A Taq-Man-based multiplex quantitative PCR for the simultaneous detection and quantification of Angiostrongylus vasorum, Crenosoma vulpis, and species of respiratory capillarids in canids.

In recent years, Angiostrongylus vasorum, Crenosoma vulpis, Eucoleus aerophilus (syn. Capillaria aerophila) and Eucoleus boehmi (syn. Capillaria boehmi), commonly referred to as canine lungworms, have gained a growing interest worldwide as the result of their geographical expansion. Each of these nematode species differs considerably in its biology and pathogenicity. Despite their impact on dogs' health, these parasites are often underdiagnosed owing to diagnostic challenges. Here, we describe the development and validation of a Taq-Man-based multiplex quantitative PCR (qPCR) for the simultaneous detection of the main species of canine lungworms in faeces of infected dogs. Using 10-fold serial dilutions of synthetic gene block fragments containing individual sequence targets of each lungworm species, the analytical sensitivity of the assay ascertained was 1.84 ng/µl for A. vasorum, 3.08 ng/µl for C. vulpis and 0.79 ng/µl for Eucoleus spp. The sensitivity of the assays and their ability to detect mixed species infections were compared with microscopy-based techniques (faecal floatation and Baermann technique) applied to faecal samples submitted for lungworm testing through an accredited diagnostic laboratory at the Institute of Parasitology, University of Zurich, Switzerland, and from community dogs as part of a research project on canine endoparasites in Cambodia. The multiplex qPCR displayed high diagnostic sensitivity (42/46, 91.3%; 95% Confidence Interval (CI): 79.1-97.1%) and a diagnostic specificity of 100% (45/45, 95% CI: 90.6-100%), and was able to detect 42.9% additional mixed lungworm species infections compared with microscopy-based methods. Kappa statistics showed substantial agreement between the qPCRs and microscopy for mixed infections (κ = 0.72, 95% CI: 0.4-1) and Eucoleus spp. (κ = 0.65, 95% CI: 0.45-0.85) and almost perfect agreement for C. vulpis (κ = 0.85, 95% CI: 0.63-1) and A. vasorum (κ = 0.92, 95% CI: 0.84-1). This multiplex qPCR enables timely, accurate, and sensitive diagnosis of canine lungworm species in faecal samples and can be used to monitor the geographical distribution and emergence of these parasitic species, globally.

Canine gastrointestinal parasites perceptions, practices, and behaviours: A survey of dog owners in Australia.

Many species of canine gastrointestinal (GI) parasites are known to be zoonotic meaning that dog owners' management and practices are key to preventing exposure of humans and dogs as well as contamination of the environment. As Australia has one of the highest rates of pet ownership in the world, we administered an online questionnaire to dog owners across the nation to assess their perceptions, practices, and behaviours towards canine GI parasites. Descriptive analysis was performed to summarise perceptions and management practices. Factors associated with the suitability of parasiticide treatments applied were investigated using uni- and multivariable ordinal regression. Just over a half of dog owners considered parasites as very or extremely important for their dog's health (59%) and less than a half as very or extremely important for human health (46%). Although the majority of dog owners stated that they deworm their dogs (90%), only the 28% followed best practice guidelines, i.e. administered a monthly prophylactic treatment all-year round. A large proportion of respondent dog owners administered prophylactic treatment at an inappropriate frequency (48%) or did not treat for canine GI parasites at all (24%). Attending vet visits at least once a year or once every six months and having a very comfortable or prosperous financial position were significantly associated with following best deworming prophylaxis guidelines. This study demonstrates that a proportion of dog owners in Australia is not complying with best practice regarding the control of canine GI parasites and is potentially exposing themselves and their dogs to the risk of infections. Veterinarians are called to implement dog owner's education, raise their awareness on the threats canine parasitic diseases pose to both dogs and humans and finally, encourage them to follow a monthly prophylactic treatment for canine GI parasites all year round.

Faecal prevalence, distribution and risk factors associated with canine soil-transmitted helminths contaminating urban parks across Australia.

Dog parks provide an ideal urban space where dogs and their owners can exercise, play, and socialise in a safe environment. However, these parks can also increase the risk of exposure to a series of infectious agents including canine soil-transmitted helminths (cSTHs) such as hookworms, roundworms, Strongyloides stercoralis, and Trichuris vulpis, which are endemic to Australia. In this study, we collected 1581 canine faecal samples in 190 urban parks across Australia and subjected these to faecal floatation and multiplex real-time PCRs (qPCR) to detect a range of cSTHs. In total, 44.2% of the parks sampled were contaminated with at least one species of cSTH, with hookworms being the most prevalent parasites (10.2%) followed by Trichuris spp. (1.3%) and Strongyloides spp. (1.2%). This is the first large-scale study investigating the contamination of urban parks with cSTHs in Australia, and the first nation-wide study to demonstrate the occurrence of Strongyloides spp. in canine faecal samples from urban areas in the country. This study reveals a high rate of contamination with cSTHs in dog parks in urban Australia, most of which having proven zoonotic potential. Preventive measures, including awareness-raising educational programmes promoting responsible pet ownership, should be encouraged to minimise the health risks associated with cSTHs for both dogs and humans.

Attitudes and practices of veterinarians in Australia to acute pain management in cats.

OBJECTIVES: Up-to-date information on the current practices and attitudes of veterinarians in Australia to acute pain management in cats was sought in 2017-2018 in the first nationwide survey in over 20 years. METHODS: An online survey was created, consisting of 54 questions in four sections, with 18 feline-specific questions. Veterinarians throughout Australia were invited to participate in the survey through advertisements in the veterinary press, electronic and regular mail, and through in-clinic visits and promotional materials. RESULTS: A total of 614 veterinarians completed the survey, with 513 (83.6%) completing the feline-specific section. The demographics of the respondents of this survey were an accurate representation of the registered veterinarians in Australia at the time. Multiple different opioids and non-steroidal anti-inflammatory drugs (NSAIDs) were widely available in practices to manage pain, with analgesic efficacy reported as the primary factor influencing drug selection. Opioids were most commonly used in the preoperative period and NSAIDs were most commonly used postoperatively. Despite the wide availability in clinic (>99%), only 55% of respondents reported regularly using local anaesthetic techniques in cats. Pain assessment of hospitalised patients was primarily performed by veterinarians (91.1%); however, 84.7% of respondents did not routinely use a validated pain scale. CONCLUSIONS AND RELEVANCE: Veterinary practitioners in Australia are adequately equipped with analgesic medication to treat pain in their patients. In the management of acute pain in cats, they frequently use a multimodal approach and practice a variety of analgesic protocols. Based on the results of this survey, potential areas for improvement in feline pain management include an increase in duration of postoperative analgesia and more routine use of validated pain scales, performed by nurses and veterinarians alike, to assess individual analgesic needs.

Field application of a novel multiplex qPCR assay reveals the occurrence of the zoonotic hookworm Ancylostoma braziliense in Nigerian dogs.

A number of gastrointestinal parasites have been reported to infect dogs in Nigeria, some of which have zoonotic potential. Of these, hookworms are the most prevalent, with both Ancylostoma caninum and Uncinaria stenocephala reported in the country. In this study, we subjected 203 hookworm microscopy-positive samples of the 885 individual faecal samples collected from dogs in Nigeria to a recently developed multiplex qPCR for the detection and characterisation of canine hookworm species. The qPCR demonstrated a diagnostic sensitivity of 98% (95% CI 95-99.4) allowing the detection of A. caninum and A. braziliense in 81.3% (165/203, 95% CI 75.3-86.1) and 51.2% (104/203, 95% CI 44.4-58) of the microscopy-positive faecal samples of dogs from Nigeria, respectively and 34.5% (70/203, 95% CI 28.3-41.3) of mixed infections with both hookworm species. The finding of A. braziliense is particularly worrisome given this is a well-known agent of persistent cutaneous larva migrans, commonly referred to as "creeping eruptions" in humans. Although this parasite has been diagnosed in locals and in people travelling in Nigeria suffering from dermatological illnesses, this represents the first molecular identification of A. braziliense in its canine reservoir in the country. These results update the occurrence and distribution of hookworm species affecting dogs in Nigeria highlighting the suitability of the newly developed multiplex qPCR assay as a high-throughput tool for the surveillance of zoonotic hookworms, globally.

Emergence of canine parvovirus subtype 2b (CPV-2b) infections in Australian dogs.

Tracing the temporal dynamics of pathogens is crucial for developing strategies to detect and limit disease emergence. Canine parvovirus (CPV-2) is an enteric virus causing morbidity and mortality in dogs around the globe. Previous work in Australia reported that the majority of cases were associated with the CPV-2a subtype, an unexpected finding since CPV-2a was rapidly replaced by another subtype (CPV-2b) in many countries. Using a nine-year dataset of CPV-2 infections from 396 dogs sampled across Australia, we assessed the population dynamics and molecular epidemiology of circulating CPV-2 subtypes. Bayesian phylogenetic Skygrid models and logistic regressions were used to trace the temporal dynamics of CPV-2 infections in dogs sampled from 2007 to 2016. Phylogenetic models indicated that CPV-2a likely emerged in Australia between 1973 and 1988, while CPV-2b likely emerged between 1985 and 1998. Sequences from both subtypes were found in dogs across continental Australia and Tasmania, with no apparent effect of climate variability on subtype occurrence. Both variant subtypes exhibited a classical disease emergence pattern of relatively high rates of evolution during early emergence followed by subsequent decreases in evolutionary rates over time. However, the CPV-2b subtype maintained higher mutation rates than CPV-2a and continued to expand, resulting in an increase in the probability that dogs will carry this subtype over time. Ongoing monitoring programs that provide molecular epidemiology surveillance will be necessary to detect emergence of new variants and make informed recommendations to develop reliable detection and vaccine methods.

Demographics, lifestyle and veterinary care of cats in Australia and New Zealand.

Objectives The aim of this survey was to provide up-to-date information on the demographics, lifestyle and veterinary care of cats in Australia and New Zealand. Methods An online survey consisting of 19 questions was created using SurveyMonkey. Cat owners were invited to participate through advertisements in veterinary clinics and social media. Results The average number of cats in a household was two. The majority of cats lived in free-standing houses (78%) in the suburbs (66%). The majority of cats were desexed (94% [49% female neutered; 45% male neutered]). A total of 57% of cats had been strays or came from an animal shelter. A total of 40% of owners had intended not to let the cats outside when they first acquired them. A total of 63% of cats were described as indoor-outdoor cats. Although owners described 34% of cats as 'indoor only', 58% of those cats had access to the outdoors. The majority of respondents' cats were vaccinated annually (63%) and visited a vet at least annually (79%). The most common reasons to take a cat to the vet were vaccinations or the cat being unwell. The most common reasons not to regularly take the cat to the vet were that the cat was never unwell, cost or stress for the cat. Conclusions and relevance Consideration of the lifestyle of cats is important to optimise veterinary care. Cats across Australia and New Zealand have a variety of different and changing lifestyles. Therefore, careful owner questioning is required at each visit to maximise healthcare outcomes for cats.

Seroprevalence of feline immunodeficiency virus and feline leukaemia virus in Australia: risk factors for infection and geographical influences (2011-2013).

OBJECTIVES: Our aim was to: (i) determine the current seroprevalence of feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV) in three large cohorts of cats from Australia; and (ii) investigate potential risk factors for retroviral infection. METHODS: Cohort 1 (n = 2151 for FIV, n = 2241 for FeLV) consisted of cats surrendered to a shelter on the west coast of Australia (Perth, Western Australia [WA]). Cohort 2 (n = 2083 for FIV, n = 2032 for FeLV) consisted of client-owned cats with outdoor access recruited from around Australia through participating veterinary clinics. Cohort 3 (n = 169 for FIV, n = 166 for FeLV) consisted of cats presenting to Murdoch University Veterinary Hospital for a variety of reasons. Fresh whole blood was collected and tested using a commercially available point-of-care lateral flow ELISA kit that detects p27 FeLV antigen and antibodies to FIV antigens (p15 and p24) (cohorts 1 and 2), or one of two lateral flow immunochromatography kits that detect p27 antigen and antibodies to FIV antigen (p24 and/or gp40) (cohort 3). Data recorded for cats in cohort 2 included signalment, presenting complaint and postcode, allowing investigation of risk factors for FIV or FeLV infection, as well as potential geographical 'hot spots' for infection. RESULTS: The seroprevalence of FIV was 6% (cohort 1), 15% (cohort 2) and 14% (cohort 3), while the seroprevalence of FeLV was 1%, 2% and 4% in the same respective cohorts. Risk factors for FIV infection among cats in cohort 2 included age (>3 years), sex (male), neutering status (entire males) and location (WA had a significantly higher FIV seroprevalence compared with the Australian Capital Territory, New South Wales and Victoria). Risk factors for FeLV infection among cats in cohort 2 included health status ('sick') and location (WA cats were approximately three times more likely to be FeLV-infected compared with the rest of Australia). No geographical hot spots of FIV infection were identified. CONCLUSIONS AND RELEVANCE: Both FIV and FeLV remain important infections among Australian cats. WA has a higher seroprevalence of both feline retroviruses compared with the rest of Australia, which has been noted in previous studies. A lower neutering rate for client-owned male cats is likely responsible for the higher seroprevalence of FIV infection in WA cats, while the reason for the higher seroprevalence of FeLV in WA cats is currently unknown.

Antiviral effect of mefloquine on feline calicivirus in vitro.

Feline calicivirus (FCV) is an important viral pathogen of domestic cats causing clinical signs ranging from mild to severe oral ulceration or upper respiratory tract disease through to a severe fatal systemic disease. Current therapeutic options are limited, with no direct acting antivirals available for treatment. This study screened a panel of 19 compounds for potential antiviral activity against FCV strain F9 and recent field isolates in vitro. Using a resazurin-based cytopathic effect (CPE) inhibition assay, mefloquine demonstrated a marked inhibitory effect on FCV induced CPE, albeit with a relatively low selectivity index. Orthogonal assays confirmed inhibition of CPE was associated with a significant reduction in viral replication. Mefloquine exhibited a strong inhibitory effect against a panel of seven recent FCV isolates from Australia, with calculated IC50 values for the field isolates approximately 50% lower than against the reference strain FCV F9. In vitro combination therapy with recombinant feline interferon-ω, a biological response modifier currently registered for the treatment of FCV, demonstrated additive effects with a concurrent reduction in the IC50 of mefloquine. These results are the first report of antiviral effects of mefloquine against a calicivirus and support further in vitro and in vivo evaluation of this compound as an antiviral therapeutic for FCV.

In vitro inhibition of field isolates of feline calicivirus with short interfering RNAs (siRNAs).

Feline calicivirus (FCV) is a common infection of domestic cats. Most infections are mild and self-limiting; however more severe disease manifestations, such as FCV-associated virulent systemic disease, may be associated with significant morbidity and mortality. There is currently a lack of effective antiviral treatments for these disease manifestations. In this study, a panel of eight siRNAs were designed to target four conserved regions of the FCV genome. siRNAs were screened for in vitro antiviral efficacy against the reference strain FCV F9 by determination of extracellular virus titres and morphological assessment of protection from cytopathic effect. Three of the siRNA (FCV3.7, FCV4.1, and FCV4.2) demonstrated a marked antiviral effect with a greater than 99% reduction in extracellular viral titre. Titration of these effective siRNAs demonstrated a clear concentration-response relationship, with IC50 values of approximately 1 nM, and combination treatment with multiple siRNAs demonstrated additive or synergistic effects. To assess the potential usefulness of the compounds in a clinical setting, siRNAs were screened against a panel of six recent Australian FCV isolates from cats with FCV-related disease. The siRNAs shown to be effective against the reference strain FCV F9 were active against the majority of the isolates tested, although some variability was noted. Taken together these data suggest potential therapeutic application of antiviral RNAi for treating FCV-associated disease in cats.

Combination siRNA therapy against feline coronavirus can delay the emergence of antiviral resistance in vitro.

Virulent biotypes of feline coronavirus (FCoV), commonly referred to as feline infectious peritonitis virus (FIPV), can result in the development of feline infectious peritonitis (FIP), a typically fatal immune mediated disease for which there is currently no effective antiviral treatment. We previously reported the successful in vitro inhibition of FIPV replication by synthetic siRNA mediated RNA interference (RNAi) in an immortalised cell line (McDonagh et al., 2011). A major challenge facing the development of any antiviral strategy is that of resistance, a problem which is particularly acute for RNAi based therapeutics due to the exquisite sequence specificity of the targeting mechanism. The development of resistance during treatment can be minimised using combination therapy to raise the genetic barrier or using highly potent compounds which result in a more rapid and pronounced reduction in the viral replication rate, thereby reducing the formation of mutant, and potentially resistant viruses. This study investigated the efficacy of combination siRNA therapy and its ability to delay or prevent viral escape. Virus serially passaged through cells treated with a single or dual siRNAs rapidly acquired resistance, with mutations identified in the siRNA target sites. Combination therapy with three siRNA prevented viral escape over the course of five passages. To identify more potent silencing molecules we also compared the efficacy, in terms of potency and duration of action, of canonical versus Dicer-substrate siRNAs for two previously identified effective viral motifs. Dicer-substrate siRNAs showed equivalent or better potency than canonical siRNAs for the target sites investigated, and may be a more appropriate molecule for in vivo use. Combined, these data inform the potential therapeutic application of antiviral RNAi against FIPV.

Identification and characterisation of small molecule inhibitors of feline coronavirus replication.

Feline infectious peritonitis (FIP), a feline coronavirus (FCoV) induced disease, is almost invariably fatal with median life expectancy measured in days. Current treatment options are, at best, palliative. The objectives of this study were to evaluate a panel of nineteen candidate compounds for antiviral activity against FCoV in vitro to determine viable candidates for therapy. A resazurin-based cytopathic effect inhibition assay, which detects viable cells through their reduction of the substrate resazurin to fluorescent resorufin, was developed for screening compounds for antiviral efficacy against FCoV. Plaque reduction and virus yield reduction assays were performed to confirm antiviral effects of candidate compounds identified during screening, and the possible antiviral mechanisms of action of these compounds were investigated using virucidal suspension assays and CPE inhibition and IFA-based time of addition assays. Three compounds, chloroquine, mefloquine, and hexamethylene amiloride demonstrated marked inhibition of virus induced CPE at low micromolar concentrations. Orthogonal assays confirmed inhibition of CPE was associated with significant reductions in viral replication. Selectivity indices calculated based on in vitro cytotoxicity screening and reductions in extracellular viral titre were 217, 24, and 20 for chloroquine, mefloquine, and hexamethylene amiloride respectively. Preliminary experiments performed to inform the antiviral mechanism of the compounds demonstrated all three acted at an early stage of viral replication. These results suggest that these direct acting antiviral compounds, or their derivatives, warrant further investigation for clinical use in cats with FIP.

Risk factors for feline infectious peritonitis in Australian cats.

The objective of this study was to determine whether patient signalment (age, breed, sex and neuter status) is associated with naturally-occurring feline infectious peritonitis (FIP) in cats in Australia. A retrospective comparison of the signalment between cats with confirmed FIP and the general cat population was designed. The patient signalment of 382 FIP confirmed cases were compared with the Companion Animal Register of NSW and the general cat population of Sydney. Younger cats were significantly over-represented among FIP cases. Domestic crossbred, Persian and Himalayan cats were significantly under-represented in the FIP cohort, while several breeds were over-represented, including British Shorthair, Devon Rex and Abyssinian. A significantly higher proportion of male cats had FIP compared with female cats. This study provides further evidence that FIP is a disease primarily of young cats and that significant breed and sex predilections exist in Australia. This opens further avenues to investigate the role of genetic factors in FIP.

In vitro inhibition of feline coronavirus replication by small interfering RNAs.

Infection with virulent biotypes of feline coronavirus (FCoV) can result in the development of feline infectious peritonitis (FIP), a typically fatal immune mediated disease for which there is currently no effective antiviral treatment. In this study we demonstrate the ability of small interfering RNA (siRNA) mediated RNA interference (RNAi) to inhibit the replication of virulent FCoV strain FIPV WSU 79-1146 in an immortalised feline cell line. A panel of eight synthetic siRNAs targeting four different regions of the FCoV genome were tested for antiviral effects. Efficacy was determined by qRT-PCR of intracellular viral genomic and messenger RNA, TCID50 infectivity assay of extracellular virus, and direct IFA for viral protein expression. All siRNAs demonstrated an inhibitory effect on viral replication in vitro. The two most effective siRNAs, targeting the untranslated 5' leader sequence (L2) and the nucleocapsid gene (N1), resulted in a >95% reduction in extracellular viral titre. Further characterisation of these two siRNAs demonstrated their efficacy when used at low concentrations and in cells challenged with high viral loads. Taken together these findings provide important information for the potential therapeutic application of RNAi in treating FIP.