Schmallenberg virus: experimental infection in goats and bucks.

BACKGROUND: Schmallenberg virus (SBV) is an emerging Orthobunyavirus of ruminant livestock species currently circulating in Europe. SBV causes a subclinical or mild disease in adult animals but vertical transmission to pregnant dams may lead to severe malformations in the offspring. Data on the onset of clinical signs, viremia and seroconversion in experimentally infected adult animals are available for cattle and sheep but are still lacking for goats. For a better understanding of the pathogenesis of SBV infection in adult ruminants, we carried out experimental infections in adult goats. Our specific objectives were: (i) to record clinical signs, viremia and seroconversion; (ii) to monitor viral excretion in the semen of infected bucks; (iii) to determine in which tissues SBV replication took place and virus-induced lesions developed. RESULTS: Four goats and two bucks were inoculated with SBV. Virus inoculation was followed by a short viremic phase lasting 3 to 4 days and a seroconversion occurring between days 7 and 14 pi in all animals. The inoculated goats did not display any clinical signs, gross lesions or histological lesions. Viral genomic RNA was found in one ovary but could not be detected in other organs. SBV RNA was not found in the semen samples collected from two inoculated bucks. CONCLUSIONS: In the four goats and two bucks, the kinetics of viremia and seroconversion appeared similar to those previously described for sheep and cattle. Our limited set of data provides no evidence of viral excretion in buck semen.

Lack of transplacental transmission of Bartonella bovis.

Transplacental transmission of Bartonella spp. has been reported for rodents, but not for cats and has never been investigated in cattle. The objective of this study was to assess vertical transmission of Bartonella in cattle. Fifty-six cow-calf pairs were tested before (cows) and after (calves) caesarean section for Bartonella bacteremia and/or serology, and the cotyledons were checked for gross lesions and presence of the bacteria. None of the 29 (52%) bacteremic cows gave birth to bacteremic calves, and all calves were seronegative at birth. Neither placentitis nor vasculitis were observed in all collected cotyledons. Bartonella bovis was not detected in placental cotyledons. Therefore, transplacental transmission of B. bovis and multiplication of the bacteria in the placenta do not seem likely. The lack of transplacental transmission may be associated with the particular structure of the placenta in ruminants or to a poor affinity/agressiveness of B. bovis for this tissue.

Evidence of transplacental transmission of bluetongue virus serotype 8 in goats.

During the incursion of bluetongue virus (BTV) serotype 8 in Europe, an increase in the number of abortions in ruminants was observed. Transplacental transmission of BTV-8 in cattle and sheep, with subsequent foetal infection, is a feature of this specific bluetongue serotype. In this study, BTV-8 ability to cross the placental barrier at the beginning of the second third of pregnancy and at the end of pregnancy was investigated in goats in two separate experiments. In the first experiment, nine goats were experimentally infected with BTV-8 at 61 days of pregnancy. Foetuses were collected 21 dpi. BTV-8 was evidenced by real time RT-PCR and by viral isolation using blood from the umbilical cord and the spleens of 3 out of the 13 foetuses. All dams were viraemic (viral isolation) at the moment of sampling of the foetuses. Significant macroscopic or histological lesions could not be observed in foetuses or in their infected dams (notably at the placenta level). In the second experiment, 10 goats were infected with BTV-8 at 135 days of pregnancy. Kids were born by caesarean section at the programmed day of birth (15 dpi). BTV-8 could not be detected by rt-RT-PCR in blood or spleen samples from the kids. This study showed for the first time that BTV-8 transplacental transmission can occur in goats that have been infected at 61 days of pregnancy, with infectious virus recovered from the caprine foetuses. The observed transmission rate was quite high (33%) at this stage of pregnancy. However, it was not possible to demonstrate the existence of BTV-8 transplacental transmission when infection occurred at the end of the goat pregnancy.

Encephalomyocarditis virus 2A protein is required for viral pathogenesis and inhibition of apoptosis.

The encephalomyocarditis virus (EMCV), a Picornaviridae virus, has a wide host spectrum and can cause various diseases. EMCV virulence factors, however, are as yet ill defined. Here, we demonstrate that the EMCV 2A protein is essential for the pathogenesis of EMCV. Infection of mice with the B279/95 strain of EMCV resulted in acute fatal disease, while the clone C9, derived by serial in vitro passage of the B279/95 strain, was avirulent. C9 harbored a large deletion in the gene encoding the 2A protein. This deletion was incorporated into the cDNA of a pathogenic EMCV1.26 strain. The new virus, EMCV1.26Δ2A, was capable of replicating in vitro, albeit more slowly than EMCV1.26. Only mice inoculated with EMCV1.26 triggered death within a few days. Mice infected with EMCV1.26Δ2A did not exhibit clinical signs, and histopathological analyses showed no damage in the central nervous system, unlike EMCV1.26-infected mice. In vitro, EMCV1.26Δ2A presented a defect in viral particle release correlating with prolonged cell viability. Unlike EMCV1.26, which induced cytopathic cell death, EMCV1.26Δ2A induced apoptosis via caspase 3 activation. This strongly suggests that the 2A protein is required for inhibition of apoptosis during EMCV infection. All together, our data indicate that the EMCV 2A protein is important for the virus in counteracting host defenses, since Δ2A viruses were no longer pathogenic and were unable to inhibit apoptosis in vitro.

Encephalomyocarditis virus mortality in semi-wild bonobos (Pan panicus).

BACKGROUND: Fatal myocarditis from encephalomyocarditis virus (EMCV) infection has previously been identified in sporadic and epidemic forms in many species of captive non-human primates probably including one bonobo (Pan paniscus). METHODS: We investigated the deaths of two bonobos that were suspicious of EMCV using a combination of histopathology, immunohistochemistry and, for one of the two bonobos, reverse transcription PCR. RESULTS: Histopathological examination of heart tissue from the two bonobos showed changes characteristic of EMCV. Immunohistochemical studies confirmed the presence of EMCV antigen in heart tissue of both and in kidney and intestine of one of the bonobos. EMCV RNA was also isolated from the serum of the bonobo tested. CONCLUSION: Together, these findings confirm that EMCV was responsible for deaths of the two bonobos. Strict separation of bonobos in particular and captive primates in general from potential sources of EMCV contamination should be maintained to prevent mortality caused by EMCV.

Severe necrotizing encephalitis in a Yorkshire terrier: topographic and immunohistochemical study.

Necrotizing encephalitis of the Yorkshire terrier is a chronic non-suppurative encephalitis that was reported in approximately 15 cases worldwide. We report the case of a 10-year-old female Yorkshire terrier with gross evidence of severe cortical degeneration and necrosis. Microscopically, affected areas were mainly located in the cortical white matter and in the mesencephalon without implication of the cerebellum. Cavitation necrosis, demyelination, gemistocytic astrocytosis, marked perivascular lymphocytic cuffing with a diffuse lymphocytic/histiocytic/gitter cell infiltration characterized the lesions. Immunohistochemical analysis identified the major infiltration of T lymphocytes and macrophages with implication of some cytotoxic lymphocytes and IgG-producing plasma cells; depositions of IgG in the affected white matter were also observed. Specific stains did not reveal fungal, protozoal or bacterial organisms and reverse transcriptase-polymerase chain reaction analysis for distemper virus was also negative. The lympho-histiocytic inflammation suggests a T-cell-mediated and a delayed-type immune reaction as a possible pathogenic mechanism for this brain disorder.

Health status of cloned cattle at different ages.

The procedure of somatic cloning is associated with important losses during pregnancy and in the perinatal period, reducing the overall efficacy to less than 5% in most cases. A mean of 30% of the cloned calves die before reaching 6 months of age with a wide range of pathologies, including, for the most common, respiratory failure, abnormal kidney development, liver steatosis. Heart and liver weight in relation to body weight are also increased. Surviving animals, although mostly clinically normal, differ from controls obtained by artificial insemination (AI) within the first 1-2 months, to become undistinguishable from them thereafter. Hemoglobin concentrations, for instance, are lower, and leptin concentrations are elevated. In response to the lack of prospective studies addressing the health of adult clones, a long-term, 3-4-year study is currently being conducted to assess the health of mature bovine clones at INRA. Preliminary results over 1 year of study do not show any statistical difference between groups for hematological parameters.

Compensatory responses in mice carrying a null mutation for Ins1 or Ins2.

Intrauterine growth retardation and postnatal acute diabetes result from insulin deficiency in double homozygous null mutants for Ins1 and Ins2 (Duvillié B, et al., Proc. Natl. Acad. Sci. USA 94:5137-5140, 1997). The characterization of single homozygous null mutants for Ins1 or Ins2 is described here. Neither kind of mutant mice was diabetic. Immunocytochemical analysis of the islets showed normal distribution of the endocrine cells producing insulin, glucagon, somatostatin, or pancreatic polypeptide. Analysis of the expression of the functional insulin gene in Ins1-/- or Ins2-/- mice revealed a dramatic increase of Ins1 transcripts in Ins2-/- mutants. This compensatory response was quantitatively reflected by total pancreatic insulin content similar for both types of mutants and wild-type mice. Moreover, both mutants had normal plasma insulin levels and normal glucose tolerance tests. The determination of beta-cell mass by morphometry indicated beta-cell hyperplasia in the mutant mice. The beta-cell mass in Ins2-/- mice was increased almost threefold, which accounts for the increase of Ins1 transcripts in Ins2-/-mutants. This study thus contributes to evaluate the potential of increasing the beta-cell mass to compensate for low insulin production.

Lymphoid hypoplasia and somatic cloning.

BACKGROUND: Adult somatic cloning by nuclear transfer is associated with high rate of perinatal mortality but there is still no evidence that nuclear transfer itself is responsible for these failures. We report on a longlasting defect linked to somatic cloning. METHODS: Skin cells grown from an ear biopsy specimen from a 15-day-old calf were used as a source of nuclei. The donor animal was a clone of three females obtained from embryonic cells. Clinical examination, haematological, and biochemical profiles, and echocardiography of the somatic clone were done from birth to death. FINDINGS: After 6 weeks of normal development, the somatic cloned calf had a sudden and rapid fall in lymphocyte count and a decrease in haemoglobin. The calf died on day 51 from severe anaemia. Necropsy revealed no abnormality except thymic atrophy and lymphoid hypoplasia. INTERPRETATION: Somatic cloning may be the cause of long-lasting deleterious effects. Our observation should be taken into account in debates on reproductive cloning in human beings.

Genetic manipulation of insulin action and beta-cell function in mice.

Transgenic and gene targeting approaches have now been applied to a number of genes in order to investigate the metabolic disorders that would result by manipulating insulin action or pancreatic beta-cell function in the mouse. The availability of such mutant mice will allow in the future to develop animal models in which the pathophysiologies resulting from polygenic defects might be reconstituted and studied in detail. Such animal models hopefully will lead to better understanding of complex polygenic diseases such as non-insulin-dependent diabetes mellitus (NIDDM).

Phenotypic alterations in insulin-deficient mutant mice.

Two mouse insulin genes, Ins1 and Ins2, were disrupted and lacZ was inserted at the Ins2 locus by gene targeting. Double nullizygous insulin-deficient pups were growth-retarded. They did not show any glycosuria at birth but soon after suckling developed diabetes mellitus with ketoacidosis and liver steatosis and died within 48 h. Interestingly, insulin deficiency did not preclude pancreas organogenesis and the appearance of the various cell types of the endocrine pancreas. The presence of lacZ expressing beta cells and glucagon-positive alpha cells was demonstrated by cytochemistry and immunocytochemistry. Reverse transcription-coupled PCR analysis showed that somatostatin and pancreatic polypeptide mRNAs were present, although at reduced levels, accounting for the presence also of delta and pancreatic polypeptide cells, respectively. Morphometric analysis revealed enlarged islets of Langherans in the pancreas from insulin-deficient pups, suggesting that insulin might function as a negative regulator of islet cell growth. Whether insulin controls the growth of specific islet cell types and the molecular basis for this action remain to be elucidated.

Targeted disruption of the insulin receptor gene in the mouse results in neonatal lethality.

Targeted disruption of the insulin receptor gene (Insr) in the mouse was achieved using the homologous recombination approach. Insr+/- mice were normal as shown by glucose tolerance tests. Normal Insr-/- pups were born at expected rates, indicating that Insr can be dispensable for intrauterine development, growth and metabolism. However, they rapidly developed diabetic ketoacidosis accompanied by a marked post-natal growth retardation (up to 30-40% of littermate size), skeletal muscle hypotrophy and fatty infiltration of the liver and they died within 7 days after birth. Total absence of the insulin receptor (IR), demonstrated in the homozygous mutant mice, also resulted in other metabolic disorders: plasma triglyceride level could increase 6-fold and hepatic glycogen content could be five times less as compared with normal littermates. The very pronounced hyperglycemia in Insr-/- mice could result in an increased plasma insulin level of up to approximately 300 microU/ml, as compared with approximately 25 microU/ml for normal littermates. However, this plasma level was still unexpectedly low when compared with human infants with leprechaunism, who lack IR but who could have extremely high insulinemia (up to > 4000 microU/ml). The pathogenesis resulting from a null mutation in Insr is discussed.