The Application of 3base™ Technology to Diagnose Eight of the Most Clinically Important Gastrointestinal Protozoan Infections.

Globally, over 3.5 billion people are infected with intestinal parasites each year, resulting in over 200,000 deaths. Three of the most common protozoan pathogens that affect the gastrointestinal tract of humans are Cryptosporidium spp., Giardia intestinalis, and Entamoeba histolytica. Other protozoan agents that have been implicated in gastroenteritis in humans include Cyclospora cayetanensis, Dientamoeba fragilis, Blastocystis hominis, and the microsporidia Enterocytozoon bieneusi and Encephalitozoon intestinalis. Genetic Signatures previously developed a 3base™ multiplexed Real-Time PCR (mRT-PCR) enteric protozoan kit (EP001) for the detection of Giardia intestinalis/lamblia/duodenalis, Cryptosporidium spp., E. histolytica, D. fragilis, and B. hominis. We now describe improvements to this kit to produce a more comprehensive assay, including C. cayetanensis, E. bieneusi, and E. intestinalis, termed EP005. The clinical performance of EP005 was assessed using a set of 380 clinical samples against a commercially available PCR test and other in-house nucleic acid amplification tests where commercial tests were not available. All methods provided at least 90% agreement. EP005 had no cross-reactivity against 82 organisms commonly found in the gut. The EP005 method streamlines the detection of gastrointestinal parasites and addresses the many challenges of traditional microscopic detection, resulting in cost savings and significant improvements in patient care.

The mitochondrial genome of Angiostrongylus mackerrasae is distinct from A. cantonensis and A. malaysiensis.

The native rat lungworm (Angiostrongylus mackerrasae) and the invasive rat lungworm (Angiostrongylus cantonensis) occur in eastern Australia. The species identity of A. mackerrasae remained unquestioned until relatively recently, when compilation of mtDNA data indicated that A. mackerrasae sensu Aghazadeh et al. (2015b) clusters within A. cantonensis based on their mitochondrial genomes (mtDNA). To re-evaluate the species identity of A. mackerrasae, we sought material that would be morphologically conspecific with A. mackerrasae. We combined morphological and molecular approaches to confirm or refute the specific status of A. mackerrasae. Nematodes conspecific with A. mackerrasae from Rattus fuscipes and Rattus rattus were collected in Queensland, Australia. Morphologically identified A. mackerrasae voucher specimens were characterized using amplification of cox1 followed by the generation of reference complete mtDNA. The morphologically distinct A. cantonensis, A. mackerrasae and A. malaysiensis are genetically distinguishable forming a monophyletic mtDNA lineage. We conclude that A. mackerrasae sensu Aghazadeh et al. (2015b) is a misidentified specimen of A. cantonensis. The availability of the mtDNA genome of A. mackerrasae enables its unequivocal genetic identification and differentiation from other Angiostrongylus species.

Transcriptome Analysis and In Situ Hybridization for FcaGHV1 in Feline Lymphoma.

Lymphoma is one of the most common malignancies in domestic cats. The lymphomagenic potential of Felis catus gammaherpesvirus 1 (FcaGHV1), a common infection in domestic cats, is unknown. In other species, including humans, cellular transformation by gammaherpesviruses is typically mediated by viral genes expressed during latency. We analysed tumour RNA, from diffuse large B-cell lymphomas (DLBCL) appearing in cats coinfected with FcaGHV1 and feline immunodeficiency virus (FIV) (n = 10), by high throughput transcriptome sequencing and reverse transcription PCR. A limited repertoire of FcaGHV transcripts was identified in five tumors, including homologs of oncogenic latency-associated transcripts, latency-associated nuclear antigen (LANA, ORF73) and vFLIP (F7), lytic genes (ORF50, ORF6, ORF59, F10), and an ORF unique to FcaGHV1, F20. In situ hybridization of FIV-associated DLBCLs (n = 9), post-transplant lymphomas (n = 6) and high-grade B and T-cell intestinal lymphomas (n = 8) identified a single case in which FcaGHV1 nucleic acid was detectable. These results demonstrate that FcaGHV1 transcripts can be detected in some FIV-associated lymphomas, but at low copy number, precluding assessment of a potential role for FcaGHV1 in lymphomagenesis. Future investigation of the FcaGHV1 transcriptome in clinical samples might employ viral enrichment and greater sequencing depth to enhance the retrieval of viral reads. Our results suggest prioritization of a subset of intestinal T-cell tumors, large granular lymphocyte lymphoma, for study.

A Novel Hepadnavirus Identified in an Immunocompromised Domestic Cat in Australia.

High-throughput transcriptome sequencing allows for the unbiased detection of viruses in host tissues. The application of this technique to immunosuppressed animals facilitates the detection of viruses that might otherwise be excluded or contained in immunocompetent individuals. To identify potential viral pathogens infecting domestic cats we performed high-throughput transcriptome sequencing of tissues from cats infected with feline immunodeficiency virus (FIV). A novel member of the Hepadnaviridae, tentatively named domestic cat hepadnavirus, was discovered in a lymphoma sample and its complete 3187 bp genome characterized. Phylogenetic analysis placed the domestic cat hepadnavirus as a divergent member of mammalian orthohepadnaviruses that exhibits no close relationship to any other virus. DNA extracted from whole blood from pet cats was positive for the novel hepadnavirus by PCR in 6 of 60 (10%) FIV-infected cats and 2 of 63 (3.2%) FIV-uninfected cats. The higher prevalence of hepadnavirus viraemia detected in FIV-infected cats mirrors that seen in human immunodeficiency virus-infected humans coinfected with hepatitis B virus. In summary, we report the first hepadnavirus infection in a carnivore and the first in a companion animal. The natural history, epidemiology and pathogenic potential of domestic cat hepadnavirus merits additional investigation.

Molecular Diagnosis of Felis catus Gammaherpesvirus 1 (FcaGHV1) Infection in Cats of Known Retrovirus Status with and without Lymphoma.

The pathogenicity of Felis catus gammaherpesvirus 1 (FcaGHV1), a common infection of domestic cats, is unknown. To explore an association between FcaGHV1 detection and feline lymphoma, a retrospective, cross-sectional, disease-association study was conducted. The infection status of all cats for feline immunodeficiency virus and feline leukaemia virus was determined. Neither a molecular diagnosis of FcaGHV1 nor whole-blood FcaGHV1 load was related to outcome in 122 lymphoma cases compared with 71 controls matched for age and sex. Molecular analysis of lymphoma-derived DNA paired with autologous uninvolved tissue did not suggest restriction of FcaGHV1 DNA to tumour tissue. FcaGHV1 DNA detection was associated with significantly shorter survival in lymphoma cases, an observation that could not be adequately explained by treatment differences. In addition, regressive feline leukaemia virus infection was identified as a risk factor for lymphoma. A history of fighting or roaming was identified as a novel epidemiological risk factor for FcaGHV1 detection, lending support to intercat aggression as a potential route of transmission. Studies investigating the cellular location and expression of FcaGHV1 are indicated to assist in ruling out a lymphomagenic role for this virus. Prospective investigation of FcaGHV1 DNA detection as a prognostic marker in feline lymphoma is warranted.

Comparative pathogenesis of eosinophilic meningitis caused by Angiostrongylus mackerrasae and Angiostrongylus cantonensis in murine and guinea pig models of human infection.

This study investigated comparatively the pathogenicity of experimental infection of mice and guinea pigs, with Angiostrongylus mackerrasae and the closely related species A. cantonensis. Time course analyses showed that A. mackerrasae causes eosinophilic meningitis in these hosts, which suggests that the species has the potential to cause meningitis in humans and domestic animals. Both A. mackerrasae and the genetically similar A. cantonensis caused eosinophilic meningitis in mice at two time points of 14 and 21 days post infection (dpi). The brain lesions in mice infected with A. mackerrasae were more granulomatous in nature and the parasites were more likely to appear degenerate compared with lesions caused by A. cantonensis. This may indicate that the mouse immune system eliminates A. mackerrasae infection more effectively. The immunologic responses of mice infected with the two Angiostrongylus species was compared by assessing ex vivo stimulated spleen derived T cells and cytokines including interferon-gamma, interleukin 4 and interleukin 17 on 14 and 21 dpi. The results were similar for mice infected with A. cantonensis and A. mackerrasae. Serum from the infected animals with either A. cantonensis or A. mackerrasae recognized total soluble antigen of A. cantonensis female worms on Western blot.

The mitochondrial genome of Angiostrongylus mackerrasae as a basis for molecular, epidemiological and population genetic studies.

BACKGROUND: Angiostrongylus mackerrasae is a metastrongyloid nematode endemic to Australia, where it infects the native bush rat, Rattus fuscipes. This lungworm has an identical life cycle to that of Angiostrongylus cantonensis, a leading cause of eosinophilic meningitis in humans. The ability of A. mackerrasae to infect non-rodent hosts, specifically the black flying fox, raises concerns as to its zoonotic potential. To date, data on the taxonomy, epidemiology and population genetics of A. mackerrasae are unknown. Here, we describe the mitochondrial (mt) genome of A. mackerrasae with the aim of starting to address these knowledge gaps. METHODS: The complete mitochondrial (mt) genome of A. mackerrasae was amplified from a single morphologically identified adult worm, by long-PCR in two overlapping amplicons (8 kb and 10 kb). The amplicons were sequenced using the MiSeq Illumina platform and annotated using an in-house pipeline. Amino acid sequences inferred from individual protein coding genes of the mt genomes were concatenated and then subjected to phylogenetic analysis using Bayesian inference. RESULTS: The mt genome of A. mackerrasae is 13,640 bp in size and contains 12 protein coding genes (cox1-3, nad1-6, nad4L, atp6 and cob), and two ribosomal RNA (rRNA) and 22 transfer RNA (tRNA) genes. CONCLUSIONS: The mt genome of A. mackerrasae has similar characteristics to those of other Angiostrongylus species. Sequence comparisons reveal that A. mackerrasae is closely related to A. cantonensis and the two sibling species may have recently diverged compared with all other species in the genus with a highly specific host selection. This mt genome will provide a source of genetic markers for explorations of the epidemiology, biology and population genetics of A. mackerrasae.

A survey of Angiostrongylus species in definitive hosts in Queensland.

Despite the recent sporadic reports of angiostrongyliasis in humans, dogs and wildlife in eastern Australia there has been no systematic study to explore the epidemiology of Angiostrongylus spp. in definitive and intermediate hosts in the region. Little is known about the epidemiology of Angiostrongylus species in the definitive host in southeast Queensland, since the only survey conducted in this region was performed in the late 1960s. In this study, free-living populations of Rattus spp. were sampled and examined for the presence of adult and larval Angiostrongylus in the lungs, and of larvae in faeces. The prevalence of infection with Angiostrongylus spp. was 16.5% in Rattus spp. trapped in urban Brisbane and surrounds. This prevalence is much higher than estimates of earlier studies. This highlights the possible risk of zoonotic infection in children, dogs and wildlife in this region and indicates the necessity for public awareness as well as more detailed epidemiological studies on this parasite in eastern Australia.

The Prevalence of Angiostrongylus cantonensis/mackerrasae Complex in Molluscs from the Sydney Region.

Angiostrongylus cantonensis and Angiostrongylus mackerrasae are metastrongyloid nematodes that infect various rat species. Terrestrial and aquatic molluscs are intermediate hosts of these worms while humans and dogs are accidental hosts. Angiostrongylus cantonensis is the major cause of angiostrongyliasis, a disease characterised by eosinophilic meningitis. Although both A. cantonensis and A. mackerrasae are found in Australia, A. cantonensis appears to account for most infections in humans and animals. Due to the occurrence of several severe clinical cases in Sydney and Brisbane, the need for epidemiological studies on angiostrongyliasis in this region has become apparent. In the present study, a conventional PCR and a TaqMan assay were compared for their ability to amplify Angiostrongylus DNA from DNA extracted from molluscs. The TaqMan assay was more sensitive, capable of detecting the DNA equivalent to one hundredth of a nematode larva. Therefore, the TaqMan assay was used to screen molluscs (n=500) of 14 species collected from the Sydney region. Angiostrongylus DNA was detected in 2 of the 14 mollusc species; Cornu aspersum [14/312 (4.5%)], and Bradybaenia similaris [1/10 (10%)], which are non-native terrestrial snails commonly found in urban habitats. The prevalence of Angiostrongylus spp. was 3.0% ± 0.8% (CI 95%). Additionally, experimentally infected Austropeplea lessoni snails shed A. cantonensis larvae in their mucus, implicating mucus as a source of infection. This is the first Australian study to survey molluscs using real-time PCR and confirms that the garden snail, C. aspersum, is a common intermediate host for Angiostrongylus spp. in Sydney.

Emergence of neural angiostrongyliasis in eastern Australia.

Despite an apparent increase in cases of angiostrongyliasis in humans and animals in Australia, the epidemiology of infection with the two species of Angiostrongylus that co-exist in this country, namely A. cantonensis and A. mackerrasae, is poorly understood. This knowledge gap is particularly important with respect to A. mackerrasae, a species evidently native to Australia, as its ability to cause disease in humans is unknown. Likewise, there is little information on the roles of native and introduced species of rodents and molluscs as hosts of Angiostrongylus species in Australia. This review focuses on the gaps in the knowledge about the two species, highlighting the need for epidemiological and pathogenesis studies on the native lungworm A. mackerrasae.

Molecular identification of novel intermediate host species of Angiostrongylus vasorum in Greater London.

Angiostrongylus vasorum is a parasitic nematode that can cause serious and potentially fatal disease in dogs and other canids. The aim of this study was to determine the intermediate slug species infected in nature by sampling sites in Greater London and Hertfordshire located within a known hyperendemic region. Overall, A. vasorum larvae were recovered from 6/381 slugs (1.6%) by tissue digestion, and their identity was confirmed by PCR. Infected slugs originated from three different sites in the Greater London area: one in Waltham Forest and two in Bromley. Slugs parasitised by A. vasorum were identified by a combination of external morphological characteristics and molecular techniques and belonged to three different families: the Arionidae, the Milacidae and the Limacidae. This includes two new host records for the parasite: Arion distinctus and Tandonia sowerbyi. This is the first record of A. vasorum in the family Milacidae, indicating that the parasite has a broader intermediate host range than previously recognised.