RÉSUMÉ
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.
Sujet(s)
Infections à Chlamydia/médecine vétérinaire , Chlamydia , Transfert d'embryon/médecine vétérinaire , Maladies des chèvres/embryologie , Maladies des chèvres/microbiologie , Animaux , Chlamydia/génétique , Chlamydia/isolement et purification , Infections à Chlamydia/transmission , ADN bactérien/analyse , Embryon de mammifère/microbiologie , Capra , Réaction de polymérisation en chaîne/médecine vétérinaire , Zone pellucide/microbiologieRÉSUMÉ
The detection of significant bacterial loads of Coxiella burnetii in flushing media and tissue samples from the genital tracts of nonpregnant goats represents a risk factor for in utero infection and transmission during embryo transfer. The aim of this study was to investigate (1) whether cells of early goat embryos isolated from in vivo-fertilized goats interact with C. burnetii in vitro, (2) whether the embryonic zona pellucida (ZP) protects early embryo cells from infection, and (3) the efficacy of the International Embryo Transfer Society (IETS) washing protocol for bovine embryos. The study was performed in triple replicate: 12 donor goats, certified negative by ELISA and polymerase chain reaction, were synchronized, superovulated, and subsequently inseminated by Q fever-negative males. Sixty-eight embryos were collected 4 days later by laparotomy. Two-thirds of the resulting ZP-intact and ZP-free 8- to 16-cell embryos (9-9, 11-11, and 4-4 in replicates 1, 2, and 3, respectively) were placed in 1 mL minimum essential medium containing 10(9)C. burnetii CBC1 (IASP, INRA Tours). After overnight incubation at 37 °C and 5% CO2, the embryos were washed according to the IETS procedure. In parallel, the remaining third ZP-intact and ZP-free uninfected embryos (3-3, 5-5, and 2-2 in replicates 1, 2, and 3, respectively) were subjected to the same procedures, but without C. burnetii, thus serving as controls. The 10 washing fluids for all batches of each replicate were collected and centrifuged for 1 hour at 13,000 × g. The washed embryos and pellets were tested by polymerase chain reaction. Coxiella burnetii DNA was found in all batches of ZP-intact and ZP-free infected embryos after 10 successive washes. It was also detected in the first five washing fluids for ZP-intact embryos and in the first eight washing fluids for ZP-free embryos. None of the control batches (embryos and washing fluids) were found to contain bacterial DNA. These results clearly indicate that caprine early embryonic cells are susceptible to infection by C. burnetii. The bacterium shows a strong tendency to adhere to the ZP after in vitro infection, and the washing procedure recommended by the IETS for bovine embryos failed to remove it. The persistence of these bacteria makes the embryo a potential means of transmission to recipient goats. Further studies are needed to investigate whether the enzymatic treatment of caprine embryos infected by C. burnetii would eliminate the bacteria from the ZP.
Sujet(s)
Coxiella burnetii , Transfert d'embryon/méthodes , Capra , Fièvre Q/prévention et contrôle , Fièvre Q/transmission , Animaux , Coxiella burnetii/isolement et purification , Transfert d'embryon/médecine vétérinaire , Embryon de mammifère , Femelle , Maladies des chèvres/épidémiologie , Maladies des chèvres/prévention et contrôle , Maladies des chèvres/transmission , Capra/embryologie , Capra/microbiologie , Mâle , Grossesse , Fièvre Q/épidémiologie , Fièvre Q/médecine vétérinaire , SuperovulationRÉSUMÉ
The objective of this study was to investigate methods of decontaminating early goat embryos that had been infected in vitro with bluetongue virus (BTV). Embryos were isolated from in vivo-fertilized BTV-free goats. Zona pellucida (ZP)-intact 8 to 16 cell embryos were cocultured for 36 h in an insert over a Vero cell monolayer infected with BTV serotype 8. The embryos were then treated with one of five different washing procedures. The treatment standard (TS) comprised phosphate-buffered saline (PBS) + 0.4% BSA (five times over for 10 s), Hank's +0.25% trypsin (twice for 45 s), and then PBS + 0.4% BSA again (five times for 10 s). The four other washing procedures all included the same first and last washing steps with PBS but without BSA (five times for 10 s) and with PBS + 0.4% BSA (five times for 10 s), respectively. The intermediate step varied for each washing procedure. Treatment 1 (T1): 0.25% trypsin (twice for 45 s). Treatment 2 (T2): 0.25% trypsin (twice for 60 s). Treatment 3 (T3): 0.5% trypsin (twice for 45 s). Treatment 4 (T4): 1% hyaluronidase (once for 5 min). After washing, the embryos were transferred and cocultured with BTV indicator Vero cell monolayers for 6 h, to detect any cytopathic effects (CPE). The effectiveness of the different washing techniques in removing the virus was evaluated by RT-qPCR analysis. The TS, T1, T3, and T4 trypsin or hyaluronidase treatments did not eliminate BTV; Treatment 2 eliminated the virus from in vitro infected goat embryos.
Sujet(s)
Virus de la langue bleue , Fièvre catarrhale du mouton/prévention et contrôle , Décontamination/méthodes , Embryon de mammifère/virologie , Capra/embryologie , Animaux , Fièvre catarrhale du mouton/transmission , Virus de la langue bleue/génétique , Chlorocebus aethiops , Techniques de coculture/médecine vétérinaire , Techniques de culture d'embryons/méthodes , Techniques de culture d'embryons/médecine vétérinaire , Transfert d'embryon/méthodes , Transfert d'embryon/médecine vétérinaire , ARN viral/analyse , Prélèvement d'organes et de tissus/médecine vétérinaire , Cellules VeroRÉSUMÉ
The three objectives of this study were to investigate whether cells of early goat embryos isolated from in vivo fertilized goats interact with bluetongue virus (BTV) in vitro, whether the embryonic zona pellucida (ZP) protects early embryo cells from BTV infection, and whether the 10 wash cycles recommended by the International Embryo Transfer Society (IETS) for bovine embryos effectively decontaminates caprine embryos exposed to Bluetongue Virus (BTV) in vitro. Donor goats and bucks were individually screened and tested negative for the virus by RT-PCR detection of BTV RNA in circulating erythrocytes. ZP-free and ZP-intact 8-16 cell embryos were co-cultured for 36 h in an insert over a Vero cell monolayer infected with BTV. Embryos were washed 10 times in accordance with IETS recommendations for ruminant and porcine embryos, before being transferred to an insert on BTV indicator Vero cells for 6 h, to detect any cytopathic effects (CPE). They were then washed and cultured in B2 Ménézo for 24 h. Non-inoculated ZP-free and ZP-intact embryos were submitted to similar treatments and used as controls. The Vero cell monolayer used as feeder cells for BTV inoculated ZP-free and ZP-intact embryos showed cytopathic effects (CPE). BTV was found by RT-qPCR in the ten washes of exposed ZP-free and ZP-intact embryos. In the acellular medium, the early embryonic cells produced at least 10(2.5) TCID(50)/ml. BTV RNA was detected in ZP-free and ZP-intact embryos using RT-qPCR. All of these results clearly demonstrate that caprine early embryonic cells are susceptible to infection with BTV and that infection with this virus is productive. The washing procedure failed to remove BTV, which indicates that BTV could bind to the zona pellucida.
Sujet(s)
Virus de la langue bleue/pathogénicité , Fièvre catarrhale du mouton/transmission , Transfert d'embryon/médecine vétérinaire , Embryon de mammifère/virologie , Maladies des chèvres/transmission , Animaux , Virus de la langue bleue/génétique , Virus de la langue bleue/isolement et purification , Chlorocebus aethiops , Techniques de culture d'embryons/médecine vétérinaire , Transfert d'embryon/méthodes , Femelle , Maladies des chèvres/virologie , Capra , Mâle , ARN viral/sang , RT-PCR , Cellules Vero , Zone pellucide/virologieRÉSUMÉ
Twenty sperm samples from five dogs were frozen in liquid nitrogen at -196 degrees C in 16 different media, two control media containing 20% egg yolk and 6% low-density lipoproteins (LDL); 10 test media containing 6% LDL (the active cryoprotective ingredient of chicken egg yolk) combined with 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 mmol of glutamine respectively at 4%, 5%, 7%, and 8% LDL. Following thawing, sperm mobility was assessed using an image analyser, HAMILTON THORN CERROS 12. The percentage of mobile spermatozoa was 62.05% in the 6% LDL + 20 mmol glutamine medium compared with 48.90% in the egg yolk-based medium (p < 0.05) or 57.55% for the 6% LDL medium (p < 0.05). Furthermore, in most cases, the motility parameters (average path velocity, curvilinear velocity, straight line velocity) in the 6% LDL + 20 mmol glutamine medium, were superior, to a statistically significant extent, to those in the control media. Finally, the 6% LDL + 20 mmol glutamine combination provides spermatozoa with better protection during freezing than egg yolk or the 6% LDL medium alone in terms of acrosome integrity (fluorescein isothiocyanate--Pisum sativum agglutinin test: p < 0.05), the flagellar plasma membrane (hypo-osmotic test: p < 0.05 for 6% LDL), the DNA (acridine orange test; no significant difference) and the integrity of the acrosome (Spermac test: no significant difference).
Sujet(s)
Cryoconservation/médecine vétérinaire , Chiens/physiologie , Glutamine/pharmacologie , Lipoprotéines LDL/pharmacologie , Conservation de semence/médecine vétérinaire , Animaux , Cryoconservation/méthodes , Cryoprotecteurs/pharmacologie , Relation dose-effet des médicaments , Mâle , Conservation de semence/méthodes , Spermatozoïdes/cytologie , Spermatozoïdes/effets des médicaments et des substances chimiques , Spermatozoïdes/physiologieRÉSUMÉ
A medium containing LDL (Low Density Lipoproteins, the cryoprotective component of chicken egg yolk) was compared with egg yolk for the preservation canine spermatozoa during the freeze-thaw process. Twenty sperm samples taken from 10 dogs were frozen in liquid nitrogen at -196 degrees C in seven different media: one control medium containing 20% egg yolk, and six test media containing 4%, 5%, 6%, 7%, 8%, and 10% LDL, respectively. Following thawing, sperm motility was assessed using a Hamilton-Thorne Sperm Analyser equipped with the CEROS 12 software. The percentage of motile spermatozoa was 55.3% in the 6% LDL medium (optimal concentration) compared with 27.7% in the egg yolk based medium (p<0.05). In comparison with the egg-yolk medium, the LDL medium also resulted in an improved preservation of spermatozoa during the freezing process (p<0.05) in terms of acrosomal integrity (FITC-PSA test), flagellar plasma membrane integrity (HOS test), and DNA integrity (Acridine Orange test). In addition, six Beagle bitches were inseminated twice, via the intra-uterine route, at an interval of 24h; 200x10(6) spermatozoa that had been previously frozen in the 6% LDL medium were used per insemination. All of the bitches became pregnant (gestation rate of 100%). In conclusion, the 6% LDL medium provides improved protection of the spermatozoa during the freeze-thaw process and a marked improvement in the motility parameters of canine spermatozoa in comparison with the control medium containing egg yolk alone. Finally, the use of LDL as a cryoprotectant for canine semen does not interfere with fertility.
Sujet(s)
Cryoconservation/médecine vétérinaire , Chiens/physiologie , Lipoprotéines LDL/pharmacologie , Conservation de semence/médecine vétérinaire , Sperme/effets des médicaments et des substances chimiques , Orange acridine , Acrosome/effets des médicaments et des substances chimiques , Animaux , Cryoconservation/méthodes , Altération de l'ADN , Jaune d'Åuf , Femelle , Fécondité , Insémination artificielle/médecine vétérinaire , Mâle , Grossesse , Sperme/physiologie , Conservation de semence/méthodesRÉSUMÉ
Zona-pellucida-free embryos at 8-16 cell stage were co-cultured for 6 days in an insert over a mixed cell monolayer infected with CAEV-pBSCA. Embryos were washed and transferred to an insert on CAEV indicator goat synovial membrane cells for 6 h, then they were washed and cultivated in B2 Ménézo for 24 h, finally, embryo cells were dissociated and cultivated on a feeder monolayer for 8 days. After 5 weeks, multinucleated giant cells typical of CAEV infection were observed in indicator GSM cell monolayers. In the acellular medium, the early embryonic cells produced at least 10(3.25) TCID50/ml over 24 h. The monolayer of cultivated embryonic cells developed cytopathic lesions within 8 days, and CAEV RNA, CAEV proviral DNA and protein p28 of the capsid were detected. All of these results clearly demonstrate that caprine early embryonic cells are susceptible to infection with CAEV and that infection with this virus is productive.
Sujet(s)
Virus de l'arthrite-encéphalite caprine/croissance et développement , Infections à lentivirus/virologie , Animaux , Virus de l'arthrite-encéphalite caprine/composition chimique , Virus de l'arthrite-encéphalite caprine/génétique , Protéines de capside/métabolisme , Cellules cultivées , Techniques de coculture , ADN viral/génétique , Embryon de mammifère/virologie , Génome viral/génétique , Capra , Immunohistochimie , Réaction de polymérisation en chaîne , Provirus/génétique , Provirus/isolement et purification , Sensibilité et spécificité , Réplication viraleRÉSUMÉ
The post-antibiotic effect in vitro (PAE) of cephalexin was determined according to a broth dilution method against 5 isolates of Staphylococcus intermedius obtained from cases of canine pyoderma. Two durations of exposure and 3 concentrations were tested. The PAE increased when time of exposure or concentration increased. The mean PAE ranged from 0.7 to 3.3 h. The PAE of cephalexin against Staph-ylococcus intermedius may be clinically relevant when selecting a dosage regimen to treat pyoderma in dogs.
RÉSUMÉ
Samples from gingival scrapings of dogs were examined for the presence of CDC Groups EF-4 bacteria. Isolation procedures were performed in 5% sheep blood agar supplemented with thiostrepton and trimethoprim (10 mg/l). Fifty nine EF-4 strains were isolated from 92% of 49 dogs. Among the Group EF-4 bacteria, the majority of isolates belonged to the arginine-negative (biovar "b") Group EF-4 (42 strains recovered in 82% of dogs). Seventeen arginine-positive strains (biovar "a") were recovered only from 35% of dogs. The strains were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The analysis of electrophoretic protein pattern of these bacteria supported the results of conventional testing, confirmed the distinction between the biovars "a" and "b" of Group EF-4 and supported the division of biovar EF-4b into two subgroups of either producing or non-producing acid from gluconate.
Sujet(s)
Chiens/microbiologie , Gencive/microbiologie , Bactéries à Gram négatif/isolement et purification , Animaux , Électrophorèse sur gel de polyacrylamide/méthodes , Femelle , Bactéries à Gram négatif/croissance et développement , Humains , MâleRÉSUMÉ
The serological response to Mycobacterium bovis BCG vaccination was studied in 3 cats, 3 dogs and 3 puppies. The animals received two doses of 0.1 mg of BCG and were studied over a period of 8 months simultaneously by ELISA with antigen A60 (Anda Biologicals, Strasbourg, France) and immunoblotting with BCG. The two methods detected an increase of antibodies at 3 or 5 weeks. In the cats, specific antibody titer remained high and stable for more than one year, in the dogs they diminished quickly within 23 weeks. Carnivores elicit a serological response against specific protein antigens in the bands corresponding to 18 and 25kDa, 30 and 45kDa, and especially 35 and 42kDa bands, and to minor bands at higher molecular weight. Non-specific bands at 50-55 kDa and 70 kDa, assigned to heat shock proteins, were enhanced by BCG vaccination. Cat immunosera recognized on A60 immunoblots the specific homologous bands at 20-25 kDa and 32 kDa, but also two other dominant bands: one was partially specific (65 kDa) and the other was absent from the M. bovis and M. paratuberculosis profiles. The suitability of A60 ELISA for detection of mycobacterial infection in carnivores may be highlighted by immunoblot analysis.
Sujet(s)
Anticorps antibactériens/analyse , Antigènes bactériens/immunologie , Vaccin BCG/immunologie , Chats/immunologie , Chiens/immunologie , Vaccination/médecine vétérinaire , Animaux , Test ELISA , Immunotransfert , Masse moléculaireRÉSUMÉ
Gingival scrapings of 62 dogs and cats were examined for the presence of Pasteurella. Isolation was performed in a medium supplemented with thiostrepton. Twenty-eight and 37 strains were obtained from 21 dogs and 26 cats, respectively, and classified in recently described species or subspecies of the genus Pasteurella (P.): P. multocida subspecies multocida and septica, P. canis, P. dagmatis and P. stomatis. Twenty-one strains were classified as atypical P. stomatis and one strain obtained from a cat remained unclassified. All strains were susceptible to the antibiotics studied. P. multocida and P. stomatis (including atypical strains) represented 65 and 30% of feline isolates, and 14 and 68% of canine isolates, respectively. Assuming that P. multocida, P. canis and P. dagmatis are potentially pathogenic for humans, and that P. stomatis has a low pathogenicity or non-pathogenic, 77 and 28% of examined cats and dogs harboured one or several pathogenic strains. This difference could explain the fact that Pasteurella infections in man are lower in dog bites rather than cat bites.