Contrasting effects of heat shock during in vitro maturation on development of in vitro‐fertilized and parthenogenetic bovine embryos

This study investigated the influence of heat shock during in vitro maturation on embryo development following in vitro fertilization (IVF) or parthenogenesis (Part). Immature bovine cumulus–oocyte complexes were exposed to heat shock (41.0°C) during the first 12 hr of in vitro maturation (IVM), followed by 12 hr at 38.5°C. Control group consisted of in vitro maturation for 24 hr at 38.5°C. Oocytes were in vitro‐fertilized or activated with ionomycin and cultured in vitro for 192 hr post‐in vitro insemination or parthenogenetic activation (hpia). There was an interaction (p < .01) between temperature of IVM and method of oocyte activation (IVF or Part) for cleavage at 48 hpia. Heat shock had a negative impact (p < .01) on cleavage of IVF embryos, whereas no (p > .05) effect was found in the Part embryos. Embryo development towards blastocyst stage at 168 and 192 hpia decreased in both IVF and Part embryos derived from heat‐shocked oocytes. Heat shock increased (p < .05) the apoptotic index in Part blastocysts, but no effect (p > .05) was found in IVF counterparts. Heat shock also down‐regulated the expression of AQP3 (p < .01) and up‐regulated the expression of HSP70.1 (p < .01) in Part blastocysts, whereas it down‐regulated the expression of ATP1A1 (p < .05) in IVF blastocysts. In conclusion, the effects of heat shock during IVM on early embryo cleavage and blastocyst apoptosis are influenced by the method of oocyte activation and expression of some genes can be disturbed in embryos derived from heat‐shocked oocytes.     

Characterisation of the 33kDa piroplasm surface antigen of Theileria orientalis/sergenti/buffeli isolates from West Java, Indonesia.

The immunodominant 33/35kDa antigen of a Theileria isolate from West Java, Indonesia, was characterised and immuno-affinity purified by use of a monoclonal antibody, KUL-a4, and was shown to be representative of the T. orientalis/sergenti/buffeli group. The aminoterminal sequence of the purified 35kDa peptide (20 residues) was determined by automated Edman degradation and found to correspond to the predicted amino acid sequence of a prospective p33 gene previously sequenced from the same isolate. The cleavage site of a putative signal peptide was identified and conforms the (-3, -1) rule for signal peptidases. The existence of dimeric and trimeric forms of the p33/35 antigen is hypothesised from Western blot profiles. KUL-a4 appeared specific for the T. orientalis/sergenti/buffeli group. It did not recognise in indirect fluorescence antibody test (IFAT), intraerythrocytic bodies of Anaplasma marginale or piroplasms and schizonts of T. mutans, T. parva and T. annulata, whereas cattle antisera raised to these species showed cross-reactivity in IFAT. It however, appeared weakly cross-reactive in Western blot and ELISA, with the 34kDa piroplasm antigen of one T. annulata (Gharb) isolate. The present study indicates that the isolated antigen belongs to the p33/34 antigen family described within the T. sergenti/orientalis/buffeli group, and documents the group-specificity of one of its epitopes.     

Bartonella species and their ectoparasites: selective host adaptation or strain selection between the vector and the mammalian host?

A wide range of blood-sucking arthropods have either been confirmed or are suspected as important vectors in Bartonella transmission to mammals, including humans. Overall, it appears that the diversity of Bartonella species DNA identified in ectoparasites is much broader than the species detected in their mammalian hosts, suggesting a mechanism of adaptation of Bartonella species to their host-vector ecosystem. However, these mechanisms leading to the fitness between the vectors and their hosts still need to be investigated.     

New Bruce-ladder multiplex PCR assay for the biovar typing of Brucella suis and the discrimination of Brucella suis and Brucella canis

Rapid and specific identification of Brucella suis at the biovar level is necessary because some of the biovars that infect animals are pathogenic for humans. None of the molecular typing methods described so far are able to discriminate B. suis biovars in a single test and differentiation of B. suis from Brucella canis by molecular approaches can be difficult. This article describes a new multiplex PCR assay, Suis-ladder, for fast and accurate identification of B. suis at the biovar level and the differentiation of B. suis, B. canis and Brucella microti. An advancement of the original Bruce-ladder PCR protocol which allows the correct discrimination of all known Brucella species is also described.     

Can the protozoan parasite Bonamia ostreae infect larvae of flat oysters Ostrea edulis?

Bonamia ostreae is an intracellular protistan parasite affecting flat oysters Ostrea edulis. It can be detected in juveniles but mortalities mainly affect oysters which are more than 2 years old. The parasite is usually observed inside haemocytes and sometimes free, notably in gill epithelia suggesting a parasite release through this organ. However, the infective form and ways of entry and release remain undetermined. Flat oysters incubate their larvae in their pallial cavity for 8-10 days before releasing them into the water column. Flat oysters in Bay of Quiberon in South Brittany (France) are known to be infected with B. ostreae since 1979 and is the most important area in France for O. edulis spat collection. Flat oysters incubating larvae were sampled in this area during summertime between 2007 and 2009. Both adults and larvae were preserved and assayed by PCR and in situ hybridisation (ISH). PCR tests revealed the presence of parasite DNA in some adults and larvae. Specific labelling could be detected by ISH in gills, digestive system, gonad and mantle in adults and in the epithelium surrounding the visceral cavity of some larvae. Our results demonstrate that larvae can be infected with B. ostreae. Larvae might thus contribute to the spread of the parasite during their planktonic life. In addition, their transfer for aquaculture purpose should be controlled especially when they are exported from infected zones.     

Ostreid herpesvirus 1 detection and relationship with Crassostrea gigas spat mortality in France between 1998 and 2006

ABSTRACT: Since its molecular characterisation, Ostreid herpesvirus 1 (OsHV-1) has been regularly detected in Crassostrea gigas in France. Although its pathogenicity was demonstrated on larval stages, its involvement during mortality outbreaks at the juvenile stage was highly suspected but not evidenced. To investigate mortality outbreaks, the French National Network for Surveillance and Monitoring of Mollusc Health (REPAMO) carried out two surveys in juvenile C. gigas. The first survey lasted from 1998 to 2006 and was an epidemiological inquiry occurring when oyster farmers reported mortality outbreaks. The second survey, a longitudinal one, was set up in 1998 to complete the network observations on OsHV-1. Data analysis showed a specific pattern of mortality outbreaks associated with OsHV-1 detection. Ostreid herpesvirus 1 detection mainly appeared during the summer, suggesting the influence of the seawater temperature on its occurrence. It mostly presented a patchy distribution in the field in contrast to the nursery. Significant relationship between OsHV-1 detection and spat mortality was found, preferentially in sheltered and closed environments. The longitudinal survey confirmed most of the network observations. Although subsequent works particularly epidemiological surveys would be useful to confirm the causal link between the detection of OsHV-1 and the mortality outbreaks in juvenile C. gigas, the role of OsHV-1 in oyster mortality is progressing.     

Effects of epidurally administered morphine or buprenorphine on the thermal threshold in cats

Objective: To determine the antinociceptive effects of epidural administration of morphine or buprenorphine in cats by use of a thermal threshold model. ANIMALS: 6 healthy adult cats. PROCEDURES: Baseline thermal threshold was determined in duplicate. Cats were anesthetized with isoflurane in oxygen. Morphine (100 microg/kg diluted with saline [0.9% NaCl] solution to a total volume of 0.3 mL/kg), buprenorphine (12.5 microg/kg diluted with saline solution to a total volume of 0.3 mL/kg), or saline solution (0.3 mL/kg) was administered into the epidural space according to a Latin square design. Thermal threshold was determined at various times up to 24 hours after epidural injection. RESULTS: Epidural administration of saline solution did not affect thermal threshold. Thermal threshold was significantly higher after epidural administration of morphine and buprenorphine, compared with the effect of saline solution, from 1 to 16 hours and 1 to 10 hours, respectively. Maximum (cutout) temperature was reached without the cat reacting in 0, 74, and 11 occasions in the saline solution, morphine, and buprenorphine groups, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: Epidural administration of morphine and buprenorphine induced thermal antinociception in cats. At the doses used in this study, the effect of morphine lasted longer and was more intense than that of buprenorphine.     

Specific detection of Histomonas meleagridis in turkeys by a PCR assay with an internal amplification control

Histomoniasis or blackhead is a disease of gallinaceous birds, caused by the protozoon Histomonas meleagridis. Since traditional diagnostics for the detection of this disease are complex and far less sensitive than molecular tools, a PCR would provide a more rapid and sensitive alternative. However, intestinal material and droppings, which are preferably used in epidemiological studies of histomoniasis, often contain PCR inhibitory substances. To detect these false negative results, the use of an internal amplification control is essential. Nevertheless, the recently developed PCR tests lack this internal control. Therefore, a new PCR assay with H. meleagridis specific primers was developed which does include an internal amplification control. The diagnostic value of the PCR assay was evaluated in comparison to three other conventional H. meleagridis specific PCR tests (HIS5, HM1 and HM2). None of the organ samples originating from uninfected turkeys, showed positive PCR results in any of the tests. Among the lesion-positive, inhibition-free samples, 95.4% were positive by our PCR assay, while only 50, 66.7 and 83.3% of the lesion-positive organs tested positive by the HM1, the HIS5 and the HM2 PCR respectively. In conclusion, our PCR offers the use of the internal control to detect false negative results and an increased sensitivity, and thus should be useful for routine diagnosis of H. meleagridis in poultry.