Mariano Carossino

During the 2020–2021 irruption year, pine siskins ( Spinus pinus ) experienced widespread mortality across North America, attributed to Salmonella enterica subsp. enterica serovar Typhimurium. In March 2021, 3 pine siskins were admitted to the Louisiana State University Wildlife Hospital (Baton Rouge, LA, USA) because of lethargy, respiratory distress, diarrhea, and inability to fly. All birds died on or shortly after presentation. Postmortem examination revealed necrotizing and granulomatous esophagitis (3), hepatitis (3), splenitis (2), enteritis (1), cloacitis (1), and stomatitis/rhinitis (1) with intralesional gram-negative bacilli. Immunohistochemistry revealed abundant extracellular and intracellular Salmonella sp. lipopolysaccharide antigen in affected organs. Salmonella Typhimurium was isolated from livers (3), cloacal swabs (2), and a spleen (1). One case had concurrent trichomonosis with erosive pharyngitis and necrotizing esophagitis. These cases are a subset of the larger 2020–2021 salmonellosis outbreak in passerines and are among the few reported from Louisiana.

Cervical cancer is a major cause of mortality worldwide and has been attributed to long-lasting (persistent) infections with the human papillomavirus (HPV). Although more than 50 types of HPVs are known the infect the genital tract, fewer than a dozen of them are regularly linked to cervical cancers leading to these types being designated as “high risk” HPVs. Worldwide, over 90% of cervical cancers arise from persistent high-risk human papillomavirus (HPV) infection and the infectious nature of the virus procures the need for preventative vaccines against cervical cancer. To address this, we have developed a live-attenuated herpes simplex virus type 1 (HSV-1) VC2-vectored vaccine expressing a catalytically inactive mutant of HPV-16 E7 (E7m) fused to the viral capsid protein VP26. Incorporation of E7m into the VC2 backbone resulted in stable incorporation of VP26-E7m fusion protein in cell free viral particles and detectable intracellular expression within infected cells. The recombinant virus (E7m VC-2) maintained growth kinetics comparable to parental VC2 in vitro, though plaque size was modestly reduced, suggesting a minimal fitness cost associated with transgene insertion. To evaluate prophylactic efficacy, mice received a one-dose vaccine schedule of E7m VC-2 prior to challenge with syngeneic HPV-16 E6/E7-expressing tumor cells. In mice that did develop tumors, growth kinetics were attenuated relative to unvaccinated animals. Importantly, neither E7m VC-2 nor adjuvanted E7m protein alone achieved statistically significant protection versus untreated controls. Immunohistochemical analysis of tumor tissue revealed markedly increased CD8+ T cell infiltration in E7m VC-2 vaccinated mice, demonstrating that vaccination promotes a cytotoxic immune microenvironment capable of restricting tumor progression. Three-dose vaccination of mice with E7m VC-2 induced a robust Th1-polarized immune response characterized by strong interferon-gamma (IFN-γ) secretion in response to HPV16 E7 expressing syngeneic cell lines, consistent with the induction of cell-mediated antitumor immunity and superior to responses elicited by adjuvanted E7m protein alone. With an extended three-dose vaccination schedule E7m VC-2 conferred significantly greater protection against tumor establishment relative to untreated controls (80% vs. 20% tumor-free). Collectively, these findings establish proof-of-concept for the HSV-1 VC2 platform as an effective vector for HPV-targeted cancer immunotherapy, delivering statistically significant prophylactic protection alongside potent induction of tumor-reactive CD8+ T cells. Future studies will incorporate both E6 and E7 antigens to broaden antigenic coverage, evaluate therapeutic efficacy in established tumor models, and optimize dosing regimens with the long-term goal of advancing this Louisiana-developed platform toward IND-enabling studies and clinical translation.

Eastern equine encephalitis virus (EEEV; family Togaviridae , taxon species Alphavirus eastern ) is a mosquito-borne alphavirus that is endemic to the Western Hemisphere and well established in freshwater hardwood swamps. EEEV is environmentally maintained through a mosquito-and-avian cycle, primarily by Culiseta melanura mosquitoes. Transmission to mammals, which are considered dead-end hosts, is well known and seasonal through C. melanura or other bridging mosquito vectors. EEE is well described in horses and humans, with relatively few reports in ruminants. Information regarding infection and disease localization, and particularly disease in the caprine species, is even more sparse. Here, we describe a case of naturally occurring EEEV infection and disease in a 4.5-y-old American pygmy goat wether with neurologic abnormalities, confirmed postmortem by reverse-transcription real-time PCR testing and indirect immunohistochemistry. The histologic alterations were confined to the CNS and were moderate-to-marked, multifocal, lymphocytic polioencephalomyelitis and meningitis, with satellitosis, neuronal necrosis, neuronophagia, and glial nodules.

Equine Coital Exanthema (ECE) is a contagious venereal disease caused by Equid alphaherpesvirus 3 (EHV‑3). Although ECE is self-limiting, it causes significant economic losses to the global equine industry due to the temporary segregation of affected mares and stallions from breeding activities. This report describes the epidemiological, clinical, histological, and molecular findings from an outbreak of ECE in Uruguayan horses. Seven days following the introduction of a stallion, five mares developed extensive papular and vesicular dermatitis in the vaginal and perineal regions. The stallion exhibited minimal preputial mucosal ulcerative lesions. Histological examination of skin biopsies from three affected mares revealed ulcerative dermatitis with intranuclear eosinophilic inclusion bodies in keratinocytes. Molecular confirmation of EHV‑3 infection was achieved by qPCR analysis of perineal skin samples from a mare with typical ECE lesions.

The circular polymerase extension reaction (CPER) has emerged as a high-fidelity, versatile and rapid tool for generating infectious cDNA clones of positive-sense, single-stranded RNA viruses. Here, we implemented this strategy to generate infectious cDNA clones of two closely related members of the family Arteriviridae : equine arteritis virus KY84 strain (EAV KY84) and porcine reproductive and respiratory syndrome virus VR2332 strain (PRRSV VR2332). Overlapping cDNA fragments spanning the entire viral genomes were generated and assembled with a linker sequence incorporating critical expression elements, yielding a circularized, full-length viral cDNA genome ready for direct transfection into permissive cells. Following transfection, infectious rEAV KY84 and rPRRSV VR2332 strains were recovered and characterized. Similarly, we generated reporter viruses by inserting the mCherry gene downstream of the nsp1/nsp2 cleavage junction in EAV KY84 ORF1a and the GFP gene downstream of ORF1b of PRRSV VR2332. All CPER-generated viruses showed high identity to their parental strains by next-generation sequencing. Growth kinetics of CPER-generated non-reporter and rEAV KY84-RFP mCherry viruses were comparable to their parental strains; however, the reporter rPRRSV VR2332-GFP showed a significant reduction in viral titres despite its stability for at least 15 passages. In contrast, mCherry expression from rEAV KY84-mCherry was negligible at passage 15 and associated with the deletion of the reporter gene. Taken together, the CPER-based method can be used as a rapid tool to generate infectious cDNA clones of arteriviruses, allowing the easy generation of mutants or reporter viruses that can facilitate the investigation of viral gene functions and their interactions with the host.

The circular polymerase extension reaction (CPER) has emerged as a high-fidelity, versatile, and rapid tool for generating infectious complementary DNA (cDNA) clones of positive-sense, single-stranded RNA viruses. Here, we implemented this strategy to generate infectious cDNA clones of two closely related members of the family Arteriviridae: equine arteritis virus KY84 strain (EAV KY84) and porcine reproductive and respiratory syndrome virus VR2332 strain (PRRSV VR2332). Overlapping cDNA fragments spanning the entire viral genomes were generated and assembled with a linker sequence incorporating critical expression elements, yielding a circularized, full-length viral cDNA genome ready for direct transfection into permissive cells. Following transfection, infectious rEAV KY84 and rPRRSV VR2332 strains were recovered and characterized. Similarly, we generated reporter viruses by inserting the mCherry gene downstream of the nsp1/nsp2 cleavage junction in EAV KY84 ORF1a and the GFP gene downstream of ORF1b of PRRSV VR2332. All CPER-generated viruses showed high identity to their parental strains by next-generation sequencing. Growth kinetics of CPER-generated non-reporter and rEAV KY84-RFP mCherry viruses were comparable to their parental strains; however, the reporter rPRRSV VR2332-GFP showed a significant reduction in viral titers despite its stability for at least 15 passages. In contrast, mCherry expression from rEAV KY84-mCherry was negligible at passage 15 and associated with the deletion of the reporter gene. Taken together, the CPER-based method can be used as a rapid tool to generate infectious cDNA clones of arteriviruses, allowing the easy generation of mutants or reporter viruses that can facilitate the investigation of viral gene functions and their interactions with the host.

Salmonella spp. is a major zoonotic pathogen. Although reptiles are mostly considered subclinical carriers, clinical disease may develop following immunosuppression. Clinical salmonellosis in reptiles has been extensively reported; however, the condition has been rarely described in bearded dragons (Pogona vitticeps). We retrospectively analyzed six cases of salmonellosis in bearded dragons and characterized the pathological, immunohistochemical, and bacteriological findings. Clinical signs and gross findings were mostly non-specific. Histological findings mainly consisted of fibrinonecrotizing enterocolitis (83.3%); necrotizing or granulomatous hepatitis (66.7%); pneumonia including bronchopneumonia or interstitial pneumonia in one case each (33.3%); tubulointerstitial nephritis with tubular necrosis (16.7%); and coelomitis (16.7%). Salmonella enterica subsp. houtenae was cultured in three cases (33.3%), whereas S. enterica subsp. enterica serovar Rissen, S. enterica subsp. enterica serovar Cotham, and S. enterica subsp. diarizonae were cultured in one case each. Intralesional bacteria were detected via immunohistochemistry in kidneys and colon in two cases (33.3%). The predominance of lesions in the intestines and liver likely reflects initial intestinal colonization followed by hematogenous dissemination to the liver. Hepatic lesions are thought to represent different stages along a continuum, progressing from acute necrosis to discrete granuloma formation. Renal and respiratory involvement was infrequent, as reported in other reptile species. Some of the isolated Salmonella subspecies (S. diarizonae and S. houtenae) are well-recognized causes of clinical disease in other reptile species but not previously identified in bearded dragons. This study provides a comprehensive pathological, immunohistochemical, and bacteriological characterization of salmonellosis in bearded dragons, thus raising awareness and assisting in the identification of this condition.