Toxoplasma gondii in wild and domestic animals from New Caledonia.

Samples (serum or meat juice) collected from 205 animals in New Caledonia in April 2009 were tested for antibodies against Toxoplasma gondii by ELISA using the multi-species ID Screen® Toxoplasmosis Indirect kit (IDVET, Montpellier). Antibodies to T. gondii were detected in 2% (1/49) of the pigs, in 3.3% (1/30) of the cattle, in 13.8% (4/29) of Rusa deers, in 16% (4/25) of the horses, in 32.8% (21/64) of the dogs, and in 50% (4/8) of cats. Statistically, no significant difference was observed between T. gondii seroprevalence and age or sex. No survey on the prevalence of T. gondii in animals has ever been conducted in New Caledonia and this is the first serological evidence of T. gondii in Rusa deer (Cervus timorensis russa). These results indicate an important circulation of T. gondii exists in the animal populations of New Caledonia. In view of humans being exposed, it is advisable to insist on sanitary education and on respect for good hygienic and food practice.

Hepatitis E virus genotype 3f in pigs in New Caledonia.

BACKGROUND: Hepatitis E virus (HEV) is highly prevalent in farm pigs worldwide and an increasing body of data from industrialised countries suggests that it is an agent of a porcine zoonosis. METHODS: We used in-house real-time reverse transcription polymerase chain reaction to study HEV infection in 4-26-week-old pigs on a pig farm in New Caledonia, Oceania, for which no data are available. RESULTS: HEV RNA was detected in faeces from 6 of 92 (6.5%) pigs tested and all were 9-16 weeks old. Phylogenetic analysis showed that the HEV open reading frame 1 and 2 sequences recovered in this study formed a single cluster among HEV genotype 3 subtype f. CONCLUSIONS: Our work shows for the first time that pigs are a reservoir for HEV in New Caledonia. Further studies are needed to assess the prevalence and phylogenetic relationships of HEV in pigs and humans in this French overseas collectivity.

Survey of Canine Dirofilaria immitis Infection in New Caledonia.

Canine dirofilariosis is a frequent parasitic disease in New-Caledonia. A survey of canine heartworm (Dirofilaria immitis) infection among dogs from the cities of Tontouta, Nandaï and Nouméa, was performed in March 2009 using two antigen test kits; the microwell ELISA test: DiroCHE (Synbiotics Europe) and the Rapid Immuno Migration (RIM) test: WITNESS DIROFILARIA (Synbiotics Europe). Blood samples were collected from 64 dogs: 49 strays and 15 military working dogs. The military dogs received a permanent chemoprophylaxis (moxidectin). In 11 stray dogs, both tests were positive (22.4%). All the military dogs were negative, showing efficiency of chemoprophaxis. Results were discrepant in 6 dogs, negative with one test and doubtful with the other. Antigen heartworm test kits are available and reliable diagnostic tools. They are useful to evaluate the efficiency of chemoprophylaxis and to detect infected animals in order to treat them and to prevent the spreading of the disease.

[Sanitary inspection of poultry]. / Inspection sanitaire des volailles.

The purpose of this technical notice is to provide public health agents with guidelines for sanitary inspection of poultry. Focusing on zoonosis-related hazards, this step-by-step notice presents the symptoms and lesions that must be checked for to approve or to reject poultry for human consumption.

[Meat inspection. 2.--Bovine]. / Inspection sanitaire des animaux de boucherie. 2.--Bovins.

The purpose of this second technical notice is to provide public health agents with guidelines for inspection of bovine meat. Focusing on hazards related to zoonotic agents, this notice successively presents the symptoms and lesions that must be checked for before approving or rejecting meat for human consumption.

[Meat inspection. 1. - sheep and goats]. / Inspection sanitaire des animaux de boucherie 1- petits ruminants.

The purpose of this initial technical notice is to provide public health agents with guidelines for inspection of mutton and goat meat. Focusing on hazards related to zoonotic agents, this notice successively presents the symptoms and lesions that must be checked for to determine to approve or reject meat for human consumption.

[Meat inspection. 3.--Swine]. / Inspection sanitaire des animaux de boucherie 3--porcins.

The purpose of this third technical notice is to provide public health agents with guidelines for inspection of swine meat. Focusing on hazards related to zoonotic agents, this notice presents a step-by-step description of sanitary inspection procedures necessary to approve or reject swine meat for human consumption.

[West Nile virus infection: serological investigation among horses in France and in Africa]. / Infection par le virus West Nile: enquêtes sérologiques sur des chevaux en France et en Afrique.

This study was carried out in 2003 to detected serological evidence of West Nile virus infection in 190 Army horses kept nearby French troops stationed in Southeast France and in Africa (Chad, Côte d'Ivoire and Senegal). Both IgG and IgM antibodies were searched for using an ELISA assay. Specifiity of IgG antibodies was determined by western blot and plaque reduction seroneutraization. Finding showed that 79% of the Army horses (n=96) tested in Africa presented specific IgG antibodies. All horses that were seropositive for IgG were seronegative for IgM. None of the Army horses (n=94) tested in the Southeast France were seropositive for West Nile virus. This study indicates that West Nile virus has circulated in all three African countries but not recently. It also underscores the value of western blotting as a rapid, specific confirmation technique that could eliminate the need to use plaque reduction seroneutralization.

Germline mutations induced by N-nitroso-N-ethylurea do not affect the inserted copia retrotransposon in a Drosophila melanogaster wa mutant.

The white-apricot (wa) mutant of Drosophila melanogaster is characterized by a copia retrotransposon inserted in the second intron of the white locus. After germinal exposure to the alkylating agent N-ethyl-N-nitrosourea, we have obtained new phenotypes in the offspring, mainly lighter eye colour, but not revertants to the original phenotype. Subsequent genetic crosses showed that only 3 out of 13 new mutant phenotypes were allelic. Three white gene regions were analysed by Southern blot in order to determine the nature of the mutations. These three regions were the 5' regulatory region, the copia insertion site and the 3' coding region. The results obtained indicate that the treatment does not induce the total or partial excision of copia in the white locus. Two of the new allelic mutants present a 5' or 3' deletion in the white locus. The other new phenotypes seem to be caused by mutations being induced in other loci acting as modifiers, most of them located on the X chromosome.

The Fanconi anaemia genome stability and tumour suppressor network.

Fanconi anaemia (FA) is a rare autosomal recessive disease characterized by increased spontaneous and DNA crosslinker-induced chromosome instability, progressive pancytopenia and cancer susceptibility. An increasing number of genes are involved in FA, including the breast cancer susceptibility gene BRCA2. Five of the FA proteins (FANCA, FANCC, FANCE, FANCF and FANCG) assemble in a complex that is required for FANCD2 activation in response to DNA crosslinks. Active FANCD2 then interacts with BRCA1 and forms discrete nuclear foci. FANCD2 is independently phosphorylated by ATM (the protein whose gene is mutated in ataxia telangiectasia) in response to ionizing radiation. In addition, the FA proteins are interconnected with other nuclear and cytoplasmic factors all related to cellular responses to carcinogenic stress and to caretaker and gatekeeper functions. In this review, the most recently published data on the molecular biology of the FA pathway and its molecular crosstalk with ATM, BRCA1 and BRCA2, proteins involved in xenobiotic and reactive oxygen species metabolism, apoptosis, cell cycle control and telomere stability, are summarized. The currently available data indicate that FA is a central node in a complex nuclear and cytoplasmic network of tumour suppressor and genome stability pathways fully committed to prevent cancer.

Use of the Drosophila wing spot test in the genotoxicity testing of different herbicides.

Four herbicides, namely propanil, maleic hydrazide, glyphosate, and 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), were investigated for genotoxicity in the wing spot test of Drosophila melanogaster. The herbicides were administered by chronic feeding to 3-day-old larvae. Two different crosses, a standard (ST) and a high-bioactivation (HB) cross, involving the flare-3 (flr(3)) and the multiple wing hairs (mwh) markers, were used. The HB cross uses flies characterized by an increased cytochrome P-450-dependent bioactivation capacity, which permits a more efficient biotransformation of promutagens and procarcinogens. In both crosses, the wings of the two types of progeny, which are inversion-free marker heterozygotes and balancer heterozygotes, were analyzed. Maleic hydrazide and glyphosate proved to be more genotoxic in the ST cross, whereas propanil appeared to be slightly more genotoxic in the HB cross. On the other hand, the herbicide 2,4,5-T increased the mutation frequency for only the small single spots in the ST cross.

Sequence analysis of the boundaries of the tandem duplication from the white-ivory mutant of Drosophila melanogaster and two chemically induced revertants.

We have previously shown that revertants obtained from the white-ivory mutants of Drosophila melanogaster, both spontaneous and induced, have lost a DNA fragment of 2.9 kb that is duplicated in tandem in the white-ivory mutation. To prove the accuracy of the deletion in revertants obtained after treatment with alkylating agents, we have sequenced DNA fragments previously amplified by PCR. These fragments correspond to the ends of the remaining 2.9 kb copy of these revertants and the internal region of the junction of both copies, which constitutes the duplication in the white-ivory mutant. These sequences are compared with those from white-ivory mutants. Our results show slight differences from the published sequence of the white-ivory mutation and with the wild-type sequence of the white locus. The sequences of the two revertants analysed show that excision of the duplicated fragment is very precise. We hypothesize the mechanism of excision in terms of intrachromosomal recombination induced by double-strand break repair after treatment with alkylating agents.

Analysis of genomic damage in the mutagen-sensitive mus-201 mutant of Drosophila melanogaster by arbitrarily primed PCR (AP-PCR) fingerprinting.

DNA repair mechanisms are important to maintain the stability of the genome. In Drosophila melanogaster, the mus-201 gene is required in the excision repair process. To study the contribution of the mus-201 gene in the stability of the Drosophila genome, we have used the arbitrarily primed PCR fingerprinting method (AP-PCR). We have analysed the changes in the genomic DNA fingerprints from the progeny of wild-type males crossed with mus-201 repair-deficient or repair-proficient females. After induction of DNA damage with 2-acetylaminofluorene (2-AAF) in the wild-type parental males, quantitative and qualitative differences in the AP-PCR fingerprints were detected between the two crosses, and the estimate of the genomic damage detected by AP-PCR has clearly shown that the mus-201 repair deficiency is associated with an increase of genomic damage. The predominant type of alterations detected by AP-PCR under the mus-201 repair-deficient conditions agree with the results obtained in microsatellite PCR analysis, suggesting that the role of the mus-201 gene, necessary in excision repair, is not associated to the mismatch repair process. The work reported here demonstrates that the AP-PCR is a suitable technique to analyse genetic alterations in D. melanogaster and, consequently, can be used to compare the susceptibility to genomic damage of different DNA repair mutants.

Overcoming false negatives due to the genomic context in polymerase chain reaction amplification.

In some instances defined genomic regions are so poorly amplified that they seem to be unamplifiable. A protocol was developed which allows good PCR amplifications by the use of restriction digestion combined with the elution of a pool of restriction fragments of defined size range from agarose gel after electrophoresis. We describe the application of the method in the PCR amplification of a region of the white locus of Drosophila melanogaster that otherwise may be considered as a negative result.

Molecular analysis of mutants obtained by treatment with alkylating agents in a quadruplicated white-ivory strain of Drosophila melanogaster.

The use of a white-ivory (wi) strain of Drosophila melanogaster carrying four copies of this allele, (wi)4, has proved to be useful in detecting somatic mutation in genotoxicity testing. Nevertheless, until now very little information exists about the nature of the genetic effects detected in such a strain. This work presents molecular data on the changes that have taken place in different germinal mutants obtained after treatment with alkylating agents. Three different phenotypes were obtained: wild-type red eyes, dark red eyes and eyes lighter than (wi)4. Our results show that, in at least one of the four copies of the allele, the wild-type red eye phenotypes are due to a precise excision of the 2.96 kb duplicated region characteristic of the wi allele. These data agree with previous results obtained in a strain carrying only a single copy of the wi allele. The dark red eye mutants analysed seemed to be generated as a cluster and all proved to be caused by deletions at the 3'-end of the duplicated wi region in two of the copies of the (wi)4 genome. Finally, the light eye mutants (obtained at high frequencies) failed to show alterations at the molecular level, although we cannot discard the possibility that they might have originated by the loss of some of the wi copies of the (wi)4 strain.

Induced somatic and germinal reversion of the white-spotted-1 insertional mutant phenotype in Drosophila melanogaster.

The white-spotted-1 (Wsp1) mutant of Drosophila melanogaster is characterized by the presence of an 8.7 kb retrotransposon (B104) inserted in the regulatory region of the white locus. The frequency of reversion in both somatic tissue and the germline after exposure to three different alkylating agents has been analysed. To determine if germinal revertants were induced by precise excision of the insertional element we analysed several phenotypic revertants using PCR and Southern blot techniques. The results indicate that, under our experimental conditions, the mutagens used did not induce excision of B104 in the white gene. In addition, the revertant phenotypes obtained were due to the existence of second site modifiers acting on expression of white. Such modifiers map near the white locus and, at least in one case, may correspond to suppressor-of-white-spotted.

Molecular study of the germinal reversions induced at the white-ivory locus in Drosophila melanogaster.

The white-ivory somatic mutation test of Drosophila melanogaster is based on the reversion of the X-linked eye colour recessive mutation white-ivory to wild-type. Although the exact mechanism of white-ivory reversion is not quite understood, it has been suggested that such reversion, both in somatic and germ-line cells, could be due to the precise excision of the tandemly duplicated 2.96 kb DNA fragment characteristic of the white-ivory mutation. We have attempted to confirm this hypothesis analysing, at the molecular level, different germinal revertants induced by chemical treatment with three well known alkylating agents: ethyl methanesulphonate, methyl methanesulphonate and N-nitroso-N-ethylurea. The molecular analysis of these germ-line revertants, using Southern blot hybridization and polymerase chain reaction techniques, shows that such reversions are associated with the deletion of the 2.96 kb tandemly duplicated DNA sequence of the white-ivory locus.

Differential aldehyde sensitivity of newly replicated chromatin from Drosophila melanogaster embryos.

By using a series of formalin concentrations we have found that high aldehyde levels in the fixation buffer of Miller spreads are correlated with the appearance of nonnucleosomal stretches in newly replicated chromatin of embryos from Drosophila melanogaster. These nucleosome-free gaps are found 0-500 nm behind the replication fork and do not correspond to naked DNA. The analysis of the distribution of nucleosome-free gaps on newly replicated DNA has revealed some structural details about the maturation of nucleosomes and provides direct evidence that parental nucleosomes have an altered structure at the replication fork. Finally, these stretches of nonnucleosomal chromatin are located in a trans disposition inside the active replicon, although there exists a considerable variability.

Restriction mapping of phage lambda vectors using non-radioactive methods.

In order to take advantage of non-radioactive methods, we have developed two plasmids (p lambda LE and p lambda RE) for mapping restriction sites of long inserts cloned in phage lambda vectors. These plasmids are constructed by cloning the left 402-bp and right 560-bp phage lambda genome ends, respectively. To map restriction sites, the cloned sequences in p lambda LE and p lambda RE are labeled with digoxygenin and hybridized to partially digested lambda DNA. The ladder of bands detected with these probes can be used to construct restriction maps in the same way as those obtained using radioactively labeled cos complementary oligodeoxyribonucleotides [Rackwitz et al., Gene 30 (1984) 195-200].