Bioassay-based detection of Toxoplasma gondii in free-range chickens from Khorramabad, Iran: comparison of direct microscopy and semi-nested PCR.

This study aimed to investigate the occurrence of anti-Toxoplasma gondii antibodies in free-range chickens from Khorramabad, western Iran, and also to compare the performance of direct microscopy and semi-nested PCR in mice bioassayed with tissues from seropositive chickens. We investigated 97 serum samples from free-range chickens, using the modified agglutination test (MAT). Tissues from all seropositive chickens (MAT ≥ 1:10) were bioassayed in mice. All inoculated mice were examined by direct microscopy and a semi-nested PCR targeting the 529 bp repeat element (RE) of the parasite. Anti-T. gondii antibodies were detected in 21.6% of chicken sera. Eighteen of 21 (85.7%) seropositive chickens were positive in mouse bioassay using molecular DNA detection. However, biological forms of the parasite were isolated only from 11 (52.3%) seropositive chickens. Compared with semi-nested PCR, the sensitivity of direct microscopy was 62.1%. It can be concluded that although direct microscopy is a rapid and specific method for the detection of T. gondii, it does not detect the parasite in all experimentally infected mice. The low sensitivity of direct microscopy highlights the need for molecular techniques, such as RE-based semi-nested PCR, to increase the sensitivity of the mouse bioassay.

Noninvasive Blood-Based Cancer Detection in Veterinary Medicine.

The past decade has seen incredible advances in blood-based cancer detection in people and in dogs - yet this represents only a glimpse of the benefits these tests can provide to patients. The clinical uses of this technology range from screening asymptomatic individuals for early detection to use as an aid in diagnosis when cancer is suspected, to cancer monitoring both during and after treatment. This article summarizes the benefits of early cancer detection and examines use cases and methods of blood-based cancer detection in dogs, including quantitative, qualitative, and alternative approaches.

Clinical experience with next-generation sequencing-based liquid biopsy testing for cancer detection in dogs: a review of 1,500 consecutive clinical cases.

OBJECTIVE: To review ordering patterns, positivity rates, and outcome data for a subset of consecutive samples submitted for a commercially available, blood-based multicancer early-detection liquid biopsy test for dogs using next-generation sequencing at 1 laboratory. SAMPLE: 1,500 consecutively submitted blood samples from client-owned dogs with and without clinical suspicion and/or history of cancer for prospective liquid biopsy testing between December 28, 2021, and June 28, 2022. PROCEDURES: We performed a retrospective observational study, reviewing data from 1,500 consecutive clinical samples submitted for liquid biopsy testing. Outcome data were obtained via medical record review, direct communication with the referring clinic, and/or a patient outcome survey through October 16, 2022. RESULTS: Sixty-four percent (910/1,419) of reportable samples were submitted for cancer screening, 26% (366/1,419) for aid in diagnosis, and 10% (143/1,419) for other indications. The positivity rate was 25.4% (93/366) in aid-in-diagnosis patients and 4.5% (41/910) in screening patients. Outcome data were available for 33% (465/1,401) of patients, and outcomes were classifiable for 428 patients. The relative observed sensitivity was 61.5% (67/109) and specificity was 97.5% (311/319). The positive predictive value was 75.0% (21/28) for screening patients and 97.7% (43/44) for aid-in-diagnosis patients, and the time to diagnostic resolution following a positive result was < 2 weeks in most cases. CLINICAL RELEVANCE: Liquid biopsy using next-generation sequencing represents a novel tool for noninvasive detection of cancer in dogs. Real-world clinical performance meets or exceeds expectations established in the test's clinical validation study.

Cancer detection in clinical practice and using blood-based liquid biopsy: A retrospective audit of over 350 dogs.

BACKGROUND: Guidelines-driven screening protocols for early cancer detection in dogs are lacking, and cancer often is detected at advanced stages. HYPOTHESIS/OBJECTIVES: To examine how cancer typically is detected in dogs and whether the addition of a next-generation sequencing-based "liquid biopsy" test to a wellness visit has the potential to enhance cancer detection. ANIMALS: Client-owned dogs with definitive cancer diagnoses enrolled in a clinical validation study for a novel blood-based multicancer early detection test. METHODS: Retrospective medical record review was performed to establish the history and presenting complaint that ultimately led to a definitive cancer diagnosis. Blood samples were subjected to DNA extraction, library preparation, and next-generation sequencing. Sequencing data were analyzed using an internally developed bioinformatics pipeline to detect genomic alterations associated with the presence of cancer. RESULTS: In an unselected cohort of 359 cancer-diagnosed dogs, 4% of cases were detected during a wellness visit, 8% were detected incidentally, and 88% were detected after the owner reported clinical signs suggestive of cancer. Liquid biopsy detected disease in 54.7% (95% confidence interval [CI], 49.5%-59.8%) of patients, including 32% of dogs with early-stage cancer, 48% of preclinical dogs, and 84% of dogs with advanced-stage disease. CONCLUSIONS/CLINICAL IMPORTANCE: Most cases of cancer were diagnosed after the onset of clinical signs; only 4% of dogs had cancer detected using the current standard of care (i.e., wellness visit). Liquid biopsy has the potential to increase detection of cancer when added to a dog's wellness visit.

Blood biomarkers for canine cancer, from human to veterinary oncology.

In recent decades, interest in circulating tumour biomarkers is increasing both in human and veterinary oncology. An ideal tumour biomarker would allow early diagnosis of neoplasia, identify it specifically, accurately, establish a prognosis and predict its behaviour, especially regarding different therapeutic solutions. It would also allow to monitor its evolution over time and all this in a non-invasive and inexpensive way. Actually, no biomarkers meeting all of these criteria have been identified in veterinary medicine, particularly due to a lack of specificity of the main protein tumour biomarkers studied to date. However, great hope is currently placed in biomarkers grouped under the name of liquid biopsy, which could prove to be effective tools for common clinical use in the near future. This review gives an update on blood cancer biomarkers studied in dogs, such as ions, proteins, nucleic acids and also circulating cells, of which some might become more prominent in the coming years to help improve the management of animal care.

Liquid biopsy can detect BRCA2 gene variants in female dogs with mammary neoplasia.

Mammary tumours (MT) are one of the most prevalent malignancies in female dogs and women. Currently, molecular analyzes have shown that each tumour type presents its own genetic signature. In this context, liquid biopsy allows a comprehensive genetic characterisation of the tumour, enabling early diagnosis and personalised treatment of patients. In women, deleterious mutations inherited in BRCA2 gene are associated with an increased risk of breast cancer, resistance to therapies and worse prognosis. In female dogs, there are many divergent data on the involvement of BRCA2 gene with mammary carcinogenesis and what its pathogenic potential is. Therefore, the objective was to identify BRCA2 gene variants in 20 plasma DNA samples, from 10 newly diagnosed dogs with mammary cancer (RD), five control (CTR) and five mastectomized patients. Eleven single nucleotide polymorphisms (SNPs) were detected, most of them in the exon 11 and two indels (deletion/insertion) in the BRCA2 gene. However, there was no statistically significant difference in the SNPs/indels detected between the groups. In addition, only one SNP (p.T1425P) and one deletion (p.L2307del) were considered deleterious using in silico computational models. Interestingly, most common variants were present in the plasma of all groups, except for the Ile2614Thr, Ile2614Val, Thr1425Pro and p.L2307del variants. Thus, we observed that SNPs are common in the BRCA2 gene of female dogs with MT, with a similar condition identified in women with breast cancer. Liquid biopsy approach in dogs with MT is useful for genetic and therapeutic proposals.

CDC6: A novel canine tumour biomarker detected in circulating extracellular vesicles.

Circulating nucleic acids and extracellular vesicles (EV) represent novel biomarkers to diagnose cancer. The non-invasive nature of these so-called liquid biopsies provides an attractive alternative to tissue biopsy-based cancer diagnostics. This study aimed to investigate if circulating cell cycle-related E2F target transcripts can be used to diagnose tumours in canine tumour patients with different types of tumours. Furthermore, we assessed if these mRNAs are localised within circulating EV. We isolated total RNA from the plasma of 20 canine tumour patients and 20 healthy controls. Four E2F target genes (CDC6, DHFR, H2AFZ and ATAD2) were selected based on the analysis of published data of tumour samples available in public databases. We performed reverse transcription and quantitative real-time PCR to analyse the plasma levels of selected E2F target transcripts. All four E2F target transcripts were detectable in the plasma of canine tumour patients. CDC6 mRNA levels were significantly higher in the plasma of canine tumour patients compared to healthy controls. A subset of canine tumour patient and healthy control plasma samples (n = 7) were subjected to size exclusion chromatography in order to validate association of the E2F target transcripts to circulating EV. For CDC6, EV analysis enhanced their detectability compared to total plasma analysis. In conclusion, our study reveals circulating CDC6 as a promising non-invasive biomarker to diagnose canine tumours.

The diagnostic value of the droplet digital PCR for the detection of bovine deltapapillomavirus in goats by liquid biopsy.

In the present study, the highly pathogenic bovine deltapapillomavirus (δPV) was investigated by liquid biopsy in blood samples of 168 clinically normal goats using both droplet digital PCR (ddPCR) and quantitative real-time PCR (qPCR). Overall, ddPCR discovered BPV E5 DNA in ~ 61.3% of the blood samples examined, while real-time qPCR revealed the virus in ~ 10.7% of the same samples. Moreover, ddPCR showed BPV E5 DNA in ~ 78.8% of blood samples from goats that were in close contact with cattle and in 20% of blood samples from goats living in closed pens without any contact with cattle. In addition, ddPCR unmasked a single BPV genotype in ~ 59.2% and multiple genotypes in ~ 40.8% of goats harbouring BPV DNA, while real-time qPCR detected single genotypes in ~ 17% and multiple genotypes in ~ 1%. Of the BPV co-infections detected by ddPCR, 28 (~67%) involved two genotypes, eight (~19%) three genotypes and six (~14%) four genotypes. In contrast, real-time qPCR revealed BPV co-infection by two genotypes in only one sample and failed to detect co-infection by three or four genotypes. BPV2 and BPV13 were the most prevalent viruses responsible for single and multiple co-infections, respectively. The present study showed that ddPCR is promising method for circulating bovine papillomavirus DNA detection and quantification and suggested that animal husbandry practices contribute to cross-species transmission of BPVs.

Novel Noninvasive Diagnostics.

Molecular diagnostics have revolutionized human oncology to allow early detection, targeted therapy, monitoring throughout treatment, and evidence of recurrence. By identifying genetic signatures associated with cancers, liquid biopsy techniques have been developed to diagnose and monitor cancer in noninvasive or minimally invasive ways. These techniques offer new opportunities for improving cancer screening, diagnosis, and monitoring the impact of therapy on the patients over time. Liquid biopsy also drives drug development programs. Similar diagnostics hold promise for comparable results in the veterinary field. Several noninvasive/minimally invasive techniques have been described in veterinary medicine that could be referred to as liquid biopsy.