Quantitative risk assessment of the introduction of rabies into Japan through animals accidentally placed in international freight containers.

Japan has been free from rabies since 1958 and various preventive measures are in place to protect the country from the introduction of the disease. With an increasing number of freight containers arriving in Japan every year, there is a concern that rabies might be reintroduced into Japan through animals arriving in international freight containers. A stochastic simulation model was built assuming the following entry and exposure pathway as being the most likely route of rabies entry: a rabies-infected animal is accidentally placed in a freight container in the country of origin; it survives transportation from the moment the container is sealed in the country of origin until it is opened at the destination in Japan; and it escapes from the container when it is opened at the destination in Japan. Input parameter values were based on surveys of container handling and warehouse agencies and scientific data from the literature. The annual probability of rabies introduction through this pathway worldwide was 5.47 × 10-6 (90 % PI: 9.72 × 10-7-1.33 × 10-5), or rabies would enter Japan every 368,864 (90 %PI: 75,267 - 1,027,568) years. Among sub-regions, the annual probability was highest for South-eastern Asia (4.54 × 10-6 (90 % PI: 8.04 × 10-7-1.11 × 10-5)), followed by Eastern Asia and Southern Asia. The rabies introduction risk from other sub-regions was negligible. The result of scenario analysis indicated that even if any of the main parameters changes, the risk of rabies introduction still remains very low, suggesting that unintentional movement of animals through international freight containers is not a very important pathway of rabies introduction into Japan.

Development of a cross-priming isothermal amplification assay based on the glycoprotein B gene for instant and rapid detection of feline herpesvirus type 1.

A cross-priming isothermal amplification (CPA) assay was developed for detection of feline herpesvirus type 1 (FHV-1). In this assay, the target fragment of the FHV-1 glycoprotein B gene is amplified rapidly by Bst DNA polymerase at a constant temperature (63 °C, 45 min), using a simple thermostat. The assay had no cross-reactions with four types of feline viruses, and the detection limit was 100 copies/µl. The positive rate of clinical samples from CPA was 100% consistent with qPCR but higher than ordinary PCR, indicating its superiority to ordinary PCR. Visualization was achieved using SYBR Green I dye.

Rabies risk and use of post-exposure prophylaxis associated with dog bites in Tennessee.

The canine variant of the rabies virus has been eliminated in the United States. Among the public and many healthcare providers, however, dog bites are still associated with risk for rabies transmission. This study examined the risk of rabies in biting dogs and the use of rabies post-exposure prophylaxis (rPEP) for dog bite victims in Tennessee. The study included a retrospective analysis of laboratory testing requisitions for dogs from 2002 to 2016, collection of clinical data on confirmed rabies-positive dogs from 2008 to 2016 and analysis of hospital discharge data for rPEP from 2007 to 2014. Among dogs submitted for rabies testing, those having a recent history of biting were significantly less likely to test positive for rabies than dogs with no reported bite (OR = 0.01; 95% CI [0.003-0.04]). The most common clinical signs reported among rabies-positive dogs were anorexia, dysphagia, ataxia, limb paresis or paralysis, and lethargy; aggressiveness was uncommon. Among hospital patients with an animal-related injury who received rPEP, more than half (52%) presented with dog bites. These data show that laboratory submissions for rabies testing and prescriptions for rPEP do not reflect the epidemiology of rabies in Tennessee. Education and outreach targeting the public and healthcare providers should emphasize the animal species and situations associated with a greater risk for rabies transmission, such as bites from rabies reservoir species or animals exhibiting signs of neurologic disease.

Epidemiologic survey of feline leukemia virus in domestic cats on Tsushima Island, Japan: management strategy for Tsushima leopard cats.

The Tsushima leopard cat (TLC) Prionailurus bengalensis euptilurus, a subspecies of P. bengalensis, is designated a National Natural Monument of Japan, and lives only on Tsushima Island, Nagasaki Prefecture, Japan. TLCs are threatened by various infectious diseases. Feline leukemia virus (FeLV) causes a serious infectious disease with a poor prognosis in cats. Therefore, the transmission of FeLV from Tsushima domestic cats (TDCs) to TLCs may threaten the TLC population. We investigated the FeLV infection status of both TDCs and TLCs on Tsushima Island by screening blood samples for FeLV p27 antigen and using PCR to amplify the full-length FeLV env gene. The prevalence of FeLV was 6.4% in TDCs and 0% in TLCs. We also demonstrated that the virus can replicate in the cells of TLCs, suggesting its potential cross-species transmission. The viruses in TDCs were classified as genotype I/clade 3, which is prevalent on a nearby island, based on previous studies of FeLV genotypes and FeLV epidemiology. The FeLV viruses identified on Tsushima Island can be further divided into 2 lineages within genotype I/clade 3, which are geographically separated in Kamijima and Shimojima, indicating that FeLV may have been transmitted to Tsushima Island at least twice. Monitoring FeLV infection in the TDC and TLC populations is highly recommended as part of the TLC surveillance and management strategy.

Recombinase Polymerase Amplification Assay-A Simple, Fast and Cost-Effective Alternative to Real Time PCR for Specific Detection of Feline Herpesvirus-1.

Feline herpesvirus 1 (FHV-1), an enveloped dsDNA virus, is one of the major pathogens of feline upper respiratory tract disease (URTD) and ocular disease. Currently, polymerase chain reaction (PCR) remains the gold standard diagnostic tool for FHV-1 infection but is relatively expensive, requires well-equipped laboratories and is not suitable for field tests. Recombinase polymerase amplification (RPA), an isothermal gene amplification technology, has been explored for the molecular diagnosis of infectious diseases. In this study, an exo-RPA assay for FHV-1 detection was developed and validated. Primers targeting specifically the thymidine kinase (TK) gene of FHV-1 were designed. The RPA reaction was performed successfully at 39°C and the results were obtained within 20 min. Using different copy numbers of recombinant plasmid DNA that contains the TK gene as template, we showed the detection limit of exo-RPA was 102 copies DNA/reaction, the same as that of real time PCR. The exo-RPA assay did not cross-detect feline panleukopenia virus, feline calicivirus, bovine herpesvirus-1, pseudorabies virus or chlamydia psittaci, a panel of pathogens important in feline URTD or other viruses in Alphaherpesvirinae, demonstrating high specificity. The assay was validated by testing 120 nasal and ocular conjunctival swabs of cats, and the results were compared with those obtained with real-time PCR. Both assays provided the same testing results in the clinical samples. Compared with real time PCR, the exo-RPA assay uses less-complex equipment that is portable and the reaction is completed much faster. Additionally, commercial RPA reagents in vacuum-sealed pouches can tolerate temperatures up to room temperature for days without loss of activity, suitable for shipment and storage for field tests. Taken together, the exo-RPA assay is a simple, fast and cost-effective alternative to real time PCR, suitable for use in less advanced laboratories and for field detection of FHV-1 infection.

Rabies outbreak in Greece during 2012-2014: use of Geographical Information System for analysis, risk assessment and control.

The objectives of this work were (i) geographical analysis of the 2012-2014 outbreak of rabies in Greece using GIS and (ii) comparative analysis of animal cases with data of potential human exposure to rabies together with environmental data, in order to provide information for risk assessment, effective monitoring and control. Most animal cases (40/48) involved red foxes, while domestic animals were also diagnosed with rabies. Overall, 80% of the cases were diagnosed in central northern Greece; 75% of the cases were diagnosed in low altitudes (<343·5 m), within a distance of 1 km from human settlements. Median distance from livestock farms was 201·25 m. Most people potentially exposed to rabies (889/1060) presented with dog bite injuries. Maximum entropy analysis revealed that distance from farms contributed the highest percentage in defining environmental niche profiles for rabid foxes. Oral vaccination programmes were implemented in 24 administrative units of the country during 2013 and 2014, covering a total surface area of ~60 000 km2. Rabies re-occurrence in Greece emphasizes the need for ongoing surveillance in cross-border areas and in areas with intense human activity.

Assessment of the IFN-ß response to four feline caliciviruses: Infection in CRFK cells.

Feline calicivirus (FCV) is a highly contagious pathogen with a widespread distribution. Although the cat genome has been sequenced, little is known about innate immunity in cats, which limits the understanding of FCV pathogenesis. To investigate the IFN-ß response during FCV infection in CRFK cells, we first cloned and identified the feline IFN-ß promoter sequence and the positive regulatory domain (PRD) motifs, which shared a high similarity with human and porcine IFN-ß promoters. Next, we found that infections with FCV strains F9, Bolin and HRB-SS at the 100 or 1000 TCID50 doses could not activate the IFN-ß promoter at 12 and 24h post-infection. Only strain 2280 infection at a 1000 TCID50 dose could induce the IFN-ß promoter mainly through IRF3 and partially through NF-κB, at 24h post-infection. However, the IFN response occurred much later and was smaller in magnitude compared with that following Sendai virus (SeV) infection. Further, we found that induction of the IFN-ß promoter by FCV 2280 infection depended on dsRNA and not on viral proteins. Finally, we examined whether the IFN-ß response had an antiviral effect against FCV replication. The over-expression of IFN-ß before exposure to the virus reduced viral yields by a range of 2.2-3.2 log10TCID50, but its over-expression at 12h post-infection did not inhibit FCV replication. Our results indicate that some FCV strains cannot induce IFN-ß expression in vitro, which may be a potential factor for FCV survival in cats. Whether this is important in evading the host interferon response in vivo must be investigated.

Prevalence of upper respiratory pathogens in four management models for unowned cats in the Southeast United States.

Upper respiratory infection (URI) is a pervasive problem in cats and impacts the capacity and cost of sheltering programs. This study determined the pattern of respiratory pathogens in cats with and without clinical signs of URI in four different models for managing unowned cats, namely, (1) short-term animal shelters (STS), (2) long-term sanctuaries (LTS), (3) home-based foster care programs (FCP), and (4) trap-neuter-return programs for community cats (TNR). Conjunctival and oropharyngeal swabs from 543 cats, approximately half of which showed clinical signs of URI, were tested for feline herpes virus-1 (FHV), feline calicivirus (FCV), Chlamydia felis, Bordetella bronchiseptica, Mycoplasma felis, and canine influenza virus by real-time PCR. FHV (59%, 41%) and B. bronchiseptica (33%, 24%) were more prevalent in both clinically affected and nonclinical cats, respectively, in STS than other management models. FCV (67%, 51%) and M. felis (84%, 86%) were more prevalent in LTS than any other management model. Clinically affected cats in FCP were more likely to carry FHV (23%, 6%), C. felis (24%, 10%), or M. felis (58%, 38%) than were nonclinical cats. Clinically affected cats in TNR were more likely to carry FCV (55%, 36%) or C. felis (23%, 4%) than were nonclinical cats. The prevalence of individual pathogens varied between different management models, but the majority of the cats in each model carried one or more respiratory pathogens regardless of clinical signs. Both confined and free-roaming cats are at risk of developing infectious respiratory disease and their health should be protected by strategic vaccination, appropriate antibiotic therapy, effective biosecurity, feline stress mitigation, and alternatives to high-density confinement.

Assessment of feline blood for transfusion purposes in the Dublin area of Ireland.

The prevalence of A, B and AB blood types and of feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV) infection was determined in cats in Ireland, in order to determine risk factors for blood taken for transfusion purposes. EDTA blood samples were available from 137 non-pedigree cats and 39 pedigree cats (91 females and 85 males, aged four months to 15.0 years) in the Dublin area of Ireland. Of the 176 EDTA blood samples obtained, 112 (from 92 healthy cats and 20 sick cats) were tested for the presence of both FIV antibodies and FeLV antigens. Blood typing was performed using an immunochromatographic cartridge (CHROM; Alvedia). Testing for FIV and FeLV was performed by ELISA (SNAP FIV/FeLV Combo Test; Idexx Laboratories). Of the 39 pedigree cats, the majority (38 [97.4 per cent]) was type A, and only one (2.6 per cent) was type B. Of the 137 non-pedigree cats, the majority (116 [84.7 per cent]) was type A, 20 (14.6 per cent) were type B, and one (0.7 per cent) was type AB. Of the 92 healthy cats tested, the prevalence of FIV and FeLV positivity was 4.35 and 1.09 per cent, respectively. None of the 20 sick cats tested was FIV-positive; two (10 per cent) of the 20 sick cats were FeLV-positive.

Assessment of viremia associated with experimental primary feline herpesvirus infection or presumed herpetic recrudescence in cats.

OBJECTIVE: To detect feline herpesvirus type 1 (FHV-1) in blood of cats undergoing experimental primary herpetic disease or with spontaneous disease presumed to be caused by FHV-1 reactivation. ANIMALS: 6 young specific-pathogen-free (SPF) cats and 34 adult cats from a shelter. PROCEDURES: Conjunctiva and nares of SPF cats were inoculated with FHV-1, and cats were monitored for 21 days. Periodically, blood was collected for CBC, serum biochemical analyses, and detection of FHV-1 DNA via PCR assay. For shelter cats, a conjunctival swab specimen was collected for FHV-1 PCR assay, and blood mononuclear cells were tested via virus isolation (with or without hydrocortisone) and FHV-1 PCR assay. RESULTS: All SPF cats developed clinical and clinicopathologic evidence of upper respiratory tract and ocular disease only. Via PCR assay, FHV-1 DNA was detected in blood of all SPF cats at least once between 2 and 15 days after inoculation. Feline herpesvirus type 1 DNA was detected in conjunctival swabs of 27 shelter cats; 25 had clinical signs of herpetic infection. However, virus was not isolated from mononuclear cell samples of any shelter cat regardless of passage number or whether hydrocortisone was present in the culture medium; FHV-1 DNA was not detected in any mononuclear cell sample collected from shelter cats. CONCLUSIONS AND CLINICAL RELEVANCE: A brief period of viremia occurred in cats undergoing primary herpetic disease but not in cats undergoing presumed recrudescent herpetic disease. Viremia may be important in the pathogenesis of primary herpetic disease but seems unlikely to be associated with recrudescent disease.

Assessment of infectious organisms associated with chronic rhinosinusitis in cats.

OBJECTIVE: To determine detection rates for feline herpesvirus type 1 (FHV-1), Mycoplasma spp, fungi, and bacteria in flush samples and biopsy specimens from the nasal cavities of cats with and without chronic rhinosinusitis (CRS). DESIGN: Prospective study. ANIMALS: 10 CRS-affected cats and 7 cats without signs of respiratory tract disease. PROCEDURES: Nasal flush samples and biopsy specimens were collected from all cats for bacterial (aerobic and anaerobic), fungal, and mycoplasmal cultures; additional biopsy specimens were collected for virus isolation and polymerase chain reaction (PCR) assay (to detect FHV-1 DNA). RESULTS: Aerobic bacteria were detected in flush samples from 5 of 7 control cats; culture of flush samples from CRS-affected cats yielded aerobic bacteria (9/10 cats), anaerobic bacteria (3/10), and Mycoplasma spp (2/10). No fungal organisms were isolated from any cat. Potential pathogens were isolated significantly more often from CRS-affected cats than from control cats. Bacterial culture of biopsy specimens yielded aerobic bacteria (2/7 control cats and 4/10 CRS-affected cats) and anaerobic bacteria (2/10 CRS-affected cats). Although FHV-1 was not detected in nasal biopsy specimens from control or CRS-affected cats, FHV-1 DNA was detected via PCR assay in specimens from 4 of 7 control cats and 3 of 10 CRS-affected cats. CONCLUSIONS AND CLINICAL RELEVANCE: Compared with findings in control cats, anaerobic bacteria, Mycoplasma spp, and a variety of potentially pathogenic organisms were detected more commonly in samples from cats with CRS. In both groups, FHV-1 was detected via PCR assay as a nonviable organism or in noncultivable amounts.

A discrete-event computer model of feline herpes virus within cat populations.

The purpose of this work was to study the epidemiology of feline herpes virus (FHV), which causes a respiratory disease within natural populations of domestic cats. A stochastic model was constructed using discrete-events simulation. Two habitats (rural vs. urban) were simulated, featuring different demographic, spatial and social patterns. The evolution of immunity in individuals was reproduced, allowing for the random recrudescence of latent infections (influenced by environment and reproduction). Hypotheses concerning the circulation of FHV were examined regarding the role of host density and the possibility of reinfection of host. Uncertainty analyses were performed on the basis of replicated Monte Carlo sampling. The results were in good agreement with serologic data from a long-term study conducted on five populations in France. The model satisfactorily reproduced the variability of natural immunity, and the epidemic features observed. The simulations have shown that FHV can persist in small populations (because of its capacity of reactivation leading to epidemics). However, the impact on demography was not dramatic. The most important parameters in determining change in epidemiology of FHV were: transmission rate corresponding to 'friendly' contacts, and the recrudescence rate of FHV. However, an interaction between these two parameters did not allow estimation of their values.