Veterinaria Italiana

Development and validation of a real time RT-qPCR assay for detection of the emerging Bluetongue virus serotype 5 from field samples

Andrea Palombieri — 2026-03-11

Bluetongue (BT) is a WOAH-notifiable economically important disease of ruminants caused by Bluetongue virus (BTV), transmitted by Culicoides spp. biting midges. Over the past two years, Italy has experienced a marked re-emergence of BT, with thousands of outbreaks reported due to the simultaneous circulation of several BTV strains belonging to serotypes 3, 4, and 8. Moreover, in September 2025, BTV-5 was detected in Sardinia, marking its first occurrence in Europe. Following the first identification by Whole Genome Sequencing, the development of a reliable real-time RT-qPCR-based assay capable of typing the novel BTV-5 ITA 2025 strain was essential, as currently available molecular typing methods targeting BTV segment 2, which encodes the outer capsid protein VP2, are unable to detect this newly emerging strain. Therefore, in this study we developed, optimised, and validated a real-time RT-PCR assay for the detection and typing of BTV-5 ITA 2025 in field samples. The assay is characterised by high sensitivity and specificity, as well as good reproducibility, and can be effectively applied for BTV-5 ITA 2025 diagnosis in the current epidemiological context, supporting surveillance and control strategies.

First comprehensive histopathological and seroepidemiological investigations of Toxoplasma gondii infection in meat goats in Algeria

Hichem Dahmani — 2026-03-30

This study assessed Toxoplasma gondii infection in 92 slaughtered goats from four ecologically distinct regions of Algeria: Laghouat, Medea, Bouira, and Djelfa. Serological analysis was performed using a multispecies indirect ELISA, and histopathological examination targeted heart and diaphragm tissues to detect cysts or suggestive inflammatory lesions. The overall seroprevalence was low (4.3%), with no significant associations with age, sex, or breed. Geographic region significantly influenced seroprevalence: Bouira exhibited the highest rate (18.8%, p < 0.01), while other regions ranged from 0–8.3%. Histopathological analysis revealed a higher prevalence of lesions compatible with T. gondii (10.9%) in both heart and diaphragm. Young goats had higher lesion rates (18.8%) than adults (6.7%), though differences were not statistically significant. Regional differences were pronounced: Bouira showed 37.5% of animals with lesions (p < 0.001), El-Djelfa 16.7% (p < 0.04), while Laghouat remained free of lesions. These findings highlight a geographically clustered distribution of infection. Comparison of ELISA with histopathology indicated high concordance: 4 goats were positive across all methods, while 6 goats were ELISA-negative but tissue-positive, suggesting early, chronic, or low-antibody infections. Overall agreement between ELISA and tissue examination was 93.5%, with a Cohen’s kappa of 0.54, indicating moderate to substantial agreement. The study demonstrates that intensive, closed-system goat farming effectively limits exposure to T. gondii, as evidenced by low seroprevalence and limited tissue infection. Importantly, combining serological and histopathological approaches provides a more comprehensive assessment of infection, capturing cases that may be missed by either technique alone. This dual diagnostic strategy enhances epidemiological understanding and improves surveillance, particularly in areas with environmental hotspots like Bouira.

Metagenomic sequencing of zoonotic viruses: evaluation of a CRISPR-Cas–based rRNA depletion system

Gianpiero Zamperin — 2026-03-04

Pathogen-agnostic diagnostics are crucial for the early detection of emerging viruses. Shotgun metagenomic sequencing enables unbiased detection of viral genomes but is frequently constrained by the abundance of host and microbial ribosomal RNA (rRNA), which reduces sensitivity and increases sequencing costs. CRISPR-Cas9–based rRNA depletion has emerged as an alternative to enzymatic methods; however, its performance for the characterization of zoonotic viruses across diverse animal hosts and tissues remains underexplored. We compared CRISPR-Cas9 (Jumpcode CRISPRclean™ Plus) and RNase H–based enzymatic depletion (Ribo-Zero Plus, Illumina) using 12 samples positive for rabies lyssavirus, influenza A virus, West Nile virus or norovirus, from multiple host species and tissues, including both high-quality and degraded RNA. CRISPR-Cas9 efficiently reduced rRNA content (14.5%) but recovered fewer viral reads than Ribo-Zero, which achieved up to 60.7× enrichment. Both methods produced complete viral consensus genomes when RNA quality and viral load were sufficient. However, based on the data generated here, enzymatic depletion currently remains more efficient and cost-effective for viral metagenomics. Further optimization of CRISPR-Cas9 workflows could enhance its utility for viral surveillance and diagnostics.

Molecular Evidence of Epizootic Haemorrhagic Disease Virus and Bluetongue Virus Circulation in Wild Ruminants in Namibia.

Umberto Molini — 2026-03-04

Bluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV) are Culicoides-borne orbiviruses affecting domestic and wild ruminants. Information on their circulation in Namibian wildlife is limited. This study investigated the molecular detection and serotype distribution of BTV and EHDV in wild ruminants from a commercial game farm in the Khomas Region, Namibia, where wildlife and livestock coexist.

Between June and September 2019, spleen samples from 62 clinically healthy animals (kudu, oryx, and red hartebeest) were analysed by real-time RT-PCR using pan-BTV and pan-EHDV assays, followed by serotype-specific tests for selected BTV types. Two animals (3.23%) tested positive for EHDV. BTV RNA was detected in 24/62 animals (38.71%), with Ct values ranging from 28.3 to 38.4. BTV-3 and BTV-4 were the most frequently identified serotypes, while one sample was positive for BTV-1; six BTV-positive samples remained untyped. High Ct values and low RNA loads likely limited sequencing success.

Although restricted to a single farm and a limited serotype panel, this study provides preliminary molecular evidence of BTV and EHDV circulation in Namibian wild ruminants, highlighting the need for broader epidemiological investigations at the wildlife–livestock interface.