50) per ml) of infectious cytopathic viruses when assayed onto the indicator goat synovial membrane (GSM) cells. Our findings demonstrate the large cell tropism of CAEV and suggest that GC could serve as a reservoir for the virus during the sub-clinical phase of infection. Furthermore, given the high seroprevalence of CAEV in the all industrialised countries and the large number of ovaries derived from unknown serological status animals used for in vitro goat embryo production, one can conclude that these feeder cell cultures might be a potential source of early transmission of CAEV to goat embryos.


Assuntos
Vírus da Artrite-Encefalite Caprina/fisiologia , Células da Granulosa/virologia , Animais , Vírus da Artrite-Encefalite Caprina/genética , Vírus da Artrite-Encefalite Caprina/metabolismo , Células Cultivadas , Feminino , Cabras , Células da Granulosa/citologia , Replicação Viral
16.
Virus Res ; 87(1): 69-77, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12135791

RESUMO

Caprine oviduct epithelial cells (COEC) are commonly used in in vitro goat embryo production protocols to stimulate early embryonic development. These feeder cells are usually collected from slaughterhouses from unknown serological status animals for caprine arthritis-encephalitis virus (CAEV) infection which is frequent in many regions of the world. Tissues derived from this source may be contaminated with CAEV and the use of such material in in vitro fertilisation systems may contribute to transmission of this pathogen to the cultured embryos and dissemination via embryo transfer (ET). The aim of this study was to determine the permissiveness of COEC to CAEV replication in vitro. Cells were isolated from goats from certified CAEV-free herds and then were inoculated with two CAEV strains: the molecularly-cloned isolate of CAEV (CAEV-pBSCA) and the French field isolate (CAEV-3112). Cytopathic effects (CPE) were observed on cell culture monolayers inoculated with both CAEV strains. Expression of CAEV proteins was shown both by immunocytochemistry using anti-p24 gag specific antibodies and by immunoprecipitation using a hyperimmune serum. The CAEV proteins were correctly and properly processed by artificially-infected COEC and the titers of virus released into the supernatant reached 10(6) TCID(50)/ml 6 days post-inoculation. Although the macrophage lineage cells are the main centre of infection in the virus-positive animal, these findings suggest that epithelial cells may be important in the viral life cycle probably as a reservoir allowing the viral persistence, dissemination and pathogenesis. These results suggest also that the use in in vitro fertilisation systems of co-culture feeder cells that support efficient replication of CAEV to high titers could represent a serious risk for permanent transmission of virus to the cultured embryos and to the surrogate dam involved.


Assuntos
Vírus da Artrite-Encefalite Caprina/crescimento & desenvolvimento , Tubas Uterinas/citologia , Animais , Células Cultivadas , Células Epiteliais/virologia , Feminino , Cabras
17.
Naunyn Schmiedebergs Arch Pharmacol ; 363(6): 680-94, 2001 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-11414663

RESUMO

Somatostatin (somatotropin release-inhibiting factor, SRIF) receptor subtypes are expressed by several retinal neurons, suggesting that SRIF acts at multiple levels of the retinal circuitry, although functional data on this issue are scarce. Of the SRIF receptors, the sst2A isoform is expressed by rod bipolar cells (RBCs) of the rabbit retina, and in isolated RBCs we studied the role of sst2 receptors in modulating both K+ current (IK) and the intracellular free [Ca2+] ([Ca2+]i) using both voltage-clamp and Ca2+-imaging techniques. SRIF and octreotide (a SRIF agonist that binds to sst2 receptors) inhibited that component of IK corresponding to the activation of large-conductance, Ca2+- and voltage-dependent K+ channels (IBK) and reduced the K+-induced [Ca2+]i accumulation, suggesting that SRIF effects on IBK may have been secondary to inhibition of Ca2+ channels. Octreotide effects on IBK or on [Ca2+]i accumulation were prevented by RBC treatment with L-Tyr8-Cyanamid 154806, a novel sst2 receptor antagonist, indicating that SRIF effects were mediated by sst2 receptor activation. The present data indicate that SRIF may modulate the information flow through second-order retinal neurons via an action predominantly at sst2 receptors, contribute to the proposition that SRIF be added to the growing list of retinal neuromodulators, and suggest that one of its possible roles in the retina is to regulate transmitter release from RBCs.


Assuntos
Cálcio/metabolismo , Receptores de Somatostatina/fisiologia , Células Fotorreceptoras Retinianas Bastonetes/efeitos dos fármacos , Somatostatina/farmacologia , Animais , Eletrofisiologia , Octreotida/farmacologia , Técnicas de Patch-Clamp , Coelhos , Receptores de Somatostatina/agonistas , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células Fotorreceptoras Retinianas Bastonetes/patologia
18.
Acta Neurol Belg ; 92(2): 83-7, 1992.
Artigo em Inglês | MEDLINE | ID: mdl-1604963

RESUMO

We report the second case of thrombotic thrombocytopenic purpura occurring during ticlopidine therapy. A cerebral hemorrhage was the first sign of the thrombocytopenia. Even if thrombocytopenia is a rare occurrence with ticlopidine therapy, we stress that platelets must be repeatedly monitored.


Assuntos
Hemorragia Cerebral/etiologia , Transtornos Puerperais/induzido quimicamente , Púrpura Trombocitopênica Trombótica/induzido quimicamente , Ticlopidina/efeitos adversos , Adulto , Terapia Combinada , Feminino , Humanos , Troca Plasmática , Púrpura Trombocitopênica Trombótica/complicações , Púrpura Trombocitopênica Trombótica/terapia
19.
Theriogenology ; 57(2): 931-40, 2002 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-11991395

RESUMO

To improve the knowledge on the risk of transmission of the caprine arthritis-encephalitis virus (CAEV) during embryo manipulations, we conducted a double-nested polymerase chain reaction (PCR) for CAEV proviral-DNA on flushing media recovered from the oviducts 48 h after the beginning of estrus and on blood from 89 donor does. Sixty-four does had negative blood and flushing media by PCR. Among the 25 CAEV infected goats (blood PCR positive), 11 were PCR flushing media positive (P < 0.01). Cell lysate from flushing media samples that were PCR positive were serially diluted 10 times at 1:100. Starting with the second 1:100 dilution all the cell lysate samples were PCR negative. The mean number of embryos recovered was not significantly different between goats with flushing media PCR positive and goats with flushing media PCR negative (6.0 +/- 5.4 versus 7.8 +/- 4.4, respectively; mean +/- S.D.) nor between goats with blood PCR positive and goats with blood PCR negative (7.0 +/- 5.0 versus 5.9 +/- 5.3; mean +/- S.D.). The presence of CAEV infected cells in oviductal flushing media from infected donor does was indicated for the first time during this study. The absence of flushing media PCR positive for goat blood PCR negative seemed to allow the use of the blood PCR test to confidently predict the absence of CAEV provirus in the oviductal fluid.


Assuntos
Vírus da Artrite-Encefalite Caprina/isolamento & purificação , Embrião de Mamíferos , Tubas Uterinas/citologia , Cabras/virologia , Animais , Vírus da Artrite-Encefalite Caprina/genética , DNA Viral/análise , Eletroforese em Gel de Ágar , Feminino , Transmissão Vertical de Doenças Infecciosas , Infecções por Lentivirus/transmissão , Infecções por Lentivirus/veterinária , Infecções por Lentivirus/virologia , Leucócitos Mononucleares/virologia , Reação em Cadeia da Polimerase , Coleta de Tecidos e Órgãos/métodos , Coleta de Tecidos e Órgãos/veterinária
20.
Theriogenology ; 59(7): 1515-23, 2003 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-12559456

RESUMO

Transmission of caprine arthritis-encephalitis virus (CAEV) is not completely understood and the vertical route of infection from the goat to the embryo or to the fetus needs to be investigated. This route of infection involves the presence of CAEV in the genital tract tissues. Prior studies have detected CAEV-infected cells in genital secretions and in flushing media recovered during embryo collection from infected goats. To specify the origin of these cells, we conducted a double-nested polymerase chain reaction (PCR) test on embryo flushing media and on mammary gland, mammary lymph node, synovial membrane, pelvic lymph node, uterus and oviduct tissues from 25 CAEV-infected (blood PCR positive) embryo donor goats for the presence of CAEV proviral DNA. The presence of proviral DNA was found in 22 of 25 mammary gland samples, 14 of 25 uterus samples, and in 16 of 25 oviduct samples. Nineteen of 25 goats had at least one positive genital tract sample. Flushing media from 11 goats were PCR positive. All goats with positive-flushing media were oviduct positive. Of this group of does, except for 1 of the 11, infection of flushing media correlated with infection of almost all the other tissues examined. The frequency of positive tissues for flushing media-positive goats (61/66; 92%) was significantly higher than that for flushing media-negative goats (50/84; 60%) (P<0.01). This study demonstrated the presence of CAEV-infected cells in the goat genital tract. The presence of CAEV-infected cells in the uterus and oviducts suggests potential for vertical transmission of CAEV from doe to embryo or fetus.


Assuntos
Vírus da Artrite-Encefalite Caprina/isolamento & purificação , DNA Viral/análise , Doenças das Cabras/transmissão , Infecções por Lentivirus/veterinária , Provírus/genética , Animais , Vírus da Artrite-Encefalite Caprina/genética , Sequência de Bases , DNA Viral/isolamento & purificação , Feminino , Amplificação de Genes , Doenças das Cabras/virologia , Cabras , Transmissão Vertical de Doenças Infecciosas/veterinária , Infecções por Lentivirus/transmissão , Infecções por Lentivirus/virologia , Glândulas Mamárias Animais/virologia , Oviductos/virologia , Reação em Cadeia da Polimerase/veterinária , Gravidez , Provírus/isolamento & purificação , Superovulação , Coleta de Tecidos e Órgãos/veterinária , Útero/virologia
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