Exploring multiplex qPCR as a diagnostic tool for detecting microfilarial DNA in dogs infected with Dirofilaria immitis: A comparative analysis with the modified Knott's test.

Current recommendations to diagnose cardiopulmonary dirofilariosis in dogs caused by Dirofilaria immitis involves tandem antigen and circulating microfilariae tests. The modified Knott's test is an important tool in heartworm diagnosis, allowing identification of circulating microfilariae. However, the subjective nature of the modified Knott's test affects its accuracy and diagnostic laboratories usually do not provide a quantitative outcome. Quantitative enumeration of microfilariae enables clinicians to track treatment progress and acts as a proxy for detecting emerging macrocyclic lactone resistance. There is a need for better diagnostic tools suitable for routine use to efficiently and accurately quantify the presence of D. immitis microfilaremia. The aim of this study was to determine whether the quantitative modified Knott's test can be substituted by multiplex quantitative polymerase chain reaction (qPCR) targeting D. immitis and associated Wolbachia endosymbiont DNA in canine blood samples. To do this, genomic DNA samples (n = 161) from Australian dogs, collected as part of a previous 2021 study, were assessed in a TaqMan qPCR targeting DNA of D. immitis, Wolbachia sp. and Canis lupus familiaris. Of the 161 genomic DNA samples, eight were considered positive for D. immitis microfilariae. The qPCR assay demonstrated good efficiency (E = 90 to 110%, R2 > 0.94). Considering the performance and efficient use of bench time, this TaqMan qPCR assay is a suitable alternative to the modified Knott's test for quantitative enumeration of microfilariae (Cohen's kappa coefficient [κ]: κ = 1 using D. immitis qPCR marker, κ = 0.93 using Wolbachia qPCR marker). The qPCR data demonstrated a comparable result to that of the quantitative modified Knott's test in a 2022 survey of D. immitis in Australian dogs (n = 23) before and after macrocyclic lactone (ML) administration. Improving the detection and diagnosis of canine heartworm infections will assist veterinarians in better managing and controlling disease outcomes and will be valuable for tracking the spread of ML resistance in Australia.

Exploration of the sensitivity to macrocyclic lactones in the canine heartworm (Dirofilaria immitis) in Australia using phenotypic and genotypic approaches.

Canine heartworm disease is a potentially deadly cardiopulmonary disease caused by the mosquito-borne filarial nematode Dirofilaria immitis. In Australia, the administration of macrocyclic lactone (ML) drugs has successfully reduced the prevalence of D. immitis infection. However, the recent re-emergence of D. immitis in dogs in Queensland, Australia and the identification of ML-resistant isolates in the USA poses an important question of whether ML-resistance has emerged in this parasite in Australia. The aim of this study was to utilise phenotypic and genotypic approaches to examine the sensitivity to ML drugs in D. immitis in Australia. To do this, we surveyed 45 dogs from Queensland and New South Wales across 3 years (2019-2022) for the presence of D. immitis infection using an antigen test, quantitative Modified Knott's test, and qPCR targeting both D. immitis and the D. immitis symbiont Wolbachia. A phenotype observed by utilising sequential quantification of microfilariae for 23/45 dogs was coupled with genetic testing of filtered microfilariae for SNPs previously associated with ML-resistance in isolates from the USA. Sixteen (16/45) dogs tested positive for D. immitis infection despite reportedly receiving 'rigorous' heartworm prevention for 12 months prior to the study, according to the owners' assessment. The phenotype and genotypic assays in this study did not unequivocally demonstrate the presence of ML-resistant D. immitis in Australia. Although the failure of 16 dogs to reduce microfilaremia by >90% after ML treatment was considered a suspect phenotype of ML-resistance, no genotypic evidence was discovered using the genetic SNP analysis. The traditional quantitative Modified Knott's test can be substituted by qPCR targeting D. immitis or associated Wolbachia endosymbiont DNA for a more rapid measurement of microfilariae levels. More definitive phenotypic evidence of resistance is critically needed before the usefulness of SNPs for the detection of ML-resistance in Australia can be properly assessed.

Adaptation of gltA and ssrA assays for diversity profiling by Illumina sequencing to identify Bartonella henselae, B. clarridgeiae and B. koehlerae.

Introduction. Bartonellosis is an emerging zoonotic disease caused by bacteria of the genus Bartonella. Mixed Bartonella infections are a well-documented phenomenon in mammals and their ectoparasites. The accurate identification of Bartonella species in single and mixed infections is valuable, as different Bartonella species have varying impacts on infected hosts.Gap Statement. Current diagnostic methods are inadequate at identifying the Bartonella species present in mixed infections.Aim. The aim of this study was to adopt a Next Generation Sequencing (NGS) approach using Illumina sequencing technology to identify Bartonella species and demonstrate that this approach can resolve mixed Bartonella infections.Methodology. We used Illumina PCR amplicon NGS to target the ssrA and gltA genes of Bartonella in fleas collected from cats, dogs and a hedgehog in Israel. We included artificially mixed Bartonella samples to demonstrate the ability for NGS to resolve mixed infections and we compared NGS to traditional Sanger sequencing.Results. In total, we identified 74 Ctenocephalides felis, two Ctenocephalides canis, two Pulex irritans and three Archaeopsylla e. erinacei fleas. Real-time PCR of a subset of 48 fleas revealed that twelve were positive for Bartonella, all of which were cat fleas. Sanger sequencing of the ssrA and gltA genes confirmed the presence of Bartonella henselae, Bartonella clarridgeiae and Bartonella koehlerae. Illumina NGS of ssrA and gltA amplicons further confirmed the Bartonella species identity in all 12 flea samples and unambiguously resolved the artificially mixed Bartonella samples.Conclusion. The adaptation and multiplexing of existing PCR assays for diversity profiling via NGS is a feasible approach that is superior to traditional Sanger sequencing for Bartonella speciation and resolving mixed Bartonella infections. The adaptation of other PCR primers for Illumina NGS will be useful in future studies where mixed bacterial infections may be present.

Cat fleas (Ctenocephalides felis clade 'Sydney') are dominant fleas on dogs and cats in New South Wales, Australia: Presence of flea-borne Rickettsia felis, Bartonella spp. but absence of Coxiella burnetii DNA.

The cat flea (Ctenocephalides felis) is the most common flea species parasitising both domestic cats and dogs globally. Fleas are known vectors of zoonotic pathogens such as vector-borne Rickettsia spp. and Bartonella spp. and could theoretically transmit Coxiella burnetii, the causative agent of Q fever. A total of 107 fleas were collected from 21 cats and 14 dogs in veterinary clinics, a feline rescue organisation and a grooming salon in New South Wales, Australia, to undergo PCR detection of Bartonella spp., Rickettsia spp. and C. burnetii DNA. Morphological identification confirmed that the cat flea (C. felis) is the most common flea in New South Wales, Australia, with only a single stick fast flea, Echidnophaga gallinacea recorded. The examined fleas (n = 35) at the cox1 locus revealed five closely related C. felis haplotypes (inter-haplotype distance < 0.5%). Multiplex TaqMan qPCR targeting the gltA (Rickettsia spp.) and ssrA (Bartonella spp.) genes was positive in 22.9% (95% CI: 11.8-39.3%) and 11.4% (95% CI: 3.9-26.6%) of samples, respectively. None of the DNA isolated from fleas was positive on TaqMan qPCRs targeting the C. burnetii IS1111, Com1 and htpAB genes. Co-infection of C. felis with Bartonella henselae and Bartonella clarridgeiae was demonstrated using gltA and ssrA Illumina next-generation amplicon sequencing. These findings reinforce the importance of flea control on domestic dogs and cats to effectively control the transmission of Rickettsia felis and Bartonella spp. The flea, however, is unlikely to be a vector of C. burnetii between companion animals and humans.