Renal candidiasis associated with papillary necrosis in a captive tiger (Panthera tigris).

A 14-year-old male tiger developed anorexia with elevated blood urea nitrogen and creatinine levels. The patient had a palpable abdominal mass and demonstrated neutrophilic leukocytosis and anaemia. Leukocytes, yeast and bacteria were present in the urine. The animal was non-responsive to therapy and was subsequently euthanised. Extensive acute renal papillary necrosis (RPN) with pyelonephritis, chronic nephritis and polycystic renal disease were evident during gross and microscopic pathology examinations. The histologic occurrence of fungal spores and pseudohyphae morphologically consistent with Candida species were observed within the necrotic papillary regions of the kidney and within multiple foci of mild parakeratotic hyperkeratosis present in the gingiva and tongue. Candida albicans along with a slight growth of Escherichia coli were recovered from kidney cultures. Possible contributory factors for the renal candidiasis and associated RPN include predisposing oral candidiasis, polycystic renal disease, ischaemic nephrosclerosis, age-associated or other forms of immunodeficiency and therapy with meloxicam, a non-steroidal anti-inflammatory drug. The absence of apparent lower urinary tract involvement coupled with the presence of intravascular renal 'Candida emboli' suggest that chronic oral candidiasis was the probable source of the kidney infection.

Validation of a quantitative PCR assay for the detection of 2 Flavobacterium columnare genomovars.

Flavobacterium columnare is the causative agent of columnaris disease in a variety of fish hosts. Using modifications to previously established protocols, a quantitative PCR (qPCR) assay was validated for the detection of 2 predominant F. columnare genomovars. The oligonucleotide primer and probe combination was designed to amplify a 203-bp region of the chondroitin AC lyase gene (GenBank AY912281) of F. columnare. There were no significant differences in amplification between genomovars. Comparable quantities of genomic DNA from 10 F. columnare strains, 5 representatives of each genomovar, produced similar results. Serial dilutions of purified PCR product demonstrated the limit of sensitivity for the assay was ~ 10 copies per reaction. The presence of gill and spleen tissue did not significantly affect the sensitivity of the assay. Comparably, bacterial DNA detected from the liver and kidney was less sensitive than pure bacterial DNA. However, detection from these tissues was within one order of magnitude of other tissues, indicating this reduction may have minimal analytic significance. This validated assay was used to approximate the minimum infectious dose for F. columnare isolate 94-081 in channel catfish and assess bacterial loads in gill and kidney tissues 48 h post-infection.

Taxonomic and Functional Metagenomic Profile of Sediment From a Commercial Catfish Pond in Mississippi.

Metagenomic analyses of microbial communities from aquatic sediments are relatively few, and there are no reported metagenomic studies on sediment from inland ponds used for aquaculture. Catfish ponds in the southeastern U.S. are eutrophic systems. They are fertilized to enhance algae growth and encourage natural food production, and catfish are fed with commercial feed from spring to fall. As result, catfish pond sediment (CPS) contains a very dense, diverse microbial community that has significant effects on the physiochemical parameters of pond dynamics. Here we conducted an in-depth metagenomic analysis of the taxonomic and metabolic capabilities of a catfish pond sediment microbiome from a southeastern U.S. aquaculture farm in Mississippi using Illumina next-generation sequencing. A total of 3.3 Gbp of sequence was obtained, 25,491,518 of which encoded predicted protein features. The pond sediment was dominated by Proteobacteria sequences, followed by Bacteroidetes, Firmicutes, Chloroflexi, and Actinobacteria. Enzyme pathways for methane metabolism/methanogenesis, denitrification, and sulfate reduction appeared nearly complete in the pond sediment metagenome profile. In particular, a large number of Deltaproteobacteria sequences and genes encoding anaerobic functional enzymes were found. This is the first study to characterize a catfish pond sediment microbiome, and it is expected to be useful for characterizing specific changes in microbial flora in response to production practices. It will also provide insight into the taxonomic diversity and metabolic capabilities of microbial communities in aquaculture. Furthermore, comparison with other environments (i.e., river and marine sediments) will reveal habitat-specific characteristics and adaptations caused by differences in nutrients, vegetation, and environmental stresses.

Morphological, Histological, and Molecular Description of Unicauda fimbrethilae n. sp. (Cnidaria: Myxosporea: Myxobolidae) from the Intestinal Tract of Channel Catfish Ictalurus punctatus.

The channel catfish Ictalurus punctatus is a known host for 10 species of Henneguya, but few other myxozoan genera are described from this species. Unicauda is a genus of myxozoan parasites within the family Myxobolidae that consists of 10 valid species from freshwater fish. Herein, we describe a novel species of Unicauda from the intestinal tract of farm-raised channel catfish in Mississippi. Myxospores were consistent with the genus Unicauda but exhibited a unique branching at the terminal end of the caudal process that has not previously been reported. Myxospores measured 90.39 ± 14.97 µm (mean ± SD; range = 70.88-126.02 µm) in total length. The spherical spore body measured 7.31 ± 0.26 µm (6.75-7.84 µm) in length and 7.01 ± 0.63 µm (6.1-8.01 µm) in width. The 2 polar capsules measured 3.45 ± 0.33 µm (3.02-4.03 µm) in length and 2.65 ± 0.32 µm (2.18-3.11 µm) in width. The single caudal process measured 82.98 ± 14.97 µm (63.39-118.63 µm) in length from the base of the spore body to the end of the most terminal projection. Terminal projections measured 26.83 ± 8.8 µm (12.34-42.29 µm) in length and 0.95 ± 0.23 µm (0.52-1.6 µm) in width. The 18S rRNA gene sequence obtained did not match any published sequences. Given the uniqueness of the myxospore morphology, histological presentation, and gene sequence data, we describe this as an unreported species, Unicauda fimbrethilae n. sp.

Serotype and antimicrobial resistance patterns of Salmonella isolates from commercial birds and poultry environment in Mississippi.

To obtain information about Salmonella from commercial birds and poultry environments within Mississippi, 50 Salmonella enterica isolates were collected and characterized by intergenic sequence ribotyping (ISR) serotyping and by determining antimicrobial resistance. ISR assigned serotype to all 50 Salmonella enterica isolates whereas the Kauffman-White-LeMinor antibody-based scheme assigned serotype to 48. Agreement between both methods was K = 89.58. Within the set, 12 serotypes were detected. The antimicrobial resistance patterns (ARP) of 12 serotypes, namely Enteritidis, Typhimurium, Kentucky, Bredeney, Mbandaka, Saintpaul, Montevideo, Cubana, Lille, Senftenberg, Johannesburg, and one serotype UN0094, were determined using minimum inhibitory concentration values. The antibiograms demonstrated differences between Salmonella serotypes and among isolates of the same serotype. All isolates were 100% susceptible to enrofloxacin and trimethoprim/sulfamethoxazole. The number of antimicrobials to which the isolates were resistant ranged from two to nine. Twenty-two different ARPs were identified and ARP1, with resistance to spectinomycin and sulfadimethoxine, was most frequently observed. Forty isolates (80%) were resistant to three or more antimicrobials and were thus designated multidrug resistant. Detection of a unique serotype, and variation in antibiograms within the set, demonstrates that it is important to survey isolates periodically from a region to follow epidemiologic trends.

Comparative analysis of Edwardsiella isolates from fish in the eastern United States identifies two distinct genetic taxa amongst organisms phenotypically classified as E. tarda.

Edwardsiella tarda, a Gram-negative member of the family Enterobacteriaceae, has been implicated in significant losses in aquaculture facilities worldwide. Here, we assessed the intra-specific variability of E. tarda isolates from 4 different fish species in the eastern United States. Repetitive sequence mediated PCR (rep-PCR) using 4 different primer sets (ERIC I & II, ERIC II, BOX, and GTG5) and multi-locus sequence analysis of 16S SSU rDNA, groEl, gyrA, gyrB, pho, pgi, pgm, and rpoA gene fragments identified two distinct genotypes of E. tarda (DNA group I; DNA group II). Isolates that fell into DNA group II demonstrated more similarity to E. ictaluri than DNA group I, which contained the reference E. tarda strain (ATCC #15947). Conventional PCR analysis using published E. tarda-specific primer sets yielded variable results, with several primer sets producing no observable amplification of target DNA from some isolates. Fluorometric determination of G+C content demonstrated 56.4% G+C content for DNA group I, 60.2% for DNA group II, and 58.4% for E. ictaluri. Surprisingly, these isolates were indistinguishable using conventional biochemical techniques, with all isolates demonstrating phenotypic characteristics consistent with E. tarda. Analysis using two commercial test kits identified multiple phenotypes, although no single metabolic characteristic could reliably discriminate between genetic groups. Additionally, anti-microbial susceptibility and fatty acid profiles did not demonstrate remarkable differences between groups. The significant genetic variation (<90% similarity at gyrA, gyrB, pho, phi and pgm; <40% similarity by rep-PCR) between these groups suggests organisms from DNA group II may represent an unrecognized, genetically distinct taxa of Edwardsiella that is phenotypically indistinguishable from E. tarda.

A real-time polymerase chain reaction assay for quantification of Edwardsiella ictaluri in catfish pond water and genetic homogeneity of diagnostic case isolates from Mississippi.

A quantitative polymerase chain reaction (qPCR) assay was developed for the detection and quantification of Edwardsiella ictaluri in channel catfish Ictalurus punctatus pond water using modifications to a published E. ictaluri-specific qPCR assay and previously established protocols for the molecular detection of myxozoan parasites in catfish ponds. Genomic DNA equivalents indicative of the number of bacteria in a sample were determined and standard curves correlating to bacterial numbers were established. The assay was found to be highly repeatable and reproducible, with a linear dynamic range of five orders of magnitude. There was no interference of the assay from the presence of large quantities of nontarget DNA. Known quantities of bacteria were added to sample volumes of 40 or 500 mL of pond water collected from several different ponds. The minimum level of detection was approximately 100 cell equivalents (CE) in 40 (2.5 CE/mL) or 500 mL of pond water (0.2 CE/mL). Sample volumes of 40 mL yielded the most consistent results, which were not significantly different from those obtained from broth culture alone. Cell equivalents determined by qPCR in 40-mL pond water samples spiked with known quantities of bacteria were within one order of magnitude of the actual number of cells added. Repetitive element-based polymerase chain reaction analysis of archived isolates demonstrated the genetic homogeneity of E. ictaluri, and consistent amplification of these isolates by qPCR analysis demonstrated the stability of the PCR target. The assay described here provides a reliable method for the detection and quantification of E. ictaluri in pond water and will be an invaluable tool in epidemiological studies. Additionally, the assay provides a way to evaluate the effects that vaccination, antibiotic treatments, and restricted feeding practices have on E. ictaluri populations during an outbreak. Information obtained with these tools will aid in optimizing disease management practices designed to maximize productivity while minimizing losses.

Variation in susceptibility to Henneguya ictaluri infection by two species of catfish and their hybrid cross.

Proliferative gill disease (PGD) in channel catfish Ictalurus punctatus is caused by the myxozoan parasite Henneguya ictaluri. There is no effective treatment for PGD, and mortalities can exceed 50% in severe outbreaks. One approach to controlling losses would be to utilize a less susceptible ictalurid species in pond culture; alternatively, one could identify the traits that convey resistance and exploit them in a selective breeding program. Challenge studies have found less severe inflammatory responses in the gill tissue of blue catfish I. furcatus and fewer mortalities than in channel catfish. However, it remains unclear whether infection and subsequent plasmodial development progress the same way in the two species. To investigate this, we compared the dynamics of H. ictaluri infection in blue catfish, channel catfish, and channel catfish x blue catfish hybrids in continuous long-term (5-7-d) and short-term (24-h) pond challenges. After long-term challenge, 66.2% of the channel catfish and 63.6% of the hybrid catfish developed characteristic PGD lesions, compared with 3.7% of the blue catfish. Quantitative polymerase chain reaction analysis detected H. ictaluri in larger percentages of channel and hybrid catfish than blue catfish (98.7% and 95.7% versus 45.9%), with significantly greater parasite DNA equivalents in channel and hybrid catfish than blue catfish. Similar findings were obtained in the short-term exposures. Histologically, channel and hybrid catfish developed severe PGD accompanied by large numbers of developing plasmodia. While mild PGD was observed in some blue catfish, the progression of lesions lagged behind that in channel and hybrid catfish. Most importantly, developing plasmodia were not observed in blue catfish, and parasite DNA was not detected 14 d after removal from the source of infection. Our findings indicate that the resistance of blue catfish to H. ictaluri infection can be overcome by large numbers of infective actinospores but that infection appears to be eliminated before plasmodial development occurs.

Multichannel microwave interferometer for the levitated dipole experiment.

A four-channel microwave interferometer (center frequency: 60 GHz) has been constructed to measure plasma density profiles in the levitated dipole experiment (LDX). The LDX interferometer has a unique design owing to the unique geometry of LDX. The main design features of the interferometer are: (1) the transmitted beam traverses the plasma entirely in O-mode; (2) the interferometer is a heterodyne system employing two free-running oscillators; (3) four signals of data are received from just on transmitted beam; (4) phase shifts are detected in quadrature. Calibration tests demonstrate that the interferometer measures phase shifts with an uncertainty of approximately 5 degrees . Plasma densities in LDX corresponding to phase shifts of up to 5pi are routinely and successfully measured.

Application of a real-time PCR assay for the detection of Henneguya ictaluri in commercial channel catfish ponds.

Proliferative gill disease (PGD) in channel catfish Ictalurus punctatus is caused by the myxozoan parasite Henneguya ictaluri. Prolonged exposure of channel catfish to the actinospore stage of the parasite results in extensive gill damage, leading to reduced production and significant mortality in commercial operations. A H. ictaluri-specific real-time (Q)PCR assay was used to determine parasite levels in commercial channel catfish ponds and evaluate the risk of losing fish newly stocked into the system. Previous research has shown the H. ictaluri actinospore to be infective for approximately 24 h; therefore, determining the parasite load (ratio of parasite DNA to host DNA) in sentinel fish exposed for 2 separate 24 h periods with a minimum of 1 wk between sampling indirectly represents the rate at which infective actinospores are being released by the oligochaete host and if that rate is changing over time. Alternatively, QPCR analysis of pond water samples eliminates the need for sentinel fish. Water samples collected on 2 separate days, with a minimum of 1 wk between sampling, not only determines the approximate concentrations of actinospores in the pond but if these concentrations are remaining stable. Increases in parasite load (r = 0.69, p = 0.054) correlated with percent mortality in sentinel fish, as did increases in mean actinospore concentrations (r = 0.63, p = 0.003). Both applications are more rapid than current protocols for evaluating the PGD status of a catfish pond and identified actinospore levels that correlate to both high and low risk of fish loss.

A real-time polymerase chain reaction assay for the detection of the myxozoan parasite Henneguya ictaluri in channel catfish.

Proliferative gill disease (PGD), caused by the myxozoan parasite Henneguya ictaluri, is the most prevalent parasitic infection affecting commercial channel catfish (Ictalurus punctatus) aquaculture. There are currently no effective chemotherapeutic or biological control measures for PGD, which often peaks during the spring and fall when water temperatures are between 16-25 degrees C. The current diagnostic techniques of gross examination of gill clip wet mounts and histopathology are subject to false-negatives during the early stages of infection, and the quantifiable nature of end-point polymerase chain reaction (PCR) is subjective. Consequently, a rapid and more sensitive quantitative real-time PCR assay was developed for the detection of H. ictaluri during the early stages of infection in channel catfish. A 23 base-pair TaqMan probe was designed based on previously published H. ictaluri PCR protocols. The sensitivity of the assay was the equivalent of a single H. ictaluri actinospore, and in a pond challenge study, quantitative real-time PCR proved to be more sensitive than gross examination, microscopic examination of gill clip wet mounts, and histopathologic examination of gill tissue sections. Future applications of this assay will focus on developing methodologies to be used in conjunction with current pond-monitoring protocols to evaluate potential treatments and better manage this significant seasonal disease.

Streptococcus ictaluri arthritis, osteolysis, myositis, and spinal meningitis in channel catfish broodstock.

This report details findings of an investigation into complaints by commercial fingerling producers of low-grade mortalities, poor reproductive success, emaciation, skin lesions, and severely arched backs among broodstock of channel catfish Ictalurus punctatus. Gross lesions involved the jaw, fin bases, and vertebral column. Jaw and fin lesions consisted of small hemorrhagic ulcers and exudate-filled tracts that communicated with underlying joints and destroyed their articular surfaces. Spinal changes, recognized grossly by severe arching of the proximal vertebral column, resulted from the collapse and displacement of vertebral bodies, causing compression of the spinal cord. Affected vertebrae were lysed by pyogranulomatous inflammation that infiltrated into adjacent muscle and the spinal canal. A Streptococcus-like organism was visualized in exudate and isolated repeatedly from lesions, but only once from the kidney of a single fish. Preliminary analysis of a 16S ribosomal DNA sequence showed a close relationship to S. iniae, S. parauberis, and S. canis. Koch's postulates were fulfilled after challenge with and reisolation of the agent from identical lesions that appeared at fin bases at 2 weeks postinjection. Historical evidence and gross and microscopic findings imply the presence of a pathogen of low virulence that is capable of producing severe localized infections after a brief period of septicemia. The disease presents as a chronic debilitating syndrome that is sufficient to inhibit mobility, feeding, and reproductive activity in affected fish. Complete biochemical and molecular characterization of the bacterium (published elsewhere) has established the agent as a novel species, S. ictaluri.

Culture of Piscirickettsia salmonis on enriched blood agar.

Piscirickettsia salmonis is the etiologic agent of piscirickettsiosis, an economically significant disease of fish. Isolation of P. salmonis by culturing on fish cell lines has been the standard technique since the initial isolation of the organism. The ability to grow P. salmonis on artificial media would relieve facilities of the cost of maintaining cell lines, permit isolation at fish culture sites with fewer contamination problems, and allow easier transport of isolates to diagnostic facilities for confirmation assays. This report describes the successful culture of P. salmonis on enriched blood agar.

Improved pulsed-field gel electrophoresis procedure for the analysis of Flavobacterium columnare isolates previously affected by DNA degradation.

Flavobacterium columnare is a fresh water bacterium that causes columnaris diseases in over 36 fish species. Intra-species typing of F. columnare can be performed by pulsed-field gel electrophoresis (PFGE). However, this method is hampered by the degradation of chromosomal DNA in about 10% of strains. In the current study, DNA degradation problems caused by extracellular DNases were overcome by fixation of cells with formaldehyde prior to isolation. The results substantiate that after problems due to DNases are overcome, PFGE analysis is a reproducible highly discriminating epidemiological method for studying F. columnare isolates regardless of fish host.

Hematologic and plasma biochemical values of healthy hybrid tilapia (Oreochromis aureus x Oreochromis nilotica) maintained in a recirculating system.

Tilapia are cultured worldwide and are increasing in popularity among aquaculturalists in the United States; however, data regarding normal health parameters are limited. Few hematologic and plasma biochemical values of clinically normal tilapia have been reported, but these data may be key for identifying and managing disease issues in recirculating systems. Therefore, blood was collected from clinically normal hybrid tilapia (Oreochromis aureus x Oreochromis nilotica) housed in recirculating systems for the purpose of establishing normal hematologic and plasma biochemical reference ranges. Using standard clinical techniques the following hematologic values were determined: packed cell volume, plasma protein, leukocyte counts, leukocyte differentials, and thrombocyte counts. Additionally, the following plasma biochemical values were determined: albumin, total protein, globulins, albumin/globulin ratio, aspartate aminotransferase, alkaline phosphatase, glucose, uric acid, calcium, phosphorus, magnesium, sodium, potassium, chloride, urea nitrogen, and creatinine. The condition of the sample was also noted (lipemic, hemolysis, and icterus). The reference ranges reported in this study can be used in the management of cultured tilapia in recirculating systems.

The relationship of soil province to molecular characterization and phylogenetic analysis of Cryptosporidium parvum isolated from calves in Georgia.

Although Cryptosporidium spp. are found throughout the world and in multiple environmental conditions, few data are available that explore the possibility of an association between specific environmental parameters and the species or strain of Cryptosporidium. This study examines the potential association between a particular Cryptosporidium species/strain found in calves and soil provinces in Georgia, USA. Necropsy cases spanning the years 1996-2002 were tested. No significant differences (P=0.962, chi(2) test of homogeneity) between numbers of positive cases were noted among soil provinces. Phylogenetic analysis of the sequences for the PCR products revealed sequence similarity of the products with Cryptosporidium parvum strain C1. Although, clinical Cryptosporidiosis in calves was not found to be affected by soil province and may be caused by a single genotype, other genotypes may be responsible for subclinical infection and warrant further investigation.

Occurrence of Piscirickettsiosis-like syndrome in tilapia in the continental United States.

From 2001 to 2003, tilapia (Oreochromis sp.) farms in Florida, California, and South Carolina experienced epizootics of a systemic disease causing mortality. The fish exhibited lethargy, occasional exophthalmia, and skin petechia. The gills were often necrotic, with a patchy white and red appearance. Grossly, the spleen and kidneys were granular with whitish irregular nodules throughout. Granulomatous infiltrates were observed in kidney, spleen, testes, and ovary tissues, but not in the liver. The granulomas contained pleomorphic coccoid bacteria, measuring 0.57 +/- 0.1 x 0.8 +/- 0.2 microm, that were Giemsa-positive, acid-fast-negative, and Gram-negative. The bacteria had a double cell wall, variable electron-dense and -lucent areas, and were present in the cytoplasm and within phagolysosomes. The syndrome was associated with cold stress and poor water conditions. These findings are consistent with an infectious process caused by a Piscirickettsia-like bacterium described previously in tilapia in Taiwan and Hawaii. This report involves the first identified cases of a piscirickettsiosis-like syndrome affecting tilapia in the continental United States.

West Nile virus in farmed alligators.

Seven alligators were submitted to the Tifton Veterinary Diagnostic and Investigational Laboratory for necropsy during two epizootics in the fall of 2001 and 2002. The alligators were raised in temperature-controlled buildings and fed a diet of horsemeat supplemented with vitamins and minerals. Histologic findings in the juvenile alligators were multiorgan necrosis, heterophilic granulomas, and heterophilic perivasculitis and were most indicative of septicemia or bacteremia. Histologic findings in a hatchling alligator were random foci of necrosis in multiple organs and mononuclear perivascular encephalitis, indicative of a viral cause. West Nile virus was isolated from submissions in 2002. Reverse transcription-polymerase chain reaction (RT-PCR) results on all submitted case samples were positive for West Nile virus for one of four cases associated with the 2001 epizootic and three of three cases associated with the 2002 epizootic. RT-PCR analysis was positive for West Nile virus in the horsemeat collected during the 2002 outbreak but negative in the horsemeat collected after the outbreak.