Development of a hemi-nested RT-PCR method for the specific determination of European Bat Lyssavirus 1. Comparison with other rabies diagnostic methods.

A simplified hemi-nested reverse transcriptase polymerase chain reaction (hnRT-PCR) has been developed to determine specifically the European Bat Lyssavirus 1 (EBLV-1) nucleoprotein gene. The specificity of this method was determined by using the seven genotypes of lyssavirus by RT-PCR, Southern blot and sequence analysis. Compared to the rabies diagnostic methods, the hnRT-PCR showed a higher sensitivity for the detection of small amounts of EBLV-1 virus. In view of these results, we suggest this new hnRT-PCR should be performed for the epidemiological survey of bat colonies, also providing rapid detection and genotyping of EBLV-1 until now encountered in all naturally infected bats in France.

Genetic analysis of European bat lyssavirus type 1 isolates from France.

European bat lyssavirus type 1a (EBLV-1a) was first identified in central France from a serotine bat (Eptesicus serotinus) collected at the end of 2002. Rabies was diagnosed by reference rabies diagnosis methods and molecular tools. Phylogenetic analysis of 14 viral isolates obtained from French bats infected with EBLV-1 between 1989 and the end of 2002 against 47 nucleoprotein sequences showed a north-west to east distribution of EBLV-1a virus and a south to north distribution of EBLV-1b virus, isolates of which could be divided into two groups: group 1 in north-eastern France and group 2 in central and north-western France.

Oral rabies vaccination of foxes with one or two delayed distributions of SAG2 baits during the spring.

During the spring of 1997, various protocols of rabies vaccine bait (SAG2) distribution for foxes were compared: in the first test zone, a first distribution was organised at the end of April, followed by a second distribution two weeks later; in the second test zone, there was a first distribution at the same period as for the previous zone, followed by a second distribution four weeks later, at the end of May. In two control zones, a classical single bait distribution was organised during the same periods as for the second distribution in the respective test zones. No statistical differences were observed for adult foxes or fox cubs sampled in the test and control zones neither for baits uptake nor for seroconversion rate. However, seroconversion rates observed in fox cubs population were significantly higher (P < 0.01) in areas vaccinated at the end of May (43 and 56%) compared with those vaccinated at mid-May (24 and 20%). The vaccinal efficacy of baits was also significantly (P < 0.05) increased for the fox cubs in the areas vaccinated at the end of May (46 and 57%) compared with those vaccinated at mid-May (24 and 25%). This increase in immunological response by fox cubs when vaccinating in late spring must be related to their development. In the early spring, fox cubs are generally too young to have access to baits or to be vaccinated when eating them. For most of these fox cubs, a second distribution will not constitute a booster. Therefore, in order to increase the efficient access of fox cubs to vaccine baits, Spring distribution of baits should preferably be organised during May or June rather than in April.

Oral immunisation of swine with a classical swine fever vaccine (Chinese strain) and transmission studies in rabbits and sheep.

Seven experiments including a total of 47 pigs, 11 wild boars, 26 rabbits, 10 hares and 16 sheep were carried out to assess the efficacy, safety and transmission of the Chinese vaccine strain of the classical swine fever virus (CSFV) administrated by the oral route. Within 3 weeks after oral vaccination, a clear seroconversion occurred in the pigs. Six weeks after vaccination, vaccinated pigs were fully protected against a virulent challenge. The C-strain was not isolated from tonsils, spleen, lymph nodes, thymus, saliva, urine and faeces of pigs within 4 days after oral vaccination. In one experiment, susceptible pigs were placed in direct contact with vaccinated pigs. None of these contact-exposed pigs became serologically positive for CSFV antibodies. It is concluded that the C-strain induces protection in pigs when administrated by the oral route and is not shed by vaccinated pigs. Serum anti-CSFV antibodies developed in seven out of eight wild boars vaccinated by the oral route. No vaccine virus was detected in the spleen and tonsils of these animals. The results in wild boar were in accordance with those obtained in domestic pigs. Sheep did not show any clinical signs after oral vaccination while rabbits had moderate hyperthermia and growth retardation. No clinical response to oral immunisation in hares was detected. At the end of the experiment, no sheep had detectable serum antibodies against CSFV, whereas a few vaccinated rabbits and hares became seropositive. None of the contact-exposed rabbits and hares seroconverted. These data indicate that the C-strain is safe for sheep and as expected, moderately or not pathogenic for rabbits and hares. These efficacy and safety studies on oral vaccination with the C-strain under experimental conditions provide essential information for further studies in wild boars under experimental and field conditions, including assays with baits to control a CSF epidemic.

Comparison of the effectiveness of two protocols of antirabies bait distribution for foxes (Vulpes vulpes).

In a plateau and hill region of France (the Doubs), two protocols of rabies vaccine bait distribution targeted at foxes were compared: helicopter distribution of vaccine baits alone (control zone) and a combined aerial distribution by helicopter with an additional deposit of vaccine baits at fox den entrances by foot (test zone). In the test zone covering an area of 436 km2, baits were distributed by helicopter at a rate of 13.4 baits/km2. Additionally, an average of 11.4 vaccine baits at the entrances of 871 fox dens were terrestrially distributed by 110 persons (9,964 baits). In this test zone, 90% of the young foxes were marked with tetracycline which permitted estimation of the bait consumption; however, only 38% had significant titre of rabies antibodies and less than one fox cub per 2.4 of those having consumed at least one bait were immunized. In the control zone, these percentages were significantly lower: respectively, 35 and 17% and one fox cub per 4.2. The relative lack of benefit between bait uptake and rate of immunological response may be due to maternal immunity which could have interfered with fox cub active immunization. A booster effect following a second distribution of baits by foot may be suggested in both adult foxes and their offspring. That these baits needed to be terrestrially distributed in order to obtain a booster effect is uncertain. Terrestrial distribution at fox den entrances is difficult to do and entails additional expenses not incurred in aerial distribution. The cost of terrestrial vaccination is 3.5 times higher than classical aerial vaccination and takes 63.5 times longer. A cost effective analysis of this type of supplementary terrestrial intervention determined that bait deposit at den entrances can be recommended for restricted areas, where residual focii exist, as a complement to the aerial distribution of baits.