Jerry R. Heidel

Investigation of Koi Herpesvirus Latency in Koi

ABSTRACT Koi herpesvirus (KHV) has recently been classified as a member of the family of Alloherpesviridae within the order of Herpesvirales. One of the unique features of Herpesviridae is latent infection following a primary infection. However, KHV latency has not been recognized. To determine if latency occurs in clinically normal fish from facilities with a history of KHV infection or exposure, the presence of the KHV genome was investigated in healthy koi by PCR and Southern blotting. KHV DNA, but not infectious virus or mRNAs from lytic infection, was detected in white blood cells from investigated koi. Virus shedding was examined via tissue culture and reverse transcription-PCR (RT-PCR) testing of gill mucus and feces from six koi every other day for 1 month. No infectious virus or KHV DNA was detected in fecal secretion or gill swabs, suggesting that neither acute nor persistent infection was present. To determine if KHV latent infections can be reactivated, six koi were subjected to a temperature stress regime. KHV DNA and infectious virus were detected in both gill and fecal swabs by day 8 following temperature stress. KHV DNA was also detectable in brain, spleen, gills, heart, eye, intestine, kidney, liver, and pancreas in euthanized koi 1 month post-temperature stress. Our study suggests that KHV may become latent in leukocytes and other tissues, that it can be reactivated from latency by temperature stress, and that it may be more widespread in the koi population than previously suspected.

Results of total DNA measurement in koi tissue by Koi Herpes Virus real-time PCR

Koi Herpes Virus (KHV) has been classified recently as a member of the Alloherpesviridae within the Herpesvirales order (Waltzek et al., 2005). Although one of the unique features of Herpesviridae, the sister family of Herpesvirales, is latent infection, it has not been demonstrated consistently that KHV of Alloherpesviridae can cause latent infection and be reactivated from latency. To investigate if KHV genomic DNA is present in koi exposed to KHV infection, 10 healthy fish were investigated from a koi population with a history of a KHV outbreak. No gross lesions or microscopic changes were observed at necropsy or by histological examination. No infectious virus was isolated from either the blood plasma or tissues. However, KHV DNA was detected in the white blood cells of nine of the ten fish by real-time PCR and PCR-Southern blot. KHV DNA was also detected in the brain, eye, spleen, gills hematopoietic kidney, trunk kidney, and intestine of nine of the ten fish by PCR-Southern blot. Interestingly, KHV DNA was also detected in the intestinal contents from seven of ten koi. Portions of major capsid gene DNA, amplified from two of the ten koi WBCs, were found to be identical to KHV-U. This study demonstrated that KHV genomic DNA can be detected in normal koi exposed previously to KHV and suggests that KHV becomes latent in fish.

Analysis of koi herpesvirus latency in wild common carp and ornamental koi in Oregon, USA.

Koi herpesvirus (KHV) infection is associated with high mortalities in both common carp (Cyprinus carpio carpio) and koi carp (Cyprinus carpio koi) worldwide. Although acute infection has been reported in both domestic and wild common carp, the status of KHV latent infection is largely unknown in wild common carp. To investigate whether KHV latency is present in wild common carp, the distribution of KHV latent infection was investigated in two geographically distinct populations of wild common carp in Oregon, as well as in koi from an Oregon-based commercial supplier. Latent KHV infection was demonstrated in white blood cells from each of these populations. Although KHV isolated from acute infections has two distinct genetic groups, Asian and European, KHV detected in wild carp has not been genetically characterized. DNA sequences from ORF 25 to 26 that are unique between Asian and European were investigated in this study. KHV from captive koi and some wild common carp were found to have ORF-25-26 sequences similar to KHV-J (Asian), while the majority of KHV DNA detected in wild common carp has similarity to KHV-U/-I (European). In addition, DNA sequences from IL-10, and TNFR were sequenced and compared with no differences found, which suggests immune suppressor genes of KHV are conserved between KHV in wild common carp and koi, and is consistent with KHV-U, -I, -J.

SERUM CHEMISTRY OF BOWHEAD WHALES (BALAENA MYSTICETUS)

Sera of 19 male and female bowhead whales (Balaena mysticetus) collected near Barrow, Alaska (USA) between 30 August and 13 October 1992 were evaluated for 18 serum chemistry values. Male bowhead whales had significantly greater creatinine and sodium concentrations, and significantly lower glucose concentrations than females. Pregnant females had greater triglyceride levels than non-pregnant females. The mean concentrations of creatinine, blood urea nitrogen, alkaline phosphatase, total bilirubin, total protein, sodium, potassium, chloride, phosphorus, and calcium were similar to those previously reported from bowhead whales. High aspartate aminotransferase and creatine kinase levels were attributed to muscle damage associated with harpooning.

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 manufacturers 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.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 manufacturers 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.

Keratinizing Ameloblastoma in a 9-Month-Old Llama (Lama Glama)

A 9-month-old male llama (Lama glama) was presented because of a rapidly growing mass on the right side of the face. Radiographs revealed a marked expansion of the right caudal face region with bone lysis involving the maxilla and the nasal, lacrimal, zygomatic, and palatine bones. Cytologically, the mass consisted of atypical round to polygonal cells with round nuclei and basophilic cytoplasms that formed acini and rows. Histologically, the mass consisted of anastomosing cords and sheets of neoplastic odontogenic epithelial cells embedded in a loose fibrovascular connective tissue. Single layers of peripheral, polarized, palisading, columnar epithelial cells were seen at the edges of some cords. Within the centers of the cords, epithelial cells showed rapid progression to keratin production. The histologic diagnosis was keratinizing ameloblastoma. Ameloblastomas are neoplasms of odontogenic epithelium that tend to be locally aggressive and can cause substantial destruction of bony structures. Because ameloblastomas do not tend to metastasize, they can be successfully treated by complete surgical excision, performed before extensive bony destruction occurs. Ameloblastoma, although expected to be rare, should be on the list of differential diagnoses for facial swelling in llamas.

Gastric smooth muscle hamartoma in a cat

An 11-year-old cat presented for evaluation of intermittent vomiting, constipation and hyporexia of 3 weeks duration. Ultrasonographic and endoscopic examination revealed a soft tissue mass adjacent to the lower gastro-esophageal sphincter. Surgical excision of the mass was successfully performed resulting in a resolution of clinical signs. Histologically the mass was consistent with a smooth muscle hamartoma. At follow-up 7 months after surgery, the cat remained free from clinical signs.

Visceral nodular cartilaginous metaplasia in rockfishes (Sebastes spp.) in the eastern North Pacific Ocean

Multiple, discrete, nodular foci of cartilaginous metaplasia were found in the spleens and kidneys of rockfishes taken from the northeastern Pacific Ocean during a survey to determine the incidence and the nature of diseases in these animals. These nodules sometimes occurred in association with granulomatous inflammation and distinct granulomas. Many of these fish were infected by Ichythophonus spp. or acid-fact bacteria (presumably Mycobacteria spp.). Some of the metaplastic foci contained encapsulated accumulations of eosinophilic vesicles and basophilic granular debris, described by other authors as “cysts of unknown etiology,” which have been observed at different sites in a variety of temperate and tropical fish species.

Mineralization of the Bulbus Arteriosus in Adult Rainbow Trout (Oncorhynchus Mykiss)

of the reported cases. More case reports of neoplasia in llamas are needed before any age-related hypothesis can be considered in this species. Acknowledgements. We thank Barbara Lehmann and Suzanne Hindman for technical assistance. This work was supported by funds from the Department of Pathobiology, University of Pennsylvania, and the College of Veterinary Medicine, Oregon State University.

The effects of venting and decompression on Yellow Tang (Zebrasoma flavescens) in the marine ornamental aquarium fish trade

Each year, over 45 countries export 30 million fish from coral reefs as part of the global marine ornamental aquarium trade. This catch volume is partly influenced by collection methods that cause mortality. Barotrauma in fish resulting from forced ascent from depth can contribute to post-collection mortality. However, implementing decompression stops during ascent can prevent barotrauma. Conversely, venting (puncturing the swim bladder to release expanded internal gas) following ascent can mitigate some signs of barotrauma like positive buoyancy. Here, we evaluate how decompression and venting affect stress and mortality in the Yellow Tang (Zebrasoma flavescens). We examined the effects of three ascent treatments, each with decompression stops of varying frequency and duration, coupled with or without venting, on sublethal effects and mortality using histology and serum cortisol measurements. In fish subjected to ascent without decompression stops or venting, a mean post-collection mortality of 6.2% occurred within 24 h of capture. Common collection methods in the fishery, ascent without decompression stops coupled with venting, or one long decompression stop coupled with venting, resulted in no mortality. Histopathologic examination of heart, liver, head kidney, and swim bladder tissues in fish 0d and 21d post-collection revealed no significant barotrauma- or venting-related lesions in any treatment group. Ascent without decompression stops resulted in significantly higher serum cortisol than ascent with many stops, while venting alone did not affect cortisol. Future work should examine links in the supply chain following collection to determine if further handling and transport stressors affect survivorship and sublethal effects.