Salmonella Pullorum (S. Pullorum) and Salmonella Gallinarum (S. Gallinarum) are two biovars of Salmonella enterica serovar Gallinarum, responsible for pullorum disease and fowl typhoid, which are the most prevalent and pathogenic forms of salmonellosis in poultry in developing countries. Traditional differentiation methods for S. Pullorum and S. Gallinarum are based on distinct clinical manifestations and biochemical traits, given their indistinguishable nature via serological assays alone. Molecular differentiation methods such as allele-specific PCR and dual PCR combined with gel electrophoresis or enzyme digestion have also been used to discriminate S. Pullorum and S. Gallinarum, but the detection efficiency is not high. This investigation introduces a Fluorescence Resonance Energy Transfer (FRET) PCR assay targeting the pegB gene, exclusively found in specific Salmonella serovars such as S. Pullorum and S. Gallinarum, and exhibiting conserved single-nucleotide polymorphisms across these two biovars. High-resolution melting curve analysis demonstrates distinct dissolution profiles, facilitating the precise discrimination of S. Pullorum and S. Gallinarum. This FRET-PCR assay exhibits a detection limit of 10 copies per reaction and has been rigorously validated utilizing 17 reference strains and 39 clinical isolates. The innovation presented herein provides a valuable tool for the rapid differentiation of S. Pullorum and S. Gallinarum, thereby enhancing diagnostic efficiency and molecular surveillance of poultry Salmonella. The developed pegB-targeting FRET-PCR assay presents a promising alternative to current cumbersome and time-consuming diagnostic modalities, offering significant potential for expedited identification and control of Salmonella in poultry and mitigating economic losses associated with Salmonella contamination in poultry production.

Conventional diagnosis of dermatophytosis relies on fungal culture and microscopic examination, methods that are often time-consuming and lack sensitivity. This study aimed to develop and compare real-time PCR assays for the simultaneous detection and differentiation of three major dermatophytes in dogs: Microsporum canis, Nannizzia gypsea, and Trichophyton mentagrophytes. Three qPCR platforms targeting the chitin synthase 1 (CHS1) gene—SYBR Green, EvaGreen, and dual-emission fluorescence resonance energy transfer (FRET)—were evaluated. The FRET assay demonstrated the highest performance, achieving a detection limit of a single gene copy per reaction and producing distinct melting profiles that enabled accurate species discrimination (M. canis ~56.1 °C, N. gypsea ~53.0 °C, T. mentagrophytes ~51.8 °C). In contrast, SYBR Green and EvaGreen assays showed reduced sensitivity and cross-reactivity with non-target fungi. All assays were validated using three ATCC reference strains, ten clinical isolates of the target dermatophytes, and nine additional fungal species, including Nocardia, Aspergillus, Fusarium, Sporothrix, and Candida. Overall, FRET-qPCR exhibited a 100% specificity and a detection limit of one copy of target gene per reaction, offering a rapid, reliable tool for accurate diagnosis and molecular surveillance of dermatophytosis in companion animals.

Mycobacterium avium subsp. hominissuis (MAH) is a zoonotic pathogen with a broad host range and diverse clinical manifestations. We report here the first documented case of MAH-induced fatal vasculitis in zebra finch (Taeniopygia guttata). Histopathological examination revealed acid-fast bacilli within macrophages and endothelial cells, primarily affecting the heart and aorta. Mycobacterial DNA was detected in cloacal swabs from affected finches and environmental samples from their housing facility. PCR targeting the rpoB gene and insertion elements IS1245 and IS901, followed by sequencing, confirmed MAH infection. MAH DNA was identified in 4 of 13 finch cloacal swabs and 7 of 28 environmental samples. This study describes a novel, highly pathogenic manifestation of MAH in birds and underscores the potential for avian involvement in environmental and zoonotic transmission.

A herd of approximately 300 Spanish meat goats in central Alabama experienced sporadic ocular, respiratory, and reproductive diseases over two years, prompting diagnostic investigation at Auburn University’s JT Vaughan Large Animal Teaching Hospital. Five representative doelings exhibiting ocular lesions were examined. Clinical signs included conjunctivitis, corneal opacity, uveitis, and, in one severe case, systemic illness. Initial treatment with topical and systemic antibiotics provided incomplete resolution, raising suspicion of infectious keratoconjunctivitis of atypical etiology. Comprehensive diagnostic testing was performed, including aerobic and Mycoplasma cultures, Giemsa staining, and molecular assays. Moraxella bovoculi was cultured; however, Giemsa staining revealed Chlamydia elementary bodies, and a FRET-qPCR with DNA sequencing confirmed high Chlamydia pecorum loads (up to 1.1 × 10⁷ copies/swab). Mycoplasma testing was negative. Extended treatment with systemic and topical oxytetracycline led to gradual clinical improvement, with C. pecorum DNA declining over 22,000-fold and becoming undetectable after five weeks. This case represents the first documented report of C. pecorum–associated keratoconjunctivitis in goats in the United States. The findings underscore the diagnostic importance of molecular assays for detecting intracellular pathogens that may be missed by culture. The protracted treatment course highlights the therapeutic challenges posed by chlamydial infections due to their intracellular persistence. Additionally, the concurrent detection of M. bovoculi suggests the potential for mixed infections influencing disease severity. These results emphasize C. pecorum as an emerging pathogen of caprine ocular disease with implications for herd health and management.