Michael J. Mauel

Genetic variants of Ehrlichia phagocytophila, Rhode Island and Connecticut.

Primers were used to amplify a 561-bp region of the 16S rRNA gene of Ehrlichia phagocytophila from Ixodes scapularis ticks and small mammals collected in Rhode Island and Connecticut. DNA sequences for all 50 E. phagocytophila-positive samples collected from 1996 through 1998 in southwestern Connecticut were identical to the sequence reported for E. phagocytophila DNA from confirmed human cases. In contrast, the sequences from 92 of 123 E. phagocytophila-positive Rhode Island samples collected from 1996 through 1999 included several variants differing by 1-2 nucleotides from that in the agent infecting humans. While 11.9% of 67 E. phagocytophila-positive ticks collected during 1997 in Rhode Island harbored ehrlichiae with sequences identical to that of the human agent, 79.1% had a variant sequence not previously described. The low incidence of human ehrlichiosis in Rhode Island may in part result from interference by these variant ehrlichiae with maintenance and transmission of the true agent of human disease.

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.

Bacterial Pathogens Isolated from Cultured Bullfrogs (Rana Castesbeiana)

A commercial bullfrog (Rana castesbeiana) operation in south Georgia had multiple epizootics of systemic bacterial infections over a 3-year period, 1998–2000. A number of potential pathogens (Aeromonas hydrophila, Chryseobacterium (Flavobacterium) meningosepticum, Chryseobacterium (Flavobacterium) indolgenes, Edwardsiella tarda, Citrobacter freundii, Pseudomonas spp., and (Streptococcus iniae) were isolated from various tissues. Clinically, frogs demonstrated acute onset of torticolis, stupor, and indifference to stimuli. Cutaneous hyperemia, subcutaneous and muscular hemorrhage, and peripheral edema were consistent gross findings. Histologically, clusters of lymphocytes, monocytes, and occasional acidophiles with scattered granulomas occurred in liver, kidney, and spleen. This is the first report of S. inae and C. meningosepticum as potential disease agents in R. castesbeiana. These findings suggest that a variety of bacteria may be associated with redleg and that culture results must be obtained for accurate diagnosis.

Endometritis in Postparturient Cattle Associated with Bovine Herpesvirus-4 Infection: 15 Cases

Suppurative, ulcerative endometritis associated with bovine herpesvirus-4 (BHV-4) infection was identified in 15 postparturient dairy cows from 5 separate dairies. Characteristic eosinophilic to amphophilic intranuclear viral inclusion bodies were identified within degenerate endometrial lining epithelium and endothelial cells. Bovine herpesvirus-4 was confirmed as the etiology by a combination of fluorescent antibody assays, viral isolation, heminested PCR, ultrastructural examination of the uterus and inoculated tissue culture cells, and negative-stain electron microscopy of tissue culture supernatant. Viral particles measuring 70–95 nm were demonstrated in uterine epithelial and endothelial cells by electron microscopy. Bacteria including Arcanobacterium pyogenes, Escherichia coli, and an α-Streptococcus isolate were isolated from all uteri. Bovine herpesvirus-4-associated endometritis has been previously reported in sporadic cases in Europe but has not been previously reported in the United States. Endometritis associated with BHV-4 appears to be an emerging syndrome in Georgia dairy herds.

Septicemic salmonellosis in two cats fed a raw-meat diet.

Salmonella gastroenteritis and septicemia were diagnosed in two cats presented for necropsy. Both cats resided in the same household and were fed a home-prepared, raw meat-based diet. Salmonella was isolated from multiple organs in both cats and from samples of raw beef incorporated into the diet fed to one of the cats. Subtyping of the bacterial isolates yielded Salmonella newport from one cat and from the diet it had been fed. This report provides evidence that the practice of feeding raw meat-based diets to domestic cats may result in clinical salmonellosis.

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 × 0.8 ± 0.2 μm, 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.

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°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.

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

Abstract 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 × 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.

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

Abstract:  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.