Communications: Tolerance of Rainbow Trout to Dissolved Oxygen Supplementation and a Yersinia ruckeri Challenge

Abstract

Dissolved oxygen accounted for a significant proportion of the variation in mortality rate of rainbow trout Oncorhynchus mykiss acclimated for 10 weeks to a range of dissolved oxygen levels and challenged with Yersinia ruckeri. Moderate levels of oxygen supersaturation (150%) resulted in greater cumulative mortality (17.9%) among fish exposed to a 0.5-h static bath challenge with Y. ruckeri (1 × 10⁷ colony-forming units/mL). In contrast, hypoxic (70%) and normoxic (100%) levels of dissolved oxygen resulted in 12.8% and 10% cumulative mortality. Higher oxygen levels provided no additional “protection” against infection by Y. ruckeri in this study.     

Assessment of Detoxifying Markers for Florfenicol in Rainbow Trout Liver

Florfenicol (FF) is employed in fish farms to contest or prevent bacterial infections. However, this pharmaceutical may produce reactive oxygen species that may cause biochemical changes in antibiotic-treated fish. The aim of this study was to evaluate the effect of FF on Rainbow Trout Oncorhynchus mykiss treated for 10 d with 7.5 and 15 mg/kg FF followed by a withdrawal period of 5 d. Superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase, glutathione S-transferase, glyoxalase I and glyoxalase II, total glutathione, lactic dehydrogenase, and alkaline phosphatase were investigated in the livers of treated and untreated fish. A general impairment of antioxidant enzymes and metabolic indicators was measured in FF-treated Rainbow Trout. Onset of oxidative damage may have occurred during the antibiotic treatment as a consequence of the effect of FF toxicity at mainly the highest dose. Nevertheless, the rise in levels of total glutathione and glutathione S-transferase even after the withdrawal period may shield the antibiotic-mediated oxidative processes.

Received December 22, 2015; accepted May 26, 2016 Published online October 28, 2016     

Gill Ectoparasites of Juvenile Rainbow Trout and Brown Trout in the Upper Colorado River

Abstract

During 1996 and 1997, 112 rainbow trout Oncorhynchus mykiss and 204 brown trout Salmo trutta, all young of the year, were sampled from a 40-km study area of the upper Colorado River and were examined for gill parasites. Ambiphrya, Chilodonella, Ichthyobodo, Apiosoma, Trichodina, Trichodinella, Tripartiella, Epistylis, and an unidentified cochliopodid amoeba were the representative protozoan genera observed on fish examined. Significant month–year–species interactions (P = 0.0295) were revealed, reflecting the changes in infestation prevalence among months, years, and species of salmonid. Greater ectoparasite richness was observed in downstream sections of the study area, most notably near Hot Sulphur Springs, Colorado. Peaks of infestation intensity and ectoparasite richness occurred in August and September of both years, presumably because of high mean water temperatures and low flows during that time.     

Validation of a Commercial Enzyme Immunoassay for Detection of Clostridium difficile Toxins in Feces of Horses with Acute Diarrhea

Background: Clostridium difficile infection (CDI) is a recognized cause of colitis in the horse. Identification of its toxins is important for management of individual cases and for prevention of transmission and zoonosis. In humans, CDI diagnosis is performed with enzyme immunoassays, none of which have been validated for horses. Hypothesis/Objectives: (1) Establish which test for CDI diagnosis was more frequently used by diagnostic laboratories, (2) determine the identified test's performance, sensitivity, and specificity, and (3) validate its use in diarrheic horses. Animals: Samples were obtained from 72 horses presented with acute diarrhea and hospitalized at the Ontario Veterinary College, University of Guelph. Methods: A survey was conducted to establish which of the tests for CDI diagnosis in horses is most commonly used throughout North America. A questionnaire was sent to all laboratories registered in the Veterinary Infection Control Society and the American Association of Veterinary Laboratory Diagnosticians. The performance of the test was evaluated by comparison to a cell cytotoxicity assay (CTA), the accepted Gold Standard for C. difficile toxin detection. Results: The Techlab C. difficile Tox A/B II ELISA was the most frequently used test. Compared with the CTA, no significant difference was observed, and a good level of agreement (93%) was obtained. The diagnostic performance of the ELISA test was adequate (84% sensitivity and 96% specificity). Conclusions and Clinical Importance: Results demonstrate that the Techlab C. difficile Tox A/B II ELISA is a reliable, adequate, and practical tool for identification of C. difficile toxins in horse feces.     

Development of a Simple Enzyme Immunoassay for the Determination of Ovine Luteinizing Hormone

The present study describes the development and validation of a simple sensitive and specific sandwich enzyme immunoassay (EIA) for the quantification of ovine luteinizing hormone (LH) in plasma. Microtitre plates were coated with the capture antibody 518b7 anti-bovine LH. A second peroxidase-labelled anti-ovine LH antibody was used as tracer. A simple 3-step procedure was used for the sample analysis; (1) incubation of standards and samples with the pre-coated antibody plates for 2 h at 37^°C; (2) incubation with the peroxidase-labelled antibody for 1 h at room temperature; and (3) colour development with TMB substrate. A linear dose–response curve was obtained in the range 0–10 ng/ml (r ² > 0.99). The detection limit was 0.05 ng/ml, and the intra-assay and inter-assay coefficients of variation were 7% and 11.7%, respectively. The theoretical stability of microplates and reagents was calculated, this being greater than one year. Low or undetectable cross-reactivities were recorded for follicle-stimulating hormone, bovine thyroid-stimulating hormone, equine chorionic gonadotrophin and a gonadotrophin-releasing hormone (GnRH) analogue. The EIA was biologically validated by the determination of plasma LH concentrations of nine Rasa Aragonesa ovariectomized and estradiol-implanted ewes after a double GnRH challenge. In conclusion, this enzyme immunoassay provides an efficient, simple and sensitive method for the routine analysis of ovine LH.     

Serological Evidence of Infectious Hematopoietic Necrosis in Rainbow Trout from a French Outbreak of Disease

Abstract

Following the detection of infectious hematopoietic necrosis virus (IHNV) in France in April 1987, a serological survey was conducted of the rainbow trout Oncorhynchus mykiss (formerly Salmo gairdneri) from an infected cultured stock previously known to be contaminated with viral hemorrhagic septicemia virus (VHSV) for 3 years. The work lasted from April to December 1987, at which time all the remaining fish were slaughtered. Serum samples were assayed by a plaque-reduction test and a simplified neutralization test that is more suitable for processing large numbers of serum samples. Such investigations revealed that IHNV neutralization by trout antibodies depended on trout complement, as did neutralization of VHSV. Incubation for 16 h at 4°C increased the sensitivity of the test compared to incubation for 1 h at 20°C. During the course of clinical IHN from April to June, young fish did not display any neutralizing activity, but in September, 29 of 50 of them exhibited significant anti-IHN neutralizing antibody titers ranging from 21 to over 160, and 18 of 46 of these same fingerlings did so in December. Similarly, fish that had undergone VHS infection in August began to develop anti-VHSV antibodies in December (5 of 50), demonstrating that one fish can harbor neutralizing antibodies to both IHNV and VHSV, and that these antibodies had required 14 weeks to appear under fish culture conditions at 10°C. As could be expected from seroneutralization tests, neutralizing antibodies to IHNV did not result in protection against VHS. Sera from 13 of 20 adult fish sampled in mid-June revealed neutralizing antibodies to IHNV, suggesting that they harbored the virus prior to the clinical infection that affected their progeny. Only two of the fish showed low anti-VHSV antibody titers. Similarly, neutralizing antibodies to IHNV were detected in 53 of 73 other adult fish sampled in late October, 10 months after they had spawned and 7 months after mortality had occurred among their progeny. Given the prevalence, level, and persistence of neutralizing antibody titers, the seroneutralization test would be worth investigating more thoroughly to define the conditions that could make it a reliable tool for checking the virus status of trout carriers.     

Loma sp. in Salmonids from the Eastern United States: Associated Lesions in Rainbow Trout

Abstract

A microsporidian of the genus Loma was noted in the gills of rainbow trout Oncorhynchus mykiss from a state hatchery (Buford Trout Hatchery) in Georgia. Mortalities of varying severity occur at this hatchery every fall, and the microsporidian was noted during an experiment from August 1991 to January 1992 to determine the effects of water source on disease. Infections first appeared to be systemic in the October sample; xenomas were observed in heart, spleen, and peripheral vessel walls. The presence of unidentified intracellular material preceded the appearance of xenomas in all tissues, but whether this material was associated with inflammation or represented immature stages of the parasite has yet to be determined. These structures were also noted in the intestine and liver, although xenomas were not noted in these organs. Mature xenomas did not elicit an inflammatory response but appeared to be short-lived. When the xenoma wall ruptured and released spores, an inflammatory response was again observed. The prevalence and severity of the infection were determined in fish maintained in troughs with well water, Chattahoochee River water, or hatchery (treated river) water. The infection tended to be more prevalent and more severe in fish maintained in the hatchery or river water than in those maintained in the well water. Stress induced by poor water quality may increase mortality from this parasite. This report extends the range of Loma sp. into the eastern United States.     

Validation of a Commercially Available Immunoassay for the Measurement of Bovine Cardiac Troponin I

Background: Commercially available cardiac troponin I (cTnI) assays developed for use in humans have not yet been validated for use in cattle.
Hypotheses: The ADVIA Centaur TnI-Ultra immunoassay can be used for the detection of bovine cTnI. In healthy cattle, serum cTnI is undetectable or is present only in trace amounts.
Methods: Purified bovine cTnI and cTnI-free bovine serum were used for the evaluation of assay performance including intra- and inter-assay precision, sensitivity, interference, linearity, and recovery. Effects of storage at 23, 4, −20, and −80 °C for 2 days, and at −20 and −80 °C for 7 and 14 days and repeated freeze-thaw cycles on recovery of cTnI were analyzed. Serum cTnI concentrations in 30 healthy dairy cows were determined.
Results: Intra- and inter-assay precisions (mean ± SD) were 4.48 ± 2.26 and 13.36 ± 6.59%, respectively. The assay demonstrated linearity at 0.5, 2, 15, and 30 ng/mL cTnI. Mean recovery was 100.81, 85.26, 87.72, and 114.42%, respectively. Skeletal muscle homogenate added to serum of known cTnI concentration did not alter the concentration of the analyte ( P > .05). Concentration of cTnI significantly decreased when samples were stored at 4 and 23 °C for 2 days ( P < .05). Repeated freeze-thaw cycles and storage at −20 °C for 7 days had no significant influence on cTnI concentration ( P > .05). Serum cTnI concentration in healthy cattle was ≤0.03 ng/mL.
Conclusion and Clinical Importance: ADVIA Centaur can be used reliably for the detection of serum cTnI concentration in cattle.     

Three Myxosporeans Found in the Cranial and Branchial Tissues of Rainbow Trout in California

Abstract

Three myxosporeans were encountered in the cranial tissues of a California population of rainbow trout Oncorhynchus mykiss examined for the presence of Myxobolus cerebralis, the causative agent of whirling disease. Typical spores of M. cerebralis and a previously undescribed species of Myxobolus were found in the cranial tissues prepared by the pepsin HCl-trypsin digestion method. Henneguya zschokkei was also detected in digest preparations of cranial tissues, but was more numerous when branchial cartilage was included in the preparations. Microscopic examinations of tissues of individual rainbow trout showed occasional infections with both myxobolid species. Myxobolus cerebralis trophozoites and spores were found in the cranial and gill cartilage, and Myxobolus sp. was found in the brain and spinal cord. Henneguya zschokkei was also found within granulomas in the connective tissues below the gill arch. Both M. cerebralis and H. zschokkei were associated with a chronic inflammatory response in their respective tissues. In contrast, the Myxobolus sp. spores were found in pockets within the nervous tissues with no detectable host response. The spore measurements, calculated from fresh digests of infected tissues for the three myxosporeans (N = 20), for length × width × thickness in micrometers (SD) were 11.7 (0.6) without tails and 42.6 (5.2) with tails × 7.7 (0.8) × 7.0 (0.1) for H. zschokkei, 9.9 (0.4) × 8.4 (0.1) × 6.5 (0.3) for M. cerebralis, and 12.7 (0.7) × 10.5 (1.0) × 9.5 (0.8) for Myxobolus sp. Examined under scanning electron microscopy, the latter two species were morphologically similar although distinctive in size.     

Plasma and Tissue Levels of Trimethoprim in The Rainbow Trout,Salmo Gairdneri,After Absorption From Fresh and Salt Water

This is a comparative study of uptake of trimethoprim from 1) fresh water, 2) salt water after 7 days of adaption and 3) salt water without previous adaptation. The rate and extent of absorption were found to vary significantly. The salt water adapted group reached a plasma concentration of approx. 1 µg/ml after 10 h, the unadapted salt water group after 24 to 48 h and the fresh water group did not reach this concentration. The results are discussed in relation to the non-ionic diffusion theory and to the alterations taking place in euryhaline species of fish during adaptation to salt water.     

Differences between Plasma and Serum Samples for the Evaluation of Blood Chemistry Values in Rainbow Trout,Channel Catfish,Hybrid Tilapias,and Hybrid Striped Bass

Abstract

Blood chemistry analytes are determined for fish from either serum or plasma samples. Serum and plasma are similar in that they both represent the fluid component of blood; however, plasma contains clotting factors that are not present in serum. This study was conducted to determine whether the type of sample—plasma or serum—had an effect on measured blood analytes in rainbow trout Oncorhynchus mykiss, channel catfish Ictalurus punctatus, hybrid tilapia Oreochromis spp., and hybrid striped bass (striped bass Morone saxatilis × white bass M. chrysops). Paired plasma and serum samples were analyzed for the following standard biochemical analytes: total protein, albumin, globulin, creatinine, total bilirubin, alkaline phosphatase, aspartate aminotransferase, sodium, potassium, chloride, calcium magnesium, phosphorus, glucose, and cholesterol. For all four taxa, values for potassium were lower in the serum and magnesium and phosphorus values were higher in the serum. Glucose values were lower in the serum from rainbow trout, hybrid striped bass, and channel catfish; whereas cholesterol values were higher in the serum of rainbow trout, channel catfish, and hybrid tilapias. The differences observed between serum and plasma were distinct from changes occurring with hemolysis and, therefore, do not represent release of erythrocyte constituents. The differences most likely represent metabolic utilization of blood constituents while the blood was clotting. This work indicates that plasma should be used preferentially to serum for biochemical analysis because analyte levels determined from serum may not accurately reflect those found in circulating blood.     

A Competitive Dipstick Enzyme Immunoassay for Diagnosis of Early Pregnancy in Bovine

Early pregnancy diagnosis in bovines is one of the important aspects in efficient dairy farm management. In order to develop a competitive enzyme immunoassay (EIA) employing low cost reagents, anti‐progesterone antiserum and progesterone‐penicillinase enzyme conjugate were prepared. Using this anti‐serum and conjugate along with pencillinV–starch–iodine substrate system, the competitive EIA was standardized. In the experiment, danazol, a weak androgen used to extract the progesterone bound to proteins in milk, was included after standardizing the optimum concentration. Incubation period and temperature and pH of the reaction mixture were also optimized. The developed test was validated with milk samples obtained from dairy farm and individual animal owners. Confirmation of the pregnancy was made by per rectum examination of the genital tract around 60 days post‐insemination. The user friendly test procedure showed sensitivity and specificity of 83.3% and 87.5%, respectively as compared with residual binding method which was earlier developed in the laboratory with sensitivity and specificity of 100% and 87.5% respectively.     

Comparison of Hatchery and Field Performance between a Whirling-Disease-Resistant Strain and the Ten Sleep Strain of Rainbow Trout

Abstract

A whirling-disease-resistant strain of rainbow trout Oncorhynchus mykiss (GRHL strain) derived from a backcross of an F1 hybrid of two strains (German strain × Harrison Lake strain) with German strain females, was compared with the Ten Sleep (TS) strain of rainbow trout. The GRHL strain had consistently superior growth and feed conversion in two consecutive hatchery trials. Hatching and mortality rates were similar between strains. Both strains were stocked into two Utah reservoirs (Hyrum, Porcupine), and a third, Causey Reservoir, was monitored as a control for seasonal variation in prevalence of Myxobolus cerebralis. A total of 1,323 salmonids captured by gill net in spring and fall sampling between 2006 and 2008 were tested for M. cerebralis via pepsin-trypsin digest methods. Only eight of these (<1% per species) had clinical signs consistent with whirling disease. In both reservoirs, GRHL survived better than the TS and had higher growth rates. The prevalence of M. cerebralis was significantly lower for GRHL (18.1%) than TS (50.0%) in Porcupine Reservoir. In Hyrum Reservoir the trend was similar, but prevalence was lower and did not significantly differ between GRHL (9.6%) and TS (23.1%). For infected fish, no significant differences were observed between strains in myxospore counts in either Hyrum (GRHL = 911–28,244 spores/fish [spf], TS = 1,822–155,800 spf) or Porcupine (GRHL = 333–426,667spf, TS = 333–230,511 spf) reservoirs. Unmarked rainbow trout in both reservoirs had significantly higher myxospore counts than stocked fish of either strain. There were significant differences in M. cerebralis prevalence and myxospore loads among other naturally reproducing salmonids in the reservoirs. The trend in susceptibility was cutthroat trout Oncorhynchus clarkii > kokanee Oncorhynchus nerka > brown trout Salmo trutta. The GRHL performed well in both hatchery and field settings and is recommended for stocking programs.

Received December 28, 2011; accepted February 2, 2012     

Multiplication of Infectious Hematopoietic Necrosis Virus in Rainbow Trout following Immersion Infection: Whole-Body Assay and Immunohistochemistry

Abstract

The sites of replication of infectious hematopoietic necrosis virus (IHNV) in infected tissues were detected in fingerling rainbow trout Oncorhynchus mykiss by in situ histologic techniques following immersion infection. Virus antigens in tissues were detected by a neutralizing mouse monoclonal antibody and a one-step anti-mouse biotin-streptavidin conjugated to horseradish peroxidase. The efficiency of infection and virulence of the virus determined by mortality rates showed high virulence of the selected IHNV isolates, and viral replication in individual fish showed that virus content of the fish increased rapidly from the second day to the seventh day postinfection. The earliest viral lesions following infection were detected in the epidermis of the pectoral fins, opercula, and ventral surface of the body. Virus lesions became evident in kidneys on the third day. By the fifth day, when there was a significant increase in virus titer, foci of viral replication were detected in gill tissue and in the anterior internal tissues below the epidermis. Subsequently, extensive virus replication and tissue destruction were observed in the spleen, dorsal adipose tissues, ventricle, and pseudobranch. Replication in the liver, the muscularis layers of the digestive tract, and the general body musculature followed later. These infection experiments indicated that the epidermis and gills of fish constitute important sites of early IHNV replication.     

Observations on the Occurrence of Bacterial Gill Disease and Amoeba Gill Infestation in Rainbow Trout Cultured in a Water Recirculation System

Abstract

Spontaneous outbreaks of bacterial gill disease (BGD) occurred in 17-44-g rainbow trout Oncorhynchus mykiss after they were placed in a water recirculation system. In each of five groups stocked from September 1991 through July 1992, BGD occurred within 6–8 d after stocking. In each instance, BGD was followed by a secondary amoeba infestation. The spontaneous BGD outbreaks did not occur among previously stocked groups that had recovered from earlier BGD disease outbreaks. Examination of gill tissue by Gram stain and indirect fluorescent antibody technique showed increased numbers of filamentous bacteria associated with BGD after the rainbow trout were stocked into the system. Bacterial numbers decreased after a 1-h treatment with chloramine-T at concentrations of 9–15 mg/L but increased within 2 d after treatment. Although the chloramine-T treatments controlled mortality related to BGD, the amoeba infestation persisted. Histological examination of gills showed some focal hyperplasia before the rainbow trout were placed into the recirculation system, but hyperplasia became more extensive and lamellar fusion and mild telangiectasis developed within a week after placement in the system. The density at which the fingerling rainbow trout were stocked and the suspended solids present in tank water may have contributed to the BGD outbreaks. Exposure of juvenile rainbow trout to tank water from the recirculation system before they were placed into the system did not afford them protection against BGD after stocking.     

Field Evaluation of Sensitivity and Specificity of a Polymerase Chain Reaction (PCR) for Detection of Nucleospora salmonis in Rainbow Trout

Abstract

We determined the sensitivity and specificity of a nested polymerase chain reaction (PCR) for detection of the microsporidian parasite Nucleospora salmonis in kidney tissue of rainbow trout Oncorhynchus mykiss. Kidney tissues were sampled on three dates from 162 juvenile rainbow trout obtained from a California State fish hatchery where the organism was endemic. Kidney tissues were used to prepare imprints stained with May–Grünwald Giemsa and for extraction of genomic DNA for a nested PCR test for N. salmonis. Positive PCR results for N. salmonis were obtained from 1 of 100, 2 of 32, and 27 of 30 kidneys collected on the first, second, and third sample dates, respectively. Kidney tissues from 3 of 27 trout in the third sample that tested positively by PCR also had microscopic evidence of parasites in stained kidney imprints. No parasites were detected in the remaining 159 kidney samples examined microscopically. Sensitivity and specificity of the PCR assay were estimated by using maximum likelihood estimation based on cross-classified test results. This method yielded estimates of sensitivity of 99.99% and specificity of 99.87%. This field evaluation supports experimental evidence that the nested PCR test will be a valuable diagnostic tool for prevention and control of N. salmonis as well as for risk assessment associated with fish movements.     

Multiplication of Infectious Hematopoietic Necrosis Virus in Rainbow Trout following Immersion Infection: Organ Assay and Electron Microscopy

Abstract

Sequential spread of infectious hematopoietic necrosis virus (IHNV) to tissues of rainbow trout Oncorhynchus mykiss was examined following immersion infection with two different isolates of IHNV, a pathogenic strain and a nonpathogenic strain from rainbow trout. Virus strain 193–110 was highly pathogenic to 1-month-old rainbow trout and caused 100% mortality within 13 d, whereas strain RB-76 was much less virulent, causing 50% mortality by the 19th day. Virus titers of 1-month-old fingerling fish dying soon after infection were significantly higher than titers of those dying later. Assays of dissected tissues showed that gills of infected 2-month-old fingerlings contained virus as early as 16 and 20 h postinfection, with definite replication occurring at 48 h. The early presence of the virus in the gills followed shortly by appearance of the virus in the kidneys and spleen indicated that the virus spreads rapidly to the target organs. Virus was detected in many other organs at lower levels on the third day and increased to higher levels during the following days. Heart tissue had high titers later in the infection. When 4-month-old rainbow trout were infected with strain 193–110, the mortality was reduced and delayed, whereas those infected with strain RB-76 produced no mortality. Assays on the day of death of these older fingerlings infected with strain 193–110 revealed that fish dying soon after infection also had higher titers than those dying later. Electron microscopic examination offish organs showed the presence of typical IHNV particles budding off from various tissue cells of affected organs, including gill tissue. The destructive effect of the virus was particularly noticeable in the disarrangement of heart muscle organelles.     

Localization of the N-methyl-D-aspartate R1 Receptor in the Pituitary Gland of Immature Rainbow Trout (Oncorhynchus mykiss)

A study was conducted on the selective immunostaining of pituitary cells and pars nervosa of immature rainbow trout by antibody raised against the R1 subunit of the rat ionotropic N-methyl-D-aspartate (NMDA) receptor. In the pars distalis, the mammotrophs of the rostral pars distalis exhibited the most marked imunoreactivity, with the somatotrophs of the proximal pars distalis showing a consistently lower degree of immunoreactivity. No imunoreactivity was associated with the corticotrophs, thyrotrophs or gonadotrophs. In the pars intermedia, the melanotrophs showed no evidence of immunoreactivity, whereas the putative somatomammotrophs exhibited a wide inter-animal range of immunoreactivity. Some imunostaining was also evident in the anterior and posterior part of the pars nervosa, at the interface of the pars distalis or pars intermedia with the pars nervosa.