The rabies situation in Western Europe.

This analysis of the present rabies situation in Western Europe is based on completed questionnaires from 16 countries. The questionnaire focuses on the years 2005 and 2006. Additional information was obtained from the Rabies Bulletin Europe, and from the OIE's publication on "Historical Perspective of Rabies in Europe and the Mediterranean Basin". Rabies in domestic animals and wild carnivores has become very rare in Western Europe. Only Germany reported a low number of rabid foxes for 2005. Bat rabies was observed in four countries. One imported human case was seen in the UK, while Germany reported four cases in transplant recipients. Wildlife vaccination campaigns were conducted by Austria, Germany and France. Reporting of human post-exposure prophylaxis is inconsistent due the lack of information. Similarly, data on domestic animal rabies prophylaxis seem to be difficult to collect.

Can rabies be eradicated?

Rabies, an acute progressive encephalitis, is an ancient zoonosis. Its distribution encompasses all continents, except Antarctica. Agents consist of at least 11 species orgenotypes of rhabdoviruses, in the Genus Lyssavirus. Susceptible natural hosts include all mammals. Primary reservoirs reside in the Orders Carnivora and Chiroptera. A plethora of variants, maintained by a diversity of abundant hosts, presents a challenge to a strict concept of true eradication. Globally, the domestic dog remains the most significant species for viral transmission, responsible for millions of suspect human exposures and tens of thousands of fatalities. As such, this single major target provides an ideal opportunity for focused intervention programmes in humane disease prevention and control, driven by laboratory-based surveillance and guided via modern epidemiological insights. Historically, substantial technical progress throughout the 20th century led to the development of safe, affordable and efficacious animal and human vaccines, resulting in declining disease burdens in selected developed and developing countries. Regional and local disease resurgence occurs, due in part to a combination of political and economic instability, environmental perturbations, and shifting government priorities. Society must recall that despite the recent recognition of other important emerging infectious diseases, none exceed the case fatality rate of rabies. Given the clear relevance of rabies in public health, agriculture, and conservation biology, substantive international progress must continue towards enhanced public awareness, human rabies prevention, wildlife rabies control, and canine rabies elimination, with renewed collaborative vigour.

A molecular epidemiological study of rabies in Cuba.

To investigate the emergence and current situation of terrestrial rabies in Cuba, a collection of rabies virus specimens was employed for genetic characterization. These data supported the monophyletic nature of all terrestrial rabies viruses presently circulating in Cuba but additionally delineated several distinct variants exhibiting limited spatial distribution which may reflect the history of rabies spread on the island. The strain of rabies currently circulating in Cuba, which emerged on the island in the early 20th century, has very close evolutionary ties to the Mexican dog type and is a member of the cosmopolitan lineage widely distributed during the colonial period. The Cuban rabies viruses, which circulate predominantly within the mongoose population, are phylogenetically distant from viruses circulating in mongooses in other parts of the world. These studies illustrate, at a global level, the adaptation of multiple strains of rabies to mongoose species which should be regarded as important wildlife hosts for rabies re-emergence. Given the recent emergence of human cases due to bat contact in Cuba, this study also included a single insectivorous bat specimen which was found to most closely resemble the rabies viruses known to circulate in Mexican vampire bats.

A molecular epidemiological analysis of the incursion of the raccoon strain of rabies virus into Canada.

Three physically separate incursions of the raccoon strain of rabies have entered Canada, two into eastern Ontario in 1999 and one into New Brunswick in 2000. The course of these epizootics is described. Phylogenetic analysis of the index cases from these two provinces with raccoon rabies viruses representative of this strain in the United States supported the independence of these incursions into Canada via cross-border transmission from the United States. Genetic characterization of 190 isolates from these two Canadian provinces over a 550-bp region of the variable central portion of the viral P gene distinguished 14 variants in Ontario and five in New Brunswick although in both regions the variant represented by the initial case was most commonly encountered. The quasi-species nature of the Ontario virus was analysed using isolates taken at different times during the main outbreak to examine whether viral variation was increasing with time as well as changing in nature. These data provide a framework for study of future incursions of this rabies strain into Canada.

Antigenic and genetic characterization of rabies viruses isolated from domestic and wild animals of Brazil identifies the hoary fox as a rabies reservoir.

Fifty Brazilian rabies viruses, collected from many different animal species and several regions of the country, were characterized by partial sequencing of the central, variable region of the P gene, a locus useful for sensitive molecular epidemiological studies. Phylogenetic analysis of the sequences, which included comparison with other rabies strains recovered from throughout the Americas, identified three main groups of Brazilian viruses, arbitrarily designated BRL-1 to BRL-3. BRL-1 was found in terrestrial carnivores and clusters with other American strains of the cosmopolitan lineage. BRL-2 comprised two distinct isolates, recovered from two species of non-haematophagous bats, that had evolutionary links to insectivorous-bat-derived strains of North America. BRL-3 consisted of isolates from vampire bats and from livestock species probably infected via contact with vampire bats. The terrestrial group was further subdivided into three subtypes: BRL-1a was associated exclusively with dogs and cats, while BRL-1b and BRL-1c were found exclusively in hoary foxes. These observations strongly support the role of the Brazilian hoary fox as a rabies reservoir. Screening of representative Brazilian rabies viruses against a collection of anti-rabies monoclonal antibodies (mAbs) identified a small panel of mAbs that could be used to discriminate between all Brazilian subgroups as defined by genetic classification in this study.

Molecular and antigenic characterization of rabies viruses from Iran identifies variants with distinct epidemiological origins.

A molecular epidemiological study of 48 recent rabies isolates recovered from cases reported throughout Iran identified three distinct viral variants, the evolutionary origins of which were identified by phylogenetic comparison with rabies viruses originating from Europe and Asia. Members of group 1 (15 isolates) were recovered from the northern half of the country only, while those of group 2 (31 isolates) were widely dispersed; both groups clustered within the widely disseminated cosmopolitan lineage. The two isolates of group 3 were detected in the northeastern tip of the country only and belonged to the Arctic strain. Rapid variant discrimination tools, employing restriction fragment length polymorphisms applied to amplified fragments of the viral genome, were devised whilst antigenic characterization of representative viruses identified a small panel of monoclonal antibodies that were also discriminatory. The future application of such methods should provide valuable epidemiological information on rabies incidence in Iran.

Lyssavirus P gene characterisation provides insights into the phylogeny of the genus and identifies structural similarities and diversity within the encoded phosphoprotein.

A comprehensive phylogenetic analysis of the Lyssavirus genus, employing P gene sequences from 128 isolates recovered globally, is presented. While confirming prior suggestions of the genetic distinctness of the Australian bat lyssaviruses, these data also revealed a clear division within the rabies virus clade (Genotype 1) between globally distributed viruses circulating predominantly in canid species (subgroup 1-1), and American strains harbored by both chiropteran and terrestrial hosts (subgroup 1-2). Nucleotide substitution patterns within the P locus suggested differential selection operating along the length of the open reading frame (ORF) between rabies viruses of these two subgroups. Comparison of the deduced primary sequences of the encoded phosphoproteins of all isolates provided insights into the product's structural organization. Two conserved (CD1,2) and two variable (VD1,2) domains were evident; high variation in the VD2 region was reflected by differences in hydropathic profiles. Only two of five serine residues reported to function as phosphate acceptors in the P protein of the rabies challenge virus standard (CVS) strain were absolutely conserved; similarly, out of four internal methionines reported to direct internal translation initiation of the CVS strain to produce N-terminally truncated P proteins, only Met(20) was universally retained. In contrast, two sequence motifs, one believed to confer binding to the cytoplasmic dynein light chain LC8, and a lysine-rich sequence probably contributing to N protein binding, were conserved throughout the genus. Most rabies viruses of the carnivora (1-1) contain a potential C ORF in alternate frame to that of P, a feature limited or absent in most other isolates of the genus, an observation interpreted with consideration to the predicted course of lyssavirus evolution.

Report of isolations of unusual lyssaviruses (rabies and Mokola virus) identified retrospectively from Zimbabwe.

Rabies isolates that had been stored between 1983 and 1997 were examined with a panel of anti-lyssavirus nucleocapsid monoclonal antibodies. Out of 56 isolates from cats and various wild carnivore species, 1 isolate of Mokola virus and 5 other non-typical rabies viruses were identified. The Mokola virus isolate was diagnosed as rabies in 1993 from a cat. Genetic analysis of this isolate suggests that it falls in a distinct subgroup of the Mokola virus genotype. The 5 non-typical rabies viruses were isolated from honey badgers (Mellivora capensis), African civets (Civettictis civetta) and an unidentified mongoose (Herpestidae). These isolates are representatives of rarely-reported wildlife-associated strains of rabies, probably maintained by the slender mongoose (Galerella sanguinea). These findings indicate that both Mokola virus and the mongoose-associated variant may be more common in Zimbabwe than is apparent from routine surveillance.

Antigenic and genetic divergence of rabies viruses from bat species indigenous to Canada.

Antigenic characterisation of over 350 chiropteran rabies viruses of the Americas, especially from species reported rabid in Canada, distinguished 13 viral types. In close accord with this classification, nucleotide sequencing of representative isolates, at both the N and G loci, identified four principal phylogenetic groups (I-IV), sub-groups of which circulated in particular bat species. Amongst the North American bat viruses, there was a notable division between group I specimens associated with colonial, non-migratory bats (Myotis sp. and Eptesicus fuscus) and those of group II harbored by solitary, migratory species (Lasiurus sp. and Lasionycteris noctivagans). Certain species of Myotis were clearly identified as rabies reservoirs, an observation often obscured previously by their frequent infection by viral variants of other chiroptera. An additional group (III) apparently circulates in E. fuscus, whilst viruses harbored by both insectivorous and haematophagus bats of Latin America clustered to a separate clade (group IV). Comparison of the predicted N and G proteins of these viruses with those of strains of terrestrial mammals indicated a similarity in structural organisation regardless of host species lifestyle. Finally, these sequences permitted examination of the evolutionary relationship of American bat rabies viruses within the Lyssavirus genus.

Immunohistochemical recognition of a wide spectrum of lyssavirus in formalin-fixed tissues by one monoclonal antibody

Automated indirect immunoperoxidase (avidin-biotin complex) staining using monoclonal antibody #5DF12-3B6, directed against rabies N-protein, was used to detect rabies antigen in tissue samples from animals either naturally or experimentally infected with rabies. This monoclonal antibody recognized all 16 strains of rabies virus tested, as well as rabies-related lyssaviruses including Duvenhage, Lagos Bat, and Mokola. The sample infected with Mokola virus initially showed only weak staining, however, deletion of protease digestion resulted in stronger stain uptake. The test was sensitive and specific, correctly identifying rabies antigen in all but one of the samples tested (37/38), and no apparent staining in any of the negative samples tested (23/23). Tissues from 16 mammalian species were tested, including one rabies infected human tissue sample. The utility of the immunoperoxidase staining method described in this study lies in the ability of one monoclonal to recognize a broad spectrum of lyssaviruses in formalin-fixed tissues.

[Rabies-free status of Switzerland following 30 years of rabies in foxes]. / Tollwutfreiheit der Schweiz nach 30 Jahren Fuchstollwut.

The European fox rabies epizootic starting in 1939 at the eastern border of Poland reached Switzerland on March 3, 1967. Rabies spread over large parts of the country until 1977, the year it caused three human deaths. In 1978 the first field trial world-wide for the oral immunization of foxes against rabies was conducted in Switzerland. Initially, the expansion of the vaccination area led to a rapid reduction in rabies cases. However, the 1990s were characterized by a recrudescence of rabies in spite of regular oral immunization of foxes. The last endemic case of rabies was diagnosed in 1996 after an adaptation of the vaccination strategy. A total of 17,109 rabies cases, of which 73% in foxes and 14% in domestic animals were diagnosed, leading to an estimated number of some 25,000 postexposure treatments in humans. To eliminate rabies, a total of 2.8 million baits containing a modified live virus were distributed--mostly by hand--in the field.

[The development of strategies for the field application of oral immunization of foxes against rabies]. / Entwicklung von Strategien zur Feldanwendung der oralen Immunisierung von Füchsen gegen Tollwut.

Preconditions for successful field application of the idea to orally immunize foxes against rabies were a) the development of an efficacious and safe vaccine and an attractive bait and b) a suitable strategy to reach a high proportion of the vector population with vaccine-ladden baits. A geographical and epidemiological concept on how to implement the vaccination strategy and how to achieve a sufficiently high level of surveillance were further cornerstones contributing to the success of the method. Finally, political decisions and a legal basis for the application of the method of oral immunization were required to eradicate fox rabies in Switzerland.

Oral immunization against rabies: afterthoughts and foresight.

The article contains personal views on some issues that are frequently addressed in discussions about rabies control, and on some related topics that are often overlooked. The first field applications of oral wildlife rabies immunization in the Swiss Rhone Valley were preceded by many years of international cooperative studies on efficacy and safety. They were significant "faits accomplis" that facilitated similar endeavors in other countries. Some aspects of the residual pathogenicity of oral rabies vaccines are discussed. The field efficacy of oral wildlife immunization is the outcome of complex interactions between vaccine and bait attributes, bait distribution procedures, and habitat properties. Significant difficulties hinder the interpretation of field observations on efficacy. Though oral wildlife immunization is not an animal welfare act and not a conservationist achievement, it is an attempt at zoonosis control intended to protect human health and prevent economic losses.

A panel of monoclonal antibodies targeting the rabies virus phosphoprotein identifies a highly variable epitope of value for sensitive strain discrimination.

A recombinant rabies virus phosphoprotein fusion product (GST-P) was used to generate a series of monoclonal antibodies (MAbs) with anti-P reactivity. Competitive binding assays classified 27 of these MAbs into four groups (I to IV), and 24 of them were deemed to recognize linear epitopes, as judged by their reaction in immunoblots. The linear epitope recognized in each case was mapped by using two series of N- and C-terminally deleted recombinant phosphoproteins. Assessment of the reactivities of representative MAbs to a variety of lyssavirus isolates by an indirect fluorescent antibody test indicated that group I MAbs, which recognized a highly conserved N-terminal epitope, were broadly cross-reactive with all lyssaviruses assayed, while group III MAbs, which reacted with a site overlapping that of group I MAbs, exhibited variable reactivities and group IV MAbs reacted with most isolates of genotypes 1, 6, and 7 only. In contrast, group II MAbs, which recognized an epitope located within a highly divergent central portion of the protein, were exquisitely strain specific. These anti-P MAbs are potentially useful tools for lyssavirus identification and discrimination.

Study of the dog population and the rabies control activities in the Mirigama area of Sri Lanka.

The national health authorities of Sri Lanka have adopted a combined strategy of rabies vaccination and stray dog removal to control endemic dog rabies. Despite the control efforts, an increase of animal and human rabies cases has occurred since 1994. As a consequence, a project to evaluate the national rabies control program has been started and a study focussing on the dog population and rabies control activities in a limited area of Mirigama was conducted. Information on canine abundance and the accessibility of dogs for rabies vaccination was obtained by a household survey, vaccination of dogs against rabies at several vaccination points, collar-marking, and transect line recapture. The number of unvaccinated dogs was estimated by using Bayesian methodology. The estimated number of dogs per square kilometre was 87 (95% credibility interval: 80, 93) for owned dogs and 108 (100, 116) for owned and ownerless dogs. Coverage after the immunisation campaign was 57.6% (53.3, 61.9%) if vaccination at the vaccination points was considered and 66% (60.4, 72.0%) if recently provided vaccination by private veterinarians was also taken into account. The proportion of households with at least one dog vaccinated varied between 59.1 and 94.2% within the catchment area of the different vaccination points. Unvaccinated dogs were puppies (12%), ownerless dogs (57%), and owned dogs, which were not presented for vaccination (31%). In order to improve the rabies immunisation coverage among dogs and to achieve complete elimination of rabies it was recommended that the 95% catchment area of each vaccination point be assessed, the distribution of vaccination points in the vaccination area be redefined if necessary, a system for the vaccination of dogs missing the vaccination campaign for dog owner-specific reasons be established, and an inexpensive marking system be used for vaccinated dogs.

Phylogeographic patterns exhibited by Ontario rabies virus variants.

A previous study on N gene variation of rabies viruses circulating in Ontario red foxes identified four viral variants. This study confirms the geographical localization of these variants and extends the analysis to the less conserved G gene of these viruses. A greater number of regionally localized variants was revealed and their phylogenetic relationships have been examined. Ongoing surveillance on recent disease outbreaks revealed that variants do not always persist in specific areas. The distribution of these variants did however appear to be influenced by topographical features of the study area likely to affect host animal movements and contacts. The majority of G gene base changes were synonymous and limited glycoprotein sequence variation predominantly to the C-terminal transmembrane and endo-domains. These data are most readily explained by random appearance of genetic viral variants followed by their spread throughout sub-populations of the fox host according to the easiest routes of transmission.

The epidemiology of rabies in Zimbabwe. 1. Rabies in dogs (Canis familiaris).

The epidemiology of rabies in dogs in Zimbabwe is described using data from 1950, when rabies was re-introduced after a 37-year absence, to 1996. Dogs constituted 45.7% of all laboratory-confirmed rabies cases and were the species most frequently diagnosed with the disease. Slightly more cases were diagnosed from June to November than in other months. From 1950 to the early 1980s, most dog cases were recorded from commercial farming areas, but since the early 1980s most have been recorded from communal (subsistence farming) areas. This change appears to be due to improved surveillance in communal areas and not to any change in the prevalence of rabies. Dog rabies therefore appears to be maintained mainly in communal area dog populations, particularly the large communal area blocks. Urban rabies was not important except in the city of Mutare. Where dog rabies prevalence was high, the disease was cyclic with periods between peak prevalence ranging from 4-7 years. Dog rabies cases were, on the whole, independent of jackal rabies and rabies in other carnivores. There was a significant negative relationship between the annual number of rabies vaccine doses administered nationally to dogs and the annual number of dog rabies cases lagged by one year, indicating that the past levels of immunisation coverage have had a significant effect on the number of rabies cases. However, dog vaccination coverage has clearly not been adequate to prevent the regular occurrence of rabies in dogs.

The epidemiology of rabies in Zimbabwe. 2. Rabies in jackals (Canis adustus and Canis mesomelas).

The epidemiology of rabies in Canis adustus (the side-striped jackal) and Canis mesomelas (the black-backed jackal) in Zimbabwe is described using data collected from 1950-1996. Cases in the two species made up 25.2% of all confirmed cases, second only to domestic dogs. Since the species of jackal cases was not recorded on rabies submission forms, the country was divided into areas according to species dominance and jackal cases were assigned to either C. adustus or C. mesomelas dominant zones or a sympatric zone where the relative status of the species is not known. Jackal rabies in both species is maintained in the commercial farming sector. Jackal rabies in the C. adustus zone occurs as dense epidemics, which begin at a single focus and spread centrifugally. The foci were initiated by rabid dogs, but once initiated the epidemic is maintained by C. adustus independently of other species. The extent of outbreaks in the C. adustus zone was limited by geographical (landuse type and jackal species interface) boundaries. Jackal rabies in C. adustus zones showed two seasonal peaks with the main peak occurring during late summer and the second peak during winter. In the C. mesomelas zone jackal rabies was more sparse but it occurred during most years. C. mesomelas is also able to maintain rabies independently of other species, although the epidemiology of the disease in this species is unclear. Transmission of rabies cycles between the two jackal species zones does not appear to occur as epidemics terminate when crossing the C. adustus and C. mesomelas interface boundaries.