Pathogens

Phylogenetic group B2 Escherichia coli is associated with urinary tract infections and other pathologies, but the basis for this phylogenetic skew is not understood. One aspect of urinary tract infections is binding to and entering uroepithelial cells. To test whether a phylogenetic skew exists for cell invasion, we examined invasion of 10 E. coli strains from three phylogenetic groups into CRL2169 and HTB-9 cells, which are derived from grade 1 and grade 2 bladder carcinomas, respectively. The top four strains that invaded CRL2169 were from group B2: three of these strains had more flagella gene transcripts than the other seven strains. The seven strains that invaded HTB-9 were from different phylogenetic groups. For the model uropathogenic group B2 strain UTI89, which expresses pili over flagella, loss of flagella or pili impacted invasion into CRL2169 to similar extents, but loss of pili had a greater effect on invasion into HTB-9 and a murine infection model than loss of flagella. A hyperflagellated variant of a group A strain did not invade either cell line better than the parental strain. Reported transcript differences, which were confirmed experimentally, showed that CRL2169 was more differentiated. The endocytosis stimulator tanshinone enhanced invasion into HTB-9, but not into CRL2169, which suggests differences in endocytic pathways and is consistent with differences in differentiation states. If the initial or recurring event in urinary tract infection is invasion into differentiated urothelial cells, as opposed to tight junctions, then the role of flagella may have been underestimated.

This novel study detected persistent low level infections of Elephant Endotheliotropic Herpesviruses (EEHV), that can cause highly pathogenic Elephant Hemorrhagic Disease (EHD) in Loxodonta and Elephas, and co-infection of presumed less pathogenic Elephant Gammaherpesviruses (EGHV), in skin nodule biopsies, saliva and tissues collected from 43 wild L. africana (savannah elephant) in Botswana, Kenya, South Africa and Zimbabwe; in saliva from 25 wild L. cyclotis (forest elephant) in Gabon; and in saliva collected over seven years from 7 wild-born L.africana at Six Flags Safari Park, USA; and in saliva, blood and tissues from an additional 200 L. africana in USA zoos. DNA from these samples was extracted in our USA laboratories and amplified by conventional polymerase chain reaction using three-round nested primer sets designed specifically to screen for known EEHV and EGHV genes loci and to discover new species and subtypes. Sanger sequencing of purified DNA from nearly all samples yielded unambiguous positive genetic matches to previously known Loxodonta-associated EEHV2, EEHV3A, EEHV3B, EEHV6, EEHV7A, and EGHV1B, EGHV2, EGHV3B, EGHV4B, EGHV5B and discovered novel types EEHV3C-H and EEHV7B and the prototype EGHV1B. Many of the primer sets used could also have detected known Elephas-associated EEHV1A, EEHV1B, EEHV4, and EEHV5 if present in these samples, but they did not. Our extensive library of EEHV and EGHV sequences from wild and zoo Loxodonta, (as well as from 100 zoo Elephas maximus not discussed in this review), is a significant contribution to the elephant virology community, particularly for comparing subtypes types of EEHV found in pathogenic cases of EHD in zoos as well as determining and comparing species and subtypes of EEHV present in existing zoo herds, and in individual elephants being transported between zoos, and for importation of wild elephants into existing zoo herds.

Ethiopia has the biggest population of livestock in Africa, and small ruminants play a significant role in both meat consumption and revenue from the export of live animals and their skins. Infectious diseases, especially pneumonic pasteurellosis, are a key constraint in productivity, which is low despite their economic relevance for a variety of technological and non-technical reasons. Sheep are particularly susceptible to high rates of morbidity and mortality from the disease while under stress. These bacterial species were to be isolated, identified, and molecularly detected from sheep in certain regions of western Oromia, Ethiopia, that appeared to be healthy, as well as those that were clinically ill. Using multi-stage sampling, a cross-sectional study was carried out in three zones: Horro Guduru Wollega, East Wollega, and West Shawa, between January and December 2022. 384 sheep (220 healthy, 164 ill) had their nasal swabs taken, and they were analyzed bacteriologically, biochemically, and molecularly using PCR for the PHSSA, Rpt2, and CapA genes. To evaluate the relationships between risk factors and bacterial prevalence, data were examined using logistic regression and descriptive statistics. With P. multocida being the most commonly isolated species, followed by M. haemolytica and B. trehalosi, the overall prevalence of Pasteurellaceae was 21.1%. Pneumonic and young sheep had a greater prevalence, and there were significant correlations with age (OR = 2.41; 95% CI: 1.42-4.18) and illness status (OR = 3.47; 95% CI: 2.06-5.97), but not with sex or location. These results demonstrate the persistent risk of pneumonic pasteurellosis in Ethiopian sheep and the significance of species-level identification in directing focused interventions, such as management, treatment, and immunization plans. To molecularly describe isolates from other places and elucidate the pathogenic function of Pasteurella and Mannheimia species in illness development, more research is advised.

Toxoplasma gondii is a globally prevalent zoonotic parasite with multiple life stages and transmission routes, including ingestion and transplacental transmission. It is a major cause of abortion in sheep, goats and pigs, among other production animals, worldwide. While Type II strains are common in livestock in North America and Europe, non-archetypal, non-clonal genotypes are highly prevalent in South America. This study aimed to determine the molecular epidemiology of T. gondii strains causing sheep abortion in Uruguay. Phylogenomic analyses confirmed significant divergence among typed strains and revealed similarities with genotypes previously detected in the human population. Two novel strains, were isolated and characterized, uncovering the connection between their genetic makeup and phenotypes. Differences in virulence could be correlated to differences in gene copy number of the pseudo kinase ROP5 - further highlighting this virulence factor as relevant in wild strains. Whole-genome sequencing further confirmed the divergence among Uruguayan isolates, uncovering at least three distinct evolutionary origins. Overall, our findings highlight the circulation of virulent non-clonal lineages with links to human infections and underscore the importance of furthering genomic surveillance in South America to better understand Toxoplasmas transmission dynamics, pathogenic potential, and zoonotic risk.

Helminthiases remain a major global health burden, and limitations of current anthelmintic therapies highlight the need for new pharmacological targets. In this study, we examined the effects of ion-channel and cytoskeletal modulators on bovine lung protoscoleces (PSCs) of Echinococcus granulosus sensu lato. Compounds acting on ion channels (praziquantel, amiloride, and amlodipine) and cytoskeletal components (albendazole and cytochalasin D) were evaluated using a semi-automated motility assay, methylene blue exclusion to assess viability, and scanning electron microscopy (SEM) to characterize structural damage. All compounds produced concentration-dependent reductions in PSCs motility. Amlodipine was the most potent inhibitor of motility, whereas praziquantel and cytochalasin D produced pronounced tegumental alterations and strong correlations between motility impairment and parasite death. In contrast, amiloride markedly reduced motility with comparatively minor effects on viability, indicating a primarily paralytic effect. Cytoskeletal disruption induced severe structural damage and parallel declines in motility and viability. SEM analysis revealed extensive tegumental collapse, loss of glycocalyx, and microtrichial damage in PSCs exposed to cytoskeletal and calcium-modulating agents. These findings highlight cytoskeletal organization and calcium-dependent ion fluxes as key physiological vulnerabilities in E. granulosus. Comparative analysis of these pharmacological targets provides mechanistic insight into how disruptions in cytoskeletal dynamics and cation homeostasis compromise parasite motility and survival.

Incidence of norovirus has strong seasonality in temperate and continental climates. Many studies have examined its association with climate variables, but evidence remains disparate. We address this gap by performing a systematic review to summarise and interpret the strength and directionality of associations between climate variables and norovirus incidence. Embase, Scopus, Web of Science and PubMed databases were screened for peer-reviewed studies on 2nd of December 2024. Articles were included if they described any climate or meteorological variable, in a categorical or numerical format, relative to a measurement of norovirus incidence risk in a human population, or prevalence or survivability outside the human host. Bias was assessed using a modified Critical Appraisal Skills Programme checklist. If dispersion of the effect in a human population was provided, the mean size was calculated using inverse variance weighting. The effect size outside the host was summarised as D-values, representing the time required to achieve a 90% reduction in the detected amount of virus. A total 139 studies were included. Predictors of risk were ambient and water temperature, relative and absolute humidity, anomalies of ambient temperature and precipitation, atmospheric and vapour pressure. High heterogeneity in direction and size of effects was observed due to regional differences in the factors driving norovirus seasonality and differences in outcome and exposure definitions. Our review suggests that the sensitivity of norovirus to individual climate variables is region and time specific, reflecting geographical differences in the relative importance of norovirus transmission via environmental pathways versus human-to-human contact. Plain Language SummaryNorovirus, a gastrointestinal virus, has a higher number of cases during specific months of the year. Regions with similar types of climate appear to have similar time periods when the increase in the number of infections occurs, which has been linked to norovirus case numbers being correlated to individual climate variables, such as temperature or rainfall. To understand how these associations compare globally and what are their potential explanations, we screened four major scientific databases, namely Embase, Scopus, Web of Science and PubMed. After the selection process, a total 139 peer-reviewed studies were included in this study. We found that ambient and water temperature, relative and absolute humidity, anomalies of ambient temperature and precipitation, atmospheric and vapour pressure were predictors of an increase in norovirus cases. However, the strength and direction of the relationships differed from region to region. A potential explanation is that geographies also differ in how important individual routes are for the transmission of norovirus, specifically via the environment as opposed to direct human-to-human contact, whereas climate is likely to have a greater influence on the former. Key pointsO_LIThe strength and direction of associations between climate variables and norovirus incidence varies by region and time period C_LIO_LIThe strength of associations vary across the transmission routes of norovirus, e.g., environmental versus human-to-human contact C_LIO_LIClimate variables impact norovirus survival and dissemination outside the host, which may inform models of environmental virus transmission C_LI

IntroductionThe vaginal microbiome is an important factor affecting both HIV risk and reproductive health in women living with HIV, and women of African descent are disproportionately affected. Here we use 16S rRNA sequencing data to predict BV in HIV-positive women living in Africa and delineate microbial features that are important for accurate prediction in this cohort compared to two HIV negative cohorts. MethodsOur study population was comprised of a cohort of HIV-positive women living in Tanzania, and two cohorts of HIV-negative women living in the United States. Using OTU variables from data in the Tanzanian cohort, we used random forest, logistic regression, support vector machine, and multi-layer perceptron algorithms to predict BV outcome. To evaluate our models, we compared predicted BV outcome to BV outcome determined using Nugent scoring. ResultsIn evaluating model performance, all four models predicted BV outcome better for the HIV-negative cohorts than the HIV-positive cohorts. Overall, models were better at predicting negative BV outcomes for low Nugent scores than positive BV outcomes for high Nugent scores. Evaluation of significant predictor variables of BV in each cohort revealed that shared features existed between the Tanzanian and Symptomatic cohorts, but not among all three cohorts. Upon comparing performance of models in predicting BV outcome for Black women in HIV-positive and HIV-negative cohorts, we observed that all four models perform better at predicting BV in the HIV-negative cohorts. ConclusionsThe lower predictive performance observed for this Tanzanian HIV positive cohort, coupled with the difference in microbial communities important for accurate prediction suggest that distinct microbial communities in women who are living with HIV may create challenges that affect the accuracy of BV diagnosis and treatment. This study highlights the need for diagnostic tools that consider unique biological and epidemiological factors of populations to address health disparities.

AO_SCPLOWBSTRACTC_SCPLOWOrnithodoros phacochoerus are nidicolous soft ticks of the Ornithodoros moubata complex of species known to be vectors of the African swine fever (ASF) virus. These Ornithodoros ticks depend on endosymbionts to produce essential nutrients necessary for their development. However, endosymbionts are only a part of the complex microbiota hosted by the tick. This microbiota often includes primary or secondary endosymbionts, commensal species from the environment, and, most of the time, some pathogens. The present study was performed to understand the organization and spatial distribution of the microbiota of O. phacochoerus. One of the objectives was to investigate if the pathogen of interest (ASF virus) is involved in the organization of the microbiota through pathogen-induced dysbiosis or other interactions. For this purpose, 704 O. phacochoerus ticks were collected from two conservation areas in Mozambique. Sequencing was performed targeting the V3-V4 region of the 16S rRNA gene, and the resulting dataset was processed using FROGS to characterize the bacterial microbiota hosted by the ticks. The results indicate that the microbiota of Ornithodoros phacochoerus contains very low bacterial diversity, with one primary endosymbiont (Francisella-like endosymbiont), one potential secondary endosymbiont (Rickettsiella), and very few environmental or pathogenic bacterial species. We found a clear spatial structure of the microbiota, with ticks from the same sampling site showing similar patterns. On the contrary, no association with the infectious status for African swine fever virus was detected, suggesting that this pathogen does not shape Ornithodoros microbial communities. Our results on tick - microbiota - pathogen - environment interactions in nidicolous soft ticks, showed patterns that differ from most hard tick studies.

BackgroundGenetic drift and host-associated adaptation in influenza A viruses threaten the long-term reliability of RT-qPCR-based diagnostics, particularly when nucleotide mismatches arise within primer and probe binding regions. Conventional assay evaluations often emphasize sequence conservation but rarely assess functional mismatch tolerance across divergent subtypes and hosts. MethodsWe performed an in silico evaluation of a matrix (M) gene-targeted RT-qPCR assay by aligning primer and probe binding regions against 22 H1N1 isolates and representative H3N2 and H5N1 reference strains, including recent zoonotic isolates from avian and bovine hosts. Nucleotide mismatches were identified, quantified, and mapped relative to assay components and oligonucleotide termini. Mismatch burden was summarized by subtype and assay region. ResultsH1N1 isolates exhibited complete conservation across primer and probe regions. In contrast, H3N2 and H5N1 strains demonstrated subtype-specific sequence variability, with a total of eleven mismatches identified across seven non-H1N1 isolates (mean mismatch per isolate = 2.43). Probe mismatches predominated (63.6%), occurring primarily at internal positions, while primer mismatches were infrequent and largely avoided 3' terminal nucleotides. Recent H5N1 isolates (2023-2024) shared conserved internal mismatches in the probe and forward primer, whereas a historical H5N1 isolate (2016) exhibited a distinct profile including a terminal probe mismatch. Despite this variability, mismatch patterns were consistent with preserved amplification potential. ConclusionThis study demonstrates that the evaluated influenza A M gene RT-qPCR assay exhibits inherent mismatch tolerance across human and zoonotic subtypes. By shifting diagnostic evaluation from strict sequence identity to functional resilience, our findings provide a framework for designing and maintaining robust molecular assays suitable for surveillance and pandemic preparedness amid ongoing viral evolution. Graphical AbstractIn silico evaluation of an influenza A matrix gene RT-qPCR assay demonstrates subtype-specific primer and probe mismatches across H3N2 and H5N1 strains, including recent zoonotic isolates. Despite observed variability, mismatches predominantly occur at internal positions and spare primer 3' termini, supporting inherent assay mismatch tolerance and suitability for surveillance applications. O_FIG O_LINKSMALLFIG WIDTH=150 HEIGHT=200 SRC="FIGDIR/small/707407v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@e48677org.highwire.dtl.DTLVardef@1380ddcorg.highwire.dtl.DTLVardef@11606f0org.highwire.dtl.DTLVardef@121b4ab_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cytolethal distending toxin (CDT) is a virulence factor produced by several gram-negative bacteria, including Escherichia coli, the most prevalent etiological agent of urinary tract infections (UTI). CDT causes DNA damage in eukaryotic cells, impairing host defenses by disrupting epithelial barriers, suppressing acquired immunity, and promoting pro-inflammatory responses. E. coli strains encoding CDT have been previously identified in samples from UTI patients, yet the specific function of CDT in the development or progression of UTI remains undefined. In this study, we used a mouse model of ascending UTI to determine the role of CDT during infection. An E. coli mutant strain lacking the cdtABC locus was generated and combined with wild-type bacteria to co-infect mice via transurethral inoculation. At 1-day post-inoculation, competitive indices demonstrated a significant disadvantage for the cdt mutant in urine, bladder, and kidneys. Single strain infections were also performed as a further assessment of CDT impact, demonstrating that the cdt mutant had reduced kidney colonization indicative of CDT contributions to ascending infection. Histopathological analysis of the urinary bladder and kidney tissues from mice infected with CDT-encoding E. coli demonstrated higher levels of inflammation and tissue damage within the kidneys at both 1 and 7 days post-inoculation and in the bladder at 7 days post-inoculation compared to mice infected with cdt mutant bacteria. Collectively, these findings identify a critical contribution of CDT to the progression of UTI.

Cochliomyia hominivorax, New world Screwworm (NWS), has become a reemerging veterinary concern in the United States due to the recent northward expansion of fly detections as far as northern Mexico. Rapid, accurate and validated detection pipelines need to be developed in the case of an incursion into the United States. Confirmatory cases are evaluated by morphological identification with no paired test to verify identifications. With the frequency of submissions of non-ideal samples, particularly from fly traps, a molecular tool would be necessary for species identification. In this manuscript, we develop and assess a pipeline including three real-time PCR assays targeting the ribosomal RNA and five sets of Sanger primers targeting the mitochondrial genome that would be used as a paired tool with morphological identification. Two of the assessed real-time PCR assays are highly specific, sensitive and repeatable requiring <1 copy per reaction for detection. Four of the five Sanger primer sets were assessed, optimized and results evaluated for potential use in preliminary geographic analysis of specimens. This workflow will expedite screening of samples, provide a method to verify results using different tools and help understand genetic variations within the mitochondria for NWS outbreaks.

Human metapneumovirus (hMPV) is a major cause of respiratory infections, particularly among infants, older adults, and immunocompromised individuals, yet no approved vaccines or targeted antiviral therapies are currently available. pH-regulated processes, including airway epithelial physiology, endosomal acidification, and viral fusion mediated by the fusion (F) protein, are critical for hMPV infection. This study evaluates PHOH-001, an inhaled alkaline buffer, as a potential therapeutic strategy to modulate airway epithelial pH and inhibit hMPV infection. Using a recombinant hMPV expressing green fluorescent protein (rhMPV-GFP), viral replication was assessed in primary human airway epithelial cells (HAECs). PHOH-001 significantly reduced GFP expression at 72 hours post-infection in both submerged and air-liquid interface (ALI) cultures, with effects comparable to those of the endosomal acidification inhibitor bafilomycin A1. In Vero E6 cells, used as a mechanistic in vitro model, PHOH-001 increased extracellular and intracellular pH in a concentration-dependent manner and correspondingly reduced hMPV infection. In HAECs, PHOH-001 reduced viral replication, as measured by TCID50 assays of infectious virus, and inhibited syncytium formation, a key step in viral spread. Furthermore, PHOH-001 altered F protein localization and was associated with changes in actin organization, consistent with impaired viral spread. Collectively, these findings demonstrate that PHOH-001 alters multiple pH-dependent steps in hMPV infection in vitro and support airway pH modulation as a potential antiviral strategy against hMPV.

Soil-transmitted helminths (STHs) pose significant challenges to public health in endemic areas, necessitating reliable methods for their detection. Shotgun metagenomics enables simultaneous detection of STHs and microbes in a sample without prior knowledge of what is present. However, validation of shotgun metagenomics with known infection intensity or across different sequencing platforms has not been carried out for eukaryote parasites including STHs, and false positives remain a pervasive issue. We validated shotgun metagenomics as a method of STH detection in faecal samples. Using the Strongyloides ratti laboratory model of a STH infection we investigated how analytical methods (nucleotide-nucleotide matching, nucleotide-protein matching, marker gene detection, mitochondrial mapping), infection intensity and sequencing technology (short-read vs. long-read) affects sensitivity and specificity of detection. S. ratti was accurately detected at a standard laboratory dose, but low intensity infections were more difficult to detect. Only mitochondrial sequence mapping was 100% accurate at identifying S. ratti with no false positives. Overall, short-read outperformed long-read sequencing methods. We applied the same analytical methods to human faecal samples with confirmed infections for at least one of four STHs. Mitochondrial sequence mapping was also the most effective method for detecting STHs in human faecal samples, detecting 100% of Necator americanus and 92% of Ascaris spp. infections, but could not reliably detect STHs where DNA levels are expected to be low or variable. In conclusion, mitochondrial mapping was the most effective method of detection for sensitivity and specificity in both the laboratory system and human faecal samples. Our findings indicate that shotgun metagenomics should be approached cautiously using validated methods, particularly when infection intensity or DNA levels are expected to be low. Author SummarySoil-transmitted helminths (STH) such as the parasite Strongyloides, are important gastrointestinal parasites of humans and livestock. Accurate methods of detection for diagnostics and monitoring are important to implement suitable control and treatment strategies. Here we validate a shotgun metagenomics approach, where all DNA in a sample is sequenced, for detecting STH in faecal samples using a Strongyloides laboratory model for infection. Strongyloides was reliability detected in faecal samples at higher infection levels, but mitochondrial genome mapping of the sequences was the only analytical method that reliably detected Strongyloides at lower infections levels. These results were reflected in stool samples from humans infected with STH, where mitochondrial mapping was also the most reliable method. However, species that were associated with low levels of parasite material or DNA in the faeces including Strongyloides stercoralis, were more difficult to detect. We compared two sequencing methods: short-read Illumina and long-read Oxford Nanopore Technologies, but short-read outperformed long-read shotgun metagenomics. Contamination of bacteria sequences in parasite genome assemblies was problematic for analysis and contributed to false positive results. Future work should focus on specific targeting of eukaryote DNA either at the laboratory or bioinformatic stage to improve STH detection further.

BackgroundLeptospirosis is a zoonotic disease caused by pathogenic Leptospira spp., which persist in soil and water environments for extended periods of time. The mechanisms enabling this environmental survival remain elusive. Free-living amoebae (FLA) are widespread protozoa that act as reservoirs or "Trojan horses" for numerous bacterial pathogens, protecting them from stress and contributing to their persistence. Whether pathogenic Leptospira exploit similar interactions with FLA has not been resolved. Methodology/Principal FindingsUsing live confocal microscopy, flow cytometry, and gentamicin protection assays, we investigated the interactions between pathogenic (Leptospira interrogans) and saprophytic (Leptospira biflexa) leptospires with three FLA species: Acanthamoeba castellanii, Dictyostelium discoideum, and Hartmannella vermiformis. While rapid internalization was observed, entry was only partially dependent on actin-driven processes and was enhanced by the presence of live bacteria. Following internalization, bacteria persisted for at least 48h as indicated by colony-forming assays. However, no evidence of intracellular replication was detected. The number of fluorescently labeled leptospires progressively declined over time, providing further evidence of leptospires survival without multiplication. Finally, analysis of environmental soils in New Caledonia showed co-occurrence of FLA and Leptospira. Soil-derived FLA also internalized pathogenic Leptospira in vitro, showing that these interactions extend to natural isolates. Conclusions/SignificanceOur results demonstrate that free-living amoebae internalize both pathogenic and saprophytic leptospires and allow their transient persistence without replication. By providing protection and prolonging viability in soil environments, FLA may contribute to the ecological maintenance of Leptospira. These findings pinpoint FLA as potential environmental reservoirs that could play a role in shaping leptospires survival strategies relevant for transmission and host infection. Author SummaryFor bacteria living in soils and freshwater environments, survival depends on their ability to adapt to complex ecological landscapes populated by numerous predators and competitors. In such habitats, interactions with other microorganisms are unavoidable and may shape long-term survival strategies. Pathogenic Leptospira, the bacteria responsible for leptospirosis, can persist for long periods outside their hosts, yet the ecological mechanisms supporting this environmental survival remain poorly understood. In soil and freshwater ecosystems, microscopic predators known as free-living amoebae commonly feed on bacteria. However, several bacterial pathogens can survive inside these amoebae and use them as temporary shelters. Because ancestral Leptospira were soil-dwelling saprophytes, interactions with amoebae likely represent an ancient ecological relationship in which successful survival strategies may have evolved and remain conserved in present-day pathogenic species. With this perspective in mind, we used microscopy approaches and bacterial viability assays to investigate whether Leptospira interacts with amoebae. We found that several amoeba species rapidly engulf both pathogenic and non-pathogenic Leptospira. Once internalized, the bacteria remained viable for up to two days but did not multiply. We also detected both amoebae and Leptospira in the same soil samples and showed that environmental amoebae could internalize the bacteria. These findings suggest that amoebae may act as temporary shelters for Leptospira, helping them persist in soils and water and potentially contributing to the environmental stage of leptospirosis transmission.

The cryptococcal Amt family of ammonium transporters have been identified from our previous studies as one of the most highly upregulated proteins during transmigration in an in vitro blood-brain barrier (BBB) model, however, the role of this gene family has never been reported. Therefore, this study aimed to investigate the role of the Amt2 gene in the transmigration of C. neoformans across the BBB, examine its association with other common virulence factors, and assess its relevance to morphological changes in C. neoformans. The results showed that the C. neoformans mutant strain lacking the Amt2 gene (amt2{Delta}) exhibited a significantly reduced ability to transmigrate across the BBB in an in vitro model. Our findings suggest that C. neoformans primarily utilizes a transcellular mechanism for invasion, as indicated by the FITC-dextran permeability assays. Additionally, the size of polysaccharide capsule was significantly smaller in the mutant strain compared to the wild-type. In conclusion, our study proposed that the Amt2 gene plays a crucial role in both the transmigration process and capsule production in C. neoformans, without affecting morphological changes. Our study provides a foundation for future research into the underlying mechanisms of the Amt2 gene in C. neoformans pathogenesis. Author summaryCryptococcus neoformans transmigrates the blood-brain barrier through various mechanisms, with transcellular migration being the major route leading to cryptococcal meningitis. In this study, we identified the Amt2 gene, a member of the Amt family of ammonium transporters, as playing a crucial role in the funguss transmigration process. Our findings indicate that the Amt2 gene promotes capsule production and facilitates the transmigration of C. neoformans, all while not causing damage to human endothelial cells.

Rio Negro virus (RNV) is an enzootic alphavirus and a member of the Venezuelan equine encephalitis virus (VEEV) complex. Despite its wide circulation in South America, RNV remains a neglected pathogen with no established wild-type animal model to study its pathogenesis. In this study, we developed a lethal mouse model using 18-day-old wild-type C57BL/6 mice to characterize the systemic and neurological features of RNV infection. Following subcutaneous inoculation, RNV exhibited rapid systemic dissemination with a brief and low-titer viremic phase and high viral loads in lymphoid tissues, pancreas, brains, and lungs. Notably, infected mice developed progressive neurological signs, including ataxia and hindlimb paralysis, culminating in 100% lethality. Histopathological analysis revealed significant damage, highlighted by a striking collapse of the splenic architecture, inflammatory and remodeling changes in the lungs, and prominent inflammatory infiltrates with neurodegenerative changes in the brain. The splenic disruption was further evaluated by immunofluorescence analysis of the spleen, which showed a consistent loss of compartmentalization, characterized by an atypical infiltration of CD8+ T cells into B-cell follicles. The terminal stage of disease was characterized by extensive neuroinflammation and neurodegeneration. Histological examination of the brain revealed meningoencephalitis, robust astrogliosis, and widespread somatodendritic and terminal degeneration, particularly clustered around blood vessels. These findings were supported by cytokine analysis of brain homogenates, which showed a significant upregulation of IFN-{gamma}, IL-6, and MCP-1/CCL2 during symptomatic stages. Collectively, these findings establish a reproducible, non-genetically modified animal model that reveals the pathogenic potential of RNV in the context of immune immaturity characteristic of early life. By identifying these specific pathological and neuroinflammatory markers, our study provides a foundational experimental framework to investigate the mechanisms underlying RNV emergence and host-pathogen interactions within the VEEV complex. Author summaryMany viruses circulate silently in nature, hidden within animal populations, until environmental or social changes bring them into contact with humans. The Rio Negro virus is one such example. Despite being closely related to the better-known Venezuelan equine encephalitis virus and showing evidence of circulation in South America, RNV has remained largely overlooked by the scientific community and public health authorities. In this study, we established a new experimental model using infant mice that allowed us to observe how the virus spreads and causes damage. We found that the virus rapidly reaches the brain and other vital organs, causing severe inflammation in the brain, inflammatory changes in the lungs, and a breakdown of the immune systems organization in the spleen. By using a genetically unmodified model, we were able to observe the infection in a host with an intact but naturally immature immune system. This approach avoids the artificial conditions of genetic engineering while providing a more realistic window into how host age and developmental stages can influence the outcome of neglected viral infections. We believe our work is a first step toward understanding how this overlooked virus emerges and highlights the need to monitor and prepare for "silent" pathogens that could pose a risk to public health in an era of intensifying human activity and ecological pressure.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the virus that causes coronavirus disease-2019 (COVID-19) in humans, is also known to infect animals including dogs (Canis lupus familiaris) and cats (Felis catus). This study evaluated the efficacy of human COVID-19 rapid antigen and antibody tests in dogs and cats. Nasal/oral swabs from 60 animals (32 dogs, 28 cats) and serum from 40 animals (20 dogs, 20 cats) were tested. Rapid antigen tests used on respiratory swabs showed low-to-moderate sensitivity (75% dogs, 57% cats) and moderate-to-high specificity (79% dogs, 95% cats) compared to RT-PCR. Rapid antibody tests used on serum samples demonstrated low-to-moderate sensitivity (70% dogs, 50% cats) and moderate-high specificity (60% dogs, 100% cats) compared to PRNT. While imperfect, these test kits may have some utility for field surveillance studies, particularly when species-specific rapid SARS-CoV-2 assays for dogs and cats are unavailable. These test characteristics in dogs and cats are similar to the findings from studies of the same types of tests in humans which have found an average sensitivity and specificity of common commercially available kits in the US range from 50.0-84.3% and 64.5-74.3%, respectively, when used with human samples (1,2).

This study aimed to evaluate DARO Systems detection of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) against serum and oral fluid surveillance methods within a controlled study consisting of one PRRSV infected seeder pig and 46 naive nursery pigs. Findings showed DARO Systems comprehensive herd-level surveillance approach detected PRRSV earlier than traditional testing methods.

Tick-borne diseases represent a significant threat to human and animal health worldwide. In the United States, the blacklegged tick, Ixodes scapularis (I. scapularis), serves as a competent vector for several bacterial pathogens, including Ehrlichia muris eauclairensis (EME). The I. scapularis embryonic cell line (ISE6) is a valuable tool for propagating tick-borne pathogens and studying tick-pathogen interactions. In this study, we examined the cellular complexity of ISE6 cells and their response to EME infection. Single-cell RNA sequencing revealed 15 distinct cell clusters present. Although ISE6 cells are heterogeneous, they do not display transcriptional similarity to any known tick tissues. Notably, this lack of similarity did not influence their susceptibility to EME infection. Our results demonstrated that EME infection induces time-dependent transcriptional changes in ISE6 cells: early infection is characterized by upregulation of genes associated with stress adaptation, mitochondrial function, and metabolic pathways, whereas late infection leads to broad downregulation of genes involved in the cell cycle, DNA replication, and cytoskeletal organization. These findings enhance our understanding of ehrlichial interactions with ISE6 cells and reinforce the utility of this cell line as a resource for isolating and propagating arthropod endosymbionts and tick-borne pathogens. IMPORTANCEThis study provides a single-cell resolution framework for interpreting tick cell line biology during infection with a medically relevant ehrlichial pathogen. Using scRNA-seq, we show that the I. scapularis embryonic-derived ISE6 cell line comprises multiple transcriptionally distinct cell states, yet these states do not map cleanly onto canonical tick tissue signatures, even when compared against a curated reference tissue atlas. Despite this heterogeneity, EME broadly infects ISE6 cell population, indicating that susceptibility is not restricted to a specific cell type. We further define a time-dependent arthropod vector response in which early infection is marked by activation of stress and metabolic adaptation response, followed by late-stage inhibition of key signaling, transcriptional, and proliferative pathways as bacterial burden increases. Together, these findings strengthen the biological interpretation of ISE6 as an in vitro model for tick-pathogen interactions and provide a resource for future mechanistic studies of ehrlichial persistence, replication, and vector competence.

Glutathione has important roles in diverse infections, yet its involvement in the interaction between the deadly fungal pathogen Batrachochytrium dendrobatidis (Bd) and its amphibian hosts is still unclear. Using in vitro assays and a cell infection model, we examined how glutathione influences Bd virulence traits and cellular host disease resistance. For Bd, inhibition of glutathione reductase rapidly killed zoospores, indicating that glutathione is essential for this pathogen. In addition, exposure to exogenous glutathione promoted the potential for virulence through accelerated and increased zoospore release. In host amphibian cells, Bd infection decreased intracellular glutathione content and increased reactive oxygen species, suggesting that chytridiomycosis pathogenesis may involve oxidative stress. Depletion of host glutathione before exposure to Bd increased infection severity and Bd growth, whereas amphibian cells with slightly elevated glutathione levels were partially protected against Bd. However, manipulation of host glutathione levels after the establishment of Bd infection did not impact its intracellular growth, implying that the host glutathione-mediated resistance only occurs during the initial Bd invasion process. Importantly, this effect of glutathione on host resistance is not a general response to pathogens, as it was not observed in cells exposed to viral pathogen FV3. As glutathione increased both infectious zoospore production and host resistance to zoospore infection, our study suggests that this antioxidant may play an important role in the host/pathogen interaction during chytridiomycosis. Thus, environmental conditions and therapeutic approaches that affect glutathione systems in the host and/or pathogen have the potential to alter chytridiomycosis dynamics and should be further explored.