Gray Fox Populations in Indiana (Phase II): Disease Ecology
(2025-2028)
(2025-2028)
A major focus of our Phase I Project (2020-2025) was disease ecology. We found that canine distemper virus was
a major source of morbidity and mortality in gray foxes, and consequently warrants further investigation.
As with Phase I, we are partnered with Dr. Dawn Reding of Luther College and Geriann Albers of Indiana Department of Natural Resources on Phase II.
a major source of morbidity and mortality in gray foxes, and consequently warrants further investigation.
As with Phase I, we are partnered with Dr. Dawn Reding of Luther College and Geriann Albers of Indiana Department of Natural Resources on Phase II.
Our previous project (Phase I) to assess a diverse range of factors that may impact gray fox populations in Indiana provided strong evidence that disease plays a major role in population dynamics of this species. Distemper caused by the canine distemper virus (CDV) seems to be a major source of morbidity and mortality and warrants further investigation. The transmission and disease dynamics of CDV in gray foxes (and other wildlife species) are likely influenced by a complex interplay of various ecological, biological, and environmental factors. Given the wide range of hosts associated with CDV, it is likely maintained in Indiana by a metareservoir, with multiple carnivore species contributing to its persistence, transmission, and prevalence. Thus, factors that influence the movement and behavior of wildlife (e.g., temperature, availability of food resources, habitat fragmentation, breeding season) can impact contact rates and transmission of CDV. Multiple genetic strains of CDV can co-circulate in a particular area, and these strains may vary in their ability to infect and cause disease in different species of wildlife and domestic animals. Additionally, co-infections with other pathogens (e.g., tick-borne hemoparasites), exposure to environmental toxins (e.g., rodenticides or pesticides), and gut microbiome disruptions (e.g., due to stress from human disturbances or diet shifts towards anthropogenic foods) can disrupt immune function and result in higher rates of morbidity and mortality for species infected with diseases like distemper. Understanding these factors is crucial for managing and mitigating the impact of CDV on gray fox and other wildlife populations.
Interspecific interactions can result in disease transmission. Here, one of our radiomarked gray foxes from Phase I seems to be defending a dens site from multiple raccoons, a situation that could result in diseases transmission between species. Video copyright Wildlife Ecology Institute.
OUR APPROACH
We are collecting carcasses of gray foxes and several common wildlife species (e.g., raccoons, coyotes, red foxes, bobcats, striped skunks) that are likely to serve as disease vectors. To ensure seasonal coverage of samples, we plan to collect carcasses during summer for animals that died via vehicle collision, and during the fall–winter trapping and hunting seasons by purchasing carcasses of legally harvested animals from trappers and hunters. The inclusion of several common wildlife species will benefit our knowledge of the overall role of CDV in Indiana, both for the gray fox population, but also by extension to several other wildlife species.
Action 1: Canine Distemper Virus Surveillance and Sequencing
Collection of carcasses throughout different times of the year will help us identify prevalence rates and seasonal and annual trends in CDV infection. We anticipate annually to collect approximately 100 carcasses from raccoons and 50 from each of the other species (~400 total). Carcasses will be collected from road-killed, legally harvested, and dispatched animals (e.g., due to showing signs of neurological illness). We will collect and extract nucleic acids from spleen or other tissues (e.g., large intestine, lung) as available, which will be used to perform qRT-PCR to test for CDV.
From CDV-positive samples, we will use a targeted amplicon approach with Illumina sequencing to generate sequence data for portions of the viral genome, focusing primarily on the ~1,824 nucleotides of the H-gene. The sequence data will be used to 1) reveal information on the ability of different strains to circulate in multiple hosts and across species; 2) identify which carnivore species may function as reservoirs capable of spreading the infection to gray foxes, and 3) compare sequences from symptomatic (or samples with high viral loads) and likely asymptomatic individuals (or samples with low viral loads) to identify whether strain differences may explain the divergent clinical outcome.
Action 2: Toxins and Co-Infections
Accumulation of subclinical levels of toxins and co-infections with pathogens could place stress on the immune system. The resulting immunosuppression within an individual animal could increase their susceptibility to CDV infection and therefore the likelihood of severe disease and mortality. Thus, from the collected gray fox carcasses, we will also sample liver, spleen, kidney, and other organs as available to test for toxins (e.g., anticoagulant rodenticides, neonicotinoids) as well as a suite of potential co-pathogens (e.g., Toxoplasma, Leptospira, Babesia).
Through examination of blood films from the live-captured gray foxes as part of our Phase I Project, we detected a high prevalence of Babesia. We suspect that Babesia infections may be correlated with higher rates of CDV-related mortality, as this has been shown for African lions. Additionally, a previous diagnostic investigation of CDV-related mortalities of gray foxes found toxoplasmosis to be the most common comorbidity, and domestic dogs with neurological symptoms of CDV were often co-infected with Toxoplasma gondii. Because CDV is also immunosuppressive, it can be challenging to determine whether CDV leads to severe secondary infections or if underlying infections at the time of CDV exposure lead to increased susceptibility to CDV. For most of the individuals live-captured that later died of CDV during our Phase I Project, we have a blood sample taken at time of capture before CDV infection, and a spleen sample taken during necropsy after mortality. Thus, we plan to test and compare these samples using a vector-borne Next Generation Sequencing panel (~22 pathogens tested) to determine if any existing infections are associated with CDV mortality.
Action 3: Diet and Gut Microbiome Analysis
Diet can influence and be influenced by disease status. In our Phase I Project, we used DNA metabarcoding to identify the plant, invertebrate, and vertebrate food items present in the stomachs of gray foxes. Our findings supported previous observations that gray foxes are omnivores with diverse diets. However, some items detected in gray fox stomachs were unidentified because similar sequences were not found in existing databases or because sequences were identical for a range of taxa. This was particularly the case when we found complex mixtures of many taxa that we suspect were signatures of pet food. For Phase II, we are conducting DNA barcoding of several key taxonomic groups in Indiana to expand the reference databases to include taxa from Indiana. In addition, we plan to use additional primer sets and Nanopore sequencing to generate larger sequence reads that should increase taxonomic resolution, particularly for plants and complex ingredients indicative of pet food. Finally, we plan to apply diet metabarcoding to additional carnivore species (e.g., red fox, bobcat, coyote) to identify dietary niche overlap and potential competition with gray foxes in Indiana. The assessment of niche overlap and competition will provide information that is critical to assessing when, where, and how disease transmission might be occurring.
The gut microbiome contains a variety of microorganisms, most of which are commensal and help the host with digestion, immune function, and other processes. The breakdown of this microbial community due to anthropogenic stressors such as land use change, diet shifts, and infectious diseases can negatively impact the overall health and fitness of wildlife. Because CDV suppresses the host immune system, it could potentially disrupt the gut microbiome and allow for fatal secondary infections from opportunistic, pathogenic bacteria. In addition, altered gut microbiome composition due to other stressors such as habitat and diet changes could increase CDV susceptibility. Preliminary analyses comparing gray fox stomach microbiomes indicate that CDV infection is associated with lower microbial diversity and higher levels of pathogenic bacteria compared to healthy individuals. However, additional samples from gray foxes and other species is necessary to determine the potential role of host-microbiome relationships in population declines of gray foxes.
We are collecting carcasses of gray foxes and several common wildlife species (e.g., raccoons, coyotes, red foxes, bobcats, striped skunks) that are likely to serve as disease vectors. To ensure seasonal coverage of samples, we plan to collect carcasses during summer for animals that died via vehicle collision, and during the fall–winter trapping and hunting seasons by purchasing carcasses of legally harvested animals from trappers and hunters. The inclusion of several common wildlife species will benefit our knowledge of the overall role of CDV in Indiana, both for the gray fox population, but also by extension to several other wildlife species.
Action 1: Canine Distemper Virus Surveillance and Sequencing
Collection of carcasses throughout different times of the year will help us identify prevalence rates and seasonal and annual trends in CDV infection. We anticipate annually to collect approximately 100 carcasses from raccoons and 50 from each of the other species (~400 total). Carcasses will be collected from road-killed, legally harvested, and dispatched animals (e.g., due to showing signs of neurological illness). We will collect and extract nucleic acids from spleen or other tissues (e.g., large intestine, lung) as available, which will be used to perform qRT-PCR to test for CDV.
From CDV-positive samples, we will use a targeted amplicon approach with Illumina sequencing to generate sequence data for portions of the viral genome, focusing primarily on the ~1,824 nucleotides of the H-gene. The sequence data will be used to 1) reveal information on the ability of different strains to circulate in multiple hosts and across species; 2) identify which carnivore species may function as reservoirs capable of spreading the infection to gray foxes, and 3) compare sequences from symptomatic (or samples with high viral loads) and likely asymptomatic individuals (or samples with low viral loads) to identify whether strain differences may explain the divergent clinical outcome.
Action 2: Toxins and Co-Infections
Accumulation of subclinical levels of toxins and co-infections with pathogens could place stress on the immune system. The resulting immunosuppression within an individual animal could increase their susceptibility to CDV infection and therefore the likelihood of severe disease and mortality. Thus, from the collected gray fox carcasses, we will also sample liver, spleen, kidney, and other organs as available to test for toxins (e.g., anticoagulant rodenticides, neonicotinoids) as well as a suite of potential co-pathogens (e.g., Toxoplasma, Leptospira, Babesia).
Through examination of blood films from the live-captured gray foxes as part of our Phase I Project, we detected a high prevalence of Babesia. We suspect that Babesia infections may be correlated with higher rates of CDV-related mortality, as this has been shown for African lions. Additionally, a previous diagnostic investigation of CDV-related mortalities of gray foxes found toxoplasmosis to be the most common comorbidity, and domestic dogs with neurological symptoms of CDV were often co-infected with Toxoplasma gondii. Because CDV is also immunosuppressive, it can be challenging to determine whether CDV leads to severe secondary infections or if underlying infections at the time of CDV exposure lead to increased susceptibility to CDV. For most of the individuals live-captured that later died of CDV during our Phase I Project, we have a blood sample taken at time of capture before CDV infection, and a spleen sample taken during necropsy after mortality. Thus, we plan to test and compare these samples using a vector-borne Next Generation Sequencing panel (~22 pathogens tested) to determine if any existing infections are associated with CDV mortality.
Action 3: Diet and Gut Microbiome Analysis
Diet can influence and be influenced by disease status. In our Phase I Project, we used DNA metabarcoding to identify the plant, invertebrate, and vertebrate food items present in the stomachs of gray foxes. Our findings supported previous observations that gray foxes are omnivores with diverse diets. However, some items detected in gray fox stomachs were unidentified because similar sequences were not found in existing databases or because sequences were identical for a range of taxa. This was particularly the case when we found complex mixtures of many taxa that we suspect were signatures of pet food. For Phase II, we are conducting DNA barcoding of several key taxonomic groups in Indiana to expand the reference databases to include taxa from Indiana. In addition, we plan to use additional primer sets and Nanopore sequencing to generate larger sequence reads that should increase taxonomic resolution, particularly for plants and complex ingredients indicative of pet food. Finally, we plan to apply diet metabarcoding to additional carnivore species (e.g., red fox, bobcat, coyote) to identify dietary niche overlap and potential competition with gray foxes in Indiana. The assessment of niche overlap and competition will provide information that is critical to assessing when, where, and how disease transmission might be occurring.
The gut microbiome contains a variety of microorganisms, most of which are commensal and help the host with digestion, immune function, and other processes. The breakdown of this microbial community due to anthropogenic stressors such as land use change, diet shifts, and infectious diseases can negatively impact the overall health and fitness of wildlife. Because CDV suppresses the host immune system, it could potentially disrupt the gut microbiome and allow for fatal secondary infections from opportunistic, pathogenic bacteria. In addition, altered gut microbiome composition due to other stressors such as habitat and diet changes could increase CDV susceptibility. Preliminary analyses comparing gray fox stomach microbiomes indicate that CDV infection is associated with lower microbial diversity and higher levels of pathogenic bacteria compared to healthy individuals. However, additional samples from gray foxes and other species is necessary to determine the potential role of host-microbiome relationships in population declines of gray foxes.
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