A comparison of microscopic ink characteristics of 35 commercially available surgical margin inks.

OBJECTIVE: To compare microscopic characteristics of commercially available surgical margin inks used for surgical pathology specimens. STUDY DESIGN: Prospective in vitro study. SAMPLE POPULATION: Thirty-five different surgical margin inks (black, blue, green, orange, red, violet, and yellow from 5 different manufacturers). METHODS: Inks were applied to uniform, single-source, canine cadaveric full-thickness ventral abdominal tissue blocks. Tissue blocks and ink manufacturers were randomly paired and each color was applied to a length of the cut tissue margin. After drying, tissues were fixed in formalin, and 3 radial slices were obtained from each color section and processed for routine histologic evaluation, yielding 105 randomly numbered slides with each manufacturer's color represented in triplicate. Slides were evaluated by 5 blinded, board-certified veterinary anatomic pathologists using a standardized scoring scheme. Statistical analyses were performed to evaluate for ink manufacturer effects on scores, correlation among different subjective variables, and pathologist agreement. RESULTS: Black and blue had the most consistently high scores whereas red and violet had the most consistently low overall scores, across all manufacturers. All colors tested, except yellow, had statistically significant differences in overall scores among individual manufacturers. Overall score was significantly correlated to all other subjective microscopic scores evaluated. The average Spearman correlation coefficient among the 10 pairwise pathologists overall ink scores was 0.60. CONCLUSIONS: There are statistically significant differences in microscopic ink characteristics among manufacturers, with a notable degree of inter-pathologist agreement.

Emergence and pathogenicity of highly virulent Cryptococcus gattii genotypes in the northwest United States.

Cryptococcus gattii causes life-threatening disease in otherwise healthy hosts and to a lesser extent in immunocompromised hosts. The highest incidence for this disease is on Vancouver Island, Canada, where an outbreak is expanding into neighboring regions including mainland British Columbia and the United States. This outbreak is caused predominantly by C. gattii molecular type VGII, specifically VGIIa/major. In addition, a novel genotype, VGIIc, has emerged in Oregon and is now a major source of illness in the region. Through molecular epidemiology and population analysis of MLST and VNTR markers, we show that the VGIIc group is clonal and hypothesize it arose recently. The VGIIa/IIc outbreak lineages are sexually fertile and studies support ongoing recombination in the global VGII population. This illustrates two hallmarks of emerging outbreaks: high clonality and the emergence of novel genotypes via recombination. In macrophage and murine infections, the novel VGIIc genotype and VGIIa/major isolates from the United States are highly virulent compared to similar non-outbreak VGIIa/major-related isolates. Combined MLST-VNTR analysis distinguishes clonal expansion of the VGIIa/major outbreak genotype from related but distinguishable less-virulent genotypes isolated from other geographic regions. Our evidence documents emerging hypervirulent genotypes in the United States that may expand further and provides insight into the possible molecular and geographic origins of the outbreak.

Cryptococcus gattii with bimorphic colony types in a dog in western Oregon: additional evidence for expansion of the Vancouver Island outbreak.

Cryptococcus gattii was isolated from a 1.5-year-old dog with systemic cryptococcosis in Oregon. The dog had no link to Vancouver Island or British Columbia, Canada. Samples from a nasal swab and from a granulomatous mass within the cranial cavity were pooled for culture. Colonies on Sabouraud dextrose agar were mucoid and exhibited bimorphic morphology, melanin-pigmented and unpigmented. Pigmented colonies were encapsulated budding spherical yeast, whereas unpigmented colonies were of unencapsulated ovoid budding yeast. In addition to defective melanin production, the unpigmented colony type exhibited defective mating. Genetic analysis by high-resolution multilocus sequence typing revealed that the 2 isolates are genetically identical at 8 unlinked loci tested and that the 2 isolates are both the VGIIa Vancouver Island major genotype. Findings are consistent with expansion of the Vancouver Island outbreak onto the mainland Pacific Northwest region of the United States.

Anabaena/Dolichospermum as the source of lethal microcystin levels responsible for a large cattle toxicosis event.

Thirty-two 14-month old steers died during a period of four days (19-23 June 2017) after drinking from Junipers Reservoir (southeastern Oregon, USA) during a cyanobacterial bloom. Clinical and histopathological findings were consistent with acute liver disease, and microcystin-LR was present at 3000 µg/L in a reservoir water sample and at 7100 µg/L in the rumen contents of one of the mortalities. Serum biochemistry and histological examination indicated severe liver damage consistent with microcystin toxicosis. Microscopic observation of reservoir water samples, limited to frozen or poorly stored and partially degraded samples, indicated the presence of abundant Anabaena/Dolichospermum, but the presence of other toxic cyanobacteria such as Microcystis could not be excluded. Metagenomic analysis showed the presence in these samples of a single cyanobacterium whose cpcBA, rpoB and rbcL genes indicated membership in the Anabaena/Dolichospermum genus. The sequence of a complete mcy gene cluster with homology to previously identified Anabaena mcy genes was recovered. These results emphasize the capacity for Anabaena/Dolichospermum blooms to produce lethal levels of microcystin, posing a danger to public health and livestock. Further, our findings indicate that such occurrences can occur outside the far-northern latitudes in which microcystin-producing Anabaena have typically been found.

Ocular plague (Yersinia pestis) in mule deer (Odocoileus hemionus) from Wyoming and Oregon.

Although plague is relatively rare in wild ungulates, this report describes ocular lesions associated with Yersinia pestis infection in three free-ranging mule deer (Odocoileus hemionus) from Wyoming and Oregon, USA. All deer were observed antemortem and seemed to be blind. Post-mortem examination revealed gross lesions of bilateral keratoconjunctivitis and/or panophthalmitis in the first two deer, but only partial retinal detachment in the third deer. Microscopically, all deer had moderate-to-severe necrotizing and fibrinopurulent endophthalmitis and varying degrees of keratoconjunctivitis with abundant intralesional coccobacilli. The lesions in the first (D1) and third deer (D3) suggested an acute course, whereas those in the second deer (D2) were subacute to chronic. Yersinia pestis was isolated from ocular tissue swabs or ocular fluids of D1 and D2, and it was demonstrated by immunohistochemistry within ocular lesions of D1 and D3. Although plague does not seem to be a major cause of morbidity or mortality in free-ranging mule deer, keratoconjunctivitis or pinkeye is relatively common in these animals and plague should be considered as a differential diagnosis in such cases, with appropriate precautions taken to protect the human and animal health.

Aspergillus Section Nigri-Associated Calcium Oxalate Crystals in an Eurasian Eagle Owl (Bubo bubo).

An adult male Eurasian eagle owl (Bubo bubo) housed at a wildlife rehabilitation facility in southern Oregon died after a short period of progressive ill-thrift. Radiographs taken prior to death demonstrated abnormal radiopaque material in the coelom and the owl was submitted for postmortem examination. Black pigmented fungus was noted grossly, particularly in the respiratory tissues, with abundant oxalate crystal deposition associated with and without hyphal elements subsequently observed histologically. Aspergillus section Nigri was cultured from the lesions. Although there have been a few reports of aspergillosis caused by Aspergillus niger in avian species, the severity and wide tissue distribution of oxalates in this case are highly unusual.

High copy number of ovine gammaherpesvirus 2 DNA associated with malignant catarrhal fever-like syndrome in a lamb.

Domestic and wild sheep are the natural reservoirs for ovine gammaherpesvirus 2 (OvHV-2), the causative agent of sheep-associated malignant catarrhal fever (SA-MCF). Virtually all adult sheep are infected with OvHV-2 under natural flock conditions, and infection is normally subclinical. MCF-like clinical signs and typical histologic lesions in sheep have been linked during case investigations at veterinary diagnostic laboratories; however, the confirmation of naturally occurring MCF in sheep is problematic. To date, the assays for detection of OvHV-2-specific antibodies or DNA are usually positive in sheep, regardless of health status, so mere detection of antibodies or the agent is of minimal diagnostic significance in this species. We document herein a naturally occurring MCF case in a 4-mo-old domestic lamb and demonstrate that the affected animal had 100-1,000 times more OvHV-2 copy numbers in tissues than in healthy adult and age-matched sheep. These results indicate that high copy numbers of viral DNA in tissues associated with characteristic lesions can be used to confirm the diagnosis of MCF in sheep.

An observation of Clostridium perfringens in Greater Sage-Grouse.

Mortality due to infectious diseases is seldom reported in the Greater Sage-Grouse (Centrocercus urophasianus). A case of necrotic enteritis associated with Clostridium perfringens type A is described in a free-ranging adult male sage-grouse in eastern Oregon. Clostridial enteritis is known to cause outbreaks of mortality in various domestic and wild birds, and should be considered as a potential cause of mortality in sage-grouse populations.

DNA-based identification of a hepatic trematode in an elk calf.

Liver fluke infection was identified as a probable cause of clinical disease in an approximately 6-mo-old elk (Cervus elaphus) in coastal Oregon. Clinical pathology and necropsy findings are described. The alcohol-fixed flukes that were submitted for identification were similar in size to Fasciola hepatica, but their shape resembled Fascioloides magna in that they lacked a distinctive anterior cone. A few structures consistent with the eggs of F. magna were observed in liver lesions, suggesting that at least some of the worms were sexually maturing. Due to difficulties in morphologic identification associated with improper fixation technique, DNA analysis was used to compare small subunit (SSU) and internal transcribed spacer 2 ribosomal RNA gene sequences of the recovered parasites with those of F. hepatica and F. magna, confirming these small, but sexually mature flukes were F. magna. This is the first publication of the SSU gene sequence for F. magna. Phylogenetic analysis showed that it is related to, but is an outlier, to the genus Fasciola. Due to the high mortality rate associated with this disease outbreak, the overall significance of trematodiasis in the herd is unclear.

Eimeria macusaniensis infection in 15 llamas and 34 alpacas.

CASE DESCRIPTION: 15 llamas and 34 alpacas between 3 weeks and 18 years old with fecal oocysts or intestinal coccidial stages morphologically consistent with Eimeria macusaniensis were examined. Nineteen of the camelids were admitted dead, and 30 were admitted alive. Camelids admitted alive accounted for 5.5% of all camelid admissions during this period. CLINICAL FINDINGS: Many severely affected camelids had signs of lethargy, weight loss, decreased appetite, and diarrhea. Camelids with clinical infection also commonly had evidence of circulatory shock, fat mobilization, and protein loss. Nonsurviving camelids also had evidence of shock, edema, bile stasis, renal insufficiency, hepatic lipidosis, muscle damage, relative hemoconcentration, and sepsis. Postmortem examination frequently revealed complete, segmental replacement of the mucosa of the distal portion of the jejunum with coccidial meronts and gamonts. For 17 of 42 camelids, results of initial fecal examinations for E macusaniensis were negative. TREATMENT AND OUTCOME: Most camelids admitted alive were treated with amprolium hydrochloride, plasma, and various supportive treatments. Fifteen of the 30 treated camelids died or were euthanized. CLINICAL RELEVANCE: Findings suggest that E macusaniensis may be an important gastrointestinal tract pathogen in camelids of all ages. Clinical signs were frequently nonspecific and were often evident before results of fecal examinations for the parasite were positive. As with other coccidia, severity of disease was probably related to ingested dose, host immunity, and other factors. The clinical and herd relevance of positive fecal examination results must be determined.

Alcelaphine gammaherpesvirus 1-induced malignant catarrhal fever in a Watusi ( Bos taurus africanus) steer in a North American game park.

A 10-y-old Watusi ( Bos taurus africanus) steer housed at a drive-through game park in Winston, Oregon developed severe clinical illness including fever, marked nasal discharge, injected scleral and conjunctival membranes, plus oral hemorrhages and erosions. The animal responded poorly to supportive treatment and was euthanized. Additional gross findings at postmortem examination included papules and erosive lesions on the tongue, hemorrhagic large intestine, and multifocal cardiac hemorrhages. Histopathologic findings included multifocal lymphoplasmacytic vasculitis plus fibrin exudation in heart and tongue. Total DNA obtained from the splenic samples was positive for alcelaphine gammaherpesvirus 1 (AlHV-1) as tested by a multiplex PCR for malignant catarrhal fever (MCF) viruses. The AlHV-1 detection was further confirmed by amplification and sequencing of a viral DNA polymerase gene fragment, which was identical to AlHV-1 sequences in GenBank. This was the first diagnosis of clinical wildebeest-associated MCF on these premises, although wildebeest have been held at the park for over 25 y. This disease is sporadic in North America and should be considered as a differential diagnosis for febrile illness with ulcerative oral lesions in ruminants.

Induction of aberrant crypt foci in DNA mismatch repair-deficient mice by the food-borne carcinogen 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP).

Disruption of the DNA mismatch repair (MMR) pathway results in elevated mutation rates, inappropriate survival of cells bearing DNA damage, and increased cancer risk. Relatively little is known about the impact of environmentally relevant carcinogens on cancer risk in individuals with MMR-deficiency. We evaluated the effect of MMR status (Mlh1(+/+) versus Mlh1(-/-)) on the carcinogenic potential of the cooked-meat mutagen, 2-amino-1-methyl-6-phenylimidazo [4,5-b] pyridine (PhIP) in mice. PhIP exposure did not obviously increase lymphoma or small intestinal tumorigenesis in either Mlh1-deficient or -proficient mice. In contrast, the frequency of aberrant crypt foci (ACF), a preneoplastic biomarker for colon tumorigenesis, was increased by PhIP, and the increase due to PhIP was significantly greater in Mlh1(-/-) versus wild-type littermates. This apparent heightened susceptibility to induction of ACF parallels the previously reported hypermutability of Mlh1-deficient mice to PhIP and is consistent with the hypothesis that MMR-deficiency would increase the likelihood of PhIP-induced carcinogenic mutations. Further evaluation of the risk that consumption of heterocyclic amines may impart to MMR-deficient individuals therefore is warranted.

Bilateral cystic rete testis in an alpaca (Lama pacos).

A 9-year-old intact male alpaca (Lama pacos) was examined because of marked enlargement of the left scrotum. Ultrasound examination revealed a thin-walled anechoic structure in the area of the left testis. Aspirated fluid contained spermatozoa, many of which had abnormal morphology. Castration was performed and the left testis was markedly enlarged with a clear fluid-filled cyst. The cyst was lined by a single layer of squamous to cuboidal epithelial cells consistent with those originating from rete testis. The right testis was of a comparable size and shape to that of normal alpaca testis, but the rete testis was mildly to moderately dilated. Additional findings included chronic inflammation of the right testis and epididymis and epididymal fibrosis with ductal hyperplasia on the left. The diagnosis was bilateral cystic rete testis, most likely secondary to chronic inflammation.

Microanatomic features of pancreatic islets and immunolocalization of glucose transporters in tissues of llamas and alpacas.

OBJECTIVE: To describe the microanatomic features of pancreatic islets and the immunohistochemical distribution of glucose transporter (GLUT) molecules in the pancreas and other tissues of New World camelids. ANIMALS: 7 healthy adult New World camelids, 2 neonatal camelids with developmental skeletal abnormalities, and 2 BALB/c mice. PROCEDURE: Samples of pancreas, liver, skeletal muscle, mammary gland, brain, and adipose tissue were collected postmortem from camelids and mice. Pancreatic tissue sections from camelids were assessed microscopically. Sections of all tissues from camelids and mice (positive control specimens) were examined after staining with antibodies against GLUT-1, -2, -3, and -4 molecules. RESULTS: In camelids, pancreatic islets were prominent and lacked connective tissue capsules. Numerous individual endocrine-type cells were visible distant from the islets. Findings in neonatal and adult tissues were similar; however, the former appeared to have more non-islet-associated endocrine cells. Via immunostaining, GLUT-2 molecules were detected on pancreatic endocrine cells and hepatocytes in camelids, GLUT-1 molecules were detected on the capillary endothelium of the CNS, GLUT-3 molecules were detected throughout the gray matter, and GLUT-4 molecules were not detected in any camelid tissues. Staining characteristics of neonatal and adult tissues were similar. CONCLUSIONS AND CLINICAL RELEVANCE: In New World camelids, microanatomic features of pancreatic islets are similar to those of other mammals. Data suggest that the poor glucose clearance and poor insulin response to hyperglycemia in adult camelids cannot be attributed to a lack of islet cells or lack of GLUT molecules on the outer membrane of those cells.

Parelaphostrongylus odocoilei in Columbian black-tailed deer from Oregon.

Documenting the occurrence of Parelaphostrongylus odocoilei has historically relied on the morphological examination of adult worms collected from the skeletal muscle of definitive hosts, including deer. Recent advances in the knowledge of protostrongylid genetic sequences now permit larvae to be identified. Dorsal-spined larvae (DSLs) collected in 2003-2004 from the lung and feces of six Columbian black-tailed deer (Odocoileus hemionus columbianus) from Oregon were characterized genetically. The sequences from unknown DSLs were compared to those from morphologically validated adults and larvae of P. odocoilei at both the second internal transcribed spacer (ITS-2) of ribosomal DNA and the mitochondrial cytochrome oxidase II gene. We provide the first unequivocal identification of P. odocoilei in Columbian black-tailed deer from Oregon. The broader geographic distribution, prevalence, and pathology of P. odocoilei are not known in populations of Oregon deer.

Rhabdochlamydia spp. in an Oregon raptor.

PCR-based approach was used to examine the rate of Chlamydia positivity in raptors from wild bird rehabilitation centers in Oregon. Three of 82 birds were identified as positive for Chlamydia with this PCR. Sequence analysis of 16S ribosomal DNA from 2 of these birds confirmed the presence of DNA from phylum Chlamydiae. One bird was positive for Chlamydia psittaci in both choanal and cloacal swabs. The second bird, a louse-infested red-tailed hawk, had evidence of choanal colonization by "Candidatus Rhabdochlamydia" spp. Our study describes evidence of this Chlamydia-like organism in the United States. This survey also suggests that the carriage rate of C. psittaci is low in raptors in Oregon wild bird rehabilitation centers, and that care must be taken in the design of PCR primers for phylum Chlamydiae such that colonization by insect endosymbionts is not mistaken for an infection by known chlamydial pathogens.

Cryptococcosis in a llama (Lama glama).

Cryptococcosis was diagnosed in a 17-year-old male llama that had been euthanatized following an acute onset of neurologic disease. Tissues affected included the brain, spinal cord, lung, and kidney. The character of the leukocytic response varied from minimal to pyogranulomatous. Cryptococcosis has not been previously reported in a llama, although the infection has been described in 2 other species of New World camelids. The pathogenesis of cryptococcosis is briefly reviewed.

Bile acids in gastric fluids from llamas and alpacas with and without ulcers.

To investigate the relationship between duodenogastric reflux and ulceration, gastric fluid was collected from the 1st (n = 26) and 3rd (n = 45) gastric compartments of llamas and alpacas during postmortem examination. Gastric fluids were analyzed for the presence of bile acids. Additionally, the 3rd compartment was examined grossly for the presence of erosions or ulcers, and the intestines were examined for evidence of fluid distention. Detectable bile acids were found in the 3rd compartment fluid of 1 of 28 camelids without ulcers, 7 of 14 camelids with ulcers, and 3 of 3 camelids with intestinal distention. Third compartment bile content was significantly higher in camelids with ulcers (median = 2.9 micromol/L) or intestinal distention (median = 371.5 micromol/L) than those with neither (median = 0 micromol/L). Bile acids were detected in the 1st gastric compartment in only 2 camelids. These findings suggest a reflux of duodenal contents, including bile acids, into the 3rd compartment in camelids with ulcers, similar to what is seen in camelids with poor intestinal emptying. Whether bile reflux was the cause of the ulcers or occurred after or concurrent to their development remains unknown.

Hair-loss syndrome in black-tailed deer of the Pacific Northwest.

A widespread hair-loss syndrome (HLS) has affected Columbian black-tailed deer (Odocoileus hemionus columbianus) in western Oregon and Washington (USA) since 1996. In order to better characterize the condition, 21 HLS-affected black-tailed deer (BTD) were necropsied, and body condition, parasite burdens, and significant lesions were noted. All deer were in poor body condition, and at least 17 had severe internal parasite burdens. A consistent finding was the presence of large numbers of chewing lice, identified as an indeterminate species of Damalinia (Cervicola). Four animals were infested with intrafollicular Demodex sp., the first report of this genus of mites in BTD. We postulate that the hair loss is largely due to ectoparasitism by a species of chewing louse that represents a new pathogen for BTD. This loss of pelage also may be an important contributor to the poor body condition of these animals.

Diagnostic sampling and gross pathology of New World camelids.

This article provides an overview of tests and appropriate samples to send to a Veterinary Diagnostic Laboratory for the diagnosis of common diseases of New World Camelids (NWC) such as abortions, congenital anomalies, anemia, enteritis, endoparasitism, gastric ulcer, hepatic lipidosis, encephalitis, pneumonia, dermatosis, neoplasia and cryptococcosis. Unique anatomic features of NWC and common findings encountered during gross necropsy examination are briefly reviewed.