You can bring plankton to fecal indicator organisms, but you cannot make the plankton graze: particle contribution to E. coli and MS2 inactivation in surface waters.

Organisms that are associated with feces ("fecal indicator organisms") are monitored to assess the potential for fecal contamination of surface water bodies in the United States. However, the effect of the complex mixtures of chemicals and the natural microbial community within surface water ("particles") on fecal indicator organism persistence is not well characterized. We aimed to better understand how particles, including biological (e.g., potential grazers) and inert (e.g., minerals) types, affect the fecal indicator organisms Escherichia coli K-12 ("E. coli") and bacteriophage MS2 in surface waters. A gradient of particles captured by a 0.2-µm-pore-size filter ("large particles") was generated, and the additional particles and dissolved constituents that passed through the filter were deemed "small particles." We measured the ratio of MS2 and E. coli that survived over a 24-h incubation period for each condition (0%-1,000% large-particle concentration in raw water) and completed a linear regression that included large- and small-particle coefficients. Particles were characterized by quantifying plankton, total bacterial cells, and total solids. E. coli and MS2 persistence was not significantly affected by large particles, but small particles had an effect in most waters. Small particles in higher-salinity waters had the largest, negative effect on E. coli and MS2 survival ratios: Significant small-particle coefficients ranged from -1.7 to -5.5 day-1 in the marine waters and -0.89 to -3.2 day-1 in the fresh and estuarine waters. This work will inform remediation efforts for impaired surface water bodies.IMPORTANCEMany surface water bodies in the United States have organisms associated with fecal contamination that exceed regulatory standards and prevent safe recreation. The process to remediate impaired water bodies is complicated because these fecal indicator organisms are affected by the local environmental conditions. For example, the effect of particles in surface water on fecal indicator concentrations are difficult to quantify in a way that is comparable between studies and water bodies. We applied a method that overcomes this limitation to assess the effects of large particles, including natural plankton that could consume the seeded fecal indicator organisms. Even in environmental water samples with diverse communities of plankton present, no effect of large particles on fecal indicator concentrations was observed. These findings have implications for the interpretation and design of future studies, including that particle characterization of surface water may be necessary to assess the fate of fecal indicators.

Toxoplasma gondii Exposure and Dietary Habits of Two Sympatric Carnivores in the Valdivian Temperate Rainforest, Southern Chile.

Toxoplasma gondii, a parasitic protozoan, may infect most warm-blooded animals, including humans and carnivores. Our study focused on alien-invasive American minks (Neogale vison) and domestic cats (Felis catus) in the Valdivian Temperate Rainforest, Chile. The main goal was to investigate the relationship between their dietary habits and T. gondii exposure in the Valdivia River watershed. To detect T. gondii exposure, blood serum samples from 49 domestic cats and 40 American minks were analyzed using an ELISA, and stable isotope analysis of δ15N and δ13C from vibrissae was performed to determine the dietary habits of both species. Relationships between T. gondii exposure and dietary habits were explored using generalized linear mixed-effects models. American minks that were T. gondii seropositive exhibited a broader prey range compared to seropositive domestic cats, with minimal dietary overlap between the two groups. Exposure of domestic cats to T. gondii had no significant association with any isotope value or prey item in their diet. In American minks, we found a positive and significant association between the proportion of Domestic chicken (Gallus gallus domesticus) in the diet and high δ15N values with T. gondii exposure. This suggests that domestic species prey related to anthropogenic areas, and the consumption of high-trophic-level prey, may contribute to T. gondii exposure in American minks. Conversely, contrary to previous hypotheses, consumption of rodents showed no significant association with T. gondii exposure in either species. Our findings emphasize the importance of further research to investigate trophic interactions in the transmission dynamics of T. gondii in the Valdivian Temperate Rainforest.

Are novel or locally adapted pathogens more devastating and why? Resolving opposing hypotheses.

There is a rich literature highlighting that pathogens are generally better adapted to infect local than novel hosts, and a separate seemingly contradictory literature indicating that novel pathogens pose the greatest threat to biodiversity and public health. Here, using Batrachochytrium dendrobatidis, the fungus associated with worldwide amphibian declines, we test the hypothesis that there is enough variance in "novel" (quantified by geographic and phylogenetic distance) host-pathogen outcomes to pose substantial risk of pathogen introductions despite local adaptation being common. Our continental-scale common garden experiment and global-scale meta-analysis demonstrate that local amphibian-fungal interactions result in higher pathogen prevalence, pathogen growth, and host mortality, but novel interactions led to variable consequences with especially virulent host-pathogen combinations still occurring. Thus, while most pathogen introductions are benign, enough variance exists in novel host-pathogen outcomes that moving organisms around the planet greatly increases the chance of pathogen introductions causing profound harm.

Helminths in Invasive Raccoons (Procyon lotor) from Southwest Germany.

As hosts of numerous zoonotic pathogens, the role of raccoons needs to be considered in the One Health context. Raccoons progressively expand their range as invasive alien species in Europe. This study aimed to investigate the intestinal helminth fauna of raccoons in Baden-Wuerttemberg, Germany, as no such screening had ever been conducted there. In total, we obtained 102 animals from hunters in 2019 and 2020. Intestinal helminths were retrieved using the SSCT (segmented sedimentation and counting technique) and identified morphologically and by PCR-based Sanger sequencing. Fecal samples were assessed using the ELISA PetChekTM IP assay (IDEXX, Germany) and flotation technique. The artificial digestion method was employed for analyzing muscle tissue. We detected species of four nematode genera (Baylisascaris procyonis, Toxocara canis, Capillaria spp., and Trichuris spp.), three cestode genera (Atriotaenia cf. incisa/procyonis, Taenia martis, and Mesocestoides spp.), and three trematode genera (Isthmiophora hortensis/melis, Plagiorchis muris, and Brachylaima spp.). Echinococcus spp. and Trichinella spp. were not found. The invasive behavior and synanthropic habits of raccoons may increase the infection risk with these helminths in wildlife, domestic and zoo animals, and humans by serving as a connecting link. Therefore, it is crucial to initiate additional studies assessing these risks.

Bacterial and Viral Pathogens with One Health Relevance in Invasive Raccoons (Procyon lotor, Linné 1758) in Southwest Germany.

In Europe, raccoons are invasive neozoons with their largest population in Germany. Globally, this mesocarnivore acts as a wildlife reservoir for many (non-)zoonotic (re-)emerging pathogens, but very little epidemiological data is available for southwest Germany. This exploratory study aimed to screen free-ranging raccoons in Baden-Wuerttemberg (BW, Germany) for the occurrence of selected pathogens with One Health relevance. Organ tissue and blood samples collected from 102 animals, obtained by hunters in 2019 and 2020, were subsequently analysed for two bacterial and four viral pathogens using a qPCR approach. Single samples were positive for the carnivore protoparvovirus-1 (7.8%, n = 8), canine distemper virus (6.9%, n = 7), pathogenic Leptospira spp. (3.9%, n = 4) and Anaplasma phagocytophilum (15.7%, n = 16). West Nile virus and influenza A virus were not detected. Due to their invasive behaviour and synanthropic habit, raccoons may increase the risk of infections for wildlife, domestic animals, zoo animals and humans by acting as a link between them. Therefore, further studies should be initiated to evaluate these risks.

TERRESTRIAL PATHOGEN POLLUTANT, TOXOPLASMA GONDII, THREATENS HAWAIIAN MONK SEALS (NEOMONACHUS SCHAUINSLANDI) FOLLOWING HEAVY RUNOFF EVENTS.

Toxoplasmosis is a major threat to Hawaiian monk seals (Neomonachus schauinslandi) in the main Hawaiian Islands where seal habitat overlaps with substantial human and domestic cat populations. As the definitive hosts, members of the Felidae are the sole sources contaminating the environment with infectious oocysts; these oocysts can be transported into the marine environment, thereby threatening marine mammals. To understand environmental factors influencing Hawaiian monk seal exposure to Toxoplasma gondii, we examined monk seal strandings from toxoplasmosis in relationship to location and rainfall patterns throughout the main Hawaiian Islands. Using a case-control study design, we compared mortalities due to toxoplasmosis (cases) with those from other causes (controls). We found that cases were up to 25 times more likely than controls to occur after heavy runoff events. The greatest odds ratio was observed when rainfall occurred 3 wk before strandings, potentially indicating important timelines in the disease process. Our results suggest that heavy rainfall frequently delivers sufficient numbers of oocysts to infect Hawaiian monk seals. With infectious doses of as low as a single oocyst, any contaminated runoff constitutes a risk to Hawaii's endangered monk seal.

Simulation of experimental synthetic DNA tracer transport through the vadose zone.

Although multiple experimental studies have proven the use of free synthetic DNA as tracers in hydrological systems, their quantitative fate and transport, especially through the vadose zone, is still not well understood. Here we simulate the water flow and breakthrough of deuterium (D) and one free synthetic DNA tracer from a 10-day experiment conducted in a transient variably saturated 1m3 10° sloped lysimeter using the HYDRUS-2D software package. Recovery and breakthrough flux of D (97.78%) and the DNA tracer (1.05%) were captured well with the advection-dispersion equation (R2 = 0.949, NSE = 0.937) and the Schijven and Simunek two-site kinetic sorption model recommended for virus transport modeling (R2 = 0.824, NSE = 0.823), respectively. The degradation of the DNA tracer was very slow (estimated to be 10% in 10 days), because the "loamy sand" porous media in our lysimeter was freshly crushed basaltic tephra (i.e., crushed rocks) and the microbes and DNase that could potentially degrade DNA in regular soils were rare in our "loamy sand". The timing of the concentration peaks and the HYDRUS-2D simulated temporal and spatial distribution of DNA in the lysimeter both revealed the role of the solid-water-air contact lines in mobilizing and carrying DNA tracer under the experimental variably saturated transient flow condition. The free DNA was nearly non-selectively transported through the porous media, and showed a slightly early breakthrough, possibly due to a slight effect of anion exclusion or size exclusion. Our results indicate that free DNA have the potential to trace vadose zone water flow and solute/contaminant transport, and to serve as surrogates to trace viral pathogen pollution in soil-water systems. To our knowledge, this study is the first to simulate transport mechanisms of free synthetic DNA tracers through real soil textured porous media under variably saturated transient flow condition.

Novel chytrid pathogen variants and the global amphibian pet trade.

Global wildlife trade spreads emerging infectious diseases that threaten biodiversity. The amphibian chytrid pathogen Batrachochytrium dendrobatidis (Bd) has caused population declines and species extinctions worldwide except in Asia. Fire-bellied toads (Bombina orientalis), exported in large numbers from Asia, are tolerant of Bd and carry hypervirulent ancestral chytrid BdAsia-1 variants. We assayed the virulence of a new isolate of BdAsia-1 on the model Australasian frog host Litoria caerulea. Infected individuals (n = 15) all showed rapid disease progression culminating in death, whereas sham-inoculated individuals (n = 10) presented no clinical signs of disease and all survived (log rank test, χ2 = 15.6, df = 1, p < 0.0001). The virulence of the new isolate of BdAsia-1 is comparable to the one we assayed previously (χ2 = 0.0, df = 1, p = 0.91). Internationally traded wildlife, even when they appear healthy, can carry hypervirulent variants of pathogens. Once new pathogen variants escape into the environment, native species that have had no opportunity to evolve resistance to them may perish. Our study suggests that hypervirulent pathogens are being spread by the international pet trade. Notifiable wildlife diseases attributable to locally endemic pathogens often fail to generate conservation concern so are rarely subject to border surveillance or import controls. Because of the danger novel variants pose, national border control agencies need to implement disease screening and quarantine protocols to ensure the safety of their endemic fauna.

Variantes Patógenas Nuevas de Quitridios y el Mercado Mundial de Anfibios Mascota Resumen El mercado mundial de fauna dispersa enfermedades infecciosas emergentes que amenazan a la biodiversidad. El quitridio patógeno de anfibios Batrachochytrium dendrobatidis (Bd) ha causado declinaciones poblacionales y la extinción de especies en todo el mundo excepto Asia. El sapo Bombina orientalis, exportado en grandes cantidades desde Asia, es tolerante al Bd y carga genéticamente las variantes ancestrales hipervirulentas de quitridio BdAsia-1. Analizamos la virulencia de una nueva cepa de BdAsia-1 con el modelo de la rana australo-asiática hospedera Litoria caerulea. Todos los individuos infectados (n = 15) mostraron una progresión acelerada de la enfermedad que culminaba con la muerte, mientras que los individuos con inoculación simulada (n = 10) no presentaron señales clínicas de la enfermedad y todos sobrevivieron (prueba log de rango, χ2 = 15.6, df = 1, p < 0.0001). La virulencia de la nueva cepa de BdAsia-1 es comparable a la que analizamos previamente (χ2 = 0.0, df = 1, p = 0.91). La fauna comercializada internacionalmente, incluso cuando parece estar saludable, puede portar variantes hipervirulentas de los patógenos. Una vez que un patógeno nuevo se introduce al ambiente, pueden perecer las especies nativas que no han tenido la oportunidad de evolucionar la resistencia a estos patógenos. Nuestro estudio sugiere que los patógenos hipervirulentos se están dispersando mediante el mercado internacional de mascotas. Con frecuencia las enfermedades silvestres notificables que pueden atribuirse a los patógenos endémicos no generan interés para la conservación, así que rara vez están sujetas a la vigilancia fronteriza o el control de importación. Debido al riesgo que representan las variantes nuevas, las agencias nacionales de control fronterizo necesitan implementar evaluaciones patológicas y protocolos de cuarentena para asegurar la seguridad de su fauna endémica.

Seasonal and spatial occurrence of zoonotic Salmonella serotypes in griffon vultures at farmland environments: Implications in pathogen pollution and ecosystem services and disservices.

Livestock production is a large source of microbial, pharmaceutical, and antimicrobial pollution worldwide. Vultures are one group of birds with particularly high exposure to food-borne pathogens due to frequent consumption of infected livestock carcasses. The potential origin and spatial-temporal shedding patterns of livestock-adapted Salmonella serotypes of zoonotic importance were evaluated in adult and nestling Griffon vultures (Gyps fulvus). We specifically assessed the exposure source and subsequent elimination of Salmonella-infected carcasses (ecosystem services) or transmission back to livestock (ecosystem disservices) by vultures, thus contributing respectively to disease mitigation or amplification in natural and farmed environments. The results show a seasonal high occurrence and turnover of a high variety of serotypes, especially swine-adapted ones isolated at high frequency. This suggests that vultures can be reservoirs and long-distance carriers of faecal Salmonella shed in supplementary feeding stations and breeding colonies. Contrary to their conservation purposes, feeding stations can act as Salmonella hotspots and reservoirs. However, a role for vultures in the transmission back to food-producing animals seems impossible because they do not come into contact at indoor farms, while transmission to free-ranging ruminants was not supported by the presence of ruminant-adapted serotypes in the vultures. Therefore, vultures do not promote disservices associated with the re-infection of livestock with Salmonella, but can provide quantitatively important ecosystem services by removing carrion contaminated with these and other zoonotic pathogens potentially affecting their health. Sanitary vigilance of the farms authorised to provide food for avian scavengers should avoid the disposal of swine and poultry carcasses with Salmonella and the antibiotics used to treat it. Extensive free-ranging livestock and their carcasses exploited in the countryside should be a priority for the conservation of vultures and their ecological function as cleaners and disease mitigators.

New insights on the infection of pathogenic Leptospira species in American mink (Neovison vison) in southern Chile.

Leptospirosis is a zoonosis of global distribution, caused by the infection of pathogenic Leptospira, a group of bacteria capable of infecting both domestic and wild animals. Mink (Neovison vison) in southern Chile is recognized as a wild and synanthropic rodent predator (among various other prey), and Leptospira infection in them can be acquired through contact with the pathogen in the environment or by eating infected prey. Thus, the aim of this study was to provide more specifics regarding the source of the infection for the American mink under the conditions of Southern Chile. Minks were captured in the Los Ríos region, southern Chile, in an area with well-developed dairy farming. Two areas were selected for mink trapping, one with a high degree of dairy farming and a second with a low degree of dairy farming. Within them, 16 study sites were visited, and 45 American mink were trapped and euthanized to obtain kidney tissue and blood serum samples for bacteria isolation and determination of antibodies titers, respectively. Molecular characterization of the isolated strains was performed. Three minks from sites of high-dairy farming industry and only one from sites with low-degree dairy farming were detected as infected through molecular confirmation. This study shows evidence that confirms previous findings made in southern Chile, regarding mink as host of Leptospira interrogans serovar Hardjo-prajitno associated to cattle-farming areas. However, typing information ( Leptospira interrogans Copenhageni and Icterohaemorrhagiae ) suggests that the consumption of rodents may also be a potential source of infection.

Expecting the unexpected: factors influencing the emergence of fungal and oomycete plant pathogens.

In recent years, the number of emergent plant pathogens (EPPs) has grown substantially, threatening agroecosystem stability and native biodiversity. Contributing factors include, among others, shifts in biogeography, with EPP spread facilitated by the global unification of monocultures in modern agriculture, high volumes of trade in plants and plant products and an increase in sexual recombination within pathogen populations. The unpredictable nature of EPPs as they move into new territories is a situation that has led to sudden and widespread epidemics. Understanding the underlying causes of pathogen emergence is key to managing the impact of EPPs. Here, we review some factors specifically influencing the emergence of oomycete and fungal EPPs, including new introductions through anthropogenic movement, natural dispersal and weather events, as well as genetic factors linked to shifts in host range.

Caryospora-Like Coccidia Infecting Green Turtles (Chelonia mydas): An Emerging Disease With Evidence of Interoceanic Dissemination.

Protozoa morphologically consistent with Caryospora sp. are one of the few pathogens associated with episodic mass mortality events involving free-ranging sea turtles. Parasitism of green turtles (Chelonia mydas) by these coccidia and associated mortality was first reported in maricultured turtles in the Caribbean during the 1970s. Years later, epizootics affecting wild green turtles in Australia occurred in 1991 and 2014. The first clinical cases of Caryospora-like infections reported elsewhere in free-ranging turtles were from the southeastern US in 2012. Following these initial individual cases in this region, we documented an epizootic and mass mortality of green turtles along the Atlantic coast of southern Florida from November 2014 through April 2015 and continued to detect additional, sporadic cases in the southeastern US in subsequent years. No cases of coccidial disease were recorded in the southeastern US prior to 2012 despite clinical evaluation and necropsy of stranded sea turtles in this region since the 1980s, suggesting that the frequency of clinical coccidiosis has increased here. Moreover, we also recorded the first stranding associated with infection by a Caryospora-like organism in Hawai'i in 2018. To further characterize the coccidia, we sequenced part of the 18S ribosomal and mitochondrial cytochrome oxidase I genes of coccidia collected from 62 green turtles found in the southeastern US and from one green turtle found in Hawai'i. We also sequenced the ribosomal internal transcribed spacer regions from selected cases and compared all results with those obtained from Caryospora-like coccidia collected from green turtles found in Australia. Eight distinct genotypes were represented in green turtles from the southeastern US. One genotype predominated and was identical to that of coccidia collected from the green turtle found in Hawai'i. We also found a coccidian genotype in green turtles from Florida and Australia with identical 18S and mitochondrial sequences, and only slight inter-regional differences in the internal transcribed spacer 2. We found no evidence of geographical structuring based on phylogenetic analysis. Low genetic variability among the coccidia found in green turtle populations with minimal natural connectivity suggests recent interoceanic dissemination of these parasites, which could pose a risk to sea turtle populations.

Reconstructing the emergence of a lethal infectious disease of wildlife supports a key role for spread through translocations by humans.

There have been few reconstructions of wildlife disease emergences, despite their extensive impact on biodiversity and human health. This is in large part attributable to the lack of structured and robust spatio-temporal datasets. We overcame logistical problems of obtaining suitable information by using data from a citizen science project and formulating spatio-temporal models of the spread of a wildlife pathogen (genus Ranavirus, infecting amphibians). We evaluated three main hypotheses for the rapid increase in disease reports in the UK: that outbreaks were being reported more frequently, that climate change had altered the interaction between hosts and a previously widespread pathogen, and that disease was emerging due to spatial spread of a novel pathogen. Our analysis characterized localized spread from nearby ponds, consistent with amphibian dispersal, but also revealed a highly significant trend for elevated rates of additional outbreaks in localities with higher human population density-pointing to human activities in also spreading the virus. Phylogenetic analyses of pathogen genomes support the inference of at least two independent introductions into the UK. Together these results point strongly to humans repeatedly translocating ranaviruses into the UK from other countries and between UK ponds, and therefore suggest potential control measures.

Exudative cloacitis in the kakapo (Strigops habroptilus) potentially linked to Escherichia coli infection.

AIM: To investigate the initiating causes of cloacitis (inflammation of the cloaca) in kakapo (Strigops habroptilus). METHODS: Metagenomics using unbiased RNA or DNA sequencing was applied to faecal material from an 11-year-old female kakapo with exudative cloacitis, and a pool of eight birds (male and female aged 1-20 years) with no current signs or history of the disease. Faecal material from the diseased bird was collected pre- and post-treatment. For RNA sequencing, extracted RNA/DNA was subject to DNase, and the remaining RNA reverse transcribed to cDNA and subject to multiple displacement amplification prior to sequencing. RESULTS: No significant alignment to any known avian virus sequence was obtained from any faecal samples. However significant BLAST alignments to five bacteriophages known to infect enterobacteria were obtained. Strong evidence was obtained for the presence of the bacteriophage Escherichia phage TL-2011b, a bacteriophage known to occur in Escherichia coli causing outbreaks of foodborne disease in humans, in the sample from the diseased bird, but not the non-diseased pool. Differences in E. coli community structure between the diseased bird and the non-diseased pool were also apparent. CONCLUSIONS: Escherichia coli infection of human origin is suggested as a possible cause of exudative cloacitis, although confirmatory work is required to test this hypothesis.

Ecopathology of ranaviruses infecting amphibians.

Ranaviruses are capable of infecting amphibians from at least 14 families and over 70 individual species. Ranaviruses infect multiple cell types, often culminating in organ necrosis and massive hemorrhaging. Subclinical infections have been documented, although their role in ranavirus persistence and emergence remains unclear. Water is an effective transmission medium for ranaviruses, and survival outside the host may be for significant duration. In aquatic communities, amphibians, reptiles and fish may serve as reservoirs. Controlled studies have shown that susceptibility to ranavirus infection and disease varies among amphibian species and developmental stages, and likely is impacted by host-pathogen coevolution, as well as, exogenous environmental factors. Field studies have demonstrated that the likelihood of epizootics is increased in areas of cattle grazing, where aquatic vegetation is sparse and water quality is poor. Translocation of infected amphibians through commercial trade (e.g., food, fish bait, pet industry) contributes to the spread of ranaviruses. Such introductions may be of particular concern, as several studies report that ranaviruses isolated from ranaculture, aquaculture, and bait facilities have greater virulence (i.e., ability to cause disease) than wild-type isolates. Future investigations should focus on the genetic basis for pathogen virulence and host susceptibility, ecological and anthropogenic mechanisms contributing to emergence, and vaccine development for use in captive populations and species reintroduction programs.