Theileria orientalis Ikeda genotype, a parasite causing a disease in cattle that leads to significant economic challenges in Asia, New Zealand, and Australia, has been identified in seven U.S. States since 2017. Two previously validated PCR tests for Theileria followed by DNA sequencing were performed to test blood samples collected from 219 cattle in Alabama, USA, during the period of 2022–2023. Bidirectional Sanger sequencing revealed that the MPSP gene sequences (639–660 bp) from two cattle in Lee and Mobile Counties of Alabama exhibited a 100% match with those of recognized T. orientalis Ikeda strains, and showed similarities ranging from 76% to 88% with ten other T. orientalis genotypes. A high copy number of T. orientalis Ikeda was detected in the blood of infected cattle (ALP-1: 1.7 × 10⁵ and 1.3 × 10⁶/mL whole blood, six months apart; ALP-2: 7.1 × 10⁶/mL whole blood). Although the confirmed competent vector for T. orientalis Ikeda, Haemaphysalis longicornis tick, has not yet been identified in Alabama, the persistent nature of T. orientalis Ikeda infection and the detection of a high pathogen burden in seemingly healthy cattle in this study suggest that other tick species, as well as shared needles and dehorning procedures, could facilitate pathogen transmission within the herd. Continued investigations are necessary for the surveillance of T. orientalis Ikeda and Haemaphysalis longicornis ticks in Alabama and other U.S. states, along with assessing the pathogenicity of T. orientalis Ikeda infections in cattle.

Antimicrobial resistance (AMR) in non-typhoidal Salmonella is a pressing public health concern in the United States, necessitating continuous surveillance. We conducted a retrospective analysis of 251 Salmonella isolates from 11 animal species recovered between 1982 and 1999, utilizing serotyping, antimicrobial susceptibility testing, and whole-genome sequencing (WGS). Phenotypic resistance was observed in 101 isolates, with S. Typhimurium, S. Dublin, S. Agona, and S. Muenster prevailing among 36 identified serovars. Notably, resistance to 12 of 17 antibiotics was detected, with ampicillin being most prevalent (79/251). We identified 38 resistance genes, primarily mediating aminoglycoside (n = 13) and β-lactamase (n = 6) resistance. Plasmid analysis unveiled nine distinct plasmids associated with AMR genes in these isolates. Chromosomally encoded bla_SCO-1 was present in three S. Typhimurium and two S. Muenster isolates from equine samples, conferring resistance to amoxicillin/clavulanic acid. Phylogenetic analysis revealed three distinct clusters for these five isolates, indicating evolutionary divergence. This study represents the first report of bla_SCO-1 in the USA, and our recovered isolates harboring this gene as early as 1989 precede those of all other reports. The enigmatic nature of bla_SCO-1 prompts further research into its function. Our findings highlight the urgency of addressing antimicrobial resistance in Salmonella for effective public health interventions.

The modulation of gut microbiota presents a promising avenue for improving livestock health, productivity, and sustainability. Traditional strategies such as probiotics, prebiotics, and synbiotics have demonstrated effectiveness in enhancing gut health and immune function while reducing the reliance on antibiotics. Recent advances have introduced innovative approaches, including postbiotics, fecal microbiota transplantation (FMT), and precision biotics, to emerging strategies such as synthetic biology applications and bacteriophage therapy, offering targeted interventions with the potential for high specificity and efficacy. However, successful integration into agricultural systems requires navigating complex regulatory frameworks and addressing safety, scalability, and economic feasibility concerns. This chapter aims to critically assess traditional, advanced, and emerging microbiota modulation strategies, evaluating their mechanisms, applications, and limitations while highlighting their transformative potential in achieving sustainable livestock production and addressing global public health challenges. The chapter offers practical guidance for veterinarians, researchers, policymakers, and producers seeking to integrate microbiota-based strategies into modern livestock operations.

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.

Salmonella enterica serovar Infantis has emerged as a prevalent foodborne pathogen in poultry with significant global health implications. This study investigates the molecular characteristics influencing virulence in a S. Infantis rough variant collected from a poultry farm in the USA. In this study, whole genome sequencing and comparative genomics were performed on smooth and rough poultry S. Infantis isolates, while chicken embryo lethality assay was conducted to assess their virulence. Comparative genomics between isolates was analyzed using Mauve pairwise Locally Collinear Blocks to measure the genetic conservation. Embryo survival rates between the isolates were compared using the Kaplan–Meier curves. High genomic conservation was observed between the two isolates, but a frameshift mutation was detected in the Wzz(fepE) gene of the rough variant, resulting in early protein truncation. The chicken embryo lethality assay showed that the lethality rate of the smooth strain was higher than that of the rough strain (p < 0.05). This study identifies a frameshift mutation in the Wzz(fepE) gene, leading to protein truncation, which may reduce bacterial virulence by impacting O-antigen biosynthesis in the rough Salmonella Infantis variant. These findings deepen our understanding of S. Infantis pathogenesis and suggest that targeting the Wzz(fepE) gene or related pathways could be a promising strategy for developing effective vaccines and therapeutic interventions.

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.