OBJECTIVE:Leptospirosis is a globally important zoonotic disease associated with renal and hepatic injury. Cats are frequently exposed to Leptospira spp., yet their epidemiological role remains unclear. This study aimed to determine whether Leptospira DNA could be detected in feline vaginal samples, in addition to urine, using Nested-PCR. Secondary objectives were to determine the proportion of PCR-positive cats and assess renal involvement by comparing renal parameters between PCR-positive and PCR-negative cats. METHODS:Seventy-six domestic and free-roaming cats were examined in Tehran, in 2025. Urine samples were collected from all cats and vaginal swabs from female cats. Nested-PCR targeting the 16S rRNA (rrs) gene was performed on both sample types followed by confirmatory sequencing of representative amplicons. Renal status was assessed using biochemical, hematological, and urinalysis parameters in all cats. RESULTS:Leptospira DNA was detected in 15/76 cats (19.7%). Positive results were obtained from urine samples in 10/76 cats (13.1%) and from vaginal swabs in 5/61 female cats (8.2%), with no concordant positive results between urine and vaginal samples (95% CIs reported). Vaginal PCR identified Leptospira DNA in cats that were negative by urine PCR. No significant differences were observed in biochemical, hematological or urinalysis parameters between PCR-positive and PCR-negative cats. CONCLUSIONS:Leptospira DNA was detected in urine and vaginal samples of both domestic and free-roaming cats, frequently in subclinical animals without observable clinical signs. The absence of concordance between urine and vaginal PCR results suggests that vaginal sampling may provide complementary molecular detection to urine testing. Further investigation is warranted to elucidate the biological significance and clinical implications of Leptospira DNA detection in feline vaginal samples.
Ovine pneumonia is a multifactorial respiratory disease in which complex microbial communities and antimicrobial resistance complicate disease control. This study characterized pulmonary microbial profiles in 49 slaughtered Duolang sheep from Kashgar stratified by gross lung lesions (apparently healthy, n = 10; pneumonic, n = 39) using pathology, bacterial isolation, Mycoplasma ovipneumoniae PCR, antimicrobial susceptibility testing, and 16S rRNA gene sequencing. Suppurative pneumonia predominated in pneumonic lungs (31/39, 79.5%), and M. ovipneumoniae positivity was higher than in apparently healthy lungs (69.2% vs. 20.0%; OR = 9.00, 95% CI, 1.66-48.88). Among 220 bacterial isolates recovered, co-isolation of at least two non-Escherichia genera remained detectable in at least 20/39 pneumonic lungs after exclusion of Escherichia. Of 120 isolates tested, 17 showed multidrug-resistant phenotypes. In the 16S subset, pneumonic lungs showed higher richness and Shannon diversity (FDR-adjusted P = 0.045), with nominal enrichment of Pasteurellaceae and Mycoplasmataceae, whereas beta diversity did not differ. Despite overlap between pneumonia and M. ovipneumoniae status in the sequencing subset, these findings support integrated surveillance of pulmonary microbial profiles and antimicrobial resistance in ovine respiratory disease.
Vitamin C (VC) supplementation has been reported to benefit poultry health and performance; however, the antiviral effects of VC have remained largely unknown. This study evaluated the effects of oral VC supplementation on viral RNA load, histopathology, and immune-related gene expression in specific-pathogen-free (SPF) chickens challenged with IBV. Eighty one-day-old SPF chicks were randomly allocated to control group or VC group and orally administered VC every other day at doses equivalent to 0 or 400 mg/kg diet for 21 days. All birds were challenged with 0.1 mL of 10⁷ 50% embryo infectious dose of IBV strain CK/CH/I0317/22 via the intraocular and intranasal routes. Results showed that VC supplementation did not affect growth performance, clinical signs, gross lesions, or IBV-specific antibody titers. However, VC alleviated IBV-induced histopathological damage in the trachea and kidney and reduced viral RNA load in the bursa of Fabricius (P < 0.05). In addition, VC supplementation modulated the expression of genes involved in Toll-like receptor signaling, avian β-defensin responses, antioxidant defense, and inflammatory regulation in a tissue-specific and time-dependent manner. In particular, the expression of several TLR signaling- and inflammation-related genes was decreased in selected tissues, whereas avian β-defensin-1 (AvBD1), AvBD2, SOD1, GSH-Px2, and IL-10 expression was increased in selected tissues and at specific time points (P < 0.05). Collectively, these findings indicated that VC supplementation was associated with attenuated IBV-induced histopathological lesions, lower viral RNA load in the bursa of Fabricius, and tissue-specific modulation of innate immune-, inflammatory-, and antioxidant-related gene expression following IBV challenge.
Infectious diarrhea in companion animals is one of the most common multi-factorial diseases in small animal medicine. This study aimed to investigate the prevalence and different carriage patterns of different enteropathogens, including E. coli, Salmonella spp., Campylobacter spp., and yeast, in diarrheic and apparently healthy dogs and cats. A total of 71 dogs (31 diarrheic and 40 healthy) and 70 cats (42 diarrheic and 28 healthy) were subjected to microbiological examination. Diarrheagenic E. coli (DEC) had a higher prevalence in diarrheic cats (64.3) compared to diarrheic dogs (48.4). Enterotoxigenic E. coli (ETEC), Shiga-toxin producing E. coli (STEC), Necrotoxigenic E. coli (NTEC), Enterohemorrhagic E. coli (EHEC), and Atypical Enteropathogenic E. coli (aEPEC) pathotypes were detected in both species, while Typical Enteropathogenic E. coli (tEPEC) pathotype was not detected in any canine or feline cases. EHEC was detected only in apparently healthy dogs and cats in percentages of 5% and 7.1%, respectively. A higher prevalence of Salmonella spp. was detected in 40% and 21% of apparently healthy dogs and cats, respectively, while Campylobacter spp. was mainly isolated from diarrheic dogs (71%) and diarrheic cats (66.7%), with no significant correlation (P = 0.3). Regarding the associated risk factors, there was no discernible association between the potential factors, including age, sex, breeds, and previous infection, and the shedding of DEC, Salmonella spp., and Campylobacter spp for feline cases. On the contrary, a higher prevalence of DEC was detected in the winter season at 75% with a significant correlation (P = 0.004). Furthermore, previous drug administration also found to affect Salmonella spp. fecal shedding significantly (P = 0.04). Diarrheic dogs revealed a higher non-significant prevalence of mixed pathogens' carriage (51.6%), whereas enteropathogens co-detection prevalence was higher in diarrheic cats (16.7%) than in apparently healthy cats (3.6%). The diagnosis of bacterial-associated diarrhea is challenging due to the detection of diarrheagenic pathogens in both diarrheic and apparently healthy animals. Moreover, the interactions between different diarrheagenic pathogens and commensals may either initiate or hinder the occurrence of diarrhea.
Serological assays are used alongside tuberculin skin testing to improve detection of bovine tuberculosis (bTB) in camelids. Tuberculin-induced anamnestic antibody responses have been described in infected animals; however, the effect of single and repeated tuberculin exposure on antigen-specific antibody responses in clinically healthy, tuberculosis-free camelids has not been evaluated. A retrospective observational study was undertaken involving alpacas and llamas subjected to voluntary or regulatory tuberculin administration, including single or repeated injections within 12 months before serological testing using the Camelid Enferplex multiplex ELISA. Animals were clinically healthy and originated from herds with no credible suspicion or history of bTB before or after testing. An unprimed control cohort from a biosecure, tuberculosis-free herd was included for comparison. Serological reactors underwent statutory post-mortem examination. Camelids exposed to tuberculin demonstrated consistent antibody reactivity to a defined subset of antigens, in the assay-reported order: MPB70-derived peptide (MPB70pep), bovine purified protein derivative (PPDB), MPB83, CFP-10 and MPB70. This pattern occurred in clinically healthy animals with no epidemiological risk factors or clinical, pathological or bacteriological evidence of Mycobacterium bovis infection at post-mortem examination. Nevertheless, ten animals were classified as serological reactors and compulsorily removed under regulatory provisions. In contrast, unprimed control animals showed no persistent multi-antigen antibody reactivity. Tuberculin administration alone, including repeated exposure within 12 months, can induce patterned antibody responses detectable by the Camelid Enferplex assay independent of infection. In low-prevalence spillover species, such responses may have important regulatory and welfare consequences, highlighting the need to consider tuberculin exposure history, herd biosecurity, herd disease status and species-specific immunology when interpreting serological results.
Elephant endotheliotropic herpesvirus hemorrhagic disease (EEHV-HD) is a rapidly fatal syndrome of juvenile Asian elephants, but the host-response programs distinguishing progression from survival and the underlying pathophysiology remain poorly defined. The serum proteome of 62 Asian elephants (Elephas maximus) was profiled using a multi-contrast design stratified by age, clinical status, and infection history; protein abundance was analyzed by empirical Bayes linear modeling and Gene Ontology enrichment with semantic similarity reduction. Clinically affected elephants showed enrichment of inflammatory and stress-associated processes—including cytokine signaling and chromatin remodeling—with suppression of type I interferon signaling and homeostatic functions, whereas asymptomatic exposed elephants showed enrichment of metabolic pathways, including fatty acid and pyruvate metabolism, vesicle-mediated transport, and protein quality control. Disease-versus-exposure comparisons distinguished a progression program (inflammatory escalation with loss of proteostasis and cell adhesion) from a resilience program (preserved metabolic and cellular homeostasis); juvenile susceptibility was further associated with impaired lipid and calcium regulation and disrupted intracellular transport. Collectively, these patterns support a pathology-centered model in which fatal EEHV-HD reflects endothelial injury coupled with maladaptive inflammation and metabolic failure. Protein-level interpretation identified candidate drivers of inflammatory amplification, endothelial barrier disruption, coagulation imbalance, and resilience—including JAK1, IL1RL2, IFI44, KCNJ15, MSN, HECW2, ITPR3, MFN2, AKT1, BMPER, and DROSHA—providing a mechanistic bridge between serum proteomic changes and the vascular lesions, thrombocytopenia, DIC-like coagulopathy, edema, and hemorrhage of EEHV-HD. These findings nominate candidate proteomic signatures for future diagnostic and risk-stratification studies; longitudinal individual-level validation is required before clinical application. Because diagnostic screening identified all PCR-positive sick cases as EEHV1A and pooled group-level serum profiles were analyzed, these signatures should be interpreted as exploratory host-response programs specifically reflecting acute EEHV1A disease requiring individual-level validation.
The Iberian lynx (Lynx pardinus), once considered the most endangered felid species worldwide, has experienced a remarkable recovery through intensive conservation efforts. Its small population size and low genetic diversity render it particularly vulnerable to infectious diseases, including those transmitted by arthropod vectors. This systematic review aimed to synthesize the current knowledge on pathogens investigated in the Iberian lynx, with a focus on vector-borne pathogens. Following PRISMA guidelines, a comprehensive search was conducted in Web of Science, PubMed, and Scopus, yielding 43 studies published between 1998 and 2024. Among these studies, parasites were the most frequently studied pathogens (58.1%), followed by viruses (30.2%), bacteria (23.3%), and fungi (7.0%). Of the 74 pathogens included in these studies, 70 were reported in the Iberian lynx. Many of the pathogens detected in this species are of concern not only for its conservation, but also for animal and public health. The percentage of publications addressing vector-borne pathogens was 18.6% (8/43). Vector-borne bacteria such as Anaplasma phagocytophilum, Bartonella henselae, Candidatus Mycoplasma haemominutum, Candidatus Mycoplasma turicensis, Ehrlichia spp. and Mycoplasma haemofelis, as well as protozoa (Cytauxzoon spp., Leishmania infantum) and helminths (Dirofilaria immitis), have been detected in Iberian lynx populations. Despite the identification of multiple ectoparasite species, data on pathogens they harbour remain scarce, and no studies have investigated vector-borne viruses. Comprehensive monitoring of vector-borne pathogens should be considered to improve understanding of pathogen exposure and their potential clinical impact on Iberian lynx populations.
Hemotropic Mycoplasma spp., also known as hemoplasmas, are blood-borne pathogens that infect red blood cells and can cause hemolytic anemia in mammals, including dogs. Mycoplasma haemocanis (Mhc) and 'Candidatus Mycoplasma haematoparvum' (CMhp) are the two most common canine hemoplasma species. However, molecular epidemiological data and information on their genetic diversity remain scarce in Thailand. This study investigated hemoplasma PCR positivity in 472 client-owned dogs from Bangkok and Nakhon Pathom and 199 community dogs from Chiang Mai using a polymerase chain reaction (PCR) assay targeting the 16S rRNA gene. The 23S rRNA and RNase P RNA (rnpB) genes were additionally amplified from positive samples for multilocus characterization. Overall, 48 of 671 dogs were PCR-positive (7.2%; 95% confidence interval: 5.4%-9.3%). PCR positivity was higher in the community-dog/Chiang Mai cohort than in the client-owned cohorts; however, dog type and sampling location were fully confounded, and these comparisons should therefore be interpreted descriptively. Sequence analyses identified Mhc and CMhp. Sequence data suitable for analysis at both the 16S and 23S rRNA genes were obtained from 29 positive samples, and rnpB sequences were obtained from nine of these samples. To the best of our knowledge, this study provides the first 23S rRNA gene sequences of CMhp; no homologous CMhp 23S rRNA gene sequence was available in GenBank for direct comparison.
Leptospirosis is a globally distributed zoonotic disease caused by pathogenic Leptospira spp., which are classified into numerous serogroups. Accurate serogroup identification is essential for epidemiological understanding and public health interventions in endemic regions. The microscopic agglutination test (MAT) is the conventional serological method for serogroup identification; however, it is labor-intensive and depends on extensive reference strain panels. Molecular serogroup typing (MST) has been proposed as an alternative approach, but its performance for region-specific strains has not been sufficiently evaluated. In this study, we improved an MST method optimized for Japanese Leptospira isolates and applied it to determine the serogroups of pathogenic Leptospira spp. detected in kidneys of wild boars (Sus scrofa) and sika deer (Cervus nippon) collected in Yamaguchi Prefecture, Japan, between 2016 and 2025. Pathogenic Leptospira DNA was detected in 23 of the 190 wild boars (12.1%) and 22 of the 275 sika deer (8.0%) by real-time PCR targeting lipL32. The improved MST identified serogroups Autumnalis, Australis, Canicola, and Hebdomadis in both host species, with Autumnalis predominating. In contrast, MAT using standard reference strains detected antibodies mainly against Hebdomadis and Australis, indicating discrepancies in the predominant serogroups between molecular and serological results. Notably, MAT using a local Autumnalis strain successfully detected Autumnalis antibodies in both hosts. These findings demonstrate that the improved MST complements serological testing.
Fighting bulls are native cattle bred for traditional contests with economic and recreational significance in southern Thailand. These animals may be infected with several blood-borne and vector-borne pathogens, including Anaplasma spp., hemotropic Mycoplasma spp. (hemoplasmas), Babesia spp., Theileria spp., and Trypanosoma spp. Blood cytology is commonly used for diagnosis because it is simple and inexpensive; however, its diagnostic performance can be limited, particularly in carrier animals with low parasitemia. This study screened blood-borne pathogens in fighting bulls (n = 198) from southern Thailand using conventional PCR and blood cytology, evaluated the diagnostic performance of blood cytology using PCR as the reference method, and genetically characterized selected PCR-positive samples by sequencing, BLAST analysis, and phylogenetic analysis. PCR-positive rates for Anaplasma spp., hemoplasmas, Babesia spp., Theileria spp., and Trypanosoma spp. were 75.3%, 80.8%, 3.0%, 98.0%, and 0.0%, respectively. PCR detection rates for Anaplasma spp., hemoplasmas, Babesia spp., and Theileria spp. were significantly higher than those obtained by blood cytology. Compared with PCR, blood cytology showed no to slight agreement (kappa < 0.20), sensitivity below 20%, and specificity above 75%. Genetic analyses identified Anaplasma spp., including A. platys-like, A. bovis-like, and Anaplasma sp. closely related to “Candidatus Anaplasma boleense”, Mycoplasma wenyonii, “Candidatus Mycoplasma haematobovis”, Babesia bigemina, and Theileria sinensis. These findings demonstrate a high occurrence of blood-borne pathogens and indicate that blood cytology substantially underestimates pathogen detection in fighting bulls.
Alphavirus madariaga (MADV), a member of the eastern equine encephalitis virus complex, is an emerging arbovirus in South America, with limited information regarding its occurrence and molecular characterization in equine populations. This study reports the detection and genetic characterization of MADV in equines from the state of Mato Grosso, Brazil. Two horses presenting acute neurological disease with a clinical course of approximately two days were evaluated. Histopathological examination revealed nonsuppurative meningoencephalomyelitis. Molecular screening for neurotropic viruses using TaqMan RT-qPCR assays was negative for rabies virus, EHV-1, WEEV, VEEV, WNV, and flaviviruses, while MADV was detected with Ct values of 27.9 and 31.4. Partial NS1 and E1 gene fragments were amplified and sequenced. Phylogenetic analyses based on both genes consistently placed the strain, named Santo Antonio, within the MADV genotype III clade, with strong support in the E1 analysis. The cases occurred in a peri-urban area located near Santo Antônio do Leverger, approximately 30 km from Cuiabá, highlighting a potential interface between sylvatic and human-modified environments. These findings provide molecular evidence of MADV circulation in central-western Brazil and association with equines disease. Our results reinforce the role of horses as sentinels for neurotropic arbovirus surveillance before reaching human population.
Acute infections caused by multidrug-resistant (MDR) Klebsiella pneumoniae immediately necessitate the development of novel therapeutic strategies. Phage therapy offers a viable alternative for combating MDR Klebsiella infections. Here, we report the isolation and comprehensive characterization of a novel lytic bacteriophage, BUCT791, recovered from hospital sewage. Phage BUCT791 exhibited a short latent period (20 min), a large burst size (∼275 PFU/cell), rapid adsorption efficiency (>90% within 15 min), and remarkable stability across a wide range of temperatures (4-50 °C) and pH values (3-12). Whole-genome sequencing revealed a 48,388 bp double-stranded DNA genome with a GC content of 50%, encoding 74 predicted open reading frames (ORFs) and lacking any virulence or antibiotic resistance genes, confirming its biosafety for therapeutic applications. Phylogenetic analysis assigned phage BUCT791 to the genus Jedunavirus (family Myoviridae) and identified it as a member of the species Klebsiella virus FZ14. In vitro assays demonstrated that BUCT791 effectively inhibited Klebsiella growth within 2 h and significantly inhibited biofilms formation. In vivo, phage treatment markedly improved Galleria mellonella larval survival from 10% to 80%. Collectively, these findings indicate that BUCT791 possesses potent antibacterial activity and represents a promising candidate for developing safe and effective phage-based therapeutics against MDR Klebsiella pneumoniae infections. IMPORTANCE: This study reports the isolation and characterization of BUCT791, a novel lytic bacteriophage active against multidrug-resistant Klebsiella pneumoniae. BUCT791 showed strong stability, efficient bacterial killing, antibiofilm activity, and improved survival in an in vivo infection model, supporting its potential as a promising alternative strategy against MDR K. pneumoniae infections.
An Igλ light chain bias (IgL) has been identified in ferrets after a primary influenza virus infections. To further study if this phenotype is consistent between species, serum immunoglobulin Igλ and Igκ light chain (IgL) prevalence was evaluated in humans, mice, and pigs following a primary respiratory virus infection. Each IgL population was tested for its binding capacity to the immunodominant viral glycoprotein of the infecting virus. Humans infected with their initial SARS-CoV-2 virus infection had a marginal Igκ bias against the spike (S) glycoprotein, but had no observable IgL bias toward the receptor binding domain of the S protein. Mice had an Igκ bias against the influenza virus hemagglutinin (HA) after a primary infection with an H1N1 or H3N2 influenza virus. Conversely, pigs had an Igλ bias to the HA after a primary infection with either influenza virus subtype. These results highlight that IgL biases vary in different species after a primary viral respiratory infection. Overall, these results have implications for how immunological data should be interpreted between species and the selection of animal models for various applications.
Zoonotic pathogens are central to the One Health framework, and the role of companion animals in SARS-CoV-2 infection, particularly in co-infection scenarios, remains unclear. This study investigated the occurrence of SARS-CoV-2, hemopathogens, and co-infections in domiciled dogs and cats from COVID-19-positive households in Southeast Brazil. Between January 2021 and August 2022, samples were collected from 86 animals (48 dogs and 38 cats) in Belo Horizonte, Minas Gerais. SARS-CoV-2 was detected by RT-qPCR, and hemopathogens were identified using nested and species-specific PCR assays. SARS-CoV-2 was detected in 9.3% (8/86) of animals, including 14.6% (7/48) of dogs and 2.6% (1/38) of cats, and one canine sample was classified as Omicron BA.1. Hemopathogens were detected in 85.4% of dogs, mainly Bartonella spp. (70.8%) and Anaplasma sp. (58.3%), with frequent co-infections. In cats, Bartonella spp. (15.8%) and hemotropic Mycoplasma spp. (21.1%) predominated. None of the animals tested positive for ther order Piroplasmida or A. phagocytophilum. Most SARS-CoV-2-positive animals (75.0%) were co-infected, but no significant association was observed between viral infection and hemopathogens (p > 0.05). These findings demonstrate the circulation of SARS-CoV-2 and hemopathogens in companion animals and reinforce the relevance of One Health surveillance.
