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.

Zoonotic hookworms of dogs and cats - lessons from the past to inform current knowledge and future directions of research.

The early-to-mid 1900s was an era that marked the first published reports of clinical cases and experimental human infections that provided valuable evidence to inform our current knowledge on the zoonotic nature of the canid and felid hookworms. To this day, descriptions of the pathognomonic syndromes produced in humans by these early pioneers provide vital information on the putative geographical distribution of these hookworm species, even before their discovery in dogs and cats. Cases of hookworm-related cutaneous larvae migrans and Ancylostoma caninum-induced eosinophilic enteritis continue to be reported in the medical literature, most commonly in recent migrants or returned travellers who have spent time in regions in which these canine and feline hookworms remain highly endemic. Ancylostoma ceylanicum, now recognised as the second most common hookworm infecting humans in the Asia Pacific region, is also being reported in regions of Africa and South America previously assumed free of this parasite. Despite the substantial technological progress made in the field of parasite diagnostics, our knowledge on the epidemiology and population-level morbidity impacts of these zoonotic hookworms in humans has remained relatively stagnant over the last few decades, with 'covert' infections continuing to remain undetected by currently available forms of routine diagnostics. Improved diagnostic tools that enable accurate and rapid species-specific diagnosis of zoonotic hookworm infections in humans are required to provide the necessary evidence to advocate for future investments in One Health-based intervention strategies.

Novel High-Throughput Multiplex qPCRs for the Detection of Canine Vector-Borne Pathogens in the Asia-Pacific.

The Asia-Pacific hosts a large diversity of canine vector-borne pathogens (VBPs) with some of the most common and most pathogenic, generating significant mortality as well as a spectrum of health impacts on local dog populations. The VBPs Anaplasma platys, Babesia gibsoni, Babesia vogeli, Ehrlichia canis, Hepatozoon canis and haemotropic Mycoplasma spp. are all endemic throughout the region, with many exhibiting shifting geographical distributions that warrant urgent attention. Moreover, many of these species cause similar clinical signs when parasitising canine hosts, whilst knowledge of the exact pathogen is critical to ensure treatment is effective. This is complicated by frequent coinfection that can exacerbate pathology. Here, we describe the development, optimisation and validation of two novel quadruplex Taq-Man based real-time PCRs (qPCRs) for the specific and sensitive detection of the aforementioned VBPs. To ensure accurate evaluation of diagnostic performance, results of our qPCRs were evaluated on field samples from Thai dogs and compared with both conventional PCR (cPCR) results and next-generation sequencing (NGS) metabarcoding. Our qPCRs were found to be more sensitive at detecting canine VBP than cPCR and generated results similar to those achieved by NGS. These qPCRs will provide a valuable high-throughput diagnostic tool available to epidemiologists, researchers and clinicians for the diagnosis of key canine VBPs in the Asia-Pacific and further afield.

Canine gastrointestinal parasites as a potential source of zoonotic infections in Nigeria: A nationwide survey.

Dog feces may contain zoonotic parasites that contaminate the environment and serve as a potential source of infection to animals and humans. In this study, microscopic and molecular analyses were used to estimate the prevalence and intensity of gastrointestinal (GI) parasites and assess the risk factors for infection in 948 dogs in three climatically distinct zones of Nigeria. Zoonotic helminths including Strongyloides stercoralis, Ancylostoma braziliense, A. caninum and Toxocara canis were detected either as single or multiple infections in 377 (39.8 %) of dogs examined. At multiple logistic regression analyses, association was found between GI parasite infection and deworming practices and dog management. Regarding A. braziliense, A. caninum and T. canis infections, intensity of egg shedding was statistically associated with the age of the dogs and not with their sex or breed. The majority of GI parasite-positive dogs did not receive regular deworming treatment (59 %) and roamed freely (56 %) thereby constituting public health risk. This is the first nationwide survey and analyses of risk factors of GI parasites of dogs using molecular methods as confirmation of their identity. The zoonotic potential of these parasites is exacerbated by the lack of both operational national policies to control the population of free-roaming dogs and to promote responsible dog ownership, and veterinary public health programs for dogs.

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.

High-throughput multiplex qPCRs for the surveillance of zoonotic species of canine hookworms.

The canine hookworms Ancylostoma braziliense, Ancylostoma ceylanicum, Ancylostoma caninum and Uncinaria stenocephala are not only capable of producing morbidity and mortality in dogs but are also neglected tropical zoonoses. Each hookworm species differs considerably in its geographical distribution, life cycle, biology, pathogenic impacts on both canine and human hosts, zoonotic potential, and response to treatment with anthelminthics. Here we describe the development and validation of two Taq-Man based multiplex PCR assays capable of detecting and differentiating all four canine hookworm species in faeces of naturally infected dogs. The analytical sensitivity of both assays was assessed using 10-fold serial dilutions of synthetic gene block fragments containing individual sequence targets of each hookworm species. The sensitivity of the assays and ability to detect mixed species infections were compared to a conventional PCR-Restriction Fragment Length Polymorphism based-approach when applied to laboratory and field samples from endemic areas. The qPCRs detected at least one species of hookworms in 82.4% of PCR-RFLP-negative but microscopy-positive samples. The qPCRs detected an additional 68% mixed infections with different species of canine hookworms, and additional single species infection with A. caninum (47%), U. stenocephala (33%) and A. ceylanicum (0.02%) that were missed by PCR-RFLP. These multiplex qPCR assays will assist field based epidemiological surveillance studies towards an accurate and sensitive monitoring of canine hookworm infections in dogs, to inform their species-specific zoonotic risks to populations living in endemic areas, globally.

Short communication: Characterization of enterotoxin-producing Staphylococcus aureus isolated from mastitic cows.

Staphylococcus aureus is not only a common cause of bovine mastitis, but also an agent of food poisoning in humans. In an attempt to determine whether staphylococci causing bovine mastitis could also cause food poisoning, 60 isolates of presumed S. aureus were isolated in the period between March and August 2017 from 3,384 routine, composite, quarter milk samples of individual cows raised on 12 dairy farms in central Italy. Seventeen out of 60 isolates were confirmed as S. aureus after coagulase, thermonuclease, and biochemical tests. These isolates were analyzed by PCR for the presence of the nuc, sea, seb, sec, sed, and see genes. The positive isolates were nuc, 100% (17); sea, 35.29% (6); seb, 5.88% (1); sec, 5.88% (1); sed, 29.41% (5); and see, 47.06% (8). The isolates were also tested with 2 enzyme immunoassay diagnostic kits, one for the screening detection of the production of staphylococcal enterotoxins (SEA, SEB, SEC, SED, SEE) and one for the detection of specific enterotoxin produced by each isolate. Seven out of 17 (41.18%) were enterotoxin producers: 7 produced SEA (41.18%), 1 SEB (5.88%), 1 SEC (5.88%), 5 SED (29.41%), and 6 SEE (35.29%). To further characterize the isolates, they were analyzed by the Kirby Bauer test for susceptibility to 13 antimicrobials (ampicillin, ciprofloxacin, kanamycin, tetracycline, gentamicin, methicillin, nalidixic acid, erythromycin, amoxicillin/clavulanic acid, streptomycin, vancomycin, neomycin, and enrofloxacin), and we detected resistance to ampicillin (52.94%), nalidixic acid (70.59%), erythromycin (5.88%), and amoxicillin/clavulanic acid (17.65%). The isolates were sensitive to the main classes of antimicrobials used for the treatment of bovine subclinical mastitis. The presence of enterotoxin-producing isolates of S. aureus in bovine milk means that a temperature abuse or a breakdown in the thermal treatment of the milk could present a food safety risk, particularly if all enterotoxigenic isolates could potentially produce SEA in milk.