Real-time quantitative polymerase chain reaction (QPCR) to identify Myxobolus cerebralis in rainbow trout Oncorhynchus mykiss

This study describes the development of a TaqMan real-time quantitative polymerase chain reaction (QPCR) technique using the heat-shock protein 70 (Hsp 70) and 18S ribosomal DNA (18S rDNA) sequences to identify Myxobolus cerebralis and attempt to quantify infection severity within rainbow trout fry Oncorhynchus mykiss. Rainbow trout for this study were exposed to M. cerebralis under natural river conditions and examined for infection by histology, polymerase chain reaction (PCR) and QPCR analysis at 900 Celsius temperature units (CTUs) following exposure. Detection sensitivity by QPCR was shown to be equal to traditional PCR but greater than histopathology. Primer/probe combinations developed for this study were capable of specifically detecting M. cerebralis DNA in infected fish tissue and single triactinomyxon (TAM) spores with a sensitivity of 12.5 and 6.3 pg microl(-1) of DNA for the Hsp 70 and 18S rDNA sequences, respectively. A strong relationship between QPCR and infection severity was found for the Hsp 70 probe when parasite copy number and histology scores of 0-4 were compared (R2 = 0.96, p = 0.003). However, a reduction in copy number was observed at higher histology scores for the 18S probe (scores of 4 and 5) and the Hsp 70 probe (score of 5). The results of this study demonstrate that QPCR analysis is an effective tool for detecting M. cerebralis in fish tissue and may provide a relative indication of infection severity.     

Epizootic cutaneous papillomatosis in roach Rutilus rutilus: sex and size dependence,seasonal occurrence and between-population differences

Validation of a single round PCR-based assay to confirm as Myxobolus cerebralis myxospores obtained from pepsin-trypsin digest preparations is described. The assay is a modification of a PCR assay published previously, based on the amplification of a segment of the gene encoding the 18S ribosomal subunit of M. cerebralis. The sensitivity, specificity and upper and lower detection limits were determined using known M. cerebralis and non-M. cerebralis myxospores and M. cerebralis-free fish. The sensitivity of PCR confirmation was 100% (95% confidence interval of 83.2-100%). The specificity was 100% (95% confidence interval of 87.2-100%). The upper detection limit was approximately 100,000 myxospores per reaction; the lower detection limit was approximately 50 myxospores per reaction. Given the high sensitivity and specificity of the assay, substitution of this assay for histologic confirmation of M. cerebralis infection is encouraged.     

Detection by PCR of hepatopancreatic parvovirus (HPV) and other viruses in hatchery-reared Penaeus monodon postlarvae

The prevalence of hepatopancreatic parvovirus (HPV), monodon baculovirus (MBV) and white spot syndrome virus (WSSV) in samples of Penaeus monodon postlarvae (PL10 to PL20, 10 to 20 d old postlarvae) in India was studied by PCR. Samples collected from different hatcheries, and also samples submitted by farmers from different coastal states, were analyzed. HPV was detected in 34%) of the hatchery samples and 31% of the samples submitted by farmers, using a primer set designed for detection of HPV from P. monodon in Thailand. However, none of these samples were positive using primers designed for detection of HPV from P. chinensis in Korea. This indicated that HPV from India was more closely related to HPV from P. monodon in Thailand. MBV was detected in 64% of the samples submitted by the farmers and 71% of the hatchery samples. A total of 84 % of the samples submitted by farmers, and 91% of the hatchery samples, were found positive for WSSV. Prevalence of concurrent infections by HPV, MBV and WSSV was 27% in hatchery samples and 29%, in samples submitted by farmers. Only 8% of the hatchery samples and 16% of the samples submitted by farmers were negative for all 3 viruses. This is the first report on the prevalence of HPV in P. monodon postlarvae from India.     

Efficacy of passive sand filtration in reducing exposure of salmonids to the actinospore of Myxobolus cerebralis

The aquatic oligochaete Tubifex tubifex parasitized by Myxobolus cerebralis releases triactinomyxon (TAM) actinospores that can infect some species of salmonids and cause salmonid whirling disease. Silica sand was tested as a filtration medium for removal of TAMs from water containing the parasite. Laboratory tests indicated sand filtration removed > 99.99% of TAMs. In 2 different field tests, groups of 1 mo old rainbow trout Oncorhynchus mykiss were exposed for 2 wk to filtered and unfiltered water from a spring-fed pond enzootic for M. cerebralis. In November 2000, the exposure dose was estimated as between 3 and 5 TAMs fish(-1). During a March 2001 exposure, the estimated dose was between 286 and 404 TAMs fish(-1). Fish were held for 6 mo post exposure (p.e.) in laboratory aquaria for observation and evidence of clinical signs of whirling disease. We used 4 diagnostic techniques to assess the prevalence and severity of infection by M. cerebralis among fish exposed to filtered and unfiltered water. These included polymerase chain reaction (PCR) for genomic DNA of the parasite, histological evaluation for tissue damage, tissue digestion for quantification of cranial myxospores of the parasite, and total non-sampling mortality that occurred over 6 mo p.e. All diagnostic tests verified that the prevalence and severity of infection was significantly reduced among fish in treatment groups exposed to filtered water compared to those exposed to unfiltered water in both the low-dose and high-dose exposures.     

Diagnostic polymerase chain reaction assay to detect Kudoa neurophila (Myxozoa: Multivalvulida) in a marine finfish hatchery

A single-round polymerase chain reaction (PCR) diagnostic assay was developed from a small subunit ribosomal DNA (SSU rDNA) gene sequence to detect the myxozoan parasite Kudoa neurophila, the causative agent of myxozoan disease in the hatchery reared marine finfish, striped trumpeter Latris lineata (Forster). The assay was developed for use as a disease control management tool in a hatchery system specifically designed to research and produce marine finfish such as striped trumpeter juveniles for aquaculture. The assay is sufficiently species specific and sensitive enough to detect a small fragment of the parasite's SSU rDNA. At the lower limits of detection, the test is consistently positive to an estimated 0.1 spore or 60 fg of parasite DNA per 25 microl PCR reaction in serial dilution and positive to an estimated 0.1 spore in 25 mg of infected fish CNS tissue (4 spores g(-1). Specifically, the test is capable of detecting early stages of the life cycle within the fish host and consequently diagnosing an infection not normally detected using traditional histological techniques. The test is also effective for screening water supplies and prey species cultures throughout the hatchery system to determine bio-security efficacy, to assist in identification of an alternate or other primary fish host, to indicate the location of potential disease reservoirs, and to enable a targetted approach to disease prevention.     

Reduced reproductive success of hatchery coho salmon in the wild: insights into most likely mechanisms

Supplementation of wild salmonids with captive-bred fish is a common practice for both commercial and conservation purposes. However, evidence for lower fitness of captive-reared fish relative to wild fish has accumulated in recent years, diminishing the apparent effectiveness of supplementation as a management tool. To date, the mechanism(s) responsible for these fitness declines remain unknown. In this study, we showed with molecular parentage analysis that hatchery coho salmon (Oncorhynchus kisutch) had lower reproductive success than wild fish once they reproduced in the wild. This effect was more pronounced in males than in same-aged females. Hatchery spawned fish that were released as unfed fry (age 0), as well as hatchery fish raised for one year in the hatchery (released as smolts, age 1), both experienced lower lifetime reproductive success (RS) than wild fish. However, the subset of hatchery males that returned as 2-year olds (jacks) did not exhibit the same fitness decrease as males that returned as 3-year olds. Thus, we report three lines of evidence pointing to the absence of sexual selection in the hatchery as a contributing mechanism for fitness declines of hatchery fish in the wild: (i) hatchery fish released as unfed fry that survived to adulthood still had low RS relative to wild fish, (ii) age-3 male hatchery fish consistently showed a lower relative RS than female hatchery fish (suggesting a role for sexual selection), and (iii) age-2 jacks, which use a sneaker mating strategy, did not show the same declines as 3-year olds, which compete differently for females (again, implicating sexual selection).     

Population characteristics of pallid sturgeon (Scaphirhynchus albus (Forbes & Richardson, )) in the Lower Missouri River

Population characteristics of pallid sturgeon Scaphirhynchus albus in the lower Missouri River are relatively unknown. Therefore, data collected from the Nebraska Game and Parks Commission Pallid Sturgeon Population Assessment Program was synthesized to (i) document the population structure of pallid sturgeon by origin (hatchery‐reared or wild), gender, and reproductive readiness, (ii) document the minimum size and age‐at‐maturity by gender, and (iii) document the fecundity rates of the fish that were successfully spawned in the hatchery. During this 4‐year study (2008–2011), relative abundance for wild and hatchery‐reared pallid sturgeon collected with gill nets did not significantly change whereas relative abundance for wild fish using trot lines declined significantly. The proportion of hatchery‐reared pallid sturgeon increased annually, with the population being composed primarily of hatchery‐reared fish. The proportion of reproductively ready females to non‐reproductively ready females was 1 : 2.0, compared to male ratios at 1 : 0.9. Minimum fork length‐at‐maturity was estimated for females at 788 mm and for males at 798 mm. Minimum age‐at‐maturity for hatchery‐reared released fish was age‐9 for females and age‐7 for males. Highest relative fecundity, based on the ovosomatic index, was 10% with an overall mean of 7%. The number of eggs per ml (egg size) was not correlated with fork length (P = 0.0615) or weight (P = 0.0957). Relative condition factor (Kn) for females was significantly different by reproductive condition (P = 0.0014) and Kn for males did not differ between reproductive conditions (P = 0.2634). Detecting shifts in population characteristics are essential not only to understand population dynamics since hatchery inputs and natural perturbations continue to change the population structure but also to assess species recovery efforts to ensure long‐term species sustainability.     

Cross-shoal variability in the feeding habits of migrating Atlantic cod (Gadus morhua)

Prey intake and selection were related to within-shoal position for Atlantic cod (Gadus morhua) engaged in annual migration across the Newfoundland shelf in the northwest Atlantic. Comparisons made among fish occupying five regions from the front to rear of a large (>10 km across) migrating shoal indicated that leading fish, or scouts, were larger, ate more food by weight, and had a more varied diet than did fish at other positions. Also, scouts consumed more preferred prey types (fish and pelagic invertebrates) than did fish at other positions. In contrast, trailing fish consumed few fish prey but a larger proportion of benthic invertebrates. Our results are the first to document systematic heterogeneous feeding success among members of a free-ranging and migrating fish shoal in the open ocean.     

Detection of gastric Helicobacter species in free-ranging lynx (Lynx lynx) and red foxes (Vulpes vulpes) in Sweden

Specimens of gastric mucosa and liver of 25 free-ranging Eurasian lynx (Lynx lynx), and four red foxes (Vulpes vulpes) shot in Sweden during 1999-2000, were investigated for the presence of Helicobacter species. Histopathology, bacteriologic culture and urease test, Helicobacter genus-specific 16S rDNA PCR analysis, and DNA sequence analysis were applied. Numerous Helicobacter-like organisms were observed histologically in the gastric mucosa of one fox. Helicobacter spp. were detected in the stomach by PCR analysis in 17 (68%) of the lynx and in three (75%) of the foxes. Seven of the positive lynx were also positive in the urease test. PCR fragments, amplified from lynx and foxes, were sequenced and compared with those of known Helicobacter species. PCR products from lynx were closely related (>or=98% homology) to H. heilmannii, and PCR fragments from foxes demonstrated close homology to H. heilmannii and H. salomonis. No Helicobacter spp. or Helicobacter-like organisms could be cultured. The PCR analysis of the liver was negative for all animals. The pathologic significance of the presence of Helicobacter spp. in the stomach of free-ranging lynx and foxes remains uncertain.     

Using parentage analysis to estimate rates of straying and homing in Chinook salmon (Oncorhynchus tshawytscha)

We used parentage analysis based on microsatellite genotypes to measure rates of homing and straying of Chinook salmon (Oncorhynchus tshawytscha) among five major spawning tributaries within the Wenatchee River, Washington. On the basis of analysis of 2248 natural‐origin and 11594 hatchery‐origin fish, we estimated that the rate of homing to natal tributaries by natural‐origin fish ranged from 0% to 99% depending on the tributary. Hatchery‐origin fish released in one of the five tributaries homed to that tributary at a far lower rate than the natural‐origin fish (71% compared to 96%). For hatchery‐released fish, stray rates based on parentage analysis were consistent with rates estimated using physical tag recoveries. Stray rates among major spawning tributaries were generally higher than stray rates of tagged fish to areas outside of the Wenatchee River watershed. Within the Wenatchee watershed, rates of straying by natural‐origin fish were significantly affected by spawning tributary and by parental origin: progeny of naturally spawning hatchery‐produced fish strayed at significantly higher rates than progeny whose parents were themselves of natural origin. Notably, none of the 170 offspring that were products of mating by two natural‐origin fish strayed from their natal tributary. Indirect estimates of gene flow based on F_ST statistics were correlated with but higher than the estimates from the parentage data. Tributary‐specific estimates of effective population size were also correlated with the number of spawners in each tributary.     

Neospora caninum-like oocysts observed in feces of free-ranging red foxes (Vulpes vulpes) and coyotes (Canis latrans)

The aim of this study was to examine the feces of free-ranging foxes and coyotes for the presence of Neospora caninum oocysts. Feces were collected from 271 foxes and 185 coyotes in the Canadian province of Prince Edward Island, processed by sucrose flotation, and examined by light microscopy for the presence of coccidian oocysts. In 2 fox and 2 coyote samples, oocysts morphologically and morphometrically similar to oocysts of N. caninum were observed. DNA was extracted from these samples and subjected to nested polymerase chain reaction (PCR) using primers to the N. caninum-specific Nc5 genomic sequence. Through DNA sequencing, alignment of the sequences of at least 3 clones from each isolate to sequences deposited in GenBank revealed 95-99% similarity to the Nc5 sequence of N. caninum. PCR using primers specific for Hammondia heydorni failed to yield an amplification product from these DNA samples.     

Susceptibility of two strains of rainbow trout (one with suspected resistance to whirling disease) to Myxobolus cerebralis infection

The susceptibility of 2 strains of rainbow trout Oncorhynchus mykiss, 1 from North America (TL) and 1 from Germany (GR), to Myxobolus cerebralis (the cause of salmonid whirling disease) was assessed following exposure to the infectious stages (triactinomyxons). Two laboratory experiments were conducted with age-matched rainbow trout of each strain. At the beginning of the study, the 2 trout strains were aged ca. 570 degree-days in Expt 1, and ca. 999 degree-days in Expt 2. In both experiments, replicate groups of each trout strain were exposed to 10, 100, 1000 or 10000 triactinomyxons (TAMs) fish(-1) for 2 h. The fish were then held in aquaria receiving 15 degrees C well-water. Severity of infection was evaluated 5 mo after exposure by presence of clinical signs (whirling and/or black tail), prevalence of infection, severity of microscopic lesions, and spore counts. Clinical signs of whirling disease were evident only in the younger fish exposed in Expt 1: These occurred first among TL rainbow trout at the highest dose at 6 to 7 wk post exposure and then 2 wk later in fish at the 1000 TAMs dose. Black tail was also observed among GR rainbow trout at the 10000 TAMs dose only, but in fewer fish. The prevalence of infection, spore numbers, and severity of microscopic lesions due to M. cerebralis among GR rainbow trout were less at all doses compared to TL rainbow trout. Risk of infection analyses showed that TL rainbow trout were more prone to infection at the lower doses than GR trout. Mean spore counts were consistently (10- to 100-fold) less in GR than TL trout at doses of 1000 TAMs or lower. Microscopic lesions increased with increasing dose in both strains of rainbow trout. The mechanisms underlying the greater resistance of the GR strain to M. cerebralis infections are unknown, but are under investigation as part of a long-term project to determine the basis for resistance and susceptibility of salmonid fishes to whirling disease.     

Detection of Aeromonas salmonicida, causal agent of furunculosis in salmonid fish, from the tank effluent of hatchery-reared Atlantic salmon smolts.

The fish pathogen, Aeromonas salmonicida, could be detected only by bacteriological culture from the kidney of dead or moribund fish in one tank in a hatchery rearing Atlantic salmon (Salmo salar L.) smolts. However, by using a DNA probe specific for this species, allied to a PCR assay, the pathogen could be detected in water, feces and effluent samples taken from this fish tank. Also, the presence of the pathogen was found in effluent samples from two fish tanks containing apparently healthy fish. Subsequently, the presence of pathogen in these tanks was confirmed by an increase in the daily mortality rate and by a plate culture from moribund fish.     

Molecular survey of Anaplasma phagocytophilum and Ehrlichia canis in red foxes (Vulpes vulpes) from central Italy

During the 2007-2008 hunting season, 150 spleen samples were collected from free-ranging red foxes (Vulpes vulpes) in central Italy. The specimens were tested by two nested PCR assays to detect DNA of Anaplasma phagocytophilum, etiologic agent of granulocytic ehrlichiosis of animals and humans, and DNA of Ehrlichia canis, which causes the monocytic ehrlichiosis in canids. None of the foxes were PCR-positive for E. canis; 25 (16.6%) were positive for A. phagocytophilum. No specific gross alterations were detected at necropsy, and no histopathologic lesions found on PCR-positive spleen samples.     

Prevalence of Myxobolus cerebralis infections among genetic lineages of Tubifex tubifex at three locations in the Madison River, Montana

Host biodiversity can impact disease risk and influence the transmission of parasitic disease. Stream sediment-dwelling worms, Tubifex tubifex (Clitellata: Oligochaeta), are the definitive host of the parasite Myxobolus cerebralis (Myxozoa: Myxosporea), which causes whirling disease in salmonid fishes. Genetic diversity of T. tubifex is correlated with host susceptibility to M. cerebralis , and mitochondrial Lineage III is generally shown to be more likely to be infected and produce the triactinomyxon (TAM) spores than other lineages. We determined the mitochondrial lineage, relative abundance, and prevalence of infection of T. tubifex collected at 3 sites in the Madison River, Montana, where previous study had shown variation in whirling disease prevalence and severity in caged trout fry. We also compared visual identification of TAMs released from cultured worms with a molecular genetic assay (diagnostic polymerase chain reaction [PCR]) for parasite detection of both infected and uninfected worms. We estimated that mitochondrial Lineage III was most abundant at the site previously shown to have high fish disease and was also most likely to be infected. The 2 techniques for detecting parasite infection did not always agree, and the likelihood of PCR (+) and spore (-) was not significantly different from PCR (-) and spore (+). Differences in the relative infection prevalence for these 2 lineages may explain the wide range of infection in natural streams.     

Small Subunit Ribosomal RNA Sequences Unite Alternate Actinosporean and Myxosporean Stages of Myxobolus cerebralis the Causative Agent of Whirling Disease in Salmonid Fish

ABSTRACT. The alternating myxosporean and actinosporean stages of the myxozoan parasitc Myxobolus cerebralis (Hofer 1903) from its salmonid fish and aquatic oligochaete hosts, respectively, were compared for sequence homology of the small subunit (18S) ribosomal RNA genes. A 99.8% similarity between the sequences of these two stages was substantially greater than that of M. cerebralis compared to two other Myxobolus sp. from salmonid fish. Our results are the first molecular evidence confirming the alternating stages initially described by Wolf and Markiw [25] for the life cycle of M. cerebralis but found in two different taxonomic classes (Myxosporea and Actinosporea) are indeed forms of the same organism. Sequencing of rRNA genes of the actinosporean stage followed by development of specific primers for DNA amplification of the myxosporean stage, as in our study, should be applied to solve other myxozoan life cycles. Additionally, these approaches will in the future provide useful diagnostic reagents for the detection and study of this important group of fish pathogens.     

Randomly amplified polymorphic DNA analysis of four different populations of the Indian major carp, Labeo rohita (Hamilton)

The level of genetic variation provides the raw material for selective improvement of a stock. Random amplified polymorphic DNA (RAPD) assay was used to assess the genetic variation in three rivers: the Halda, the Jamuna and the Padma as well as in one hatchery population of the commercially important Indian major carp, Labeo rohita. RAPD markers were amplified from DNA samples of 35 fish from each of the four populations using six decamer random primers. The polymorphic loci proportions were 0.33, 0.28, 0.28 and 0.26 and Nei's gene diversity values were 0.06, 0.07, 0.06 and 0.05 for the Halda, the Jamuna, the Padma and the hatchery populations, respectively. The pairwise population differentiation (F_ST) values indicated a low level of genetic differentiation between the population pairs. From the unweighted pair group method of arithmetic mean (UPGMA) dendrogram based on Nei's genetic distances a correlation between genetic affinities and geographical area was found. The populations were segregated into two groups: the Halda in one group and the Jamuna, the Padma and the hatchery in another group. Overall, the RAPD technique can be introduced as a tool in the population genetics of the rohu fish to provide information on their genetic stock structure.     

Serum alpha- and gamma-tocopherols, retinol, retinyl palmitate, and carotenoid concentrations in captive and free-ranging bottlenose dolphins (Tursiops truncatus)

Concentrations of retinol, retinyl palmitate, beta-carotene, alpha-carotene, cryptoxanthin, lutein, lycopene, alpha-tocopherol, and gamma-tocopherol were measured in blood samples collected from 15 captive and 55 free-ranging bottlenose dolphins (Tursiops truncatus). From June 1991 to June 1994, blood samples were collected from captive animals residing at two locations; at Seven Seas (Brookfield Zoo, Brookfield, IL) and Hawk's Cay (Marathon Key, FL). Blood samples were collected from free-ranging animals from June 1991 to June 1996. Retinol levels were not significantly different between captive dolphin groups. However, Seven Seas animals had higher (P < 0.01) serum retinol concentrations compared to free-ranging animals (0.061 vs 0.041 microgram/ml). Retinyl palmitate was not detected in the serum of captive or free-ranging dolphins. Alpha-tocopherol levels were significantly (P < 0.05) higher for Seven Seas dolphins (16.4 micrograms/ml) than for Hawk's Cay (13.0 micrograms/ml) and free-ranging dolphins (12.5 micrograms/ml). Gamma-tocopherol concentrations were similar among captive and free-ranging dolphins. Free-ranging dolphins showed levels of circulating carotenoids (lutein and beta-carotene) while the captive animals did not. Additional carotenoids (lycopene, alpha-carotene and cryptoxanthin) were analyzed but not detected in any samples. Serum vitamin differences between captive and free-ranging dolphins may reflect the natural diet or indicate some potential biological or nutritional status significance.