Publications

4596

Abstract Candidatus Liberibacter asiaticus ( C Las), the causal agent of Huanglongbing, is transmitted by the Asian citrus psyllid ( Diaphorina citri ) in a persistent, propagative manner. Reproducible transmission studies require not only controlled inoculation assays but also standardized propagation of citrus host plants, maintenance of D. citri colonies, and reliable pathogen detection. This study presents a comprehensive set of coordinated protocols that collectively support controlled C Las transmission experimentation. The workflow includes propagation of Citrus macrophylla for colony and assay use; generation and maintenance of C Las‐infected citrus and D. citri colonies; synchronization of D. citri populations to enable C Las acquisition; and a controlled single‐ D. citri transmission assay with defined inoculation access periods, and a Dellaporta‐based nucleic acid extraction method compatible with citrus and psyllid tissues. Quantitative PCR detection is integrated throughout to verify infection status, determine C Las titer, and monitor C Las movement and systemic colonization in citrus over time. By combining standardized plant production, vector infection systems, transmission assays, and molecular diagnostics, these coordinated methods provide a unified framework for testing hypotheses about vector competence, pathogen–vector interactions, microbial co‐infections, host susceptibility, and environmental effects on transmission. Together, the procedures enable consistent and comparable assessment of C Las acquisition, dissemination, and transmission outcomes under controlled laboratory conditions. © 2026 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1 : Controlled laboratory method for assessing Candidatus Liberibacter asiaticus transmission by Diaphorina citri Support Protocol 1 : Generation of Candidatus Liberibacter asiaticus‐infected Citrus macrophylla and Diaphorina citri Basic Protocol 2 : Dellaporta total nucleic acid extraction Basic Protocol 3 : Citrus macrophylla propagation

Abstract ‘Candidatus Mycoplasma haemolamae’ (CMhl) is a hemotropic bacterium infecting South American Camelids (SAC), whose epidemiology and clinical significance are still not fully elucidated. This study investigated CMhl by qPCR in blood samples of alpacas ( Vicugna pacos ) and llamas ( Lama glama ) in Italy in 2021–2024, characterizing CMhl haplotypes based on partial 16 S rRNA sequences and defining haematological findings in a subset of CMhl-infected animals. Statistical analysis was performed to detect potential risk factors associated with CMhl positivity. Out of 206 animals (200 alpacas and 6 llamas), CMhl was detected by qPCR in 88 (42.7%) animals. CMhl haplotype #1 and #2 were detected in 42.2% and 0.5% of the animals, respectively. Binary logistic regression analysis showed that higher CMhl positivity was significantly associated with larger herd size ( P  = 0.011), Northern Italy area of origin ( P  = 0.031) and warmer seasons ( P  = 0.003). The detection of two CMhl haplotypes confirms the high diffusion of CMhl in SAC farmed in Italy. Results confirm that CMhl usually causes subclinical infections, suggesting that antimicrobial treatment is likely not necessary based on qPCR positivity alone. Results focusing on the association of CMhl positivity with warm temperatures and large herd size suggest the need of further studies on the genetic features, epidemiology and immune response in CMhl-positive SAC from Italy, with emphasis on understanding potential vector transmission.