Jairo Alfonso Mendoza-Roldan

Zoological collections represent unique environments in which captivity-related alterations of host microbiota may increase susceptibility to infectious diseases. While reptiles are recognized reservoirs of microorganisms, data on quantitative yeast colonization and antifungal susceptibility in captive lizards remain limited. This study assessed 28 lizards belonging to nine species housed in a zoological park in southern Italy. Skin and cloacal swabs (n = 56) revealed yeast growth in 75% of animals and 57.1% of samples, with positivity rates of 60.7% and 53.6% for skin and cloacal samples, respectively. MALDI-TOF MS and ITS regions sequencing identified 113 isolates belonging to 17 distinct fungal species, including Arthrographis spp., Meyerozyma spp., Candida spp., Pichia spp., Trichosporon spp. and others. Arthrographis kalrae was the predominant species (i.e., 36.3%), followed by Meyerozyma guilliermondii (i.e., 16.8%). Mean colonization loads were 4.25 CFU/swab for skin and 10.50 CFU/swab for cloacal samples. Eight species were considered clinically relevant, including the Candida parapsilosis complex, Pichia kudriavzevii, and Trichosporon asahii. Antifungal susceptibility testing performed according to Clinical and Laboratory Standards Institute (CLSI, 2022) guidelines revealed marked phenotypic heterogeneity and frequent elevation of minimum inhibitory concentration (MIC). Above-threshold MICs differed significantly according to yeast species, antifungal compound, and sampling site. Non-wild-type phenotypes and elevated MICs were observed across multiple antifungal classes, with amphotericin B >64 µg/mL and fluconazole >256 µg/mL in several isolates (i.e., 92.3 and 91.7%, respectively). Reduced susceptibility or resistance was evident in Clavispora lusitaniae, M. guilliermondii, and Wickerhamomyces anomalus. These findings support the hypothesis that captive lizards may represent asymptomatic reservoirs of opportunistic and potentially antifungal-resistant yeasts, emphasizing the importance of hygiene measures and microbiological surveillance in zoological settings.

Invasive alien snakes pose an increasing ecological and sanitary risk, being potential reservoirs and dispersers of pathogens that can affect wildlife, domestic animals, and humans. As biological invasions accelerate due to globalization and the exotic pet trade, the epidemiological role of non-native species has come under increased investigation. Recent epidemiological research of established populations—such as Burmese pythons ( Python bivittatus ) in the Greater Everglades ecosystem and California kingsnakes ( Lampropeltis californiae ) in Gran Canaria—have identified a wide and alarming diversity of microorganisms of significant zoonotic concern. Current literature extensively highlights the high prevalence of zoonotic enteric bacteria in these invasive predators. Most notably, invasive snakes frequently harbor multi-drug resistant (MDR) strains of Salmonella enterica , posing a severe public health risk via direct handling or the contamination of shared environments. Alongside Salmonella , studies have identified these reptiles as shedding sources for Campylobacte r spp., Yersinia enterocolitica , Pseudomonas aeruginosa , and Escherichia coli . Furthermore, the recent detection of the emerging, multi-drug resistant fungal pathogen Candidozyma auris ) in reptilian hosts raises critical alarms regarding the potential for wildlife to serve as environmental maintenance reservoirs for urgent global human health threats. Beyond bacterial and fungal agents, invasive snakes are potent dispersers of zoonotic parasites and vectors. Recent ecological health assessments underscore the introduction and rapid spillover of novel parasites, such as the respiratory pentastomid Raillietiella orientalis from Burmese pythons to native herpetofauna, carrying broader implications for zoonotic visceral pentastomiasis. The gastrointestinal tracts of these invaders also frequently shed zoonotic protozoa (including Cryptosporidium and Entamoeba species) and helminths (oxyurids, ascarids), which can contaminate local watersheds. Additionally, non-native snakes introduce and sustain exotic ectoparasites, such as non-endemic ticks, which can act as competent vectors for rickettsial and other blood-borne pathogens affecting human and veterinary health. These findings collectively emphasize the profound capacity of invasive snakes to introduce novel pathogens, amplify endemic disease cycles, or facilitate bidirectional spillover events within native ecosystems. To mitigate these multifaceted risks, a One-Health framework is essential. By integrating active wildlife health assessments, advanced genomic sequencing, environmental DNA (eDNA) monitoring, and public-health survelliance, this approach enables the early identification of emerging zoonotic threats. Ultimately, systematic and cross-disciplinary monitoring represents a critical preventive tool to safeguard global biodiversity, prevent the establishment of novel pathogens in naïve ecosystems, and formulate evidence-based mitigation strategies to reduce exposure risks for both humans and animals.

Background: Canine vector-borne pathogens (CVBPs) are increasingly relevant, particularly those of zoonotic concern, due to climate change and increased animal mobility. However, there is no information on the actual burden of these infections in canine populations in several Western and South-Central Asian countries, including Afghanistan. Therefore, this study aimed to investigate the molecular prevalence of CVBP infections in stray and owned dogs across regions of Afghanistan. Methods: From July 2020 to August 2024, a total of 100 dogs with outdoor lifestyles in four provinces of Afghanistan (i.e., Kabul, Kunduz, Mazar-i-Sharif, and Takhar) were blood-sampled and molecularly screened for Hepatozoon spp., Babesia spp., Leishmania spp., filarioid helminths, and Bartonella spp. Results: Overall, 81% of dogs tested positive for at least one VBP. Hepatozoon canis was the most common (68%; present in all provinces), followed by Bartonella vinsonii subsp. berkhoffii (18%; present in all provinces), Babesia vogeli (4%; present in 3 provinces), Acanthocheilonema sp. (4%; present in 3 provinces), Leishmania infantum (2%; in Kunduz), and Babesia negevi (1%; in Mazar-i-Sharif). Co-infections were detected in 12% of dogs. Conclusions: To the best of our knowledge, this is the first epidemiological study of CVBPs in Afghanistan, demonstrating the sympatric circulation of H . canis , Bartonella vinsonii berkhoffii , L. infantum , B. vogeli , B. negevi , and Acanthocheilonema sp. Overall, the findings underscore the importance of endoparasite and ectoparasite control in owned dogs, as well as the need to control feral animal populations to reduce VBP transmission.

Snakes introduced outside their native range are an emerging conservation concern because unnoticed populations may establish and impact native vertebrate communities. Here, we investigate two recent extra-range records of the western whip snake ( Hierophis viridiflavus ) in south-western Germany using mitochondrial DNA, phylogeographic network analyses, and local contextual information. Five samples from a waste dump population in Zweibrücken shared the same ND4 haplotype, belonging to a widespread Italian lineage, whereas a road-killed individual from Saarbrücken carried a previously undescribed haplotype of the western lineage. These results indicate two distinct origins. The Zweibrücken population is consistent with human-mediated introduction via long-distance waste transport from Italy, whereas the Saarbrücken record may reflect arrival from the nearby French range margin. Preliminary surveys suggest possible local impacts on native lizards. This case highlights waste movement as a potentially overlooked pathway for snake introductions, with ecological and sanitary implications.

Background: The life cycle of Leishmania infantum is maintained mainly in dogs in anthropogenic environments and in many other wild animals in the sylvatic cycle. The ecological plasticity of some wild canids facilitates their role as hosts for Leishmania spp. in different endemic regions. Although red foxes ( Vulpes vulpes ) frequently test positive for L. infantum in Europe, little is known about their clinical presentation, immune response, or treatment outcomes. This study investigated the prevalence, clinical, and immunological features of L. infantum infection in foxes from southern Italy, complemented by an in vitro evaluation of cytokine responses in fox macrophages. Methods: Wild foxes from a wildlife rehabilitation center in southern Italy were molecularly and serologically screened for L. infantum . One sick fox underwent a complete diagnostic confirmation, treatment, and follow-up through hematological, biochemical, cytological, and molecular evaluations. In addition, peripheral blood mononuclear cells (PBMCs) isolated from a healthy fox were differentiated into macrophages and experimentally infected with L. infantum to assess early host–parasite interactions and cytokine gene expression profiles at two time points (4 h and 48 h). Results: Overall, 13 out of 54 foxes (24.1%) were molecularly positive for L. infantum , with a prevalence of 25% (5/20) in necropsied animals and 22% (9/41) in live animals. One individual tested seropositive for L. infantum and Ehrlichia sp. using the SNAP Leish 4Dx ^® test (2.4%, 1/41). The sick fox treated with meglumine antimoniate and allopurinol showed marked clinical and laboratory improvement. In the in vitro evaluation, the percentage of infected macrophages decreased from 32.8% at 4 h (2.25 parasites/cell) to 21.5% at 48 h (2 parasites/cell). Cytokine gene expression at 4 h and 48 h showed an increase in interleukin-6 (IL-6) (0.164–0.552) and IL-10 (0.828–4.245), stable IL-4 (0.505–0.708), a decrease in IL-12 (1.793–1.223) and IFN-γ (1.507–0.613), and consistently low TNF-α (0.377–0.411). Conclusions: The present study confirmed a high L. infantum prevalence of infection in red foxes from southern Italy. Serological findings herein and in the literature, together with in vitro cytokine gene expression, suggested that most foxes may remain subclinically infected. The high molecular positivity and the synanthropic nature of red foxes reinforce their role as reservoirs of L. infantum in endemic areas. Graphical

Hybridization, the merging of distinct genomes, is increasingly recognized as a major evolutionary force among eukaryotic pathogens, including facultatively sexual protist parasites like Leishmania and Trypanosoma. While it may contribute to pathogen virulence and drug resistance, hybridization remains poorly characterized, particularly how genetic distance between parental cells influences genomic compatibility and which compensatory mechanisms ensure hybrid viability. Here, we report the in vitro generation of an unusual sexual hybrid between Leishmania species infecting mammals (L. infantum) and reptiles (the Sauroleishmania L. tarentolae), and used this unique genetic model system to address these open questions. Our data provide evidence of genomic compatibility between even highly divergent Leishmania species, offering new insights into the evolutionary potential of Leishmania and related pathogens. We demonstrate that the genomic shock caused by the fusion of distinct genomes can be mitigated by two key mechanisms: (i) at the genomic level, chromosome loss allows the establishment of mosaic aneuploidy in the newly formed hybrid, and (ii) at the post-transcriptional level, preferential mono-parental allelic expression acts as a secondary compensatory mechanism. Our findings establish genome instability and post-transcriptional regulation as central processes in Leishmania hybridization, which may be of broad relevance to other biological systems undergoing genetic exchange.

Background: Canine leishmaniasis caused by Leishmania infantum is recognized as one of the most important neglected vector-borne diseases of zoonotic concern worldwide. Cats may also be infected with L. infantum , though the mechanisms underlying the immune response to this protozoal infection in the feline host remain poorly understood. In this study, the early cytokine gene-expression profile was investigated by in vitro infection of feline monocyte-derived macrophages with L. infantum . Methods: Primary macrophages were matured from peripheral blood mononuclear cells of a healthy domestic cat, and cells were collected at different time points post-infection (i.e., 4, 24, and 72 h) for microscopic evaluation and quantitative real-time PCR analysis of cytokine expression [interleukin (IL)-2, IL-4, IL-6, IL-10, interferon (IFN)-γ, and tumor necrosis factor (TNF)-α]. Results: Infection rates ranged from 35.6% to 48.2%, with 2.1–2.4 intracellular parasites per infected cell. Gene-expression analysis revealed a marked upregulation of IL-2, IFN-γ, and TNF-α at 72 h, indicating a predominant Th1-type pro-inflammatory response, while IL-4, IL-6, and IL-10 remained stable or slightly downregulated. Conclusions: These findings provide unprecedented evidence that feline macrophages activate a Th1-like cytokine pattern following in vitro infection with L. infantum . Overall, the outcomes of this investigation support the hypothesis that, when compared with dogs, the milder clinical course of leishmaniasis in cats is related to a predominant Th1-like immune response, eventually contributing to the infection control in naturally infected animals. Graphical