MYXOZOAN COMMUNITY COMPOSITION AND DIVERSITY IN CLINICAL CASES OF PROLIFERATIVE GILL DISEASE IN MISSISSIPPI CATFISH AQUACULTURE.

An abundance of morphologically variable Henneguya species complicates the understanding of disease relationships between ictalurid catfish and myxozoan (Phylum: Cnidaria) parasites on North American aquaculture operations. Henneguya ictaluri, the cause of proliferative gill disease (PGD) in channel and hybrid catfish, is arguably the most important parasite of commercial catfish aquaculture in the southeastern United States. While research indicates arrested development and limited sporogenesis of H. ictaluri in channel (Ictalurus punctatus) × blue (Ictalurus furcatus) hybrid catfish, incidents of PGD persist in hybrid production systems. This work investigated the influence of fish host on myxozoan community composition and diversity within naturally infected gill tissues from diagnostic case submissions to the Aquatic Research and Diagnostic Laboratory in Stoneville, Mississippi, from 2017 to 2019. Gills collected from farm-raised catfish with clinical PGD were subjected to metagenomic amplicon sequencing of the myxozoan 18S SSU rDNA gene diagnostic variable region 3 (DVR3). Myxozoan community composition significantly differed between channel and hybrid catfish PGD cases, with channel catfish having more diverse community structures. Channel catfish gills had a greater relative abundance of H. ictaluri in 2017 and 2019, while no differences were observed in 2018. Importantly, H. ictaluri was present in all channel and hybrid catfish PGD cases across all years; however, H. ictaluri was not the most abundant myxozoan in almost half the cases examined, suggesting other myxozoan species may also contribute to PGD pathology. The detection of numerous known and unclassified myxozoan sequences in addition to H. ictaluri provides evidence PGD may involve mixed species infections. Furthermore, the presence of numerous unclassified myxozoan sequences in gill samples from clinical PGD cases indicates the number of described species from U.S. farm-raised catfish vastly underestimates the true myxozoan diversity present within the varied pond microcosms associated with catfish aquaculture.

Arrested Development of Henneguya ictaluri (Cnidaria: Myxobolidae) in ♀ Channel Catfish × â™‚ Blue Catfish Hybrids.

Henneguya ictaluri is the etiologic agent of proliferative gill disease (PGD) in farm-raised Channel Catfish Ictalurus punctatus and hybrid catfish in the southeastern United States, and significant annual losses are attributed to this disease. Research suggests that H. ictaluri infection dynamics in Blue Catfish I. furcatus and hybrid catfish (Channel Catfish × Blue Catfish) differ from those in Channel Catfish. Two separate infectivity trials were conducted to investigate H. ictaluri development in Channel Catfish, Blue Catfish, and their hybrids. On two separate occasions with two different year-classes, fish were exposed to pond water containing H. ictaluri actinospores and sampled weekly for 12 weeks (trial 1) or 14 weeks (trial 2). In trial 1, the presence of H. ictaluri was evaluated histologically and by quantitative PCR of fish tissues, including gills, blood, anterior kidney, brain, heart, liver, posterior kidney, spleen, and stomach. Henneguya ictaluri DNA was detected in significantly higher concentrations throughout multiple organ systems in the Channel Catfish compared to the hybrid catfish and Blue Catfish, with the gills having higher quantities. Myxospores were observed in Channel Catfish gill tissue at 8 weeks postexposure. No myxospores were observed in Blue Catfish or hybrid catfish. The second trial focused on gills only and yielded similar results, with Channel Catfish having significantly greater H. ictaluri DNA quantities than hybrids or Blue Catfish across all time points. Myxospores were observed in Channel Catfish beginning at 6 weeks postexposure and were found in 36% (58/162) of Channel Catfish sampled for molecular and histological analysis during weeks 6-14. Myxospores in hybrid catfish were sparse, with single pseudocysts observed in two hybrid catfish (1.2%) at 14 weeks postexposure. These results imply arrested development of H. ictaluri in hybrid catfish. As such, culture of hybrid catfish may be an effective management strategy to minimize the burden of PGD.

Population structure and phenotypic variation of Sclerotinia sclerotiorum from dry bean (Phaseolus vulgaris) in the United States.

The ascomycete pathogen Sclerotinia sclerotiorum is a necrotrophic pathogen on over 400 known host plants, and is the causal agent of white mold on dry bean. Currently, there are no known cultivars of dry bean with complete resistance to white mold. For more than 20 years, bean breeders have been using white mold screening nurseries (wmn) with natural populations of S. sclerotiorum to screen new cultivars for resistance. It is thus important to know if the genetic diversity in populations of S. sclerotiorum within these nurseries (a) reflect the genetic diversity of the populations in the surrounding region and (b) are stable over time. Furthermore, previous studies have investigated the correlation between mycelial compatibility groups (MCG) and multilocus haplotypes (MLH), but none have formally tested these patterns. We genotyped 366 isolates of S. sclerotiorum from producer fields and wmn surveyed over 10 years in 2003-2012 representing 11 states in the United States of America, Australia, France, and Mexico at 11 microsatellite loci resulting in 165 MLHs. Populations were loosely structured over space and time based on analysis of molecular variance and discriminant analysis of principal components, but not by cultivar, aggressiveness, or field source. Of all the regions tested, only Mexico (n = 18) shared no MLHs with any other region. Using a bipartite network-based approach, we found no evidence that the MCGs accurately represent MLHs. Our study suggests that breeders should continue to test dry bean lines in several wmn across the United States to account for both the phenotypic and genotypic variation that exists across regions.

Comparative Phenotypic and Genotypic Analysis of Edwardsiella Isolates from Different Hosts and Geographic Origins, with Emphasis on Isolates Formerly Classified as E. tarda, and Evaluation of Diagnostic Methods.

Edwardsiella spp. are responsible for significant losses in important wild and cultured fish species worldwide. Recent phylogenomic investigations have determined that bacteria historically classified as Edwardsiella tarda actually represent three genetically distinct yet phenotypically ambiguous taxa with various degrees of pathogenicity in different hosts. Previous recognition of these taxa was hampered by the lack of a distinguishing phenotypic character. Commercial test panel configurations are relatively constant over time, and as new species are defined, appropriate discriminatory tests may not be present in current test panel arrangements. While phenobiochemical tests fail to discriminate between these taxa, data presented here revealed discriminatory peaks for each Edwardsiella species using matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) methodology, suggesting that MALDI-TOF can offer rapid, reliable identification in line with current systematic classifications. Furthermore, a multiplex PCR assay was validated for rapid molecular differentiation of the Edwardsiella spp. affecting fish. Moreover, the limitations of relying on partial 16S rRNA for discrimination of Edwardsiella spp. and advantages of employing alternative single-copy genes gyrB and sodB for molecular identification and classification of Edwardsiella were demonstrated. Last, sodB sequencing confirmed that isolates previously defined as typical motile fish-pathogenic E. tarda are synonymous with Edwardsiella piscicida, while atypical nonmotile fish-pathogenic E. tarda isolates are equivalent to Edwardsiella anguillarum Fish-nonpathogenic E. tarda isolates are consistent with E. tarda as it is currently defined. These analyses help deconvolute the scientific literature regarding these organisms and provide baseline information to better facilitate proper taxonomic assignment and minimize erroneous identifications of Edwardsiella isolates in clinical and research settings.

Analysis of microcystin-LR and nodularin using triple quad liquid chromatography-tandem mass spectrometry and histopathology in experimental fish.

Microcystins (MCs) are hepatotoxic cyanobacterial metabolites produced sporadically in aquatic environments under favorable environmental conditions. Affinity of these toxins to covalently bind with protein phosphatases poses a challenge in their detection. Lemieux oxidation to release 2-methyl-3-methoxy-4-phenylbutyric acid (MMPB), a common moiety to all MCs congeners, has been used in detection of these compounds, however a lack of sensitivity has limited the usefulness of the method. In this study, modifications of the oxidation and solid phase extraction procedures, combined with a sensitive LC/MS/MS (liquid chromatography/mass spectrometry) detection, have resulted in 25 ng/g method detection limits in both liver and plasma samples. Samples harvested from six fingerling channel catfish (Ictalurus punctatus) dosed intraperitoneally with a sublethal MC-LR dose of 250 µg/kg were analyzed, and microcystin concentrations ranging from 370 to 670 ng/g in plasma and 566-1030 ng/g in liver were detected. Similarly, 250 µg/kg nodularin-dosed channel catfish fish were found to contain 835-1520 ng/g in plasma and 933-1140 ng/g in liver. Detection of the toxins in serum and liver combined with the presence of histopathological lesions consistent with these hepatocellular toxin in exposed fish and no positive findings in the control fish demonstrates the usefulness of this analytical procedure for the diagnosis of suspected algal toxicity cases.

Genetic and aggressiveness variation among Sclerotinia sclerotiorum dry bean isolates from Brazil fields / Variação genética e agressividade entre isolados de Sclerotinia sclerotiorum provindos de campos de feijão do Brasil

Sclerotinia sclerotiorum, infection of bean fields, has increased in Brazil. Fungicides application is the control strategy used due to lack of cultivars with complete disease resistance. To guide the use of isolates in resistance screening 25 S. sclerotiorum isolates from Brazilian dry bean fields were characterized using microsatellite markers, mycelial compatibility groups (MCGs) and aggressiveness. Microsatellite primer pairs were used to identify polymorphisms among the S. sclerotiorum isolates and MCGs were determined from interaction of all isolates grown sideby-side. Aggressiveness was derived from a straw test where fungal mycelium was placed over a cut bean stem and rated for disease progress. Data from microsatellite profiles grouped the 25 isolates into four clusters and seven MCGs were identified. No association among host cultivar and cluster or MCG of isolates was observed. For MCGs, 57% contained isolates sampled frequently over multiple locations and 43% contained isolates unique to locations. There were significant differences among isolates in aggressiveness within and between MCGs. The most aggressive isolates in resistance screening will be helpful in the identification of higher levels of resistance in bean germplasm/lines.

A infecção de Sclerotinia sclerotiorum em campos de feijoeiro tem aumentado no Brasil. A aplicação de fungicidas é a estratégia de controle utilizada devido à falta de cultivares com resistência completa á doença. Para orientar o uso de isolados visando resistência, 25 isolados de S. sclerotiorum coletados em campos de feijoeiro no Brasil foram caracterizados utilizando marcadores microssatélites, grupos de compatibilidade micelial (MCGs) e agressividade. Pares de primers de microssatélites foram utilizados para identificar polimorfismo entre os isolados de S. sclerotiorum e MCGs foram determinados a partir de interação dos isolados crescendo lado-a-lado. O teste de agressividade foi derivado a partir do straw test onde o micélio do fungo foi depositado sobre a haste cortada de feijoeiro e avaliado o progresso da doença. Os dados de microssatélites dos 25 isolados de S. Sclerotiorum foram agrupados em quatro grupos e identificados sete MCGs. Não foi observada associação entre a cultivar hospedeira e o cluster ou MCG dos isolados. Para MCGs, 57% continham isolados amostrados em vários locais e 43 % continham isolados de apenas um local. Houve diferença significativa entre os isolados na agressividade dentro e entre os MCGs. O isolado mais agressivo no screening de resistência será útil na identificação de níveis mais elevados de resistência em germoplasma/linhagens de feijoeiro.

Chronic pathology and longevity of Drepanocephalus spathans infections in juvenile Channel Catfish.

Drepanocephalus spathans (Digenea: Echinostomatidae) is a common parasite of the double-crested cormorant Phalacrocorax auritus. The cercariae of D. spathans have been shown infective to juvenile Channel Catfish Ictalurus punctatus. The developing metacercariae concentrate in the cranial regions, often occluding blood vessels at the base of the branchial arch, occasionally resulting in death. The purpose of this study was to determine how long metacercariae of D. spathans persist in experimentally challenged Channel Catfish. Two separate infectivity trials were conducted. In both trials, metacercariae persisted at least 49 d postinfection, although prevalence and intensity of infection decreased over time. In the first trial, juvenile catfish (1-3 g) were exposed over three consecutive days to 100, 100, and 80 cercariae/fish/d, respectively. Fish were sampled 7 d after the final exposure, and metacercariae were observed in 83.3% (five of six) of challenged fish. At 21 d postexposure, metacercariae were present in only 50% of exposed fish (three of six). No metacercaria were observed in fish sampled at 35 d, however, metacercariae were present in one of six (16.7%) fish sampled 49 and 70 d postexposure, respectively. A second challenge consisted of a 24-h pooled exposure of 500 cercariae per fish. Again, metacercariae were present in most (six of seven; 85.7%) fish at 7 d postexposure. At 21 d postexposure, metacercariae were only evident in one of seven (14.3%) sampled fish. No metacercariae were present in any fish at 35 d postchallenge, yet one of seven (14.3%) fish was positive at 49 d postchallenge. The second study was terminated at 63 d postchallenge, as all fish sampled (n = 14) were negative for metacercariae. These data suggest that cercariae of D. spathans are infective to juvenile Channel Catfish, although the infection appears short lived as metacercariae rarely persisted longer than 2 months.

Edwardsiella piscicida identified in the Southeastern USA by gyrB sequence, species-specific and repetitive sequence-mediated PCR.

A new Edwardsiella taxon was recently described from fishes of Europe and Asia. Phenotypically similar to E. tarda, extensive genetic and phenotypic characterization determined this new strain does not belong to any established Edwardsiella taxa, leading to the adoption of a new taxon, E. piscicida. Concurrent research in the USA also identified 2 genetically distinct taxa within the group of organisms traditionally classified as E. tarda. Comparisons of gyrB sequences between US isolates and E. piscicida from Europe and Asia identified several US isolates with >99.6% similarity to the gyrB sequence of the E. piscicida type strain (ET883) but <87% similarity to the E. tarda type strain (ATCC #15947). A discriminatory PCR was developed for the identification of E. tarda and 2 genetic variants of E. piscicida (E. piscicida and E. piscicida-like species). Using these PCR assays, a survey was conducted of 44 archived bacterial specimens from disease case submissions to the Aquatic Research and Diagnostic Laboratory (Stoneville, MS, USA) between 2007 and 2012. All 44 isolates, originally identified phenotypically and biochemically as E. tarda, were identified as E. piscicida by PCR. Repetitive sequence-mediated PCR (rep-PCR) analysis of these archived specimens suggests they are largely homogenous, similar to what has been observed for E. ictaluri. The gyrB sequence data, coupled with the E. piscicida specific-PCR and rep-PCR data, confirms that E. piscicida has been isolated from fish disease cases in the southeastern USA. Moreover, our survey data suggests E. piscicida may be more prevalent in catfish aquaculture than E. tarda.

Uromyces appendiculatus in Honduras: Pathogen Diversity and Host Resistance Screening.

Bean rust, caused by the fungus Uromyces appendiculatus, is a major constraint for common bean production worldwide. Virulence of U. appendiculatus collected from wild and cultivated Phaseolus spp. was examined in 28 locations across Honduras. Host accessions representing wild and domesticated Phaseolus spp. collected at the same sampling locations were evaluated for resistance against U. appendiculatus. In total, 91 pathotypes were identified from 385 U. appendiculatus isolates according to their virulence on each of the 12 host differentials. No significant difference in pathogen total virulence, measured as the mean disease score, was found between locations. However, significant differences were found in pathotype virulence among isolates collected from different Phaseolus spp. within a location. Moreover, when locations were compared on the basis of pathotype occurrence and frequency, differences among locations were evident. No two locations had the same pathotype composition. The most common pathotype was virulent on 9 of the 12 differential lines. A high number of resistant accessions were identified in Phaseolus coccineus and P. lunatus. Although most wild P. vulgaris accessions were highly susceptible, rust resistance was observed in P. vulgaris landraces collected from farmer's fields. Thirty-two (52%) of the accessions screened showed intermediate to high levels of resistance and, of those, 16% were P. coccineus accessions. Our findings support the hypothesis that interaction of U. appendiculatus in host populations composed of diverse Phaseolus spp. and genotypes has favored highly diverse and virulent pathotypes, creating a center for virulence diversity of the pathogen in Honduras. The high percentage of intermediate and highly resistant accessions identified in the present study supports the strategy of collecting plants from the center of diversity of a pathogen or in locations with high incidence of disease and pathogen diversity to maximize the probability of identifying new sources of resistance.

The pathology associated with visceral toxicosis of catfish.

Visceral toxicosis of catfish (VTC) syndrome was recognized in the late 1990 s and recently has been associated with exposure to Clostridium botulinum type E neurotoxin. Tentative diagnosis is based on clinical presentation and gross findings, and is confirmed by bioassay. In April 2009, channel catfish (Ictalurus punctatus) from 2 different farms presented with abnormal swimming behavior and mortalities. Nine fish were submitted to the Aquatic Research and Diagnostic Laboratory (Stoneville, Mississippi) for evaluation. Bacterial cultures from these fish were negative. Necropsy findings included intestinal intussusceptions, ascites, pale proximal intestines with engorged serosal blood vessels, splenic congestion, and a reticular pattern to the liver. Significant histopathologic findings were limited to cerebral, splenic, and hepatic congestion, splenic lymphoid depletion and perivascular edema, vascular dilation and edema of the gastrointestinal tract, and perivascular edema in the anterior and posterior kidneys. Intoxication from C. botulinum type E neurotoxin was suspected based on the clinical signs and lack of gross and microbiological evidence of an infectious disease process. The toxicosis was confirmed with a positive bioassay using serum collected from the submitted fish.

Variation in Sclerotinia sclerotiorum Bean Isolates from Multisite Resistance Screening Locations.

There is no complete resistance to Sclerotinia sclerotiorum, cause of white mold in dry bean (Phaseolus vulgaris). Variable resistance expression is one problem in screening for improved white mold resistance. With no previous information in the literature, pathogen variation in multisite screening nurseries was evaluated as one cause of diverse resistance expression. In all, 10 isolates of S. sclerotiorum used in greenhouse screening and 146 isolates collected from nine white mold field screening nurseries in major bean production areas in the United States were compared using mycelial compatibility groupings (MCGs) and an aggressiveness test. These 10 greenhouse screening isolates formed six MCGs. Among 156 field and greenhouse isolates, 64 MCGs were identified and 36 of those were each composed of a single unique isolate. Significant differences in isolate aggressiveness were found between some isolates in different MCGs but the isolates within an MCG did not differ in aggressiveness. High isolate variation found within and between field locations could influence the disease phenotype of putative white mold resistant germplasm. We next compared genotype and phenotype of isolates from screening nurseries and those from producer fields. Variability found in and among screening locations did reflect variability found in the four producer fields sampled. White mold resistance screening can be improved by knowledge of isolate genotypic and phenotypic characteristics.

Determination of florfenicol dose rate in feed for control of mortality in Nile tilapia infected with Streptococcus iniae.

A dose titration study was conducted to determine the dosage of florfenicol (FFC) in feed to control Streptococcus iniae-associated mortality in Nile tilapia Oreochromis niloticus. Six tanks were assigned to each of five treatments: (1) not challenged with S. iniae and fed unmedicated feed; (2) challenged with S. iniae by injection and fed unmedicated feed; (3) challenged with S. iniae and given FFC at 5 mg/kg of body weight (bw) in medicated feed; (4) challenged with S. iniae and given 10 mg FFC/kg bw; and (5) challenged with S. iniae and given 15 mg FFC/kg bw. Treatment was initiated the day after inoculation, and feed was administered for 10 d. Cumulative mortality was 0% in the unchallenged, untreated group; 35.8 +/- 4.4% (mean +/- SE) in the challenged, unmedicated group; 19.2 +/- 2.7% in the 5-mg/kg treated group, 12.5 +/- 3.8% in the 10-mg/kg group, and 2.5 +/- 1.1% in the 15-mg/kg group. The cumulative mortality was significantly less in each challenged, FFC-treated group than in the challenged, unmedicated controls (5 mg/ kg: P = 0.0156; 10 mg/kg: P = 0.0007; 15 mg/kg: P < 0.0001). The efficacy of the 10- and 15-mg/kg FFC dosages was studied in a separate dose confirmation study. Fish in all tanks were injected with S. iniae. At 4 h postinoculation, 10 tanks were assigned to each of three feed treatments: (1) unmedicated feed; (2) 10 mg FFC/kg bw; and (3) 15 mg FFC/kg bw. Cumulative mortality was 20.5 +/- 2.0% in the challenged, unmedicated group; 11.0 +/- 2.1% in the 10-mg/kg group; and 5.5 +/- 2.4% in the 15-mg/kg group. Mortality was significantly less in the medicated groups than in the challenged, unmedicated control group (10 mg/kg: P = 0.0270; 15 mg/kg: P = 0.0007). Fish in both studies were necropsied, cultured for bacteria, and examined for gross lesions. The minimum inhibitory concentration of FFC against S. iniae in both studies ranged from 0.5 to 1.0 microg/mL. Florfenicol was palatable, safe, and efficacious for control of Nile tilapia mortality due to S. iniae infection.

Evaluation of Resistance Screening Methods for Sclerotinia Stem Rot of Soybean and Dry Bean.

Three methods to identify levels of resistance to Sclerotinia sclerotiorum in soybean (Glycine max) and dry bean (Phaseolus vulgaris) were compared using multiple data analyses. The three methods were mycelial plug inoculations of cotyledons, cut stems, and detached leaves. Six S. sclerotiorum isolates of known relative aggressiveness were inoculated on each of three soybean and dry bean cultivars with varied response to S. sclerotiorum. For soybean, all three inoculation methods accurately identified isolate aggressiveness irrespective of cultivar, but identification of susceptible and partially resistant soybean cultivars was influenced by isolate. For dry bean, the cotyledon and cut stem methods accurately identified isolate aggressiveness, but identification of susceptible and partially resistant dry bean cultivars was influenced by isolate and inoculation method. The cut stem method had the smallest coefficient of variation and was more precise for detecting interactions. When considering root mean square residual error combined over species and experiments, coefficient of variation based on residual error, significance of isolate-by-cultivar interaction from ANOVA, rank correlation between pairs of methods, and sensitivity ratio for the three resistance screening methods under controlled environmental conditions, the cut stem method was statistically better than the cotyledon and detached leaf methods for evaluating resistance in soybean and dry bean cultivars.

Isolates of Uromyces appendiculatus with Specific Virulence to Landraces of Phaseolus vulgaris of Andean Origin.

Five isolates of the bean rust fungus Uromyces appendiculatus were shown to be specifically virulent on bean genotypes of Andean origin. This specificity was demonstrated by the virulence of five pairs of isolates on a differential set of 30 Phaseolus vulgaris landraces. Each isolate pair was from a different country in the Americas and consisted of one Andean-specific isolate and one nonspecific isolate. Of the differential P. vulgaris landraces, 15 were of Middle American origin and 15 were of Andean origin. The Andean-specific rust isolates were highly virulent on Andean landraces but not on landraces of Middle American origin. Rust isolates with virulence to Middle American landraces were also generally virulent on Andean material; no truly Middle American-specific isolates were found. Random amplified polymorphic DNA (RAPD) analysis of the rust isolates also distinguished the two groups. Four of the Andean-specific rust isolates formed a distinct group compared to four of the nonspecific isolates. Two of the isolates, one from each of the two virulence groups, had intermediate RAPD banding patterns, suggesting that plasmagomy but not karyogamy occurred between isolates of the two groups.