Molecular detection of Culicoides spp. and Culicoides imicola, the principal vector of bluetongue (BT) and African horse sickness (AHS) in Africa and Europe

Bluetongue (BT) and African Horse Sickness (AHS) are infectious arthropod-borne viral diseases affecting ruminants and horses, respectively. Culicoides imicola Kieffer, 1913, a biting midge, is the principal vector of these livestock diseases in Africa and Europe. Recently bluetongue disease has re-emerged in the Mediterranean Basin and has had a devastating effect on the sheep industry in Italy and on the islands of Sicily, Sardinia, Corsica and the Balearics, but fortunately, has not penetrated onto mainland France and Spain. To survey for the presence of C. imicola, an extensive light-trap network for the collection of Culicoides, was implemented in 2002 in southern mainland France. The morphological identification of Culicoides can be both tedious and time-consuming because its size ranges from 1.5 to 3 mm. Therefore, an ITS1 rDNA polymerase chain reaction (PCR)-based diagnostic assay was developed to rapidly and reliably identify Culicoides spp. and C. imicola. The aim of this work was to set up a rapid test for the detection of C. imicola amongst a pool of insects collected in areas at risk for BT. The sequence similarity of the rDNA (nuclear ribosomal DNA), which is greater within species than between species, is the foundation of its utilisation in species-diagnostic assays. The alignment of the 11 ITS1 sequences of Culicoides obtained from Genbank and EMBL databases helped us to identify one region in the 5' end and one in the 3' end that appear highly conserved. PCR primers were designed within these regions to amplify genus-specific fragments. In order to set up a C. imicola-specific PCR, another forward primer was designed and used in combination with the previously designed reverse primer. These primers proved to be highly specific and sensitive and permitted a rapid diagnostic separation of C. imicola from Culicoides spp.     

Effects of Tryptophan Analogs, Mitomycin c and Acridine Orange on the Production of Filtrable Hemolysin by Escherichia coli

Bacteriostatic doses of 5-methyltryptophan and of 7-azatryptophan exert a complete inhibition on the in vitro production of filtrable hemolysin by Escherichia coli. This inhibition is readily overcome by L-tryptophan, and does not seem to be specific but secondary to an interference with de novo protein synthesis and cell multiplication as is observed with chloramphenicol in sensitive strains. Although the influence of the two tryptophan analogs on hemolysin production and cell multiplication is similar, their mechanism of action at the molecular level appears to be different.

The addition of 50 µg/ml 7-azatryptophan which causes an unbalanced growth characterized by an arrest of the cellular division and an increase of cell size, blocks the production of an active hemolysin. Exposure to 4 µg/ml 5-methyltryptophan also prevents cell multiplication and hemolysin production but no sign of unbalanced growth is evident. Mitomycin C in concentrations sufficient to prevent increase in the number of viable units provokes an extreme elongation of E. coli cells and, apparently, does not stop the synthesis of hemolysin. In blocking the production of hemolysin the three inhibitors of protein synthesis used in this study were more effective than mitomycin C an agent known to affect deoxyribonucleic acid synthesis and to induce extrachromosomic genetic factors. Results of conjugation experiments also described here support the finding of other workers that the genetic factor that controls the production of the filtrable hemolysin in E. coli can be transmitted by conjugation. Acridine orange eliminated the hemolytic property from a large proportion of the population of a hemolytic strain which did not carry the R factor, but was little effective in the strains which had received both the R factor and the hemolytic character by conjugation.

     

Mortality factors, environmental contaminants, and parasites of white-tailed sea eagles from Greenland

Twelve white-tailed sea eagles (Haliaeetus albicilla groenlandicus) found dead between 1997 and 2000 in Greenland were examined to investigate the health status, including the causes of death and the burden of organochlorine contaminants and potentially toxic heavy metals. The determined causes of death were unspecific trauma (n = 6), lead poisoning (n = 2) with 36 and 26 ppm lead in the liver tissue, infectious diseases (n = 1), injuries sustained during intraspecific conflict (n = 1), and gunshot (n = 1). One lead poisoned eagle had a single lead shot pellet in its gizzard. No diagnosis could be made in one case because of decomposition of the carcass. Four of the investigated eagles were injured with lead shot or bullet fragments; one of the birds was killed with about 69 lead shots. Levels of organochlorine pesticides, polychlorinated biphenyls, mercury, and cadmium in organs were moderate. The parasite fauna consisted of one coccidian and three helminth species. The acanthocephalas Profiliocollis botulus and Corynosoma suduche as well as the nematode Stegophorus stellaepolaris are all new records for the white-tailed sea eagle.     

Quantification of porcine circovirus type 2 (PCV-2) within- and between-pen transmission in pigs

PCV-2 within- and between-pen transmission was quantified by estimating the daily transmission rate beta and the basic reproduction ratio (R(0)) using a stochastic SEIR (Susceptible, Exposed, Infectious, Removed) model fitted on experimental data. Within-pen transmission was quantified by using four groups of eight SPF (specific pathogen-free) pigs (four infected and four susceptible pigs having direct contact). Between-pen transmission was studied in two groups of 16 SPF pigs (eight infected and eight susceptible pigs having indirect contact (10 cm distance)). Pigs were monitored twice a week (blood samples) and were tested for PCV-2 antibodies (ELISA test) and viral genome load in sera (real-time PCR). Transmission parameters beta(within) and beta(between) were estimated using a maximum likelihood method and the duration of infectiousness, to compute R(0), was estimated with a parametric survival model. Different assumptions were made to determine the end of infectiousness (seroconversion, seroconversion and decline in viral genome load, permanent infectiousness). R(0[within]) (8.9 (5.1-15.4)) was greater when the end of infectiousness was assumed to be related to both seroconversion and a decline of PCV-2 genome load in sera (average duration of infectiousness = 32 days) compared with only seroconversion as the indicator of recovery (R(0[within]) = 5.5 (3.3-9.0)). Whatever the assumption, between-pen R(0) (0.58 (0.23-1.47)) was always significantly lower than within-pen R(0). Only beta(within) was sensitive to the assumption on end of infectiousness and decreased with increasing duration of infectiousness. These results showed that PCV-2 transmission is influenced by contact structure that appears worth being taken into account in an epidemic model.     

Development and evaluation of a real-time quantitative PCR assay for Culicoides imicola, one of the main vectors of bluetongue (BT) and African horse sickness (AHS) in Africa and Europe

The current microscopy method for identifying the Culicoides imicola Kieffer, 1913 species can be time and labour intensive. There is a need for the development of a rapid and quantitative tool to quantify the biting midges C. imicola ss in light trap catches. A reproducible and sensitive real-time polymerase chain reaction method that targets the internal transcribed spacer (ITS-1) of ribosomal DNA of C. imicola ss species was developed. This real-time PCR assay was first performed on 10-fold serial dilutions of purified plasmid DNA containing specific C. imicola ss ITS-1. It was then possible to construct standard curves with a high correlation coefficient (r2=0.99) in the range of 10(-2)-10(-8) ng of purified DNA. The performances of this PCR were evaluated in comparison with morphological determination on Culicoides trapped along the Mediterranean coastal mainland France. ROC statistical analysis was carried out using morphology as gold standard and the area under the ROC curve had a satisfactory value of 0.9752. The results indicated that this real-time PCR assay holds promise for monitoring C. imicola ss population in both surveillance and research programmes because of its good specificity (92%) and sensitivity (95%).     

Landscape genetics matches with behavioral ecology and brings new insight on the functional connectivity in Mediterranean mouflon

Context

In natural populations, gene flow often represents a key factor in determining and maintaining genetic diversity. In a worldwide context of habitat fragmentation, assessing the relative contribution of landscape features to gene flow thus appears crucial for sustainable management of species.

Objective

We addressed this issue in Mediterranean mouflon (Ovis gmelini musimon?×?Ovis sp.) by combining previous knowledge on behavioral ecology with landscape genetics. We also assessed how sex-specific behavioral differences translated in term of functional connectivity in both sexes.

Methods

We relied on 239 individuals genotyped at 16 microsatellite markers. We applied a model optimization approach in a causal modeling framework of landscape genetics to test for the effects on gene flow of habitat types and linear landscape features previously identified as important for movements and habitat selection in both sexes. Five resistance values were alternately assigned to these landscape characteristics leading to a comprehensive set of resistance surfaces.

Results

Isolation by resistance shaped female gene flow, supporting the central role of linear landscape features as behavioral barriers for animal movements. Conversely, no isolation by resistance was detected in males. Although a lack of statistical power cannot be discarded to explain this result, it tended to confirm that males are less influenced by landscape structures during the mating period.

Conclusions

Combining previous knowledge on behavioral ecology with results from landscape genetics was decisive in assessing functional landscape connectivity in both sexes. These results highlighted the need to perform sex-specific studies for management and conservation of dimorphic species.

     

Functional morphology of the large intestinal mucosa of horses (Equus przewalskii f. caballus) with special regard to the epithelium

The mucous membrane of the caecum and colon ascendens of adult horses was first studied using light and transmission electron microscopy. In the surface epithelium there was an inconspicuous constellation of organelles, otherwise there was a lot of mitochondria as a source of energy for absorptive performances. Moreover, enlarged intercellular spaces exist as an indication of an increased uptake of water and electrolytes. In the basal region of Lieberkühn's crypts there were single enteroendocrine cells and numerous granules in the apical epithelial cytoplasm. The functional meaning of these granules is contrarily discussed in the literature. There was no epithelial activity of alkaline phosphatase. Against that a small positive reaction of adenosine triphosphatase was observed at the lateral plasmalemata of the epithelial cells. The sense of these findings with regard to the transport of substances in the equine large intestine is discussed.