An evaluation of immunofluorescence and PCR methods for detection of rabies in archival Carnoy-fixed, paraffin-embedded brain tissue.

Direct immunofluorescence and PCR detection methods were compared for sensitivity in evaluating the rabies status of archival specimens of Carnoy-fixed, paraffin-embedded brain tissue. The material consisted of 23 samples obtained during a rabies outbreak in Finland in 1988, and one sample isolated from a bat researcher who died of rabies in Finland in 1985. These results were compared with the original diagnoses performed on the fresh tissues. The immunofluorescence assay detected 100% (12/12) of the rabies-positive archival cases. A PCR assay designed to detect a 139-bp target near the 5' end of the rabies nucleoprotein gene also detected 100% (12/12) of the samples identified as positive in the fresh tissue specimens. A PCR assay designed to detect a 304-bp target spanning the 139-bp target of the first assay detected only 67% (8/12) of the original cases. No false positives were recorded. Both immunofluorescence detection of antigen and PCR detection of a short region of the nucleoprotein gene are useful in determining the rabies status of fixed, paraffin embedded (archival) material.

No rabies detected in voles and field mice in a rabies-endemic area.

Brain samples were collected from 514 voles and wild mice in Estonia, and examined for rabies. The samples were tested with antigen ELISA, and 8.6% of them additionally by virus isolation assay. The results were negative. Our data show that in areas of north-eastern Europe, where rabies is endemic in raccoon dogs and red foxes, populations of smaller mammals may remain free of rabies.

Characterization of phocid herpesvirus-1 and -2 as putative alpha- and gammaherpesviruses of North American and European pinnipeds.

To study the relationships between herpesvirus recently isolated from different pinniped species, antigenic and genetic analyses were performed. First, herpesviruses isolated from North American harbour seals (Phoca vitulina), a Californian sea lion (Zalophus californianus) and a European grey seal (Halichoerus grypus) were examined in an enzyme immunoassay (EIA) with a panel of monoclonal antibodies which had previously been shown to allow typing of herpesviruses from European harbour seals into two distinct virus types: phocid herpesvirus type-1 and type-2 (PhHV-1 and PhHV-2). The EIA data showed that all but one of the isolates from seals ranging in United States coastal waters were PhHV-2-like while the European grey seal herpesvirus was PhHV-1-like. Genetic characterization was facilitated by PCR analysis using primers based on conserved regions of the glycoprotein B and D (gB and gD) genes of the antigenically closely related canid (CHV) and felid (FHV) herpesvirus. Specific amplified products were obtained with five isolates antigenically characterized as PhHV-1-like but not with five PhHV-2-like isolates. Sequence analysis of the PCR products confirmed greatest similarity to members of the genus Varicellovirus of the Alphaherpesvirinae and in particular to CHV. Sequence analysis of two EcoRI fragments of the PhHV-2 genome (European isolate 7848) revealed greatest similarity to gammaherpesviruses and in particular equine herpesvirus-2. Although an unambiguous subgrouping was not feasible, this is the first evidence that PhHV-2 may be a putative gammaherpesvirus of pinnipeds.

Rabies antibodies in vaccinated dogs.

Forty-seven healthy, owned dogs were vaccinated with Madivak and 85 with Rabisin. Geometric mean titres of 17.40 and 1.03 IU/ml were measured by the rapid immunofluorescent focus inhibition test 30-40 and 350-370 days, respectively, after a single injection. Four out of 130 (3.1%) and 18 out of 106 (17%) dogs had a titre of less than 0.5 IU/ml in serum 30-40 and 350-370 days after vaccination. Twenty-one dogs (19.8%) had a titre of 0.5 IU/ml 350-370 days after vaccination. There was no significant difference in antibody levels between animals vaccinated with Rabisin or Madivak. Our results indicate that a booster is always necessary after a single injection to ensure that all dogs have a lasting antibody titre.

Immunization of cattle against rabies using inactivated cell culture vaccines.

Twenty-one heads of cattle were vaccinated with Madibovin, 31 with Rabdomun and 127 with Rabisin on 4 different farms. Rabies neutralizing antibody titre (> or = 0.5 IU/ml) was detected in 80% of 163 animals tested about 1 month and in 42% of 133 animals tested about 1 year after primary vaccination. On 3 of the farms 86 animals received booster vaccination about 1 year after primary vaccination. All these animals had antibody titre (> or = 0.5 IU/ml) about 1 month after booster and antibody levels were higher than after the primary vaccination. Rabies antibody titres (> or = 0.5 IU/ml) were detected in 96% of 50 animals tested 1 year after the booster. No significant differences (p > 0.05) in antibody levels were detected between animals vaccinated with Madibovin or Rabisin (farm C) respectively with Rabisin or Rabdomun (farm D) at any collection time. Responses to rabies vaccines varied considerably between the farms. After primary vaccination of the animals on 2 farms with the same batch of Rabisin, the antibody levels clearly differed (p < 0.0001) between the farms. Our results indicate that booster is always necessary after primary vaccination to ensure that all animals are protected.

Differentiation of two rabies strains in Estonia with reference to recent Finnish isolates.

We compared 24 rabies samples collected in Estonia in 1989 to 1992, to identify the kinds of rabies strains circulating in this country. Eleven of the strains came from the islands of Saaremaa, Hiiumaa and Muhu, off the Baltic coast; 13 came from the mainland. The mainland strains, like those from the 1988 to 1989 epizootic in Finland, were antigenically different from the 11 island isolates. The island isolates reacted negatively with monoclonal antibody W-187.5 as does the SAD B19 rabies vaccine strain, currently spread as baits to wildlife in Finland and other parts of Europe. In order to unambiguously distinguish the island isolates from the SAD B19 vaccine, we developed a polymerase chain reaction (PCR) protocol for rabies, followed by a single restriction enzyme digestion. This method enabled the island isolates to be differentiated with ease from the vaccine strain SAD B19 at the level of the nucleoprotein-coding region. Additionally, this method had the ability to distinguish other polar field isolates examined, as well as the laboratory challenge virus strain CVS, from SAD B19 vaccine. Modifications of the above PCR method may be used for epidemiological investigations of new outbreaks or of outbreaks involving different species.

Rabies antibody titres in vaccinated reindeer.

One dose of inactivated, adjuvanted rabies vaccine of cell culture origin (Rabisin) induced good but short-duration immunity in close to 100% of the 50 semi-domesticated reindeer (Rangifer tarandus tarandus L.) vaccinated. Most of the animals (44) had rabies virus antibody titre > or = 1.5 IU/ml at 38 days after vaccination. Five animals had titre 0.5 IU. Antibody titres were not, however, present 1 year after primary vaccination in most animals. About 1 year (360-413 days) after primary vaccination, 22 of the 39 reindeer that could be sampled had rabies virus antibody titre < 0.5 IU/ml.

Occurrence of infectious fish diseases in fish farms in northern Finland.

A total of 47 fish located in 10 lake and river systems in northern Finland were examined for furunculosis, enteric redmouth diseases (ERM), viral fish diseases and the parasite Gyrodactylus salaris. Furunculosis was found in 2 fish farms in different watercourses, ERM in 8 fish farms in 3 watercourses and viral diseases were not found at all. G. salaris was looked for only in salmon and rainbow trout and was found in both species in 3 farms belonging to 2 watercourses.

An epidemic of sylvatic rabies in Finland--descriptive epidemiology and results of oral vaccination.

When rabies reappeared in Finland in April 1988, the country had been rabies free since 1959. Soon a picture of sylvatic rabies become evident, its main vector and victim being the raccoon dog (Nyctereutes procyonoides). Between 8 April 1988 and 16 February 1989, 66 virologically verified cases were recorded (48 raccoon dogs, 12 red foxes, 2 badgers, 2 cats, 1 dog and 1 dairy bull) in an area estimated at 1700 km2 in south-eastern Finland. The greatest distance between recorded cases was 67 km. A positive reaction with monoclonal antibody p-41 indicated that the virus was an arctic-type strain. A field trial on oral immunization of small predators was initiated in September 1988 using Tübingen fox baits according to the Bavarian model of bait distribution. Each bait contained 5*10(7) TCID50/ml modified live rabies virus (SAD-B19). The 6 months' surveillance indicate a seroconversion rate of 72% (N = 126) in the raccoon dog population, 67% (N = 56) in the red foxes and 13% (N = 16) in the badgers, when titers greater than or equal to 1.0 IU/ml are considered seropositive. In the whole follow-up period, no statistically significant difference could be detected between the raccoon dogs and red foxes in the rate of seroconversion or in the uptake of tetracycline from the baits. Notably high antibody levels were recorded in both raccoon dogs and red foxes within 4-5 months after vaccination. Of the seropositive animals, the proportion of animals with titers 3.0 IU/ml or greater was higher in raccoon dogs (73%) than in red foxes (51%) (x2 = 5.29, p less than 0.05). The trial shows that raccoon dogs can be immunized against rabies in the field with vaccine baits originally developed for controlling sylvatic rabies in foxes.

Methods for detecting arctic rabies virus in dilution series of field samples.

A modified first passage cell culture method, mouse inoculation test, and ELISA were compared for the detection of rabies in serially diluted field samples. Each original specimen contained rabies antigen, individually identified as an arctic strain by monoclonal antibodies. In 47% of the samples tested in cell cultures, the end-point titer was increased as a result of the first passage. The cell culture method gave higher titers than the mouse inoculation test or ELISA (P less than 0.05 and P less than 0.01, respectively).

Vertical transmission of woodchuck hepatitis virus.

One newborn and 24 fetal woodchuck litters from a woodchuck hepatitis virus (WHV) endemic population were examined for serological or hepatic evidence of WHV. In 18 of 24 fetal litters, there was detectable WHV DNA in the livers, either at explant culture or tissue extract. Most of those WHV DNA-positive liver extracts, which were examined by Southern blot, showed integration of WHV. However, WHV DNA replicative forms without integration were demonstrated in livers of two litters from late gestation. Woodchuck hepatitis surface antigen was detected in the sera of two other fetal litters from the late gestation period. WHV DNA was demonstrated in sera of three litters at different stages of ontogeny.