Salamander Chytrid DiseaseSalamander chytridiomycosis is an infectious disease caused by the fungus Batrachochytrium salamandrivorans (Bsal). The fungus is a close relative of B. dendrobatidis, which was described more than two decades ago and is responsible for the decline or extinction of over 200 species of frogs and toads. B. salamandrivorans has not, so far, been reported in North America. An introduction of the fungus into native North American salamander populations could have devastating e�ects. To help prevent the deadly fungus from killing native salamanders, the U.S. Fish and Wildlife Service declared 201 salamander species as injurious wildlife under the Lacey Act. The Act, which took e�ect on 28 January 2016, prohibits the import and interstate trade of listed species. See the Bsal NWDC fact sheet (https://sites.tufts.edu/nwdc/disease- fact-sheets/) for more details about this disease.

What can NWDC members do to protect wild amphibians?If you �nd dead salamanders or newts, consider collecting fresh carcasses; preserving them in 70% ethanol is best. If not available, a commercial alcohol (e.g. vodka) is acceptable. Do not freeze the specimens. Storage in a refrigerator is acceptable over short term

(up to 48 hrs). Take close-up photographs of the amphibians (Fig. 1) and send your pictures to NWDC Field Veterinarian, Dr. Walt Cottrell (wocottrell@gmail.com) or to your NWDC diagnostician. Specimens can be sent to NWDC labs for testing.

Make sure to have a disinfection protocol in placeThe following protocol should be used any time work is done in amphibian habitat. Field gear should be cleaned with soapy water and a brush to remove excess organic material. Rinse the equipment with water. After washing and rinsing, apply a 5% bleach solution by spraying or immersing the gear: use ½ cup of bleach for 9 ½ cups of water. Rinse the bleached items with water (avoid rinsing into the wetland) to minimize damage to the equipment and to prevent exposing the next wetland to residual bleach. If in the �eld, rinse with water from the next sampling site. Allow the equipment to dry completely if you are done for the day. Use bleach or �ame with a candle to disinfect calipers, measuring boards, and other sensitive equipment.

Active surveillance activities in the Northeast U.S. Dr. Evan Grant, NE Amphibian Research and Monitoring Initiative, USGS Patuxent Wildlife Research CenterA nationwide investigation led by the USGS Amphibian Research and Monitoring Initiative (ARMI) is being undertaken to determine whether Bsal is present in wild amphibian populations in the United States. Early detection of the fungus, if it is present, is crucial for implementing mitigation strategies to prevent spread and developing a better understanding of the risk to native populations. Accordingly, sampling will focus on the species shown to be most vulnerable to infection in laboratory trials, the eastern red-spotted newt (Notophthalmus viridescens). Locations of sampling

The Northeast Wildlife Disease CooperativeOffering wildlife health and disease services in the Northeast U.S.

Phone: 508-887-4933Email: nwdc@tufts.edu

http://sites.tufts.edu/nwdc

NWDC NOTESQUARTERLY NEWSLETTER FROM THE NORTHEAST WILDLIFE DISEASE COOPERATIVE

Volume 3, Number 2, April 2016

e�ort will be strati�ed according to the risk of Bsal introduction, which will maximize the probability of detecting the fungus if it is already present in wild populations. E�ort will focus on up to 20 isolated wetlands on state and federal properties where native species with populations expected to contain >30 individuals are most likely to be found, and we will begin our sampling at sites with known populations of native species. Sampling in 2016 will occur in: CT, ME, MD, MA, NH, NJ, NY, PA, and VA.

Common Loon (Gavia Immer) Death Caused by Avian Malaria InfectionOn July 6, 2015, Daniel Eastland (seasonal loon technician) found a dead Common Loon on Big Island North in Lake Umbagog, Errol, NH. There were no obvious signs indicating cause of death. Sean Flint, Wildlife Biologist, Umbagog National Wildlife Refuge submitted the carcass to Dr. Inga Sidor at the NHVDL. The loon was a reproductively-active adult female in good nutritional condition. Dr. Sidor found protozoa consistent with Plasmodium sp. in the heart and brain (Fig. 2). Proliferation of these parasites was associated with in�ammation in the heart and was suspected to be the cause of vasculitis and edema (vascular leakage) in other tissues. There was no in�ammation in the brain, but altered circulation, thrombosis or possible cerebral hypoxia may have been present. No other abnormalities were seen.

Plasmodium sp. protozoa are transmitted by mosquitoes, and clinical disease has been reported in a variety of wild and captive avian species, globally. Infectious sporozoites from the mosquito are injected into the bloodstream and colonize endothelial cells and macrophages throughout the body, including liver, kidney, lung, CNS and heart. These develop into merozoites, which are released and further infect erythrocytes; further cycles of development occur, eventually resulting in production of gametes which are infectious to the mosquito host. Typically, clinical signs in birds are caused by development of the parasites in erythrocytes, causing anemia. Mortality in some bird species, especially penguins, is associated with the extra-erythrocytic form of the parasite, which appears more signi�cant than red blood cell destruction in this case.

This case is the �rst documented mortality of a loon attributable to malaria. Plasmodium sp. infection in loons in the Midwest has been detected by blood smear examination, but previous health surveys in the 1990’s of Northeast loons did not show any evidence of this parasite. More recent molecular testing performed by Dr. Ellen Martinson of the Smithsonian Institute now indicate several circulating strains of malaria in Northeast loons. It is unclear how susceptible loons are to this parasitic disease. Further health monitoring may help to ascertain how common death due to this infection might become as the Plasmodium parasite range expands into Northeast loons. Current cooperative plans for the summer of 2016 are in place to collect fresh loon carcasses for necropsy at NHVDL, with the assistance of NH and Maine state wildlife agencies, the Loon Preservation Committee (NH), Maine Audubon, and the Biodiversity Research Institute (ME).

Canine Distemper Virus Epizootics in New EnglandOn 4 March, 2016 a gray fox in Haverhill, NH (Fig. 3) was observed to be ill and was euthanized. After rabies testing it was sent to the New Hampshire Veterinary Diagnostic Lab (NHVDL) where it was diagnosed with canine distemper (CDV).

In early March, gray foxes were observed displaying abnormal behavior in the vicinity of Washington County, VT. Three were tested by the Vermont

Department of Health and did not have rabies. On 15 March, another gray fox was observed following pedestrians in Montpelier VT (map). Local police euthanized this fox and rabies was ruled out by the Department of Health. The carcass was then submitted to NHVDL where canine distemper was con�rmed (Fig. 4).

Sporadic mammalian mortality was also reported in other VT counties during March. 14/15 foxes, 8/13 raccoons, and 1/1 skunks were found dead/sick in Washington and adjacent Orange County, suggesting that CDV might have a�ected these animals as well. Though the 2 con�rmed cases from NH and VT were adults, CDV most often a�ects juveniles. The disease can reduce local populations, but is not known to signi�cantly a�ect populations on a larger geographic scale. The magnitude, frequency, and distribution CDV epizootics are often unknown because of limited response capacity, and because rabies is the predominant public health consideration, it becomes the diagnostic end point, and CDV

testing is not performed. It is also unknown if CDV makes animals more susceptible to rabies or mange because of its ability to suppress the immune system. These factors make it highly likely that CDV is underreported, and that its impacts on populations are incompletely understood.

Canine distemper virus diversity and host range: What viruses are circulating in North America and in which animal hosts?Andrew Allison, Department of Microbiology and Immunology, Baker Institute for Animal Health, Cornell UniversityCanine distemper virus (CDV) is a highly contagious, enveloped RNA virus that belongs to the genus Morbillivirus, family Paramyxoviridae, and is found worldwide. CDV is known to cause signi�cant disease in the normal hosts that the virus usually infects, which includes members of the order Carnivora such as domestic dogs, coyotes, foxes, raccoons, and skunks. Although the normal host range of CDV has presumed to be restricted to carnivores, outbreaks of fatal disease in additional species have been documented. In 1989, cases of fatal encephalitis in javelinas or collared peccaries (Tayassu tajacu) were recognized in Arizona, with central nervous system lesions similar to those seen in CDV-infected dogs. More recently, in 2006, an outbreak of CDV among captive rhesus macaques (Macaca mulatta) in Guangxi province, China, resulted in the infection of ~10,000 monkeys with 4,250 deaths. Since the 2006 epidemic, additional CDV outbreaks in

macaques have been documented in China and also Japan. Collectively, these �ndings suggest that CDV not only has the propensity to infect a very large number of di�erent species within a single order (Carnivora), but also can jump into and cause disease in other hosts, including non-human primates and suids, and possibly other species. Although there is no evidence to suggest that CDV could become an imminent threat to public health, its wide host range (which now appears to include non-human primates) suggests that a more thorough characterization of the genetic diversity of CDV that exists in North America and the potential host ranges of these viruses is warranted.

In collaboration with the NWDC and the Southeastern Cooperative Wildlife Disease Study (SCWDS) at the University of Georgia, we are attempting to collect and analyze CDV cases from wildlife in the Eastern United States (and potentially Midwestern and Western states). Our aim is to understand in more detail the natural biodiversity of CDV that exists in North America, what the normal host ranges of these viruses are, and to potentially determine if speci�c genotypes are associated with increased virulence in certain hosts. CDV-positive tissues from any species would be greatly appreciated.

For those who are interested in submitting tissue samples from CDV cases to the project or have additional questions, please contact Andrew Allison at aba75@cornell.edu until August 1st, 2016, and at aba25@vt.edu after August 1st, 2016, or Julie Ellis at Julie.Ellis@tufts.edu.

Study of the Canada Lynx (Lynx Canadensis) Population in MaineCanada lynx live in moist boreal forest areas in the U.S. and Canada. The lynx is a specialized hunter of the snowshoe hare, and both species are associated with boreal forest. Due to the patchiness of boreal forest in the U.S. there is a lower density of lynx compared to that of lynx populations in the forests of Canada. Since 1997, the lynx has been a species of Special Concern to the Maine Department of Inland Fisheries and Wildlife (MDIFW) and has been federally listed as Threatened since 2000. Once considered “irregular and anecdotal,” the population of lynx in Maine has been increasing since the late 1990s in response to the abundance of high quality habitat for lynx and their prey. In 1999, 6 lynx were radio collared and results indicated that there was, in fact, a resident population of lynx in Maine. Over

the course of the next 12 years, MDIFW captured 185 lynx in northern Maine and radio-collared 85 of those. In 2006, the estimated population of adult lynx in core lynx range in Maine was 750-1,000, and when kittens are included, the total approaches 2,000. The population has since expanded and MDIFW is in the process of collecting data to update the 2006 estimates.

Due to the status of lynx in Maine, biologists began collecting lynx carcasses in 2000 in order to identify potential causes of mortality. In 2006, gross necropsies done by Dr. Jim Webber (University of Maine) revealed the presence of parasitic lung worms in some of the dead lynx. Due to these previous �ndings, MDIFW biologist Jennifer Vashon reinitiated a collaborative study to assess mortality factors in Maine’s lynx population. On 29 April, Jen brought 4 of some 40 stored lynx to the New Hampshire Veterinary Diagnostic Laboratory (NHVDL) to begin the process of detailed necropsy and data collection. In addition to monitoring radioed lynx, MDIFW responds to reports of dead lynx. Like all of the approximately 100 lynx recovered since 2000, these 4 animals died various ways from unknown illness to vehicular trauma. The gross necropsies documented nutritional status, type and extent of injuries, presence of external and internal parasites, stomach contents, and organ abnormalities. After gross necropsies, NHVDL pathologists, Drs. David Needle and Brian Stevens, will examine representative samples from all organs microscopically.

Results from necropsies by University of Maine and NHVDL could inform lynx conservation in Maine, and perhaps, throughout the Northeast.

Figure 1. Fire salamander with ulcers on its back. Photo by Mark Blooi, Ghent University (2016).

Salamander Chytrid DiseaseSalamander chytridiomycosis is an infectious disease caused by the fungus Batrachochytrium salamandrivorans (Bsal). The fungus is a close relative of B. dendrobatidis, which was described more than two decades ago and is responsible for the decline or extinction of over 200 species of frogs and toads. B. salamandrivorans has not, so far, been reported in North America. An introduction of the fungus into native North American salamander populations could have devastating e�ects. To help prevent the deadly fungus from killing native salamanders, the U.S. Fish and Wildlife Service declared 201 salamander species as injurious wildlife under the Lacey Act. The Act, which took e�ect on 28 January 2016, prohibits the import and interstate trade of listed species. See the Bsal NWDC fact sheet (https://sites.tufts.edu/nwdc/disease- fact-sheets/) for more details about this disease.

What can NWDC members do to protect wild amphibians?If you �nd dead salamanders or newts, consider collecting fresh carcasses; preserving them in 70% ethanol is best. If not available, a commercial alcohol (e.g. vodka) is acceptable. Do not freeze the specimens. Storage in a refrigerator is acceptable over short term

(up to 48 hrs). Take close-up photographs of the amphibians (Fig. 1) and send your pictures to NWDC Field Veterinarian, Dr. Walt Cottrell (wocottrell@gmail.com) or to your NWDC diagnostician. Specimens can be sent to NWDC labs for testing.

Make sure to have a disinfection protocol in placeThe following protocol should be used any time work is done in amphibian habitat. Field gear should be cleaned with soapy water and a brush to remove excess organic material. Rinse the equipment with water. After washing and rinsing, apply a 5% bleach solution by spraying or immersing the gear: use ½ cup of bleach for 9 ½ cups of water. Rinse the bleached items with water (avoid rinsing into the wetland) to minimize damage to the equipment and to prevent exposing the next wetland to residual bleach. If in the �eld, rinse with water from the next sampling site. Allow the equipment to dry completely if you are done for the day. Use bleach or �ame with a candle to disinfect calipers, measuring boards, and other sensitive equipment.

Active surveillance activities in the Northeast U.S. Dr. Evan Grant, NE Amphibian Research and Monitoring Initiative, USGS Patuxent Wildlife Research CenterA nationwide investigation led by the USGS Amphibian Research and Monitoring Initiative (ARMI) is being undertaken to determine whether Bsal is present in wild amphibian populations in the United States. Early detection of the fungus, if it is present, is crucial for implementing mitigation strategies to prevent spread and developing a better understanding of the risk to native populations. Accordingly, sampling will focus on the species shown to be most vulnerable to infection in laboratory trials, the eastern red-spotted newt (Notophthalmus viridescens). Locations of sampling

NWDC NOTES – Quarterly Newsletter From The Northeast Wildlife Disease Cooperative

2Volume 3, Number 2, April 2016

e�ort will be strati�ed according to the risk of Bsal introduction, which will maximize the probability of detecting the fungus if it is already present in wild populations. E�ort will focus on up to 20 isolated wetlands on state and federal properties where native species with populations expected to contain >30 individuals are most likely to be found, and we will begin our sampling at sites with known populations of native species. Sampling in 2016 will occur in: CT, ME, MD, MA, NH, NJ, NY, PA, and VA.

Common Loon (Gavia Immer) Death Caused by Avian Malaria InfectionOn July 6, 2015, Daniel Eastland (seasonal loon technician) found a dead Common Loon on Big Island North in Lake Umbagog, Errol, NH. There were no obvious signs indicating cause of death. Sean Flint, Wildlife Biologist, Umbagog National Wildlife Refuge submitted the carcass to Dr. Inga Sidor at the NHVDL. The loon was a reproductively-active adult female in good nutritional condition. Dr. Sidor found protozoa consistent with Plasmodium sp. in the heart and brain (Fig. 2). Proliferation of these parasites was associated with in�ammation in the heart and was suspected to be the cause of vasculitis and edema (vascular leakage) in other tissues. There was no in�ammation in the brain, but altered circulation, thrombosis or possible cerebral hypoxia may have been present. No other abnormalities were seen.

Plasmodium sp. protozoa are transmitted by mosquitoes, and clinical disease has been reported in a variety of wild and captive avian species, globally. Infectious sporozoites from the mosquito are injected into the bloodstream and colonize endothelial cells and macrophages throughout the body, including liver, kidney, lung, CNS and heart. These develop into merozoites, which are released and further infect erythrocytes; further cycles of development occur, eventually resulting in production of gametes which are infectious to the mosquito host. Typically, clinical signs in birds are caused by development of the parasites in erythrocytes, causing anemia. Mortality in some bird species, especially penguins, is associated with the extra-erythrocytic form of the parasite, which appears more signi�cant than red blood cell destruction in this case.

This case is the �rst documented mortality of a loon attributable to malaria. Plasmodium sp. infection in loons in the Midwest has been detected by blood smear examination, but previous health surveys in the 1990’s of Northeast loons did not show any evidence of this parasite. More recent molecular testing performed by Dr. Ellen Martinson of the Smithsonian Institute now indicate several circulating strains of malaria in Northeast loons. It is unclear how susceptible loons are to this parasitic disease. Further health monitoring may help to ascertain how common death due to this infection might become as the Plasmodium parasite range expands into Northeast loons. Current cooperative plans for the summer of 2016 are in place to collect fresh loon carcasses for necropsy at NHVDL, with the assistance of NH and Maine state wildlife agencies, the Loon Preservation Committee (NH), Maine Audubon, and the Biodiversity Research Institute (ME).

Canine Distemper Virus Epizootics in New EnglandOn 4 March, 2016 a gray fox in Haverhill, NH (Fig. 3) was observed to be ill and was euthanized. After rabies testing it was sent to the New Hampshire Veterinary Diagnostic Lab (NHVDL) where it was diagnosed with canine distemper (CDV).

In early March, gray foxes were observed displaying abnormal behavior in the vicinity of Washington County, VT. Three were tested by the Vermont

Department of Health and did not have rabies. On 15 March, another gray fox was observed following pedestrians in Montpelier VT (map). Local police euthanized this fox and rabies was ruled out by the Department of Health. The carcass was then submitted to NHVDL where canine distemper was con�rmed (Fig. 4).

Sporadic mammalian mortality was also reported in other VT counties during March. 14/15 foxes, 8/13 raccoons, and 1/1 skunks were found dead/sick in Washington and adjacent Orange County, suggesting that CDV might have a�ected these animals as well. Though the 2 con�rmed cases from NH and VT were adults, CDV most often a�ects juveniles. The disease can reduce local populations, but is not known to signi�cantly a�ect populations on a larger geographic scale. The magnitude, frequency, and distribution CDV epizootics are often unknown because of limited response capacity, and because rabies is the predominant public health consideration, it becomes the diagnostic end point, and CDV

testing is not performed. It is also unknown if CDV makes animals more susceptible to rabies or mange because of its ability to suppress the immune system. These factors make it highly likely that CDV is underreported, and that its impacts on populations are incompletely understood.

Canine distemper virus diversity and host range: What viruses are circulating in North America and in which animal hosts?Andrew Allison, Department of Microbiology and Immunology, Baker Institute for Animal Health, Cornell UniversityCanine distemper virus (CDV) is a highly contagious, enveloped RNA virus that belongs to the genus Morbillivirus, family Paramyxoviridae, and is found worldwide. CDV is known to cause signi�cant disease in the normal hosts that the virus usually infects, which includes members of the order Carnivora such as domestic dogs, coyotes, foxes, raccoons, and skunks. Although the normal host range of CDV has presumed to be restricted to carnivores, outbreaks of fatal disease in additional species have been documented. In 1989, cases of fatal encephalitis in javelinas or collared peccaries (Tayassu tajacu) were recognized in Arizona, with central nervous system lesions similar to those seen in CDV-infected dogs. More recently, in 2006, an outbreak of CDV among captive rhesus macaques (Macaca mulatta) in Guangxi province, China, resulted in the infection of ~10,000 monkeys with 4,250 deaths. Since the 2006 epidemic, additional CDV outbreaks in

macaques have been documented in China and also Japan. Collectively, these �ndings suggest that CDV not only has the propensity to infect a very large number of di�erent species within a single order (Carnivora), but also can jump into and cause disease in other hosts, including non-human primates and suids, and possibly other species. Although there is no evidence to suggest that CDV could become an imminent threat to public health, its wide host range (which now appears to include non-human primates) suggests that a more thorough characterization of the genetic diversity of CDV that exists in North America and the potential host ranges of these viruses is warranted.

In collaboration with the NWDC and the Southeastern Cooperative Wildlife Disease Study (SCWDS) at the University of Georgia, we are attempting to collect and analyze CDV cases from wildlife in the Eastern United States (and potentially Midwestern and Western states). Our aim is to understand in more detail the natural biodiversity of CDV that exists in North America, what the normal host ranges of these viruses are, and to potentially determine if speci�c genotypes are associated with increased virulence in certain hosts. CDV-positive tissues from any species would be greatly appreciated.

For those who are interested in submitting tissue samples from CDV cases to the project or have additional questions, please contact Andrew Allison at aba75@cornell.edu until August 1st, 2016, and at aba25@vt.edu after August 1st, 2016, or Julie Ellis at Julie.Ellis@tufts.edu.

Study of the Canada Lynx (Lynx Canadensis) Population in MaineCanada lynx live in moist boreal forest areas in the U.S. and Canada. The lynx is a specialized hunter of the snowshoe hare, and both species are associated with boreal forest. Due to the patchiness of boreal forest in the U.S. there is a lower density of lynx compared to that of lynx populations in the forests of Canada. Since 1997, the lynx has been a species of Special Concern to the Maine Department of Inland Fisheries and Wildlife (MDIFW) and has been federally listed as Threatened since 2000. Once considered “irregular and anecdotal,” the population of lynx in Maine has been increasing since the late 1990s in response to the abundance of high quality habitat for lynx and their prey. In 1999, 6 lynx were radio collared and results indicated that there was, in fact, a resident population of lynx in Maine. Over

the course of the next 12 years, MDIFW captured 185 lynx in northern Maine and radio-collared 85 of those. In 2006, the estimated population of adult lynx in core lynx range in Maine was 750-1,000, and when kittens are included, the total approaches 2,000. The population has since expanded and MDIFW is in the process of collecting data to update the 2006 estimates.

Due to the status of lynx in Maine, biologists began collecting lynx carcasses in 2000 in order to identify potential causes of mortality. In 2006, gross necropsies done by Dr. Jim Webber (University of Maine) revealed the presence of parasitic lung worms in some of the dead lynx. Due to these previous �ndings, MDIFW biologist Jennifer Vashon reinitiated a collaborative study to assess mortality factors in Maine’s lynx population. On 29 April, Jen brought 4 of some 40 stored lynx to the New Hampshire Veterinary Diagnostic Laboratory (NHVDL) to begin the process of detailed necropsy and data collection. In addition to monitoring radioed lynx, MDIFW responds to reports of dead lynx. Like all of the approximately 100 lynx recovered since 2000, these 4 animals died various ways from unknown illness to vehicular trauma. The gross necropsies documented nutritional status, type and extent of injuries, presence of external and internal parasites, stomach contents, and organ abnormalities. After gross necropsies, NHVDL pathologists, Drs. David Needle and Brian Stevens, will examine representative samples from all organs microscopically.

Results from necropsies by University of Maine and NHVDL could inform lynx conservation in Maine, and perhaps, throughout the Northeast.

Figure 2. Within capillaries throughout sections of the loon brain, there were small to moderate numbers of developing clusters of round to oval shaped, 1-2 µ diameter basophilic bodies, indicating presump-tive Plasmodium sp. meronts (infectious stage of the parasite).

Salamander Chytrid DiseaseSalamander chytridiomycosis is an infectious disease caused by the fungus Batrachochytrium salamandrivorans (Bsal). The fungus is a close relative of B. dendrobatidis, which was described more than two decades ago and is responsible for the decline or extinction of over 200 species of frogs and toads. B. salamandrivorans has not, so far, been reported in North America. An introduction of the fungus into native North American salamander populations could have devastating e�ects. To help prevent the deadly fungus from killing native salamanders, the U.S. Fish and Wildlife Service declared 201 salamander species as injurious wildlife under the Lacey Act. The Act, which took e�ect on 28 January 2016, prohibits the import and interstate trade of listed species. See the Bsal NWDC fact sheet (https://sites.tufts.edu/nwdc/disease- fact-sheets/) for more details about this disease.

What can NWDC members do to protect wild amphibians?If you �nd dead salamanders or newts, consider collecting fresh carcasses; preserving them in 70% ethanol is best. If not available, a commercial alcohol (e.g. vodka) is acceptable. Do not freeze the specimens. Storage in a refrigerator is acceptable over short term

(up to 48 hrs). Take close-up photographs of the amphibians (Fig. 1) and send your pictures to NWDC Field Veterinarian, Dr. Walt Cottrell (wocottrell@gmail.com) or to your NWDC diagnostician. Specimens can be sent to NWDC labs for testing.

Make sure to have a disinfection protocol in placeThe following protocol should be used any time work is done in amphibian habitat. Field gear should be cleaned with soapy water and a brush to remove excess organic material. Rinse the equipment with water. After washing and rinsing, apply a 5% bleach solution by spraying or immersing the gear: use ½ cup of bleach for 9 ½ cups of water. Rinse the bleached items with water (avoid rinsing into the wetland) to minimize damage to the equipment and to prevent exposing the next wetland to residual bleach. If in the �eld, rinse with water from the next sampling site. Allow the equipment to dry completely if you are done for the day. Use bleach or �ame with a candle to disinfect calipers, measuring boards, and other sensitive equipment.

Active surveillance activities in the Northeast U.S. Dr. Evan Grant, NE Amphibian Research and Monitoring Initiative, USGS Patuxent Wildlife Research CenterA nationwide investigation led by the USGS Amphibian Research and Monitoring Initiative (ARMI) is being undertaken to determine whether Bsal is present in wild amphibian populations in the United States. Early detection of the fungus, if it is present, is crucial for implementing mitigation strategies to prevent spread and developing a better understanding of the risk to native populations. Accordingly, sampling will focus on the species shown to be most vulnerable to infection in laboratory trials, the eastern red-spotted newt (Notophthalmus viridescens). Locations of sampling 3Volume 3, Number 2, April 2016

NWDC NOTES – Quarterly Newsletter From The Northeast Wildlife Disease Cooperative

e�ort will be strati�ed according to the risk of Bsal introduction, which will maximize the probability of detecting the fungus if it is already present in wild populations. E�ort will focus on up to 20 isolated wetlands on state and federal properties where native species with populations expected to contain >30 individuals are most likely to be found, and we will begin our sampling at sites with known populations of native species. Sampling in 2016 will occur in: CT, ME, MD, MA, NH, NJ, NY, PA, and VA.

Common Loon (Gavia Immer) Death Caused by Avian Malaria InfectionOn July 6, 2015, Daniel Eastland (seasonal loon technician) found a dead Common Loon on Big Island North in Lake Umbagog, Errol, NH. There were no obvious signs indicating cause of death. Sean Flint, Wildlife Biologist, Umbagog National Wildlife Refuge submitted the carcass to Dr. Inga Sidor at the NHVDL. The loon was a reproductively-active adult female in good nutritional condition. Dr. Sidor found protozoa consistent with Plasmodium sp. in the heart and brain (Fig. 2). Proliferation of these parasites was associated with in�ammation in the heart and was suspected to be the cause of vasculitis and edema (vascular leakage) in other tissues. There was no in�ammation in the brain, but altered circulation, thrombosis or possible cerebral hypoxia may have been present. No other abnormalities were seen.

Plasmodium sp. protozoa are transmitted by mosquitoes, and clinical disease has been reported in a variety of wild and captive avian species, globally. Infectious sporozoites from the mosquito are injected into the bloodstream and colonize endothelial cells and macrophages throughout the body, including liver, kidney, lung, CNS and heart. These develop into merozoites, which are released and further infect erythrocytes; further cycles of development occur, eventually resulting in production of gametes which are infectious to the mosquito host. Typically, clinical signs in birds are caused by development of the parasites in erythrocytes, causing anemia. Mortality in some bird species, especially penguins, is associated with the extra-erythrocytic form of the parasite, which appears more signi�cant than red blood cell destruction in this case.

This case is the �rst documented mortality of a loon attributable to malaria. Plasmodium sp. infection in loons in the Midwest has been detected by blood smear examination, but previous health surveys in the 1990’s of Northeast loons did not show any evidence of this parasite. More recent molecular testing performed by Dr. Ellen Martinson of the Smithsonian Institute now indicate several circulating strains of malaria in Northeast loons. It is unclear how susceptible loons are to this parasitic disease. Further health monitoring may help to ascertain how common death due to this infection might become as the Plasmodium parasite range expands into Northeast loons. Current cooperative plans for the summer of 2016 are in place to collect fresh loon carcasses for necropsy at NHVDL, with the assistance of NH and Maine state wildlife agencies, the Loon Preservation Committee (NH), Maine Audubon, and the Biodiversity Research Institute (ME).

Canine Distemper Virus Epizootics in New EnglandOn 4 March, 2016 a gray fox in Haverhill, NH (Fig. 3) was observed to be ill and was euthanized. After rabies testing it was sent to the New Hampshire Veterinary Diagnostic Lab (NHVDL) where it was diagnosed with canine distemper (CDV).

In early March, gray foxes were observed displaying abnormal behavior in the vicinity of Washington County, VT. Three were tested by the Vermont

Department of Health and did not have rabies. On 15 March, another gray fox was observed following pedestrians in Montpelier VT (map). Local police euthanized this fox and rabies was ruled out by the Department of Health. The carcass was then submitted to NHVDL where canine distemper was con�rmed (Fig. 4).

Sporadic mammalian mortality was also reported in other VT counties during March. 14/15 foxes, 8/13 raccoons, and 1/1 skunks were found dead/sick in Washington and adjacent Orange County, suggesting that CDV might have a�ected these animals as well. Though the 2 con�rmed cases from NH and VT were adults, CDV most often a�ects juveniles. The disease can reduce local populations, but is not known to signi�cantly a�ect populations on a larger geographic scale. The magnitude, frequency, and distribution CDV epizootics are often unknown because of limited response capacity, and because rabies is the predominant public health consideration, it becomes the diagnostic end point, and CDV

testing is not performed. It is also unknown if CDV makes animals more susceptible to rabies or mange because of its ability to suppress the immune system. These factors make it highly likely that CDV is underreported, and that its impacts on populations are incompletely understood.

Canine distemper virus diversity and host range: What viruses are circulating in North America and in which animal hosts?Andrew Allison, Department of Microbiology and Immunology, Baker Institute for Animal Health, Cornell UniversityCanine distemper virus (CDV) is a highly contagious, enveloped RNA virus that belongs to the genus Morbillivirus, family Paramyxoviridae, and is found worldwide. CDV is known to cause signi�cant disease in the normal hosts that the virus usually infects, which includes members of the order Carnivora such as domestic dogs, coyotes, foxes, raccoons, and skunks. Although the normal host range of CDV has presumed to be restricted to carnivores, outbreaks of fatal disease in additional species have been documented. In 1989, cases of fatal encephalitis in javelinas or collared peccaries (Tayassu tajacu) were recognized in Arizona, with central nervous system lesions similar to those seen in CDV-infected dogs. More recently, in 2006, an outbreak of CDV among captive rhesus macaques (Macaca mulatta) in Guangxi province, China, resulted in the infection of ~10,000 monkeys with 4,250 deaths. Since the 2006 epidemic, additional CDV outbreaks in

macaques have been documented in China and also Japan. Collectively, these �ndings suggest that CDV not only has the propensity to infect a very large number of di�erent species within a single order (Carnivora), but also can jump into and cause disease in other hosts, including non-human primates and suids, and possibly other species. Although there is no evidence to suggest that CDV could become an imminent threat to public health, its wide host range (which now appears to include non-human primates) suggests that a more thorough characterization of the genetic diversity of CDV that exists in North America and the potential host ranges of these viruses is warranted.

In collaboration with the NWDC and the Southeastern Cooperative Wildlife Disease Study (SCWDS) at the University of Georgia, we are attempting to collect and analyze CDV cases from wildlife in the Eastern United States (and potentially Midwestern and Western states). Our aim is to understand in more detail the natural biodiversity of CDV that exists in North America, what the normal host ranges of these viruses are, and to potentially determine if speci�c genotypes are associated with increased virulence in certain hosts. CDV-positive tissues from any species would be greatly appreciated.

For those who are interested in submitting tissue samples from CDV cases to the project or have additional questions, please contact Andrew Allison at aba75@cornell.edu until August 1st, 2016, and at aba25@vt.edu after August 1st, 2016, or Julie Ellis at Julie.Ellis@tufts.edu.

Study of the Canada Lynx (Lynx Canadensis) Population in MaineCanada lynx live in moist boreal forest areas in the U.S. and Canada. The lynx is a specialized hunter of the snowshoe hare, and both species are associated with boreal forest. Due to the patchiness of boreal forest in the U.S. there is a lower density of lynx compared to that of lynx populations in the forests of Canada. Since 1997, the lynx has been a species of Special Concern to the Maine Department of Inland Fisheries and Wildlife (MDIFW) and has been federally listed as Threatened since 2000. Once considered “irregular and anecdotal,” the population of lynx in Maine has been increasing since the late 1990s in response to the abundance of high quality habitat for lynx and their prey. In 1999, 6 lynx were radio collared and results indicated that there was, in fact, a resident population of lynx in Maine. Over

the course of the next 12 years, MDIFW captured 185 lynx in northern Maine and radio-collared 85 of those. In 2006, the estimated population of adult lynx in core lynx range in Maine was 750-1,000, and when kittens are included, the total approaches 2,000. The population has since expanded and MDIFW is in the process of collecting data to update the 2006 estimates.

Due to the status of lynx in Maine, biologists began collecting lynx carcasses in 2000 in order to identify potential causes of mortality. In 2006, gross necropsies done by Dr. Jim Webber (University of Maine) revealed the presence of parasitic lung worms in some of the dead lynx. Due to these previous �ndings, MDIFW biologist Jennifer Vashon reinitiated a collaborative study to assess mortality factors in Maine’s lynx population. On 29 April, Jen brought 4 of some 40 stored lynx to the New Hampshire Veterinary Diagnostic Laboratory (NHVDL) to begin the process of detailed necropsy and data collection. In addition to monitoring radioed lynx, MDIFW responds to reports of dead lynx. Like all of the approximately 100 lynx recovered since 2000, these 4 animals died various ways from unknown illness to vehicular trauma. The gross necropsies documented nutritional status, type and extent of injuries, presence of external and internal parasites, stomach contents, and organ abnormalities. After gross necropsies, NHVDL pathologists, Drs. David Needle and Brian Stevens, will examine representative samples from all organs microscopically.

Results from necropsies by University of Maine and NHVDL could inform lynx conservation in Maine, and perhaps, throughout the Northeast.

Figure 4. Microscopic view of the epithelial cells of the bile duct of a red fox, where there are numerous variably sized eosinophilic/pink intracytoplasmic inclusion bodies (sites of viral multiplication). Inclusion bodies indicated by the black arrows.

GraftonOrange

Washington

V T NH

0 10 20 30 40 50 MilesFigure 3. Map of canine distemper epizootics in NH and VT.

Salamander Chytrid DiseaseSalamander chytridiomycosis is an infectious disease caused by the fungus Batrachochytrium salamandrivorans (Bsal). The fungus is a close relative of B. dendrobatidis, which was described more than two decades ago and is responsible for the decline or extinction of over 200 species of frogs and toads. B. salamandrivorans has not, so far, been reported in North America. An introduction of the fungus into native North American salamander populations could have devastating e�ects. To help prevent the deadly fungus from killing native salamanders, the U.S. Fish and Wildlife Service declared 201 salamander species as injurious wildlife under the Lacey Act. The Act, which took e�ect on 28 January 2016, prohibits the import and interstate trade of listed species. See the Bsal NWDC fact sheet (https://sites.tufts.edu/nwdc/disease- fact-sheets/) for more details about this disease.

What can NWDC members do to protect wild amphibians?If you �nd dead salamanders or newts, consider collecting fresh carcasses; preserving them in 70% ethanol is best. If not available, a commercial alcohol (e.g. vodka) is acceptable. Do not freeze the specimens. Storage in a refrigerator is acceptable over short term

(up to 48 hrs). Take close-up photographs of the amphibians (Fig. 1) and send your pictures to NWDC Field Veterinarian, Dr. Walt Cottrell (wocottrell@gmail.com) or to your NWDC diagnostician. Specimens can be sent to NWDC labs for testing.

Make sure to have a disinfection protocol in placeThe following protocol should be used any time work is done in amphibian habitat. Field gear should be cleaned with soapy water and a brush to remove excess organic material. Rinse the equipment with water. After washing and rinsing, apply a 5% bleach solution by spraying or immersing the gear: use ½ cup of bleach for 9 ½ cups of water. Rinse the bleached items with water (avoid rinsing into the wetland) to minimize damage to the equipment and to prevent exposing the next wetland to residual bleach. If in the �eld, rinse with water from the next sampling site. Allow the equipment to dry completely if you are done for the day. Use bleach or �ame with a candle to disinfect calipers, measuring boards, and other sensitive equipment.

Active surveillance activities in the Northeast U.S. Dr. Evan Grant, NE Amphibian Research and Monitoring Initiative, USGS Patuxent Wildlife Research CenterA nationwide investigation led by the USGS Amphibian Research and Monitoring Initiative (ARMI) is being undertaken to determine whether Bsal is present in wild amphibian populations in the United States. Early detection of the fungus, if it is present, is crucial for implementing mitigation strategies to prevent spread and developing a better understanding of the risk to native populations. Accordingly, sampling will focus on the species shown to be most vulnerable to infection in laboratory trials, the eastern red-spotted newt (Notophthalmus viridescens). Locations of sampling 4Volume 3, Number 2, April 2016

NWDC NOTES – Quarterly Newsletter From The Northeast Wildlife Disease Cooperative

e�ort will be strati�ed according to the risk of Bsal introduction, which will maximize the probability of detecting the fungus if it is already present in wild populations. E�ort will focus on up to 20 isolated wetlands on state and federal properties where native species with populations expected to contain >30 individuals are most likely to be found, and we will begin our sampling at sites with known populations of native species. Sampling in 2016 will occur in: CT, ME, MD, MA, NH, NJ, NY, PA, and VA.

Common Loon (Gavia Immer) Death Caused by Avian Malaria InfectionOn July 6, 2015, Daniel Eastland (seasonal loon technician) found a dead Common Loon on Big Island North in Lake Umbagog, Errol, NH. There were no obvious signs indicating cause of death. Sean Flint, Wildlife Biologist, Umbagog National Wildlife Refuge submitted the carcass to Dr. Inga Sidor at the NHVDL. The loon was a reproductively-active adult female in good nutritional condition. Dr. Sidor found protozoa consistent with Plasmodium sp. in the heart and brain (Fig. 2). Proliferation of these parasites was associated with in�ammation in the heart and was suspected to be the cause of vasculitis and edema (vascular leakage) in other tissues. There was no in�ammation in the brain, but altered circulation, thrombosis or possible cerebral hypoxia may have been present. No other abnormalities were seen.

Plasmodium sp. protozoa are transmitted by mosquitoes, and clinical disease has been reported in a variety of wild and captive avian species, globally. Infectious sporozoites from the mosquito are injected into the bloodstream and colonize endothelial cells and macrophages throughout the body, including liver, kidney, lung, CNS and heart. These develop into merozoites, which are released and further infect erythrocytes; further cycles of development occur, eventually resulting in production of gametes which are infectious to the mosquito host. Typically, clinical signs in birds are caused by development of the parasites in erythrocytes, causing anemia. Mortality in some bird species, especially penguins, is associated with the extra-erythrocytic form of the parasite, which appears more signi�cant than red blood cell destruction in this case.

This case is the �rst documented mortality of a loon attributable to malaria. Plasmodium sp. infection in loons in the Midwest has been detected by blood smear examination, but previous health surveys in the 1990’s of Northeast loons did not show any evidence of this parasite. More recent molecular testing performed by Dr. Ellen Martinson of the Smithsonian Institute now indicate several circulating strains of malaria in Northeast loons. It is unclear how susceptible loons are to this parasitic disease. Further health monitoring may help to ascertain how common death due to this infection might become as the Plasmodium parasite range expands into Northeast loons. Current cooperative plans for the summer of 2016 are in place to collect fresh loon carcasses for necropsy at NHVDL, with the assistance of NH and Maine state wildlife agencies, the Loon Preservation Committee (NH), Maine Audubon, and the Biodiversity Research Institute (ME).

Canine Distemper Virus Epizootics in New EnglandOn 4 March, 2016 a gray fox in Haverhill, NH (Fig. 3) was observed to be ill and was euthanized. After rabies testing it was sent to the New Hampshire Veterinary Diagnostic Lab (NHVDL) where it was diagnosed with canine distemper (CDV).

In early March, gray foxes were observed displaying abnormal behavior in the vicinity of Washington County, VT. Three were tested by the Vermont

Department of Health and did not have rabies. On 15 March, another gray fox was observed following pedestrians in Montpelier VT (map). Local police euthanized this fox and rabies was ruled out by the Department of Health. The carcass was then submitted to NHVDL where canine distemper was con�rmed (Fig. 4).

Sporadic mammalian mortality was also reported in other VT counties during March. 14/15 foxes, 8/13 raccoons, and 1/1 skunks were found dead/sick in Washington and adjacent Orange County, suggesting that CDV might have a�ected these animals as well. Though the 2 con�rmed cases from NH and VT were adults, CDV most often a�ects juveniles. The disease can reduce local populations, but is not known to signi�cantly a�ect populations on a larger geographic scale. The magnitude, frequency, and distribution CDV epizootics are often unknown because of limited response capacity, and because rabies is the predominant public health consideration, it becomes the diagnostic end point, and CDV

testing is not performed. It is also unknown if CDV makes animals more susceptible to rabies or mange because of its ability to suppress the immune system. These factors make it highly likely that CDV is underreported, and that its impacts on populations are incompletely understood.

Canine distemper virus diversity and host range: What viruses are circulating in North America and in which animal hosts?Andrew Allison, Department of Microbiology and Immunology, Baker Institute for Animal Health, Cornell UniversityCanine distemper virus (CDV) is a highly contagious, enveloped RNA virus that belongs to the genus Morbillivirus, family Paramyxoviridae, and is found worldwide. CDV is known to cause signi�cant disease in the normal hosts that the virus usually infects, which includes members of the order Carnivora such as domestic dogs, coyotes, foxes, raccoons, and skunks. Although the normal host range of CDV has presumed to be restricted to carnivores, outbreaks of fatal disease in additional species have been documented. In 1989, cases of fatal encephalitis in javelinas or collared peccaries (Tayassu tajacu) were recognized in Arizona, with central nervous system lesions similar to those seen in CDV-infected dogs. More recently, in 2006, an outbreak of CDV among captive rhesus macaques (Macaca mulatta) in Guangxi province, China, resulted in the infection of ~10,000 monkeys with 4,250 deaths. Since the 2006 epidemic, additional CDV outbreaks in

macaques have been documented in China and also Japan. Collectively, these �ndings suggest that CDV not only has the propensity to infect a very large number of di�erent species within a single order (Carnivora), but also can jump into and cause disease in other hosts, including non-human primates and suids, and possibly other species. Although there is no evidence to suggest that CDV could become an imminent threat to public health, its wide host range (which now appears to include non-human primates) suggests that a more thorough characterization of the genetic diversity of CDV that exists in North America and the potential host ranges of these viruses is warranted.

In collaboration with the NWDC and the Southeastern Cooperative Wildlife Disease Study (SCWDS) at the University of Georgia, we are attempting to collect and analyze CDV cases from wildlife in the Eastern United States (and potentially Midwestern and Western states). Our aim is to understand in more detail the natural biodiversity of CDV that exists in North America, what the normal host ranges of these viruses are, and to potentially determine if speci�c genotypes are associated with increased virulence in certain hosts. CDV-positive tissues from any species would be greatly appreciated.

For those who are interested in submitting tissue samples from CDV cases to the project or have additional questions, please contact Andrew Allison at aba75@cornell.edu until August 1st, 2016, and at aba25@vt.edu after August 1st, 2016, or Julie Ellis at Julie.Ellis@tufts.edu.

Study of the Canada Lynx (Lynx Canadensis) Population in MaineCanada lynx live in moist boreal forest areas in the U.S. and Canada. The lynx is a specialized hunter of the snowshoe hare, and both species are associated with boreal forest. Due to the patchiness of boreal forest in the U.S. there is a lower density of lynx compared to that of lynx populations in the forests of Canada. Since 1997, the lynx has been a species of Special Concern to the Maine Department of Inland Fisheries and Wildlife (MDIFW) and has been federally listed as Threatened since 2000. Once considered “irregular and anecdotal,” the population of lynx in Maine has been increasing since the late 1990s in response to the abundance of high quality habitat for lynx and their prey. In 1999, 6 lynx were radio collared and results indicated that there was, in fact, a resident population of lynx in Maine. Over

the course of the next 12 years, MDIFW captured 185 lynx in northern Maine and radio-collared 85 of those. In 2006, the estimated population of adult lynx in core lynx range in Maine was 750-1,000, and when kittens are included, the total approaches 2,000. The population has since expanded and MDIFW is in the process of collecting data to update the 2006 estimates.

Due to the status of lynx in Maine, biologists began collecting lynx carcasses in 2000 in order to identify potential causes of mortality. In 2006, gross necropsies done by Dr. Jim Webber (University of Maine) revealed the presence of parasitic lung worms in some of the dead lynx. Due to these previous �ndings, MDIFW biologist Jennifer Vashon reinitiated a collaborative study to assess mortality factors in Maine’s lynx population. On 29 April, Jen brought 4 of some 40 stored lynx to the New Hampshire Veterinary Diagnostic Laboratory (NHVDL) to begin the process of detailed necropsy and data collection. In addition to monitoring radioed lynx, MDIFW responds to reports of dead lynx. Like all of the approximately 100 lynx recovered since 2000, these 4 animals died various ways from unknown illness to vehicular trauma. The gross necropsies documented nutritional status, type and extent of injuries, presence of external and internal parasites, stomach contents, and organ abnormalities. After gross necropsies, NHVDL pathologists, Drs. David Needle and Brian Stevens, will examine representative samples from all organs microscopically.

Results from necropsies by University of Maine and NHVDL could inform lynx conservation in Maine, and perhaps, throughout the Northeast.

Figure 5. MDIFW biologist, Jen Vashon, with Drs. Walt Cottrell, Brian Stevens, and David Needle (from left to right) conducting a lynx necropsy.