Comparison of intra- and inter-host genetic diversity in rabies virus during experimental cross-species transmission.

The development of high-throughput genome sequencing enables accurate measurements of levels of sub-consensus intra-host virus genetic diversity and analysis of the role played by natural selection during cross-species transmission. We analysed the natural and experimental evolution of rabies virus (RABV), an important example of a virus that is able to make multiple host jumps. In particular, we (i) analyzed RABV evolution during experimental host switching with the goal of identifying possible genetic markers of host adaptation, (ii) compared the mutational changes observed during passage with those observed in natura, and (iii) determined whether the colonization of new hosts or tissues requires adaptive evolution in the virus. To address these aims, animal infection models (dog and fox) and primary cell culture models (embryo brain cells of dog and fox) were developed and viral variation was studied in detail through deep genome sequencing. Our analysis revealed a strong unidirectional host evolutionary effect, as dog-adapted rabies virus was able to replicate in fox and fox cells relatively easily, while dogs or neuronal dog cells were not easily susceptible to fox adapted-RABV. This suggests that dog RABV may be able to adapt to some hosts more easily than other host variants, or that when RABV switched from dogs to red foxes it lost its ability to adapt easily to other species. Although no difference in patterns of mutation variation between different host organs was observed, mutations were common following both in vitro and in vivo passage. However, only a small number of these mutations also appeared in natura, suggesting that adaptation during successful cross-species virus transmission is a complex, multifactorial evolutionary process.

SAFETY AND SEROLOGICAL RESPONSE TO MULTIVALENT CANINE DISTEMPER VIRUS VACCINE IN RED FOXES (VULPES VULPES).

Canine distemper virus (CDV) vaccination using commercial vaccines has been recommended as a useful preventive tool in zoological collections worldwide for the past 30 yr. Zoological facilities have not conducted studies to assess the effectiveness and safety of the multivalent Recombitek C6 and C8 in nondomestic carnivores. They are the only CDV recombinant vaccines available in Latin America. Seventeen clinically healthy red foxes born in Buin Zoo were divided into three groups and administered 1 ml of Recombitek C6 vaccine. Group A consisted of three animals of 9 mo of age without previous vaccination (WPV) that received a single dose. Group B consisted of four animals of 10 mo of age WPV; they received a series of three doses with a 21-day interval between doses. Group C consisted of eight animals > 1 yr of age that had received a previous vaccination > 1 yr ago; they received a single-dose booster vaccination. Titers for antibodies against CDV were measured by a serum neutralization test. All animals remained clinically healthy throughout the study period and without clinical signs of disease. Only two foxes (group C) did not show any increase in the antibody titer to the vaccine. All animals of groups A and B seroconverted at 21 days after the first vaccination. Only two animals (both from group B) showed an adequate antibody protective response (titers >100) after 180 days. Absence of adverse reactions in red foxes included in this study supports the safety and apparently nondeleterious effect of CDV recombinant vaccine reported in other nondomestic carnivores. Low antibody response and lack of persistence in the serological response 6 mo after vaccination with a single dose suggested limited protective benefits in this species. Additional research is needed to confirm the antibody titer response to multiple vaccinations in this species.

Protective immune response of oral rabies vaccine in stray dogs, corsacs and steppe wolves after a single immunization.

In this study the safety and protective immunity of an oral rabies vaccine, based on the live, modified rabies virus strain VRC-RZ2, was examined in stray dogs (Canis Sp.), corsacs (Vulpes corsac) and steppe wolves (Canis lupus campestris). In the safety group (dogs, n=6; corsacs, n=3; wolves, n=3) which was vaccinated with a 10-times field dose/animal, no animals showed any signs of disease or changes in behavior or appetite during the period of clinical observation, similar to the animals in the negative control group. Saliva samples taken from animals prior and post (5th and 10th days) vaccination failed to demonstrate rabies virus antigen. Observations of immunogenicity in vaccinated carnivores (dogs, corsacs and wolves) during a 180 day period showed the titers of virus neutralizing antibodies (VNA) in the blood sera of vaccinated dogs to be within 0.59-1.37 IU/mL. On 14 days post vaccination (dpv), all the wild carnivores had detectable levels of neutralizing antibodies, with mean titers ranging from 0.50 ± 0.07 IU/mL (for wolves) to 0.59 ± 0.10 IU/mL (for corsacs). Weeks after vaccination, all the vaccinated wolves and corsacs had higher levels of neutralizing antibodies: 0.70 ± 0.10 - 0.71 ± 0.08 IU/mL at 30 dpv, 1.06 ± 0.08 - 1.28 ± 0.21 IU/mL at 60 dpv and 0.41 ± 0.09 - 047 ± 0.06 at 180 dpv. The highest level of VNA (˃1.0 IU/ml) was detected at 60 dpv, in all vaccinated animals. After challenge all vaccinated dogs remained healthy for 180 days. Control animals (unvaccinated dogs) developed symptoms of rabies on day 6 post administration of a virulent virus and died of rabies on days 11-13. Of note, the VNA titers in all the wild carnivores (corsacs and wolves) immunized with VRC-RZ2 were higher than 0.5 IU/ml (0.59 ± 0.11 IU/ml), even as early as 14 days post vaccination. These, presumably protective, titers of antibodies to rabies virus were present in the dogs and wild carnivores examined in this study for at least 180 days.

The Successful Elimination of Sylvatic Rabies Using Oral Vaccination of Foxes in Slovenia.

Sylvatic rabies was present in Slovenia between 1973 and 2013, with the red fox as the main reservoir of the rabies virus. The first oral rabies vaccination (ORV) control program in foxes started in 1988, using the manual distribution of baits. Significant improvement of fox vaccination was achieved with the aerial distribution of baits, starting in 1995 and successfully finished with the final, fifty-ninth vaccination campaign in 2019. Between 1979 and 2019, a total of 86,471 samples were tested, and 10,975 (12.69%) rabies-positive animals were identified. Within the ORV, two different vaccines were used, containing modified live virus strain Street Alabama Dufferin (SAD) B19 and SAD Bern, while the last ORV campaigns were completed in 2019, with a vaccine containing a genetically modified strain of SPBN GASGAS. Molecular epidemiological studies of 95 rabies-positive samples, originating from red foxes, badgers, cattle, dogs, martens, cats, and horses, revealed a low genetic diversity of circulating strains and high similarity to strains from neighboring countries. During the elimination program, few vaccine-induced rabies cases were detected: three in red foxes and one case in a marten, with no epidemiological relevance. Slovenia has been officially declared a country free of rabies since 2016.

Responsiveness of various reservoir species to oral rabies vaccination correlates with differences in vaccine uptake of mucosa associated lymphoid tissues.

Oral rabies vaccination (ORV) is highly effective in foxes and raccoon dogs, whereas for unknown reasons the efficacy of ORV in other reservoir species is less pronounced. To investigate possible variations in species-specific cell tropism and local replication of vaccine virus, different reservoir species including foxes, raccoon dogs, raccoons, mongooses, dogs and skunks were orally immunised with a highly attenuated, high-titred GFP-expressing rabies virus (RABV). Immunofluorescence and RT-qPCR screenings revealed clear differences among species suggesting host specific limitations to ORV. While for responsive species the palatine tonsils (tonsilla palatina) were identified as a main site of virus replication, less virus dissemination was observed in the tonsils of rather refractory species. While our comparison of vaccine virus tropism emphasizes the important role that the tonsilla palatina plays in eliciting an immune response to ORV, our data also indicate that other lymphoid tissues may have a more important role than originally anticipated. Overall, these data support a model in which the susceptibility to oral live RABV vaccine infection of lymphatic tissue is a major determinant in vaccination efficacy. The present results may help to direct future research for improving vaccine uptake and efficacy of oral rabies vaccines under field conditions.

A highly efficient recombinant canarypox virus-based vaccine against canine distemper virus constructed using the CRISPR/Cas9 gene editing method.

Canine distemper virus (CDV) is the causative agent of canine distemper (CD), which is one of the most important infectious diseases affecting wild and domestic carnivores. Vaccination represents an effective approach to prevent CDV infection among domestic carnivores. Canarypox-vectored recombinant CD vaccines (such as Recombitek CDV, PureVax Ferret Distemper, and Merial) with the CDV hemagglutinin (H) and fusion (F) genes can induce a potent immune response in dogs and ferrets. However, the vaccine's effectiveness varies with the species. In the current study, we developed a highly efficient recombinant canarypox virus termed as "ALVAC-CDV-M-F-H/C5-" that contained CDV virus-like particles (VLPs) by using the CRISPR/Cas9 gene editing method, which enabled concurrent expression of the matrix (M), H, and F genes. The recombinant strain provided faster seroconversion than the parent strain among minks as well as provided higher rates of antibody positivity than the parent strain among foxes and minks even before the administration of a second booster vaccination. We demonstrated, for the first time, that the CRISPR/Cas9 system can be applied for the rapid and efficient modification of the ALVAC-CDV-F-H genome and also that a high-dose new recombinant strain that produces CDV VLPs may present good outcomes in the prevention of CD among foxes and minks.

Serological detection of anti-Trichinella antibodies in wild foxes and experimentally infected farmed foxes in Norway.

Trichinella surveillance in wildlife has relied on the detection of muscle larvae using digestion techniques. Serology has been proposed as more suitable for large-scale epidemiological studies in wildlife. In this study, 328 individual sera from wild red foxes and 16 sera from experimentally infected farmed foxes were serologically tested with both excretory/secretory antigen (E/S) and the synthetic beta-tyvelose glycan antigen, in indirect ELISA tests. The wild red foxes (Vulpes vulpes) had previously been examined for muscle larvae, using muscle digestion, whilst the experimentally infected farmed foxes were inoculated per os with either a low dose, 500 larvae, or a high dose, 10,000 of Trichinella nativa muscle larvae. Western blot (WB) was carried out on all seropositive samples using crude larval antigen. The present study found both beta-tyvelose and E/S antigen suited for the detection of antibodies to Trichinella spp., and T. nativa in particular, in foxes. Both ELISA antigens performed well, although, the E/S antigen was superior to the beta-tyvelose antigen, with sera that had been stored at -20 degrees C for more than 10 years. Neither antigen, however, detected all of the samples proven seropositive by WB: E/S detected 21 of the 27 wild red fox sera positive by WB; beta-tyvelose detected 22 positive sera; and in total 24 of the 27 positive WB sera were identified using both antigens. Serology alone, without WB or muscle digestion, led to a two- to threefold higher seroprevalence estimate, respectively. The use of E/S antigen in conjunction with the WB was the method of choice for the screening of wild red fox populations for Trichinella. Antibody persistence to T. nativa was short in the low dose group where antibody levels were not different from background by 32 wpi. In total, 7.3% (24/328) of the wild red fox population had antibodies to Trichinella on ELISA and WB. Antibodies were identified in foxes from a further two regions in Norway compared to the original muscle digestion results.

Control and elimination of rabies in Croatia.

Despite the implementation of control measures (preventive dog vaccination), rabies has become endemic in Croatia, with red foxes being the main reservoir species. Oral rabies vaccination (ORV) campaigns supported by the European Commission have been conducted twice a year since the spring of 2011. The first campaigns were limited to the northern and eastern parts of the country, and from the autumn of 2012, the program was extended to the entire country. The Lysvulpen vaccine containing the SAD Bern strain was used for ORV. Following the vaccination campaigns, the number of rabies cases decreased, and the last positive case was recorded in February 2014. The bait uptake ranged from 24.86% to 84.62% and the immunisation rate from 11.24% to 35.64%.

Rickettsia parkeri in free-ranging wild canids from Brazilian Pampa.

Spotted fevers are tick-borne diseases associated with various Rickettsia species. Rickettsia parkeri sensu stricto (s.s.) is the agent of an emerging eschar-associated rickettsiosis in humans from the USA and South American Pampa. Considering that R. parkeri s.s. is restricted to Americas and the potential role of dogs in the epidemiology of the disease, it is thus reasonable to hypothesize that wild canids could be involved in the enzootic cycle of this rickettsiosis. The aim of this work was to investigate the potential role of the wild canids from Pampa, Cerdocyon thous (crab-eating fox) and Lycalopex gymnocercus (Pampas fox), in the ecology of R. parkeri s.s. For that, 32 live-trapped free-ranging wild canids were sampled. Ticks were observed in 30 of the 32 foxes. Of the 292 ticks collected, 22 (7.5%) were positive by PCR for the presence of R. parkeri s.s. DNA. Also, 20 (62%) wild canids showed antibodies against R. parkeri. The results suggest that wild canids are involved in the enzootic cycle of R. parkeri s.s. in the Pampa biome and could be responsible for pathogen (and its vectors) dispersal.

Factors influencing the success of aerial rabies vaccination of foxes.

Sylvatic rabies has been eradicated from most of Central Europe, but cases still occur in the Balkans. Oral rabies vaccination of foxes is an effective method for controlling the disease. The aim of this study was to evaluate the success of aerial vaccination campaigns conducted in Montenegro by identifying ecological, environmental and climatic factors that influenced the prevalence of antibodies to the rabies vaccine. To monitor the bait uptake and the serological responses to vaccination, foxes were shot by hunters. Of 175 shot foxes, 142 foxes (81.1%) had consumed baits. Of these only a total of 81 (57.0%) tested positive for rabies vaccine antibodies, possibly, due to the delayed uptake of bait in which the rabies vaccine was already inactivated. We found that low vaccination responses were associated with high fox density and bait delivery in open areas. In high fox density habitat, bait uptake might be delayed as other food and prey options for foxes are abundant. Similarly, delayed bait uptake probably occurred in open areas as such areas are less frequently used by foxes. The findings of this study suggest that efficacy of oral rabies vaccination by aerial delivery is associated with landscape features.

Cross reactive antigenicity in orally vaccinated foxes and raccoon dogs against European Bat Lyssavirus type 1 and 2.

For the first time, the effectiveness of oral rabies vaccines against European Bat Lyssaviruses Type 1 (EBLV-1) and Type 2 (EBLV-2) by means of cross-neutralization assays was investigated. Sera from orally vaccinated red foxes (Vulpes vulpes) and raccoon dogs (Nyctereutes procyonoides) with the Street Alabama Dufferin (SAD) B19 and SAD P5/88 live-modified vaccine viruses were used to study the cross reactive antigenicity against CVS-11 (genotype 1), EBLV-1 (genotype 5) and EBLV-2 (genotype 6). For comparison, similar crossneutralization assays with sera from EBLV-1 and EBLV-2 infected ferrets (Mustela putorius furo) and/or foxes were conducted. Sera from animals vaccinated with the two oral rabies vaccines were reactive against CVS-11 (homologous virus), EBLV-1 and EBLV-2 (heterologous virus). There was a positive relationship among the virus neutralising antibody titres (VNA); high VNA titres against CVS-11 also resulted in high VNA titres against each EBLV, whereas in general, the VNA-titres obtained with homologous virus were statistically higher than those with the heterologous virus except for SAD P5/88 vaccinated raccoon dogs. No significant difference was found between EBLV-1 and EBLV-2 VNA titres. A similar trend was observed when the results of the cross-neutralization data of the foxes and ferrets inoculated i.m. with EBLV-1 and/or EBLV-2 was analysed. Based on the similarity of the EBLV-VNA titres obtained in our study questions were raised on whether the genetic distance of genotype 5 & 6 within phylogroup 1 really does reflect their antigenetic characteristics or whether this is a feature of attenuated live vaccine viruses. This broad cross protection, however, demonstrated that the representatives of attenuated SAD strains of oral rabies vaccines currently used in Germany are most likely able to protect the reservoir species, red fox and raccoon dog, against EBLV-1 and EBLV-2 infection.

Comparison of two antigens for demonstration of Trichinella spp. antibodies in blood and muscle fluid of foxes, pigs and wild boars.

For the surveillance of trichinellosis, the digestion method is reliable but also labour intensive. The serological methods for the detection of Trichinella-specific antibodies using ELISA offer a sensitive and relatively specific alternative. For serological studies, sera or plasma from blood samples are the most common source of antibodies, but although the concentration of antibodies is approximately 10-fold lower, muscle fluid can be a good alternative particularly for testing of wildlife samples. In the present study, an indirect ELISA technique was evaluated on both sera and muscle fluids from experimentally infected foxes, pigs, and wild boars using both excretory/secretory (E/S) antigens and a synthetic glycan antigen, beta-tyvelose. Although the synthetic antigen appears to be less sensitive than the E/S antigens, Trichinella-specific IgG antibodies were detected in both serum samples and muscle fluid samples from pigs, wild boars and foxes infected at levels which would be important for food safety or represent a significant reservoir for further transmission.

Serological evidence for naturally occurring transmission of Neospora caninum among foxes (Vulpes vulpes).

The study describes the time course of the Neospora caninum-specific antibody response in experimentally infected foxes, in naturally N. caninum-seropositive vixens and their litters. An immunofluorescence test, a tachyzoite surface antigen based ELISA and an immunoblot assay were established for this purpose. The immunoblot patterns of naturally seropositive and experimentally infected foxes revealed a high degree of similarity and resembled those reported for other intermediate host species. Reactions against immunodominant antigens of Mr 56, 68 and >94 kDa were observed which could be linked with a period of 14 days-2 months post experimental infection with tachyzoites. Cubs born by naturally seropositive vixens were found to be persistently or transiently seropositive, in the latter case, specific antibodies were detected only up to 44 days after birth. These antibodies may thus be of maternal origin. Differences between the immunoblot patterns of persistently positive cubs, those of their mothers and of transiently positive cubs, in particular the differential response to antigens of Mr 56 and 68 kDa, prove that cubs with persistent antibodies had actively mounted an antibody response. This result provides the first evidence for the postnatal or vertical transmission of N. caninum among naturally seropositive foxes.

Intrafamilial lung xenografts from fox to dog.

The technical feasibility and rejection characteristics of intrafamilial fox-to-dog xenografts were studied. The results indicate that (1) both lungs of a smaller donor can be successfully transplanted into a single hemithorax of a larger recipient, (2) fox-to-dog lung xenografts are capable of providing total pulmonary function at tolerable right ventricular pressures, and (3) the rejection process of unmodified intrafamilial lung xenografts, was similar to and not more vigorous or more rapid than that of canine lung allografts in untreated recipients.

Where do we stand with oral vaccination of foxes against rabies in Europe?

The oral vaccination of wild animals was first attempted in 1962 after the repeated failure of poisoning or trapping to control movement of the disease in these species. Foxes were chosen for research purposes because they are a problem animal species and are exquisitely susceptible to rabies. The first successful laboratory studies with attenuated vaccine came in 1971, and the first successful field trial was carried out in Switzerland beginning in 1978. In the 1980's several European countries joined the trials. In the following years many improvements were made: the chicken head was replaced by machine-made baits for easy mass production, the hand placement of vaccine baits was to a greater extent being replaced by aerial distribution (small aircraft or helicopter), and several new vaccines were developed. Additionally, the European Union supported the oral vaccination financially. There was a great impact on the rabies situation. When the second country, Germany, joined the field trial in 1983 the total of reported rabies cases in Europe amounted to 23,002, in 1995 a total of 8,134 cases was reported. In spite of the great improvement made in the past years, in the beginning of the 1990's several severe set-backs were experienced. The paper elaborates on reasons for these set-backs and suggests a strategy to overcome them.

Partial characterization of antigenic sperm proteins in foxes (Vulpes vulpes).

The aim of this work was to identify antigenic proteins on fox spermatozoa. Fox spermatozoa proteins were injected into 3 female rabbits and into 3 male and 3 female foxes. In rabbits, a rapid humoral response was observed. Using rabbit sera for Western blotting, 23 fox sperm protein bands were recognized between 10 and 110 kd. In foxes, the time course of antibody response was studied in the same manner. The number of recognized bands was maximal on day 75 for 2 foxes, on day 90 for 3 foxes, and on day 120 for 1 fox. Western blot patterns varied from one fox to another. On the whole, 25 protein bands between 10 and 110 kd were recognized. Using fluorescein isothiocyanate (FITC) labeling on fox spermatozoa with rabbit and fox sera, we showed that several antigens recognized by the antisera were located at or near the surface of the spermatozoa. By two-dimensional electrophoresis and gel-purification, we have selected 6 highly antigenic proteins with molecular weights of 11.4, 14.7, 16.4, 16.4, 16.8, and 16.9 kd, and isoelectric points of 6.0, 6.0, 6.2, 5.5, 5.3, and 5.8, respectively, and one antigenic protein at 97 kd with an isoelectric point of 4.3 to 4.6. The results of this study can be used to characterize these 7 antigens selected more precisely by microsequencing or mass spectrometry.

Kinetics of maternal immunity against rabies in fox cubs (Vulpes vulpes).

BACKGROUND: In previous experiments, it was demonstrated that maternal antibodies (maAb) against rabies in foxes (Vulpes vulpes) were transferred from the vixen to her offspring. However, data was lacking from cubs during the first three weeks post partum. Therefore, this complementary study was initiated. METHODS: Blood samples (n = 281) were collected from 64 cubs (3 to 43 days old) whelped by 19 rabies-immune captive-bred vixens. Sera was collected up to six times from each cub. The samples were analysed by a fluorescence focus inhibition technique (RFFIT), and antibody titres (nAb) were expressed in IU/ml. The obtained data was pooled with previous data sets. Subsequently, a total of 499 serum samples from 249 cubs whelped by 54 rabies-immune vixens were fitted to a non-linear regression model. RESULTS: The disappearance rate of maAb was independent of the vixens' nAb-titre. The maAb-titre of the cubs decreased exponentially with age and the half-life of the maAb was estimated to be 9.34 days. However, maAb of offspring whelped by vixens with high nAb-titres can be detected for longer in RFFIT than that of offspring whelped by vixens with relatively low nAb-titres. At a mean critical age of about 23 days post partum, maAb could no longer be distinguished from unspecific reactions in RFFIT depending on the amount of maAb transferred by the mother. CONCLUSIONS: The amount of maAb cubs receive is directly proportional to the titre of the vixen and decreases exponentially with age below detectable levels in seroneutralisation tests at a relatively early age.

A seroepidemiological survey for orthopox virus in the red fox (Vulpes vulpes).

703 blood samples from red foxes (Vulpes vulpes) were investigated to determine the prevalence of antibody against an orthopox virus (vaccinia virus strain Elstree). A blocking-ELISA based on a neutralizing monoclonal antibody was used. In this assay 46 sera (6.5%) were positive with titers of 1:2 to 1:16. ELISA-results were confirmed by the plaque reduction test with 44 of the 46 sera reacting positively. The specificity of antibodies in 21 selected sera was also demonstrated by Western blot analysis.

[Cellular immunity in the red fox vaccinated against rabies. Studies with the lymphocyte transformation test]. / Immunité à mediation cellulaire du renard vaccine contre la rage. Etude par le test de transformation lymphoblastique.

The best conditions for performing a lymphocyte transformation test have been studied in the red fox with three phytomitogens: phytohaemagglutinin, concanavalin A and Pokeweed (phytolacca). These assays were carried out with varying different parameters of the reaction, among which concentration of mitogens, nature and duration of the lymphocyte cultures. Isolation of fox lymphocytes gave lower yields than human lymphocytes, but were similar in number to those from other domestic animal species. The best mitogenetic reactivity was observed with concanavalin A (optimal concentration being not very different from one mitogen to another) and the maximum transformation ratio was usually observed after four days of lymphocyte cultivation in round bottom plates. Mean value of stimulation ratio is about ten times less that in man, but not very different from dogs. These results allowed the use of this technique a second time to explore specific stimulations by rabies antigen. These last were observed only in 33% of foxes recently vaccinated against rabies, either with live or inactivated virus vaccine.

Effectivity of oral vaccination of foxes against rabies with Flury HEP strain 675.

A group of 14 adult foxes was orally vaccinated with 2 ml Flury HEP strain 675, titre 10(8.2 TCID50. Each fox seroconverted neutralizing antibody titres ranging between 1:40 and 1:640. These foxes survived an intramuscular challenge with 8000 mouse LD50 pathogenic rabies virus, whereas six out of seven unprotected control foxes died within 28 days. This study was accompanied by virus isolation, fluorescence microscopic examination and neutralization tests.