NOVEL SIMPLEXVIRUS (SIMPLEXVIRUS DOLICHOTINEALPHA1) ASSOCIATED WITH FATALITY IN FOUR PATAGONIAN MARA (DOLICHOTIS PATAGONUM).

Four of seven Patagonian maras (Dolichotis patagonum) at a zoological institution developed acute neurologic signs that progressed to tetraparesis and death. All affected were young adult females (10 mon-5 yr old) that presented over 11 d. Clinical signs were rapidly progressive and unresponsive to supportive therapies. Two of the four individuals were found deceased 4 d after hospitalization. Two individuals were euthanized due to poor prognosis and decline after 6 and 8 d, respectively. Simultaneously, an additional mara developed mild and self-resolving clinical signs, including a kyphotic gait and paraparesis. On gross examination, there were widespread petechiae and ecchymoses of the skeletal muscle, myocardium, skin, pericardium, urinary bladder mucosa, and spinal cord. On histopathology, all animals had necrotizing myelitis and rhombencephalitis, with intranuclear viral inclusions in three individuals. Electron microscopy confirmed herpesviral replication and assembly complexes in neurons and oligodendrocytes. Consensus PCR performed on spinal cord, brainstem, or cerebellum revealed a novel Simplexvirus most closely related to Simplexvirus leporidalpha 4. The virus was amplified and sequenced and is referred to as Simplexvirus dolichotinealpha1. It is unknown whether this virus is endemic in Patagonian mara or whether it represents an aberrant host species. Clinicians should be aware of this virus and its potential to cause severe, rapidly progressive, life-threatening disease in this species.

Helicosporidium sp. infection in a California kingsnake (Lampropeltis californiae): Spillover of a pathogen of invertebrates to a vertebrate host.

Helicosporidium is a genus of nonphotosynthetic, green algae in the family Chlorellaceae, closely related to Prototheca. It is a known pathogen of invertebrates, and its occurrence in vertebrates has not been documented. A captive, 10-month-old, male, albino California kingsnake (Lampropeltis californiae) was submitted for necropsy. Gross examination revealed hemorrhagic laryngitis and a red mottled liver. Histologically, intravascular, intramonocytic/macrophagic and extracellular, eukaryotic organisms were observed in all tissues. These organisms stained positive with Grocott-Gomori methenamine silver and periodic acid-Schiff and were variably acid-fast and gram-positive. Ultrastructural analysis revealed approximately 4 µm vegetative multiplication forms and cysts with 3 parallel ovoid cells and a helically coiled filamentous cell. A polymerase chain reaction with primers targeting Prototheca, amplicon sequencing, and Bayesian phylogenetic analysis confirmed it clustered within Helicosporidium sp. with 100% posterior probability. The genus Helicosporidium was found to nest within the genus Prototheca, forming a clade with Prototheca wickerhamii with 80% posterior probability.

Systemic Caryospora-like coccidiosis in a clutch of hatchling red-eared slider turtles (Trachemys scripta elegans).

Caryospora-like organisms (CLOs) form a clade of at least 11 genotypes of related coccidia that can cause epizootic mortality in marine turtles. The biology, transmission, host species range, and host cell tropism of these organisms are still largely unknown. The goal of this study was to characterize the host cell tropism, pathologic and ultrastructural features, and phylogeny associated with the first report of a mortality event due to CLO in the freshwater red-eared slider turtle (Trachemys scripta elegans). Sudden mortalities within a clutch of captive-raised red-eared slider hatchlings (n = 8) were recorded, and deceased animals had severe segmental to diffuse, transmural, fibrinonecrotic enterocolitis and multifocal to coalescing hepatic necrosis, among other lesions associated with numerous intracytoplasmic developing stages of intralesional coccidia. Among the different developmental stages, merozoites were ultrastructurally characterized by an apical complex. A pan-apicomplexan polymerase chain reaction (PCR) yielded a 347 bp-amplicon matching the Schellackia/Caryospora-like clade with 99.1% identity to the US3 strain from green sea turtles (Chelonia mydas) and 99.1% identity to Schellackia sp. Isolate OC116. Surviving hatchlings were treated with toltrazuril sulfone (ponazuril) but were subsequently euthanized due to the risk of spreading the parasite to other chelonids in the collection. The ponazuril-treated hatchlings (n = 4) had mild proliferative anterior enteritis, with few intraepithelial coccidia in one hatchling confirmed as CLO by PCR. This is the first report of Caryospora-like coccidiosis in non-cheloniid turtles, highlighting the relevance of this disease as an emerging highly pathogenic intestinal and extra-intestinal form of coccidiosis of turtles with potential cross-species infectivity.

Proliferative strongyloidiasis in a colony of colubrid snakes.

Strongyloides are small rhabditid nematodes primarily associated with enteric disease in a variety of animal species, including reptiles. Strongyloides spp life stages were associated with a disease outbreak in a large breeding colony of snakes. Multiple Pantherophis and Lampropeltis colubrids exhibited respiratory distress, anorexia, stomatitis, facial deformation, and waning body condition that resulted in death or necessitated euthanasia. Postmortem examinations of 13 snakes revealed epithelial hyperplasia and inflammation of the alimentary and respiratory tracts associated with varying numbers of adult and larval nematodes and embryonated or larvated ova. In a subset of snakes, aberrant nematode migration was also observed in the eye, genitourinary system, coelom, and vasculature. Histomorphology and gross examination of parasitic adult female nematodes from host tissues were consistent with a Strongyloides spp. Sedimented fecal material from 101/160 (63%) snakes housed in the affected facility was positive for nematodes and/or larvated ova. Polymerase chain reaction amplification and sequencing of portions of the 18S and 28S ribosomal ribonucleic acid (RNA) genes and the internal transcribed spacer region of adult female parasites and positive fecal samples supported the diagnosis of strongyloidiasis. Strongyloides spp possess a unique life cycle capable of alternating between parasitic (homogonic) and free-living (heterogonic) stages, resulting in the production of directly infective larvae. Commonly utilized husbandry practices in reptile collections can amplify the numbers of infective larvae generated in the captive environment, increasing the risk for rhabditid hyperinfections. This report documents morbidity, mortality, and non-enteric disease manifestations due to Strongyloides hyperinfections in a captive colubrid snake colony.

A novel herpesvirus from a wild-caught Madagascar spider tortoise shows evidence of host-viral coevolution with a duplication event in Durocryptodira.

Herpesviruses can be significant reptile pathogens. Herpesviral infection in a wild-caught, male spider tortoise (Pyxis arachnoides) under human care was detected during a routine wellness examination prior to transition between zoologic organizations. The tortoise had no clinical signs of illness. Oral swabs obtained during a physical examination as part of pre-shipment risk mitigation for infectious disease were submitted for consensus herpesvirus PCR assay and sequencing. Based on comparative sequence analysis, the novel herpesvirus identified is a member of the subfamily Alphaherpesvirinae. Studies of herpesviral phylogeny in chelonian species support branching patterns of turtle herpesviruses that closely mirror those of their hosts. The symmetry of these patterns is suggestive of close codivergence of turtle herpesviruses with their host species. The distribution of these viruses in both tortoises and emydids suggests a phylogenetic duplication event in the herpesviruses after host divergence of the Pleurodira and basal to the divergence of Americhelydia. Herpesviral infections have been documented to cause higher morbidity when introduced to aberrant host species, and significant consideration must be given to the presence of herpesviruses in the management of tortoise collections, particularly collections that include various species of testudines.

Case report: Sarcocystis speeri, Aspergillus fumigatus, and novel Treponema sp. infections in an adult Atlantic spotted dolphin (Stenella frontalis).

A complete postmortem examination, including a computed tomography scan "virtopsy" (virtual necropsy), gross necropsy, cytology, histology, and molecular diagnostics were performed to investigate the cause of death of a deceased adult male Atlantic spotted dolphin (Stenella frontalis) that stranded on Pensacola Beach, Florida, USA in February 2020. Significant findings included chronic inflammation of the meninges, brain, and spinal cord with intralesional protozoa (identified as Sarcocystis speeri via 18S rRNA and ITS-1 sequences), suppurative fungal tracheitis and bronchopneumonia (identified as Aspergillus fumigatus via ITS-2 gene sequence) and ulcerative bacterial glossitis (associated with a novel Treponema species, Candidatus Treponema stenella, identified via 23S rRNA gene sequence). This is the first reported case of S. speeri in a marine mammal. Little is understood about the epidemiology of S. speeri, including the identity of its intermediate hosts. The findings of this case suggest that S. frontalis may be a capable aberrant host and experience morbidity and mortality from this parasite. It is suspected that the novel Treponema and Aspergillus fumigatus infections were opportunistic or secondary to immunosuppression, either due to S. speeri infection or other co-morbidities.

Meningoencephalitis caused by concurrent infection with canine distemper virus and a unique Sarcocystis sp. in a gray fox.

A deceased 9-wk-old male gray fox (Urocyon cinereoargenteus) with a history of decreased ambulation and diarrhea was submitted to the Texas A&M Veterinary Medical Diagnostic Laboratory. No significant gross findings were evident on postmortem examination. Histologically, the cerebrum and brainstem had mild necrotizing meningoencephalitis with protozoal schizonts and merozoites. Additionally, glial cells contained intracytoplasmic and intranuclear viral inclusion bodies. Sections of the cerebrum were positive for canine distemper virus (CDV) and negative for Sarcocystis neurona on immunohistochemistry. Bayesian analysis revealed that this Sarcocystis sp. clustered most closely with a clade of unnamed Sarcocystis sp. found in viperid snakes, with a posterior probability of 99%. CDV likely played a significant role in the expression of clinical sarcocystosis in this gray fox.

IDENTIFICATION AND RETROSPECTIVE EVALUATION OF A FILARIOID NEMATODE SPECIES IN MANAGED GRASSHOPPER SPARROWS (AMMODRAMUS SAVANNARUM).

The grasshopper sparrow (Ammodramus savannarum) is a species of ground-dwelling passerine bird with 12 different subspecies. The Florida subspecies (Ammodramus savannarum floridanus) is classified as federally endangered, with the most common threats including habitat loss, nest predation, and floods. A managed breeding program was established at White Oak Conservation (Yulee, FL) in 2015 with eastern grasshopper sparrows (Ammodramus savannarum pratensis) as a model for breeding Florida grasshopper sparrows as part of an assurance colony. A filarioid parasite species (Aproctella sp.) was characterized by PCR after identification by blood films and postmortem examinations of both subspecies housed at White Oak Conservation. This Aproctella species was distinct from others with available sequence. Records from 157 eastern and Florida grasshopper sparrows were reviewed, and correlations between presence of filariasis and subspecies, sex, body condition score, and presence of systemic isosporosis, squamous metaplasia, coelomitis, airsacculitis, or a combination of conditions were investigated. Twenty-nine (18.5%) birds (13 of 71 Florida grasshopper sparrows; 16 of 86 eastern grasshopper sparrows) were positive for filariasis by blood film review, grossly or by tissue imprint at postmortem examination, or histologically. Filariasis was significantly correlated with systemic isosporosis, coelomitis, and airsacculitis; was not correlated with subspecies, sex, or squamous metaplasia; and had a questionable correlation with body condition score. This report provides evidence that this Aproctella species has potential to contribute to morbidity and mortality in the grasshopper sparrow. This information will be helpful for implementing effective measures against suspected vectors and for the development of best practice strategies for the health management of the species in breeding programs.

Psittacid alphaherpesvirus 5 infection in Indian ringneck parakeets in southern California.

Four Indian ringneck parakeets (Psittacula krameri; syn. ringneck parrots or rose-ringed parakeets) were submitted by 2 private owners for autopsy following a history of dyspnea and death. Gross findings were varied and included thickening of the left caudal thoracic air sac, white spots throughout the liver, mild dilation of the proventriculus, coelomic effusion, splenomegaly, and pulmonary congestion and edema. Microscopically, the submitted parakeets had significant lesions in the lower respiratory tract, including necrotizing bronchitis, parabronchitis, and interstitial pneumonia with numerous syncytia containing eosinophilic intranuclear inclusions. Electron microscopy of the lungs was compatible with a herpesviral infection and Psittacid alphaherpesvirus 5 (PsAHV5) was detected via PCR and sequencing. There has been inconsistent terminology used with Psittacid alphaherpesvirus 3 and PsAHV5; we attempt here to clarify the reported history of these viruses.

Identification of a novel Neorickettsia species in a Kemp's ridley sea turtle with granulomatous nephritis and development of a quantitative PCR assay.

An adult male Kemp's ridley turtle was found dead on the coast of Kenedy County, Texas, in August 2019 with bilateral severe, diffuse granulomatous nephritis. Pan-bacterial 16S rRNA gene polymerase chain reaction (PCR) and amplicon sequencing of affected tissue indicated the presence of a Neorickettsia. Neorickettsia is a genus of obligate intracellular Alphaproteobacteria that are transmitted by digenean trematodes. For further characterization, primers were designed to amplify and sequence the groEL gene. Phylogenetic analysis found that the organism was distinct from other known species to a degree consistent with a novel species. Immunohistochemistry using an antibody directed against a Neorickettsia surface protein showed bacterial clusters within the renal granulomas. A species-specific quantitative PCR was designed and detected the organism within the liver and colon of the index case. A quantitative PCR survey of grossly normal kidneys opportunistically collected from additional stranded sea turtle kidneys detected this organism in five of 15 Kemp's ridley turtles, two of nine green turtles, and neither of two loggerhead turtles. Recognition of this novel organism in an endangered species is concerning; additional work is underway to further characterize the potential of this organism as a pathogen of sea turtles.

Evaluation of a probe hybridization quantitative polymerase chain reaction assay for Cryptosporidium serpentis in eastern indigo snakes (Drymarchon couperi).

A probe-hybridization quantitative polymerase chain reaction assay specific for Cryptosporidium serpentis (qPCR) has been developed and shown to be extremely sensitive in the laboratory, but clinical sensitivity and specificity for this test are lacking. To approximate the sensitivity and specificity of the C. serpentis qPCR, the medical records from a captive snake colony were reviewed, and between November 2015 and June 2021, 63 eastern indigo snakes (Drymarchon couperi) were necropsied. Of these 63 snakes, 11 had qPCR performed on gastric biopsies collected at the time of necropsy, 8 had qPCR performed on samples collected by gastric swab within 35 days of necropsy, and 34 had qPCR performed on samples collected by cloacal swab within 84 days of necropsy. The qPCR results were then compared to the post-mortem histological findings, where all three sampling techniques had a 100% specificity. The sensitivity was highest in samples collected at necropsy (100%, CI: 63.06 - 100%) followed by the ante-mortem testing: gastric swab (87.50%, CI: 42.13 - 99.64%) and cloacal swab (66.67%, CI: 44.68 - 84.37%).

A novel herpesvirus detected in 3 species of chelonians.

Herpesviruses are found in free-living and captive chelonian populations, often in association with morbidity and mortality. To date, all known chelonian herpesviruses fall within the subfamily Alphaherpesvirinae. We detected a novel herpesvirus in 3 species of chelonians: a captive leopard tortoise (Stigmochelys pardalis) in western TX, USA; a steppe tortoise (Testudo [Agrionemys] horsfieldii) found near Fort Irwin, CA, USA; and 2 free-living, three-toed box turtles (Terrapene mexicana triunguis) found in Forest Park, St. Louis, MO. The leopard tortoise was coinfected with the tortoise intranuclear coccidian and had clinical signs of upper respiratory tract disease. The steppe tortoise had mucopurulent nasal discharge and lethargy. One of the three-toed box turtles had no clinical signs; the other was found dead with signs of trauma after being observed with blepharedema, tympanic membrane swelling, cervical edema, and other clinical signs several weeks prior to death. Generally, the branching order of the turtle herpesviruses mirrors the divergence patterns of their hosts, consistent with codivergence. Based on phylogenetic analysis, this novel herpesvirus clusters with a clade of viruses that infect emydid hosts and is likely of box turtle origin. Therefore, we suggest the name terrapene alphaherpesvirus 3 (TerAHV3) for the novel virus. This virus also has the ability to host-jump to tortoises, and previously documented herpesviral morbidity tends to be more common in aberrant hosts. The relationship between clinical signs and infection with TerAHV3 in these animals is unclear, and further investigation is merited.

A NEW COCCIDIAN (APICOMPLEXA: EIMERIIDAE) IN THE CRITICALLY ENDANGERED CENTRAL AMERICAN RIVER TURTLE (DERMATEMYS MAWII) IN BELIZE.

As part of a biannual health examination, coprological samples from 3-mo-old Central American river turtles, Dermatemys mawii (Gray, 1847) in a breeding program in Belize, Central America, revealed a previously undescribed coccidian (Apicomplexa) in 17 of 46 (37%) samples. Of 3 positive fecal samples transported to the University of Florida, coccidian oocysts were observed in 1 sample. Sporulated oocysts were measured and described, and using polymerase chain reaction (PCR), an approximately 400-base pair (bp) region of both the small subunit (18S) ribosomal RNA gene and 1,200-bp region of the internal transcribed spacer (ITS) gene were amplified in all 3 samples and their products were sequenced. For comparative value, the same PCR reactions and amplifications were performed on a fecal sample containing oocysts of Eimeria mitraria obtained from a red-eared slider, Trachemys scripta elegans. Results indicated a new eimerian in D. mawii, Eimeria grayi n. sp.

Rat Lungworm (Angiostrongylus cantonensis) in the Invasive Cuban Treefrog (Osteopilus septentrionalis) in Central Florida, USA.

Cuban treefrogs, Osteopilus septentrionalis, were grossly examined for parasites and parasite species confirmed by PCR. Angiostrongylus cantonensis larvae were recovered from the hind leg muscle of O. septentrionalis. This is the first report of the zoonotic rat lungworm in the Cuban treefrog and new geographic location (Volusia County) in Florida, US.

AUSTWICKIA CHELONAE IN A WILD GOPHER TORTOISE (GOPHERUS POLYPHEMUS) AND EVIDENCE OF POSITIVE SELECTION ON THE DIPHTHERIA-LIKE TOXIN GENE.

Austwickia (Dermatophilus) chelonae is a filamentous, Gram-positive Actinobacteria in the Dermatophilaceae family. It has caused fatal granulomatous disease in diverse captive reptile species on three continents, but its presence in wild or free-ranging populations was unknown. An adult female gopher tortoise (Gopherus polyphemus) was presented euhydrated, but cachectic and infested with ticks, with two firm, encapsulated masses over the cranioventral neck and right stifle. The tortoise had moderate nonregenerative anemia and evidence of inflammation; plasma biochemistry data was within normal limits. Fine needle aspirate of the neck lesion revealed abundant necrosis and aggregates of cocci. Computed tomography delineated the masses and revealed an additional mass adjacent to the left zygomatic bone. After surgical excision, histology identified chronic granulomas with intralesional filamentous bacteria. Pan-bacterial 16S rRNA PCR and sequencing of the masses identified A. chelonae. Despite treatment with oxytetracycline and ceftazidime, the tortoise deteriorated and was euthanatized. An esophageal lesion consistent with A. chelonae was seen on postmortem examination, although it was determined that the tortoise ultimately succumbed to fungal pneumonia caused by Metarhizium robertsii, an entomopathogenic biotoxin sprayed as insect control. This case reveals A. chelonae is present in free-ranging chelonians in North America. This organism produces a toxin gene similar to diphtheria toxin, one of the most potent known biotoxins, which has not been previously identified outside the genus Corynebacterium. Novel PCR primers were designed for the toxin and rpoB genes, which were amplified and sequenced from two cases and compared with two available genomes. Selection analysis revealed that the toxin gene is under positive selection, which implies it interacts significantly with the immune system, making it a good candidate for immunodiagnostic test development.

Identification of a novel mortality-associated Helicobacter species in gopher tortoises (Gopherus polyphemus), qPCR test development and validation, and correlation with mortality in a wildlife rehabilitation population.

The genus Helicobacter includes spiral-shaped bacteria in the phylum Proteobacteria, class Epsilonproteobacteria, order Campylobacteriales, that have been associated with disease in animals, including reptiles. Three wild gopher tortoise (Gopherus polyphemus) index cases presented between 2012 and 2019 with nasal discharge, lethargy, and weight loss. Cytological examination of nasal discharge from all 3 tortoises identified marked heterophilic and mild histiocytic rhinitis with abundant extracellular and phagocytized spiral shaped bacteria that stained positive with Warthin-Starry stain. Polymerase chain reaction (PCR) and sequencing of the 16S rRNA gene revealed this to be a novel Helicobacter species. Two tortoises died despite treatment attempts, and the third was moribund and was euthanized. Histological examination of the nasal mucosa (n = 3) showed granulocytic to lymphocytic rhinitis with variable mucosal hyperplasia, erosion, and ulceration; Warthin-Starry staining highlighted the presence of spiral bacteria in the untreated tortoise. Genus-specific primers were designed, and the gyrA and groEL genes were amplified by PCR and sequenced. Phylogenetic analysis shows that this organism and other previously characterized Helicobacter from tortoises form a clade. Development and cross-validation of two qPCR diagnostic assays for the gyrA and groEL genes showed significant correlation of the results of two assays (P < 0.0001). These assays were used to survey nasal wash samples from 31 rehabilitating gopher tortoises. Mortality of tortoises significantly correlated with higher Helicobacter loads detected by qPCR (P = 0.028). Appropriate quarantine protocols for tortoises during rehabilitation should consider this organism. Upper respiratory disease in tortoises may involve complex microbial ecology; factors beyond Mycoplasmopsis (Mycoplasma) agassizii should be taken into account.

A novel herpesvirus in a white stork associated with splenic and hepatic necrosis.

We identified a novel herpesvirus in a captive juvenile white stork (Ciconia ciconia) that experienced progressive weight loss followed by death. Histologic findings included severe splenic and hepatic necrosis, and intranuclear inclusion bodies in hepatocytes and unidentified splenic cells. The virus was amplified, sequenced, and subsequently accessioned as Ciconiid alphaherpesvirus 1. Phylogenetic analysis was performed and revealed that this virus is more closely related to mammalian herpesviruses than those within the genus Mardivirus. Additional sequence of viruses in this area may elucidate the ancestral virus that jumped from reptilian to mammalian hosts.

Development and validation of 2 probe-hybridization quantitative PCR assays for rapid detection of a pathogenic Coxiella species in captive psittacines.

Avian coxiellosis is an emerging cause of morbidity and mortality among captive psittacines, and the utility of a rapid detection test using easily obtained samples is paramount in a clinical setting. New sequences were obtained from 3 genes: groEL, dnaK, and rpoB. We developed probe-hybridization quantitative PCR (qPCR) assays using groEL and dnaK genes. Samples, including splenic aspirates, liver aspirates, whole blood, and choanal, conjunctival, and cloacal swabs, were collected from 4 psittacine species including 3 blue-and-gold macaws (Ara ararauna), 2 scarlet-chested parrots (Neophema splendida), 1 Timneh African grey parrot (Psittacus timneh), and 1 yellow-naped Amazon parrot (Amazona auropalliata). Retrospective review of postmortem findings from 3 of these psittacines included splenomegaly, hepatitis, and/or transmission electron microscopy confirmation consistent with previous reports of avian coxiellosis. There was 100% agreement between these assays and consensus PCR with sequencing. A Wilcoxon rank-sum test found a strong correlation between groEL and dnaK cycle threshold values (p < 0.001), validating these assays for detection of this avian Coxiella sp.

Clinical Manifestation and Molecular Characterization of a Novel Member of the Nannizziopsiaceae in a Pulmonary Granuloma From a Galapagos Tortoise (Chelonoidis nigra).

Nannizziopsiaceae is a family of fungal organisms within the order Onygenales containing two genera of important reptile pathogens, Nannizziopsis and Paranannizziopsis. A captive Galapagos tortoise (Chelonoidis nigra) from Boca Raton, Florida, United States, was presented for a clinical history of chronic progressive lethargy and inappetence. At initial presentation, the tortoise had a moderate non-regenerative anemia, leukocytosis, whip-like heterophil projections, erythrocyte fragmentation, and fibrin strands, with the latter two raising concern for disseminated intravascular coagulation. A single large encapsulated pulmonary granuloma was identified through imaging, including plain film radiography and bronchoscopy. Direct intralesional samples were obtained from transcarapacial celioscopy for fungal culture, cytology, histopathology, and polymerase chain reaction. Amplification and sequencing of the ITS2 region of the rRNA genes with Bayesian and maximum likelihood analyses placed the fungus in the family Nannizziopsiaceae within the order Onygenales, representing a novel fungal species.

Oral, Cloacal, and Hemipenal Actinomycosis in Captive Ball Pythons (Python regius).

Ball pythons (Python regius) are one of the most commonly kept and bred reptiles in captivity. In a large ball python breeding colony, a unique syndrome characterized by granulomatous inflammation of the cloaca and hemipenes (phalli) was observed in 140 of 481 (29.1%) breeding males, but only one of 1,446 breeding females. Lesions were absent in virgin males (n = 201) and virgin females (n = 293). On postmortem examination (n = 13, 12 males, 1 female), numerous well-defined mucosal and submucosal granulomas were present in the hemipenes (males) and cloaca (males and female). Extension into the coelomic cavity and liver was noted in a subset of these animals. An additional small subset of breeder animals (6/2027; 0.3%) presented with oral and mandibular swellings. Postmortem examination (n = 4, all female) showed oral lesions histologically indistinguishable from the cloacal/hemipenal lesions. Aerobic bacterial culture of a hepatic granuloma of one snake resulted in the isolation of filamentous, Gram-positive bacilli; amplification, and sequencing of the 16S rRNA gene and subsequent phylogenetic analysis of the isolate identified the bacterium as a novel species of Actinomyces. Screening of cloacal and oral granulomas using a specific, heminested 16S rRNA PCR assay confirmed the presence of the agent in all 17 snakes, as well as in cloacal swabs taken at the time of necropsy in 11/13 snakes. The Actinomyces sp. was also identified by PCR of cloacal swabs of unaffected snakes (n = 94) from the affected colony and two unrelated, grossly unaffected breeding colonies. In the affected colony, 65.5% of breeding animals (n = 23) but only 11.9% of virgin animals (n = 42) tested PCR positive, with breeding status being a significant predictor of bacterium presence (P < 0.00001). This study characterizes a granulomatous mucosal disease syndrome of breeding male ball pythons associated with a novel Actinomyces. In stark contrast to male snakes, the presence of the bacterium in both breeding and virgin females was very rarely associated with clinical disease. Though additional studies are necessary, these data suggest a role for the novel bacterium in the disease process, a predilection for clinical disease in male snakes, and the potential for sexual transmission of the disease.