Detection of infectious spleen and kidney necrosis virus (ISKNV) and turbot reddish body iridovirus (TRBIV) from archival ornamental fish samples.

Although infections caused by megalocytiviruses have been reported from a wide range of finfish species for several decades, molecular characterisation of the viruses involved has been undertaken only on more recent cases. Sequence analysis of the major capsid protein and adenosine triphosphatase genes is reported here from formalin-fixed, paraffin-embedded material from 2 archival ornamental fish cases from 1986 and 1988 in conjunction with data for a range of genes from fresh frozen tissues from 5 cases obtained from 1991 through to 2010. Turbot reddish body iridovirus (TRBIV) genotype megalocytiviruses, previously not documented in ornamental fish, were detected in samples from 1986, 1988 and 1991. In contrast, megalocytiviruses from 1996 onwards, including those characterised from 2002, 2006 and 2010 in this study, were almost indistinguishable from infectious spleen and kidney necrosis virus (ISKNV). Three of the species infected with TRBIV-like megalocytiviruses from 1986 to 1991, viz. dwarf gourami Trichogaster lalius (formerly Colisa lalia), freshwater angelfish Pterophyllum scalare and oscar Astronotus ocellatus, were infected with ISKNV genotype megalocytiviruses from 2002 to 2010. The detection of a TRBIV genotype isolate in ornamental fish from 1986 represents the index case, confirmed by molecular sequence data, for the genus Megalocytivirus.

Chytridiomycosis in dwarf African frogs Hymenochirus curtipes.

Chytridiomycosis, resulting from an infection with the fungal agent Batrachochytrium dendrobatidis (Bd), has resulted in widespread population declines in both wild and captive amphibians. The dwarf African frog (DAF) Hymenochirus curtipes is native to central Africa and is commonly sold throughout North America as an aquarium pet species. Here we document fatal chytridiomycosis resulting from cutaneous Bd infections in DAF purchased directly from a pet store and from a historical lethal epizootic occurring at an aquaculture facility in central California, USA, more than 25 yr ago. Histological lesions and PCR-amplified sequence data were consistent with the etiology of Bd. The potential epidemiological relevance of this infection in DAF is discussed.

Evaluation of an extended-release formulation of ceftiofur crystalline-free acid in koi (Cyprinus carpio).

The use of an extended release ceftiofur crystalline-free acid formulation (CCFA, Excede For Swine(®) , Pfizer Animal Health) in koi was evaluated after administration of single intramuscular (i.m.) or intracoelomic (i.c.) doses. Twenty koi were divided randomly into a control group and four treatment groups (20 mg/kg i.m., 60 mg/kg i.m., 30 mg/kg i.c., and 60 mg/kg i.c.). Serum ceftiofur-free acid equivalents (CFAE) concentrations were quantified. The pharmacokinetic data were analyzed using a nonlinear mixed-effects approach. Following a CCFA injection of 60 mg/kg i.m., time durations that serum CFAE concentrations were above the target concentration of 4 µg/mL ranged from 0.4 to 2.5 weeks in 3 of 4 fish, while serum CFAE concentrations remained below 4 µg/mL for lower doses evaluated. Substantial inter-individual variations and intra-individual fluctuations of CFAE concentrations were observed for all treatment groups. Histological findings following euthanasia included aseptic granulomatous reactions, but no systemic adverse effects were detected. Given the unpredictable time vs. CFAE concentration profiles for treated koi, the authors would not recommend this product for therapeutic use in koi at this time. Further research would be necessary to correlate serum and tissue concentrations and to better establish MIC data for Aeromonas spp. isolated from naturally infected koi.

Case report: concurrent herpesviral and presumptive iridoviral infection associated with disease in cultured shortnose sturgeon, Acipenser brevirostrum (L.), from the Atlantic coast of Canada.

Approximately 8 weeks after a chlorine insult associated with the city water supply, shortnose sturgeon, Acipenser brevirostrum (L.), from one group presented with small (3-4 mm) irregular foci of cutaneous pallor that involved the dorsocranial integument with progressive ulceration of the nascent lesions. Various bacterial organisms were isolated from the cutaneous lesions, but not from the internal viscera. Histologically, the nuclei of the intralesional and perilesional epidermal cells often exhibited margination of the chromatin that resulted in a homogenous, pale, amphophilic, tinctorial quality of the nucleoplasm consistent with a herpesvirus infection. In addition, rare lamellar epithelial cells were prominently enlarged due to an abundant, dense, basophilic cytoplasm characteristic of an iridovirus infection. Inoculation of cutaneous lesion and kidney, spleen, liver sample pools from affected shortnose sturgeon onto white sturgeon spleen (WSS-2) cell line induced cytopathic effect characterized by syncytia formation. Ultrastructural analysis of infected WSS-2 cells revealed viral particles with a characteristic herpesvirus morphology. Intranuclear hexagonal capsids had a diameter of 95-108 nm, and enveloped particles present in the cytoplasm of infected cells had a diameter of 176-196 nm. This is the first report of a herpesvirus and a possible iridovirus-like infection in shortnose sturgeon.

Molecular characterization of "Candidatus Parilichlamydia carangidicola," a novel Chlamydia-like epitheliocystis agent in yellowtail kingfish, Seriola lalandi (Valenciennes), and the proposal of a new family, "Candidatus Parilichlamydiaceae" fam. nov. (order Chlamydiales).

Three cohorts of farmed yellowtail kingfish (Seriola lalandi) from South Australia were examined for Chlamydia-like organisms associated with epitheliocystis. To characterize the bacteria, 38 gill samples were processed for histopathology, electron microscopy, and 16S rRNA amplification, sequencing, and phylogenetic analysis. Microscopically, the presence of membrane-enclosed cysts was observed within the gill lamellae. Also observed was hyperplasia of the epithelial cells with cytoplasmic vacuolization and fusion of the gill lamellae. Transmission electron microscopy revealed morphological features of the reticulate and intermediate bodies typical of members of the order Chlamydiales. A novel 1,393-bp 16S chlamydial rRNA sequence was amplified from gill DNA extracted from fish in all cohorts over a 3-year period that corresponded to the 16S rRNA sequence amplified directly from laser-dissected cysts. This sequence was only 87% similar to the reported "Candidatus Piscichlamydia salmonis" (AY462244) from Atlantic salmon and Arctic charr. Phylogenetic analysis of this sequence against 35 Chlamydia and Chlamydia-like bacteria revealed that this novel bacterium belongs to an undescribed family lineage in the order Chlamydiales. Based on these observations, we propose this bacterium of yellowtail kingfish be known as "Candidatus Parilichlamydia carangidicola" and that the new family be known as "Candidatus Parilichlamydiaceae."

Cutaneous biology and diseases of fish.

The normal structure and function of the piscine integument reflects the adaptation of the organism to the physical, chemical, and biological properties of the aquatic environment, and the natural history of the organism. Because of the intimate contact of fish with the environment, cutaneous disease is relatively more common in fish than in terrestrial vertebrates and is one of the primary disease conditions presented to the aquatic animal practitioner. However, cutaneous lesions are generally nonspecific and may be indicative of disease that is restricted to the integument or a manifestation of systemic disease. Regardless, a gross and microscopic examination of the integument is simple to perform, but is highly diagnostic and should always be included in the routine diagnostic effort of the aquatic animal practitioner, especially since various ancillary diagnostic procedures are either not practical or lack predictive value in fish. The purpose of this article is to provide an overview of normal cutaneous biology prior to consideration of specific cutaneous diseases in fish.

Hematology and clinical chemistry of cyprinid fish. Common carp and goldfish.

Evaluation of the clinical status in aquatic species is compromised by the limited diagnostic techniques that can be performed in these species. The hematologic and plasma chemistry parameters can provide predictive information, although these parameters can be highly variable owing to the influence of various intrinsic and extrinsic factors. However, these parameters are fairly stable in acclimated, well-managed fish if stress is reduced during collection and samples are properly collected and analyzed. Evaluation of any single parameter is not predictive and, therefore, not recommended. Ideally, the diagnostic protocol should include evaluation of the hematologic indices, total and differential cell counts, TPP, glucose, sodium, and chloride. The practitioner who is routinely involved in aquatic animal medicine should consider in-house evaluation of these parameters.

Replication and pathogenesis of white sturgeon iridovirus (WSIV) in experimentally infected white sturgeon Acipenser transmontanus juveniles and sturgeon cell lines.

Characteristics of the in vitro propagation of the white sturgeon iridovirus (WSIV) were examined in 6 sturgeon cell lines. One new cell line originating from gonadal tissues (WSGO) produced up to 12-fold more WSIV [approximately 22 TCID50 (50% tissue culture infective dose) cell-1], than that of a previously established reference spleen cell line (WSS-2). Infected WSGO cell cultures were examined using phase microscopy, viral infectivity assay and transmission electron microscopy (TEM). At 15 degrees C, both mature virions and infectious virus were first detected after 7 d post-infection. Capsids acquired envelopes in the cytoplasm and virions remained primarily cell-associated during the 35 d replication cycle. Cellular changes including hyper-refractility and cytoplasmic swelling with dense cytoplasmic inclusions correlated to extensive proliferation of cytoplasmic vesicles and viral assembly sites. These cytological characteristics corresponded to changes in target cells of WSIV-infected juvenile white sturgeon following bath challenge. Microscopic changes in stained tissue sections of the host epithelium were detected 4 d post-challenge, approximately 8 d prior to the onset of clinical signs. Hypertrophied Malpighian cells surrounded by a prominent pericellular cisternum characterized epithelial lesions in the skin. Similar changes to epithelial cells of the barbels, olfactory organs and esophagus were also observed. Destruction of the sensory epithelium is suggested as a cause for cessation of feeding which occurs early in the infection of white sturgeon juveniles with WSIV.

Cytokeratin-filament expression in epithelial and non-epithelial tissues of the common carp (Cyprinus carpio).

Cytokeratin expression in mammals is generally restricted to epithelial cells and has been utilized to differentiate epithelial from nonepithelial tissues in these species. Since cytokeratins have been shown to be highly conserved during vertebrate evolution, the objective of the present study has been to ascertain the expression pattern of cytokeratins in tissues of the common carp (Cyprinus carpio). A panel of 10 anti-human cytokeratin antibodies was evaluated using a streptavidin-biotin-peroxidase complex detection system. Tissues were fixed in 10% neutral-buffered formalin, 100% ethanol or methacarn. Only formalin-fixed tissues were pre-digested with trypsin prior to immunostaining. Formalin-fixed tissues generally resulted in a less intense, more diffuse staining pattern with considerable background compared with ethanol and methacarn and was therefore the least desirable fixative. The diverse staining pattern observed with the various antibodies used in this study was consistent with previous findings in other teleosts. The results confirm that cytokeratin expression in teleosts is fundamentally different from that in mammals and therefore should be used as a method to differentiate epithelial cell types in these species only with discretion.

Epitheliocystis infection in cultured white sturgeon (Acipenser transmontanus): antigenic and ultrastructural similarities of the causative agent to the chlamydiae.

A mild to moderate branchial epitheliocystis infection was diagnosed in subyearling (11 months old, 250-300 g) white sturgeon (Acipenser transmontanus) from a private culture facility with a 4-8% mortality in the population. Infected branchial epithelial cells contained the coccoid to coccobacillary epitheliocystis organisms, which appeared as cytoplasmic inclusions composed of a fine, homogeneous, dense, basophilic, granular material. The infected cells were variably enlarged with spherical to oval profiles and were randomly distributed throughout the branchial epithelium. The cytoplasmic inclusions stained positive with Macchiavello stain but negative with Brown and Brenn, periodic acid-Schiff, and Gimenez stains. Expression of chlamydial antigen was demonstrated within the cytoplasmic inclusions using a standard peroxidase-antiperoxidase immunohistochemical technique. Three stages of coordinated intracellular development were recognized by electron microscopy. The reticulate bodies were oval to spherical and 0.4-0.8 x 0.5-1.4 microns but often exhibited a pleomorphic and convoluted appearance because of variable membrane invaginations and evaginations suggestive of uneven fission and budding. Separate host cells contained intermediate bodies that were spherical to oval and 0.2-0.4 x 0.3-0.6 microns although often observed in the process of apparent uneven division. The presence of a cap or plaque composed of hexagonally arrayed fibrillar surface projections was initially recognized in this stage. A homogeneous population of 0.3-0.4 microns oval elementary bodies were observed separately in individual host cells. This developmental stage had a single, dense, compact, eccentrically located cytoplasmic condensation that occurred opposite to the location of the cap of hexagonally arrayed fibrillar surface projections. Morphologic characteristics of the epitheliocystis organism in these white sturgeon were similar to those previously described in other teleosts and expands the species catalogue of epitheliocystis infection. Furthermore, the ultrastructural similarities to the chalmydiae and the immunohistochemical detection of chlamydial antigen provides further evidence that the epitheliocystis agent is related to members of the Chlamydiales. Although the infection was considered mild to moderate and could not be definitively attributed to the mortality in this population, the potential adverse impact of epitheliocystis infection on sturgeon culture should be considered especially in intensive fish culture operations.

Effects of protein, lipid, or carbohydrate supplementation on hepatic lipid accumulation during rapid weight loss in obese cats.

Effects of restricted tube-feeding (25% of energy requirements) of protein, lipid, or carbohydrates on body weight loss; hematologic and clinical chemical variables; plasma lipid and amino acid concentrations; nitrogen balance; and hepatic histologic features and lipid concentrations were compared with values in voluntary-fasting cats (control, CON). Twelve obese cats (6.1 +/- 0.1 kg, > 40% above optimal body weight) were randomly assigned to 4 matched treatment groups (n = 3)--protein (PRO), lipid (LIP), carbohydrate (CHO), and CON--and were offered a low-palatability diet for 4 weeks. Cats of the PRO, LIP, and CHO groups were also tube-fed isocaloric amounts (88 kcal of metabolizable energy) of a casein-soybean protein mixture, corn oil, or a dextrin-dextrose mixture, respectively, during the 4 weeks. All cats fasted, rather than eat the low-palatability purified diet. Cats of the PRO group lost weight at a lower rate (P < 0.05) than did cats of other groups. After 4 weeks of fasting, serum alkaline phosphatase activities were higher than reference values in all cats of the CON and LIP groups and in 2 cats of the CHO group. At that time, 1 cat of the LIP group had lethargy, hepatomegaly, and hyperbilirubinemia. Total hepatic lipid and triglyceride concentrations increased in all groups during the study, but the increase was significantly (P < 0.05) less in cats of the PRO group, compared with those of the CON and LIP groups, and those of the CHO group, compared with those of the LIP group.(ABSTRACT TRUNCATED AT 250 WORDS)

Experimental induction of hepatic lipidosis in cats.

The effect of long-term voluntary fasting on hematologic variables, biochemical profiles, and liver histologic findings was assessed in 15 obese cats (> 40% overweight). Clinical signs and laboratory results consistent with hepatic lipidosis were observed in 12 of 15 cats after 5 to 7 weeks of fasting, and were associated with 30 to 35% reduction of initial body weight. Histologic examination of successive liver biopsy specimens revealed that obesity was not associated with liver parenchymal lipid accumulation, but that fasting resulted in lipidosis in all 15 cats. The long-term fast was associated with an early (after 2 to 4 weeks of fasting) and significant (P < 0.05) reduction in serum urea, glucose, and albumin concentrations, and RBC mass. Fasting for 5 to 7 weeks was associated with a significant (P < 0.05) increase in hepatic-associated enzyme activities and in total and direct serum bilirubin concentrations. Significant (P < 0.05) changes in serum alkaline phosphatase developed as early as 3 weeks before the onset of hyperbilirubinemia. Except for development of hepatic lipidosis, cats appeared to tolerate the fast without other adverse effect. This study confirmed that long-term fasting may induce clinical hepatic lipidosis in obese cats. Fasting appears to induce a syndrome of hepatic lipidosis that is indistinguishable from feline idiopathic hepatic lipidosis and may be an appropriate model to study the pathophysiologic features and treatment of hepatic lipidosis.

Nitrogen balance, plasma free amino acid concentrations and urinary orotic acid excretion during long-term fasting in cats.

The purpose of this study was to ascertain the changes in nitrogen balance, plasma free amino acid concentrations, urinary orotic acid excretion and body weight during long-term fasting in adult obese cats. Results from eight cats that fasted rather than eat an unpalatable diet are reported. After 5 to 6 wk of weight loss, six of the eight cats developed signs of hepatic lipidosis, and the livers of all cats were severely infiltrated with lipids. Cats lost (mean +/- SE) 33.2 +/- 1.4% of their pre-fasting body weight. Mean nitrogen balance (+/- SE) was -547 +/- 54 mg.d-1.kg-2/3 for the first week, and then the net nitrogen losses decreased to a plateau (-303 +/- 52 mg.d-1.kg-2/3) after 4 wk. Fasting was associated with a decrease in plasma concentration of essential amino acids. When plasma amino acid concentrations were considered individually, concentrations of alanine, methionine, taurine, citrulline, arginine and tryptophan decreased the most (> or = 50%), whereas concentrations of glutamine, glutamate and ornithine significantly increased. Orotic acid was not detected in the urine during the fast. After 1 wk of fasting, obese cats had reduced nitrogen excretion, but not to the same extent as has been shown in obese humans or obese rats. It is suggested that the availability of several amino acids may become limiting for liver protein synthesis during fasting and that these deficiencies may contribute to the development of hepatic lipidosis. Orotic acid was not linked to hepatic lipidosis caused by fasting in cats.

Chromatophoromas and chromatophore hyperplasia in Pacific rockfish (Sebastes spp.).

Pacific rockfish from Cordell Bank, off central California (United States), were collected and histologically examined from 1985 to 1990. Hyperplastic and neoplastic cutaneous lesions, involving dermal chromatophores, were observed in five species; yellowtail rockfish (Sebastes flavidus), bocaccio (S. paucispinis), olive rockfish (S. serranoides), widow rockfish (S. entomelas), and chilipepper rockfish (S. goodei). Yearly prevalences were highest in S. paucispinis (29-38%). Prevalence was initially low in S. flavidus, but increased more than 3-fold from 1985 (7.5%) to 1990 (25%). The majority of lesions were black, but white, yellow, orange, red, and mixed-color variants were also seen. Lesions were found in skin, fins, lips, gingiva, tongue, urogenital papilla, conjunctiva, and cornea of the eye. Flat lesions were consistent with melanophore (black), xanthophore (yellow or orange), and erythrophore (red) hyperplasia. Neoplastic lesions included melanophoromas, amelanotic melanophoromas, xanthophoromas, erythrophoromas, and mixed chromatophoromas. Although etiology has not been determined, interest is currently focused on potential exposure to chemical and radioactive carcinogens from the Farallon Island Radioactive Waste Dump, 30 km to the south.

Experimental horizontal transmission of Enterocytozoon salmonis to chinook salmon, Oncorhynchus tshawytscha.

Enterocytozoon salmonis was transmitted to chinook salmon Oncorhynchus tshawytscha by feeding tissues infected with the parasite and by cohabitation of noninfected fish with experimentally infected fish. Affected fish (dead and survivors) in both transmission trials had gross and microscopic signs of the disease and merogonic and sporogonic stages of the parasite. There were no morbidities or mortalities, or evidence of the parasite among control fish in either study. Results suggest that the parasite may be contracted by indirect contact among healthy and infected fish held in crowded ponds or net pens or by direct ingestion of spores found in the water.

An epizootic of cutaneous zygomycosis in cultured dwarf African clawed frogs (Hymenochirus curtipes) due to Basidiobolus ranarum.

Morbidity and mortality approaching 100% occurred in dwarf African clawed frogs (Hymenochirus curtipes) from a culture facility in central California. Moribund frogs exhibited preference for a terrestrial environment rather than their normal aquatic environment. Affected animals had a slight pallor of the integument but were otherwise grossly unremarkable. Microscopic examination revealed a fungal infection of the integument primarily characterized by the presence of surface and intra-epidermal conidia. Skin cultures of the infected animals yielded an organism identified as Basidiobolus ranarum, based on the formation of conidia in culture with internal cleavage to form sporangiospores. The organism was transmitted to healthy frogs by co-habitation with infected frogs but not by short-term immersion exposure of healthy frogs to homogenized broth cultures of the fungus. Benzalkonium chloride at 2.0 mg l-1 was efficacious in controlling the infection. Although Basidiobolus species are normally found in the intestinal tract of amphibians, the severity of this epizootic indicates that B. ranarum may be an important pathogen of amphibians reared in culture facilities.

Gill sphaerosporosis in goldfish (Carassius auratus).

Infections caused by a Sphaerospora sp. resembling S. chinensis are reported for the first time in goldfish (Carassius auratus) from North America. The myxosporean was found in the respiratory epithelium of the gill of pond-reared fish. Spores from stained tissue sections were spherical with an equal mean length and width of 6.3 microns. Spore valves were thickened at the suture which lies in a plane perpendicular to two prominent pyriform polar capsules. The polar capsules were 4.0 x 2.8 microns in length and width. Both monosporous and disporous development within a surrounding "pseudoplasmodium" was detected. Infections caused moderate hyperplasia and occasional necrosis of the respiratory epithelial cells of the gill.

Herpesviruses detected in papillomatous skin growths of koi carp (Cyprinus carpio).

Herpesviruses were found associated with epidermal hyperplasia of koi carp (Cyprinus carpio) in northern California (USA). Papillomas were found principally on the caudal regions of the fish including the fins. The growths occurred most commonly in the fall and winter among populations of captive carp. Infected epidermal cells were characterized by greatly enlarged nuclei depleted of chromatin but with thickened nuclear membranes. Numerous virions were detected in infected cells. Herpesvirus nucleocapsids in the cell nucleus had a diameter of 109 nm. Virions with envelopes with a diameter of 157 nm were abundant in cytoplasmic vacuoles. The characteristics of the papillomatous growths and the viruses were consistent with descriptions of Herpesvirus cyprini known in koi carp populations in Japan and extends the range of this pathogen to koi carp to North America.