Journal of Veterinary Diagnostic Investigation

Gas gangrene is a necrotizing infection of subcutaneous tissue and muscle that affects mainly ruminants and horses, but also other domestic and wild mammals. Clostridium chauvoei, C. septicum, C. novyi type A, C. perfringens type A, and C. sordellii are the etiologic agents of this disease, acting singly or in combination. Although a presumptive diagnosis of gas gangrene can be established based on clinical history, clinical signs, and gross and microscopic changes, identification of the clostridia involved is required for confirmatory diagnosis. Gross and microscopic lesions are, however, highly suggestive of the disease. Although the disease has a worldwide distribution and can cause significant economic losses, the literature is limited mostly to case reports. Thus, we have reviewed the current knowledge of gas gangrene in mammals.

Tetanus is a neurologic disease of humans and animals characterized by spastic paralysis. Tetanus is caused by tetanus toxin (TeNT) produced by Clostridium tetani, an environmental soilborne, gram-positive, sporulating bacterium. The disease most often results from wound contamination by soil containing C. tetani spores. Horses, sheep, and humans are highly sensitive to TeNT, whereas cattle, dogs, and cats are more resistant. The diagnosis of tetanus is mainly based on the characteristic clinical signs. Identification of C. tetani at the wound site is often difficult.

Clostridia can cause hepatic damage in domestic livestock, and wild and laboratory animals. Clostridium novyi type B causes infectious necrotic hepatitis (INH) in sheep and less frequently in other species. Spores of C. novyi type B can be present in soil; after ingestion, they reach the liver via portal circulation where they persist in phagocytic cells. Following liver damage, frequently caused by migrating parasites, local anaerobic conditions allow germination of the clostridial spores and production of toxins. C. novyi type B alpha toxin causes necrotizing hepatitis and extensive edema, congestion, and hemorrhage in multiple organs. Clostridium haemolyticum causes bacillary hemoglobinuria (BH) in cattle, sheep, and rarely, horses. Beta toxin is the main virulence factor of C. haemolyticum, causing hepatic necrosis and hemolysis. Clostridium piliforme, the causal agent of Tyzzer disease (TD), is the only gram-negative and obligate intracellular pathogenic clostridia. TD occurs in multiple species, but it is more frequent in foals, lagomorphs, and laboratory animals. The mode of transmission is fecal–oral, with ingestion of spores from a fecal-contaminated environment. In affected animals, C. piliforme proliferates in the intestinal mucosa, resulting in necrosis, and then disseminates to the liver and other organs. Virulence factors for this microorganism have not been identified, to date. Given the peracute or acute nature of clostridial hepatitis in animals, treatment is rarely effective. However, INH and BH can be prevented, and should be controlled by vaccination and control of liver flukes. To date, no vaccine is available to prevent TD.

Clostridium perfringens type C causes severe and lethal necrotic enteritis (NE) in newborn piglets. NE is diagnosed through a combination of pathology and bacteriologic investigations. The hallmark lesion of NE is deep, segmental mucosal necrosis with marked hemorrhage of the small intestine. C. perfringens can be isolated from intestinal samples in acute cases but it is more challenging to identify pathogenic strains in subacute-to-chronic cases. Toxinotyping or genotyping is required to differentiate C. perfringens type C from commensal type A strains. Recent research has extended our knowledge about the pathogenesis of the disease, although important aspects remain to be determined. The pathogenesis involves rapid overgrowth of C. perfringens type C in the small intestine, inhibition of beta-toxin (CPB) degradation by trypsin inhibitors in the colostrum of sows, and most likely initial damage to the small intestinal epithelial barrier. CPB itself acts primarily on vascular endothelial cells in the mucosa and can also inhibit platelet function. Prevention of the disease is achieved by immunization of pregnant sows with C. perfringens type C toxoid vaccines, combined with proper sanitation on farms. For the implementation of prevention strategies, it is important to differentiate between disease-free and pathogen-free status of a herd. The latter is more challenging to maintain, given that C. perfringens type C can persist for a long time in the environment and in the intestinal tract of adult animals and thus can be distributed via clinically and bacteriologically inapparent carrier animals.

Clostridium (Clostridioides) difficile is a gram-positive, spore-forming bacterium that is an important cause of disease in people, a variably important cause of disease in some animal species, and an apparently harmless commensal in others. Regardless of whether it is a known pathogen in a particular species, it can also be found in healthy individuals, sometimes at high prevalences and typically with higher rates of carriage in young individuals. As it is investigated in more animal species, it is apparent that this bacterium is widely disseminated in a diverse range of domestic and wild animal species. Although it can be found in most species in which investigations have been performed, there are pronounced intra- and inter-species differences in prevalence and clinical relevance. A wide range of strains can be identified, some that appear to be animal associated and others that are found in humans and animals. A large percentage of strains that cause disease in people can at least sporadically be found in animals. It is a potentially important zoonotic pathogen, but there is limited direct evidence of animal–human transmission. Although C. difficile has been studied extensively over the past few decades, it remains an enigmatic organism in many ways.

Storage procedures are known to affect the detectability of Clostridium difficile toxins in equine and human feces. We assessed the impact of different storage conditions on the detectability of C. difficile toxins in swine feces. Specimens were inoculated with toxins, 112 ng/g of toxin A (TcdA) and 16 ng/g of toxin B (TcdB) and subjected to the following 3 storage treatments: 4°C, −30°C, repetitive freezing at −30°C and thawing. Toxin determination was assessed at 1, 2, 7, 14, and 21 d with ELISA. A decrease in concentrations of TcdA with time was observed for samples stored at 4°C and repetitive freezing–thawing (p ≤0.05). On day 14, storage at 4°C resulted in decreased TcdA concentration as opposed to storage at −30°C and repetitive freezing–thawing (p ≤0.05). On day 21, storage at 4°C resulted in decreased TcdA detectability compared with storage at −30°C (p ≤0.05). The TcdB concentration was unaffected. These results on toxin detectability in swine feces should be carefully considered in in vitro studies on toxigenic C. difficile. Our results also offer valuable information for microbiologists and veterinarians monitoring the presence of virulent C. difficile in pigs.

A concurrent outbreak of infection by canine parvovirus 2b (CPV-2b) and Clostridium difficile producing A and/or B toxins occurred in Asian small-clawed otters (Amblonyx cinereus). The 5 clinically affected otters were 6- to 24-mo-old intact females that had severe diarrhea, dehydration, were acutely comatose, and died 1–4 d after the onset of clinical signs. Postmortem examination was performed in 3 of 7 otters. Macroscopically, the small intestine was diffusely reddened and contained red-to-brown, malodorous, watery digesta without formed feces (3 of 3). Histologic examination identified loss of enterocytes and necrosis of crypt epithelial cells. Denuded villi were often covered by mixed bacterial colonies with a predominance of gram-positive cocci to short rods in addition to larger gram-positive and -negative rods. There was also splenic lymphoid follicle depletion (2 of 3). Immunofluorescence assay revealed CPV antigen in enterocytes (2 of 3), mesenteric lymph nodes (3 of 3), and spleen (1 of 3). Immunohistochemistry revealed CPV antigen in enterocytes, lymphocytes, and dendritic cells of the Peyer patches and spleen (3 of 3), and lingual epithelial cells (1 of 2). CPV was isolated from tissues from 2 of 3 otters, and DNA sequencing identified CPV-2b for the 1 isolate tested. C. difficile producing A and/or B toxins were identified in the intestinal content by ELISA (3 of 3). To our knowledge, an outbreak of CPV-2b infection and C. difficile with clinically significant gastrointestinal disease has not been described previously in otters. The source of the viral infections remains unknown; however, these agents should be considered in otters and other mesocarnivores with similar clinical and pathologic findings.

The role of type A Clostridium perfringens in canine acute hemorrhagic diarrhea syndrome and foal necrotizing enteritis is poorly characterized. However, a highly significant association between the presence of novel toxigenic C. perfringens and these specific enteric diseases has been described. These novel toxigenic strains produce 3 novel putative toxins, which have been designated NetE, NetF, and NetG. Although not conclusively demonstrated, current evidence suggests that NetF is likely the major virulence factor in strains responsible for canine acute hemorrhagic diarrhea syndrome and foal necrotizing enteritis. NetF is a beta–pore-forming toxin that belongs to the same toxin superfamily as CPB and NetB toxins produced by C. perfringens. The netF gene is encoded on a conjugative plasmid that, in the case of netF, also carries another putative toxin gene, netE. In addition, these strains consistently also carry a cpe tcp-conjugative plasmid, and a proportion also carry a separate netG tcp-conjugative plasmid. The netF and netG genes form part of a locus with all the features of the pathogenicity loci of tcp-conjugative plasmids. The netF-positive isolates are clonal in origin and fall into 2 clades. Disease in dogs or foals can be associated with either clade. Thus, these are strains with unique virulence-associated characteristics associated with serious and sometimes fatal cases of important enteric diseases in 2 animal species.

Enteric disease in horses may be caused by a variety of microorganisms, including several clostridial species. Paeniclostridium sordellii (previously Clostridium sordellii) has been frequently associated with gas gangrene in humans and several animal species, including horses. However, its role in enteric diseases of animals has not been fully determined. We describe herein 7 cases of enteric disease in horses associated with P. sordellii infection. Grossly, the small and/or large intestines were necrotic, hemorrhagic, and edematous. Microscopically, there was severe mucosal necrosis and hemorrhage of the small and/or large intestine of all horses. P. sordellii was isolated and/or demonstrated by immunohistochemistry and/or PCR in the intestine of all horses. All other known causes of enteric disease in horses were ruled out in these 7 cases. P. sordellii should be considered among the differential diagnoses in cases of enteric disease in horses.

Gas gangrene occurs in several animal species and is caused by one or more clostridial species. In horses, the disease is most often caused by Clostridium perfringens type A. Although Clostridium sordellii has been associated with gas gangrene in ruminants and humans, cases of the disease associated with this microorganism have not been described in horses, to our knowledge. We report herein 8 cases of gas gangrene caused by C. sordellii in horses. These cases were characterized by myonecrosis and cellulitis, associated with systemic changes suggestive of toxic shock. The diagnosis was confirmed by gross and microscopic changes combined with anaerobic culture, fluorescent antibody test, immunohistochemistry, and/or PCR. The predisposing factor in these cases was an injection or a traumatic skin injury. C. sordellii should be considered as a possible etiologic agent in cases of gas gangrene in horses.

Clostridium perfringens type G is one of the pathogens involved in enteric diseases in poultry. NetB, a pore-forming toxin, is considered the main virulence factor responsible for necrotic enteritis during C. perfringens infection. We carried out a field study involving 14 farms to evaluate the occurrence of netB-positive C. perfringens and the impact of infection in Italian poultry flocks. Environmental samples (n = 117) and 50 carcasses were screened by microbiologic and molecular methods. Microbiologic investigations yielded 82 C. perfringens isolates. DNA was extracted from all samples and screened for α-toxin and NetB encoding genes by real-time PCR. The C. perfringens α-toxin gene was detected in 151 of 167 extracts (90.4%), and 31 of 151 (20.5%) were netB gene positive also. Sixteen isolates from a turkey flock with mild enteric disorders were also netB positive, demonstrating their occurrence not only in broiler but also in turkey flocks. A pulsed-field gel electrophoresis protocol was optimized to evaluate the diversity among isolates and revealed high genetic heterogeneity. The complete NetB toxin-coding gene of 2 C. perfringens isolates from turkey and broiler flocks were analyzed and showed very high relatedness with analogous sequences worldwide.

Two putative zinc metalloproteases encoded by Clostridium perfringens have been implicated in the pathogenesis of necrotic enteritis, an economically significant poultry disease that is caused by this anaerobic bacterium. These proteases have ~64% amino acid identity and are encoded by the zmpA and zmpB genes. We screened 83 C. perfringens isolates by PCR for the presence of these genes. The first gene, zmpB, is chromosomally located and was present in all screened strains of C. perfringens, regardless of their origin and virulence. The second gene, zmpA, is plasmid-borne and was only found in isolates derived from chickens with necrotic enteritis. We describe the generation of insertionally inactivated mutants of both zmpA and zmpB in a virulent C. perfringens isolate. For each mutant, a significant (p < 0.001) reduction in virulence was observed in a chicken necrotic enteritis disease model. Examples of each mutant strain were characterized by whole genome sequencing, which showed that there were a few off-site mutations with the potential to affect the virulence of these strains. To confirm the importance of these genes, independently derived zmpA and zmpB mutants were constructed in different virulent C. perfringens isolates and shown to have reduced virulence in the experimental disease induction model. A zmpA–zmpB double mutant also was generated and shown to have significantly reduced virulence, to the same extent as the respective single mutants. Our results provide evidence that both putative zinc metalloproteases play an important role in disease pathogenesis.

Focal duodenal necrosis (FDN) is an intestinal disease of egg-layer chickens characterized by multifocal necrosis of the duodenal loop and proximal jejunum. Affected flocks usually have decreased egg weights and drops in egg production. Previous studies have associated this condition with Clostridium perfringens infection. We tried to reproduce FDN by experimental infection of egg-laying chickens using different netB-positive and netB-negative C. perfringens strains, and duodenal homogenate obtained from FDN lesions. Chickens challenged with C. perfringens and/or duodenal homogenate developed duodenitis after challenge. Gross lesions included mucosal erosions, hyperemia, mucosal hemorrhages, and watery intestinal content. Microscopic lesions included mild enterocyte degeneration and necrosis, and mild-to-moderate hemorrhage and lymphoplasmacytic and heterophilic infiltration of the lamina propria. Two netB-positive C. perfringens strains closely related to necrotic enteritis pathogenic strains, by genomic composition, were re-isolated from lesions. Necrosis of intestinal crypts was observed in chickens challenged with duodenal homogenate with or without C. perfringens coinfection. Characteristic microscopic FDN lesions with significant necrosis and loss of villus enterocytes were not reproduced.

Clostridium perfringens type D epsilon toxin (ETX) is responsible for a severe and frequently fatal neurologic disorder in ruminant livestock. Light microscopic, immunohistochemical, and ultrastructural studies have suggested that ETX injury to the cerebral microvasculature, with subsequent severe, generalized vasogenic edema and increased intracranial pressure, is critically important in producing neurologic dysfunction. However, the effect of ETX on brain capillary endothelial cells in vitro has not been examined previously, to our knowledge. We exposed a well-characterized human blood–brain barrier cell line to increasing concentrations of ETX, and demonstrated a direct and dose-dependent endotheliotoxic effect. Our findings are concordant with the primacy of vasculocentric brain lesions in the diagnosis of acute epsilon toxin enterotoxemia in ruminant livestock.

Clostridium perfringens type D epsilon toxin (EXT) causes an important neurologic disorder of sheep, goats and, rarely, cattle. The disease can occur in peracute, acute, subacute, and chronic forms. High circulating levels of ETX produce vasculocentric brain lesions, in which microvascular endothelial injury results in diagnostically useful perivascular and intramural extravasations of plasma protein, especially in sheep, and less frequently in goats. With lower toxin doses, a more protracted clinical course tends to occur, particularly in sheep, leading to focal, bilaterally symmetrical, necrotic foci in certain brain regions. Although these morphologic features usually permit the diagnostic pathologist to make a definitive etiologic diagnosis, there are many aspects of the pathogenesis of these cerebral lesions that are not completely understood. ETX has also been shown to produce microvascular damage in the retina of rats, resulting in severe, diffuse vasogenic edema, similar to that found in brains exposed to this neurotoxin. The pathoclisis and vascular theories offer alternative explanations of the differential susceptibility of different brain regions to the same neurotoxic insult.

Clostridium botulinum is an important pathogen that causes botulism in humans and animals worldwide. C. botulinum group III strains, which produce a single toxin of type C or D or a chimeric toxin of type C/D or D/C, are responsible for botulism in a wide range of animal species including cattle and birds. We used unbiased high-throughput RNA sequencing (i.e., metatranscriptomics) to identify a strain of group III C. botulinum from a deceased Mongolian wild ass (Equus hemionus). The strain was closely related to some European strains. Genetic analysis of the recovered bacterial sequences showed that the C. botulinum strain identified might represent a type C/D strain of group III. Infection by C. botulinum producing the mosaic toxin of type C/D is the most likely cause of the death of the wild ass.

We compared inductively coupled plasma–mass spectrometry (ICP-MS) test results for the analysis of heavy metals (As, Ba, Cd, Hg, Pb, and Se) in pet foods and routine veterinary diagnostic specimens using intralaboratory and interlaboratory comparisons. Four laboratories, 1 principal laboratory and 3 collaborating laboratories, conducted instrument comparison (limit of detection [LOD], limit of quantification [LOQ], and linear dynamic range [LDR] on 24 data sets), in-house method comparison (accuracy and precision on 120 data sets), and interlaboratory comparison (reproducibility on 528 data sets using Horwitz equation analysis). Matrices tested included 2 types of pet food jerky treats (chicken and sweet potato), bovine blood, and bovine liver and kidney. The instrument comparison study confirmed that ICP-MS provided the sensitivity necessary for the analysis of all heavy metals tested at concentrations below the level of concern for routine diagnostic testing. The “in-house” method comparison samples, spiked at low (0.04 µg/g), medium (0.4 µg/g), and high (8.0 µg/g; note: the high validation level spike for mercury was 2 µg/g) concentration levels, indicated that ICP-MS can meet U.S. FDA acceptance criteria for both accuracy (90–105% recovery) and precision (< 6% coefficient of variation). The interlaboratory comparison studies showed that ICP-MS is a reproducible method for the analysis of heavy metals (HorRat value of 0.5–2.0) except for mercury in one laboratory, which used a different sample preparation method (open block rather than microwave digestion). Overall, our study showed that ICP-MS is a reproducible method for the analysis of heavy metals in spite of minor differences in methodology.

Influenza vaccines historically have been multivalent, whole virus inactivated products. The first bivalent, intranasal, live attenuated influenza vaccine (LAIV; Ingelvac Provenza), with H1N1 and H3N2 subtypes, has been approved for use in swine. We investigated the LAIV hemagglutinin (HA) sequences in diagnostic cases submitted to the Iowa State University Veterinary Diagnostic Laboratory and potential vaccine virus reassortment with endemic influenza A virus (IAV) in swine. From January 3 to October 11, 2018, IAV HA sequences demonstrating 99.5–99.9% nucleotide homology to the H1 HA or 99.4–100% nucleotide homology to the H3 HA parental strains in the LAIV were detected in 58 of 1,116 (5.2%) porcine respiratory cases (H1 HA A/swine/Minnesota/37866/1999[H1N1; MN99]; H3 HA A/swine/Texas/4199-2/1998[H3N2; TX98]). Nine cases had co-detection of HA genes from LAIV and wild-type IAV in the same specimen. Thirty-five cases had associated epidemiologic information that indicated they were submitted from 11 states representing 31 individual sites and 17 production systems in the United States. Whole genome sequences from 11 cases and another subset of 2 plaque-purified IAV were included in our study. Ten whole genome sequences, including 1 plaque-purified IAV, contained at least one internal gene from endemic IAV detected within the past 3 y. Phylogenetic analysis of whole genome sequences indicated that reassortment occurred between vaccine virus and endemic field strains circulating in U.S. swine. Our data highlight the need and importance of continued IAV surveillance to detect emerging IAV with LAIV genes in the swine population.

In February 2015, we conducted a field study of causes of mortality of northern elephant seal (Mirounga angustirostris) pups on San Miguel Island, California. Autopsies were performed on 18 freshly dead pups. Ages of pups ranged from stillborn to 6–8 wk. Gross and histologic lesions included trauma (9 of 18 pups), multifocal necrotizing myopathy (8 of 18), starvation with emaciation (7 of 18), congenital anomalies (3 of 18), bacterial infections (3 of 18), and perinatal mortality (stillbirths and neonates; 2 of 18). Trauma and emaciation or starvation were the most significant contributors to death. Bacterial infections included hemolytic Escherichia coli isolated from the lungs of 2 pups with pneumonia. Additionally, non-hemolytic Streptococcus sp. and hemolytic E. coli were isolated from the liver of an emaciated pup that had mild multifocal suppurative hepatitis. Other lesions, including a previously described necrotizing myopathy, congenital anomalies, and bacterial infections, were detected concurrently in cases with starvation and/or emaciation or trauma.

Nonadrenal diseases (NAD), including congestive heart failure (CHF), can affect the conversion of cortisone to cortisol favoring the production of cortisol’s urinary downstream metabolites 5α/5β-tetrahydrocortisol (THF) relative to tetrahydrocortisone (THE). We hypothesized that healthy dogs would have lower urinary levels of cortisol, cortisone, THF, and THE than dogs with hypercortisolism (HC) or CHF, and the latter would have higher urinary levels of THF and lower THE than dogs with HC. Four, 9, and 8 dogs with HC, CHF, and normal health, respectively, were included in a pilot prospective cross-sectional study. A single morning voided urine sample was analyzed for urinary cortisol metabolites by liquid chromatography–mass spectrometry. The percentages of conjugated urinary metabolites were significantly higher in dogs with CHF than in healthy dogs (p = 0.001), and not different in HC dogs (p = 0.07). Log-transformed urine cortisol metabolites–to–creatinine ratios in healthy dogs were significantly lower than the 2 other groups (p < 0.001). The urinary free THE:THF ratio was significantly higher (p < 0.001) than the urinary total and conjugated THE:THF ratios. Health status did not affect the total, conjugated, and free THE:THF ratios (p = 0.61). Additional studies are needed to investigate differences in cortisol metabolites between dogs with HC and NAD to accurately discriminate between the groups.

Porcine epidemic diarrhea virus (PEDV) is an emerging porcine coronavirus that causes a tremendous economic burden on the swine industry. The assessment of PEDV-neutralizing antibody levels provides a valuable tool to assess and predict herd immunity. We evaluated the performance of a PEDV imaging cytometry–based high-throughput neutralization test (HTNT) and compared the HTNT to a fluorescent focus neutralization (FFN) assay using serum samples from pigs of known PEDV infection status (n = 159). Estimates of diagnostic sensitivity and specificity for HTNT and FFN assays derived from receiver-operator characteristic (ROC) curve analyses showed that both PEDV FFN and HTNT provided excellent diagnostic performance. However, in the laboratory, imaging cytometry provided an objective and semi-automated approach that removed human subjectivity from the testing process and reduced the read-time of a 96-well plate to < 4 min. In addition, imaging cytometry facilitated the rapid collection and long-term storage of test images and data for further evaluation or client consultation. For PEDV and other pathogens, imaging cytometry could provide distinct advantages over classic virus neutralization or FFN assays for the detection and quantitation of neutralizing antibody.

We describe and illustrate herein a case of eosinophilic pulmonary granulomatosis (EPG) in a 16-mo-old, castrated male, Great Pyrenees crossbred dog. EPG appears to differ from eosinophilic pneumonias and eosinophilic bronchopneumopathy in dogs by the presence of nodules and masses in the lungs composed of eosinophils, macrophages, and various combinations of lymphocytes, plasma cells, neutrophils, and mast cells within fibrous tissue. Specific information from this dog was added to the information from the limited number of other dogs diagnosed with EPG reported in the veterinary literature, and the information analyzed. EPG appears to have no breed or sex predilection and tends to be a disease of younger dogs, often ≤ 3 y of age. Antemortem imaging of nodules and masses in the lungs, combined with cytologic evidence of eosinophils in the lesions, is highly suggestive of EPG, and the diagnosis can be confirmed postmortem. Concurrent eosinophilia and basophilia can be features of EPG, but their diagnostic value requires further investigation, as does the possible causal association with Dirofilaria immitis infection.

A captive, adult female Brent goose (Branta bernicla) with a history of severe feather picking by its mate, was presented with 0.5–2.5 cm skin nodules on the head and neck. Histologic examination revealed a well-delineated dermal mass that surrounded an intact feather follicle and was composed of lakes of proteinaceous fluid and fibrin with scattered foamy macrophages and multinucleate giant cells. No bacteria or fungi were identified with histology, microbial culture, or PCR. Sterile panniculitis is an infrequent finding in animals and traumatic panniculitis is rarely sterile.

Ulcers of the oral cavity, esophagus, and gastric compartments of South American camelids are uncommon. Multifocal-to-coalescing ulcers were identified in the oral cavity, esophagus, and/or gastric compartments of 5 alpacas submitted for postmortem examination. Fusobacterium necrophorum was isolated from the lesions in all alpacas, in combination with other aerobic and anaerobic bacteria. In 4 of these cases, F. necrophorum–associated lesions were considered secondary to neoplasia or other chronic debilitating conditions; in 1 case, the alimentary ulcers were considered the most significant autopsy finding. It is not known if this agent acted as a primary or opportunistic agent in mucosal membranes previously damaged by a traumatic event, chemical insult, immunodeficiency, or any other debilitating condition of the host.

A unilateral, malformed gnathotheca in a green-winged macaw (Ara chloropterus) was managed for 16 mo with periodic beak trims. Biopsy of the affected beak did not identify an underlying cause; however, at postmortem examination, a densely cellular neoplasm invaded the mandible from the beak, dissecting and effacing the cortex and trabecular bone and inciting periosteal proliferation. There was no evidence of metastasis. Neoplastic cells were strongly immunopositive for cytokeratin AE1/AE3, consistent with squamous cell carcinoma (SCC). A black-headed caique (Pionites melanocephalus) was presented with a similar clinical history, timeline, and gross appearance. Biopsy of the mandibular portion of the lesion resulted in a diagnosis of SCC. Medical management with toceranib at a palliative dose resulted in adverse side effects and was discontinued. Early diagnosis of mandibular SCC may allow for early intervention and development of treatment modalities. Our 2 cases suggest that the mandibular bone, not the beak, may be a preferred biopsy site in the diagnosis of mandibular SCC in psittacines.