Evaluation of Culture Time and Media in an In Vitro Testis Organ Culture System.

BACKGROUND: The complexity of spermatogenesis makes development of appropriate in vitro testis models challenging. A novel in vitro mouse testis culture system has been reported but not yet evaluated as an alternative model for male reproductive toxicity testing. We assessed the effects of media composition on sperm differentiation and testis morphology of cultured mouse testis fragments. METHODS: Testes from postnatal day 5 B6:CBA-Tg(Acrv1-EGFP)2727Redd/J male mice were cultured in knockout serum replacement (KSR) or Albumax I (Albumax) medium. Enhanced green fluorescent protein (EGFP) expression was examined on days 35, 42, 45, and 49 of culture. Histology and flow cytometry were performed for testis morphology and spermatid differentiation. RESULTS: EGFP signals were first observed in round spermatids on day 22 of culture (corresponding to postnatal day 27) and were observed until the end of culture, indicating testis-specific protein expression. A-kinase anchor protein 4 expression, a marker of elongated spermatid (step 15-16) occurred earlier in explants cultured in KSR than Albumax medium (typically day 35 and after day 42 of culture, respectively). The percentage of seminiferous tubules with elongated spermatid was higher in Albumax than KSR medium from days 45 to 49 of culture. CONCLUSION: Albumax medium may facilitate or support better morphology and spermatid production than KSR medium. Further studies need to improve spermatid production and refinement of this in vitro testis culture system that may be useful as a supplement to current male reproductive toxicity testing or an alternative model in cases where in vivo testing may be unfeasible. Birth Defects Research 109:465-474, 2017. © 2017 Wiley Periodicals, Inc.

Transcriptomics analysis of early embryonic stem cell differentiation under osteoblast culture conditions: Applications for detection of developmental toxicity.

The mouse embryonic stem cell test (mEST) is a promising in vitro assay for predicting developmental toxicity. In the current study, early differentiation of D3 mouse embryonic stem cells (mESCs) under osteoblast culture conditions and embryotoxicity of cadmium sulfate were examined. D3 mESCs were exposed to cadmium sulfate for 24, 48 or 72h, and whole genome transcriptional profiles were determined. The results indicate a track of differentiation was identified as mESCs differentiate. Biological processes that were associated with differentiation related genes included embryonic development and, specifically, skeletal system development. Cadmium sulfate inhibited mESC differentiation at all three time points. Functional pathway analysis indicated biological pathways affected included those related to skeletal development, renal and reproductive function. In summary, our results suggest that transcriptional profiles are a sensitive indicator of early mESC differentiation. Transcriptomics may improve the predictivity of the mEST by suggesting possible modes of action for tested chemicals.

Detection and surveillance of viral hemorrhagic septicemia virus using real-time RT-PCR. I. Initial comparison of four protocols.

Eight laboratories worked collectively to evaluate 4 real-time RT-PCR (rRT-PCR) protocols targeting viral hemorrhagic septicemia virus (VHSV) being considered for deployment to a USA laboratory testing network. The protocols utilized previously published primers and probe sets developed for detection and surveillance of VHSV. All participating laboratories received and followed a standard operating protocol for extraction and for each of the rRT-PCR assays. Performance measures specifically evaluated included limit of detection (defined as the smallest amount of analyte in which 95% of the samples are classified as positive), analytical specificity, assay efficiency across genotype representatives, within- and between-plate variation within a laboratory, and variation between laboratories using the same platform, between platforms, and between software versions. This evaluation clearly demonstrated that the TaqMan®-based assay developed by Jonstrup et al. (2013; J Fish Dis 36:9-23) produced the most consistent analytical performance characteristics for detecting all genotypes of VHSV across the 8 participating laboratories.

Detection and surveillance of viral hemorrhagic septicemia virus using real-time RT-PCR. II. Diagnostic evaluation of two protocols.

Two real-time reverse transcription polymerase chain reaction (rRT-PCR) assays under consideration for deployment to multiple testing laboratories across the USA were evaluated for diagnostic sensitivity and specificity on tissue homogenates obtained from natural and experimental viral hemorrhagic septicemia (VHS)-infected fish. Estimates for diagnostic specificity using virus isolation as the reference method were similar between laboratories regardless of the assay. Diagnostic sensitivity estimates of 0.96 (95% CI: 0.95, 0.97) for Jonstrup et al. (2013)'s assay (J Fish Dis 36:9-23) exceeded the diagnostic sensitivity of 0.85 (95% CI: 0.83, 0.87) for Phelps et al. (2012)'s assay (J Aquat Anim Health 24:238-243). The Jonstrup rRT-PCR assay is robust as demonstrated by high sensitivity and specificity estimates across laboratories and can be used as a valuable tool for targeted surveillance and for testing of suspect VHSV samples.

Rapid quantitative detection of Aeromonas hydrophila strains associated with disease outbreaks in catfish aquaculture.

A new strain of Aeromonas hydrophila has been implicated in significant losses in farm-raised catfish. Outbreaks attributable to this new strain began in Alabama in the summer of 2009 and have spread to Arkansas and Mississippi in subsequent years. These outbreaks mostly afflicted market-sized fish and resulted in considerable losses in short periods of time. The present research was designed to develop an expeditious diagnostic procedure to detect the new strains of A. hydrophila due to the rapid onset and biosecurity concerns associated with this new disease. A discriminatory quantitative polymerase chain reaction assay was developed using gene sequences unique to the virulent strains identified in a related comparative genomic study. Using this assay, suspect colonies on a culture plate can be positively identified as the new strain within 2 hr. The assay is repeatable and reproducible with a linear dynamic range covering 8 orders of magnitude and a sensitivity of approximately 7 copies of target DNA in a 15-µl reaction. In addition, the assay is able to detect and quantify the virulent strain from catfish tissues (0.025 g), pond water (40 ml), and sediments (0.25 g) with a sensitivity limit of approximately 100 bacteria in a sample. This assay provides rapid discrimination between the new virulent strain and more common A. hydrophila and is useful for epidemiological studies involving the detection and quantification of the virulent strain in environmental samples and fish tissues.

Mortality and carrier status of bluegills exposed to viral hemorrhagic septicemia virus genotype IVb at different temperatures.

The emergence of the viral hemorrhagic septicemia virus (VHSV) genotype IVb (VHSV-IVb) in the Great Lakes of North America has led to concern that the virus might spread to natural fisheries and aquaculture in the southern USA. We exposed bluegills Lepomis macrochirus to VHSV-IVb by intraperitoneal injection at six temperatures from 10 degrees C to 30 degrees C and followed the disease course by quantitative real-time reverse transcriptase polymerase chain reaction (qrt-RT-PCR). Mortality of injected fish was 90% at 10 degrees C, 35% at 14 degrees C, and 10% at 18 degrees C; no mortality attributable to VHSV was observed at temperatures of 22-30 degrees C. In survivors tested at 21 d postchallenge, viral copies and prevalence determined by qrt-RT-PCR were inversely related to temperature, and VHSV-IVb could not be detected in fish held at temperatures above 22 degrees C. Similar results were obtained for bluegills that were exposed by cohabitation with the intraperitoneally injected fish. Acclimation of the fish to 12 degrees C after 21 d at higher temperatures did not appear to cause a re-emergence of the virus. Based on our findings, the temperature range of VHSV-IVb appears to be the same as published values for VHSV genotype I, which has an optimum of 9-12 degrees C and an upper limit of 18-20 degrees C.

Replication and persistence of VHSV IVb in freshwater turtles.

With the emergence of viral hemorrhagic septicemia virus (VHSV) strain IVb in the Great Lakes of North America, hatchery managers have become concerned that this important pathogen could be transmitted by animals other than fish. Turtles are likely candidates because they are poikilotherms that feed on dead fish, but there are very few reports of rhabdovirus infections in reptiles and no reports of the fish rhabdoviruses in animals other than teleosts. We injected common snapping turtles Chelydra serpentine and red-eared sliders Trachemys scripta elegans intraperitoneally with 10(4) median tissue culture infectious dose (TCID50) of VHSV-IVb and 21 d later were able to detect the virus by quantitative real-time reverse transcriptase PCR (qrt-RTPCR) in pools of kidney, liver, and spleen. In a second experiment, snapping turtles, red-eared sliders, yellow-bellied sliders T. scripta scripta, and northern map turtles Grapetemys geographica at 14 degrees C were allowed to feed on tissues from bluegill dying of VHSV IVb disease. Turtle kidney, spleen, and brain pools were not positive by qrt-RTPCR on Day 3 post feeding, but were positive on Days 10 and 20. Map turtles on Day 20 post-feeding were positive by both qrt-RTPCR and by cell culture. Our work shows that turtles that consume infected fish are a possible vector for VHSV IVb, and that the fish rhabdoviruses may have a broader host range than previously suspected.

Detection and prevalence of the nonsyncytial American grass carp reovirus Aquareovirus G by quantitative reverse transcriptase polymerase chain reaction.

The American grass carp reovirus (AGCRV) Aquareovirus G is not strongly associated with disease in fish, but it is often detected by cell culture during routine inspections of healthy fish. The cytopathic effect of AGCRV does not involve the typical syncytia associated with most aquareoviruses. Instead, the AGCRV produces a pattern of cell rounding that is very similar to that produced by rhabdoviruses, including those that are highly regulated. We have developed a quantitative polymerase chain reaction assay that can be used to identify AGCRV in cell cultures or directly on fish tissues. The assay detects as few as two copies of the plasmid template, has a coefficient of variation of 15% among assays performed on different days, and does not cross-react with any other aquareoviruses tested. Assays performed on tissues of cultured golden shiners Notemigonus crysoleucas and fathead minnow Pimephales promelas revealed a high prevalence of infection among healthy fish but no association with disease.

Are all koi ulcer cases associated with infection by atypical Aeromonas salmonicida? Polymerase chain reaction assays of koi carp skin swabs submitted by hobbyists.

Infection by atypical Aeromonas salmonicida is regarded as the cause of ulcer disease (KUD) in koi carp Cyprinus carpio and goldfish Carassius auratus. However, other causes--including parasites, viral infection, and fungi--have been proposed. In our diagnostic work, we often fail to isolate A. salmonicida even when clear clinical signs of KUD are present. This failure may be because these fastidious and slow-growing bacteria are difficult to isolate in culture or because the bacteria are not actually present in the lesions. In this study, we used polymerase chain reaction (PCR) to detect A. salmonicida in DNA samples swabbed from koi carp ulcers. These alcohol-preserved samples were collected and submitted by hobbyists and included 40 separate cases from 12 different states. We identified atypical A. salmonicida by PCR in 52 of 62 samples submitted and in 33 of 40 unique cases. The negative findings for A. salmonicida by PCR could all be attributed to high water temperatures, prior antibiotic use, poor sample quality, or misdiagnosis of columnaris disease as KUD. Tests for Aphanomyces invadans by PCR were negative in every case. This work confirms that A. salmonicda is still the predominant cause of KUD and that our negative culture results were most likely due to technical failures rather than an absence of A. salmonicda in the ulcer lesions.