Design standards for experimental and field studies to evaluate diagnostic accuracy of tests for infectious diseases in aquatic animals

Design and reporting quality of diagnostic accuracy studies (DAS) are important metrics for assessing utility of tests used in animal and human health. Following standards for designing DAS will assist in appropriate test selection for specific testing purposes and minimize the risk of reporting biased sensitivity and specificity estimates. To examine the benefits of recommending standards, design information from published DAS literature was assessed for 10 finfish, seven mollusc, nine crustacean and two amphibian diseases listed in the 2017 OIE Manual of Diagnostic Tests for Aquatic Animals. Of the 56 DAS identified, 41 were based on field testing, eight on experimental challenge studies and seven on both. Also, we adapted human and terrestrial‐animal standards and guidelines for DAS structure for use in aquatic animal diagnostic research. Through this process, we identified and addressed important metrics for consideration at the design phase: study purpose, targeted disease state, selection of appropriate samples and specimens, laboratory analytical methods, statistical methods and data interpretation. These recommended design standards for DAS are presented as a checklist including risk‐of‐failure points and actions to mitigate bias at each critical step. Adherence to standards when designing DAS will also facilitate future systematic review and meta‐analyses of DAS research literature.     

Weaning of the wedge sole <Emphasis Type="Italic">Dicologoglossa cuneata</Emphasis> (Moreau): influence of initial size on survival and growth

The weaning phase can be decisive in fish-culture viability. In this work, the relationship between the initial size and weaning success has been studied in wedge sole (Dicologoglossa cuneata). For each age (30, 50, and 70 days after hatching, DAH), two to three sizes were selected, and all were put on the same feeding schedule for 20 days. Each batch (three replicates) was sampled at 1, 10, and 20 days. Specific growth rate (SGR) and survival were compared at the end of the co-feeding period, after 10 days on dry feed only. The best results for survival and growth were found with the smallest larvae, and vice versa. The SGRs and survival rates recorded during the co-feeding period were higher (0.8–15.6 day⁻¹ and 68.3–97.8%) than those from the dry-food phase (0.9–4.7 day⁻¹ and 56.3–66.7%). Successful weaning (survival = 65% and SGR = 9.3 day⁻¹) is possible with 30 DAH larvae (7.6–8.1 mm and 3.9–4.6 mg). In conclusion, the most effective weaning would be possible at 30 DAH, implying significant Artemia savings (25–50%).     

Establishment,characterization, virus susceptibility and transfection of cell lines from cobia,Rachycentron canadum (L.), brain and fin

Establishment and characterization of two cobia, Rachycentron canadum, cell lines derived from cobia brain (CB) and cobia fin (CF) are described. Caudal fin and brain from juvenile cobia were dissociated for 30 and 10 min, respectively, in phosphate‐buffered saline containing 0.25% trypsin at 25 °C. The optimal culture condition for both dissociated cells (primary cell culture) was at 28 °C in Leibovitz‐15 medium containing 10% foetal bovine serum. The cells have been sub‐cultured at a ratio of 1:2 for more than 160 passages over a period of 3 years. Origin of the cultured cells was verified by comparison of their sequences of mitochondrial cytochrome oxidase subunit I genes (cox I) with the cox 1 sequence from cobia muscle tissue. The cell lines showed polyploidy. No mycoplasma contamination was detected. Susceptibility to grouper iridovirus was observed for the CB cell line but not the CF cell line. Both cell lines expressed green fluorescent protein after being transfected with green fluorescent reporter gene driven by the cytomegalovirus promoter.     

Protection of turbot, Scophthalmus maximus (L.), and rainbow trout, Oncorhynchus mykiss (Richardson), against vibriosis using two different vaccines

Abstact. The potency of a whole-cell bacterin (WCB) and a toxoid enriched whole-cell vaccine (WCEB) administered intraperitoncally into rainbow trout, Oncorhynchus mykiss (Richardson), were compared. The most effective vaccine was further evaluated by bathing turbot, Scophthalmus maximus (L.). These vaccines were composed of three strains of V. anguillarun , of the serotypes 01 and 02. Both vaccines conferred the highest protection against strains of serotype 01 within 4 weeks. With the toxoid enriched vaccine giving the best results (77 RPS). When trout were revaccinated after 7 weeks with this vaccine, good protection was achieved against strains of serotypes 01 and 02. Interestingly, when the WCEB was administered by bath to turbot, acceptable levels of protection against strains of both serotypes were obtained after 4 weeks of immunization.     

Immunological response of the Indian major carps to Aeromonas hydrophila vaccine

Abstract. Fingerlings of the three major types of Indian carp, i.e. Catla catla (Hamilton), Labeo rohita (Hamilton) and Cirrhunas mrigala (Hamilton), were immunized using a haemolysin-negative mutant of Aeromonas hydrophila . Very high titres of antibodies were induced in C. catla , followed by C. mrigala and L. rohita . Immunized fish showed good protection against homologous challenge. Moderate protection against heterologous challenge was observed in C. mrigala and L. rohita .     

Effect of new ethyl and methyl carbamates on Rhipicephalus microplus larvae and adult ticks resistant to conventional ixodicides

The effects of six new synthetic carbamates on fully engorged females of four Rhipicephalus microplus strains (one reference strain susceptible to conventional ixodicides, two strains multiresistant to ixodicides and one tick field isolate) were compared. In addition, the effect of two other new synthetic carbamates was tested on larvae from the same strains. The first six tested carbamates reduced egg laying and inhibited egg hatching in the four studied strains (P < 0.05). Compared with untreated females, the eggs produced by the treated engorged female ticks of all strains had a dark, dry, opaque appearance and were less adherent. The remaining two tested carbamates induced larval mortality in all of the evaluated strains. The three studied R. microplus strains displayed 50% resistance ratios (RR₅₀) of less than 2 when compared to the susceptible reference strain. These results demonstrate that both carbamates with a larvicidal effect and carbamates that inhibit egg laying and embryo development are efficacious against tick strains that are resistant to commercial ixodicides, no cross resistance was observed.     

Salinity Effects on Early Life Stages of Southern Flounder Paralichthys lethostigma

Abstract.— In South Carolina, studies have been conducted to develop rearing techniques for southern flounder Paralichthys lethostigma a candidate for aquaculture development and stock enhancement programs. To help define environmental tolerances, a variety of salinity studies were conducted with the early life stages of this species. Eggs were buoyant at 32 ppt and sank at 29 ppt with salinities of 30–31 ppt providing varying levels of suspension in the water column. Eggs incubated at 0 and 5 ppt all died, whereas 82.5% hatched at 10 ppt but larvae died shortly thereafter. At 63 h post-fertilization, there were no differences in hatch level for eggs incubated at salinities of 15 to 35 ppt (mean hatch level 98.5%). In a 72-h study, fish 3 wk post-metamorphosis (13.7 mm TL, 50-d-old) were acclimated to seven salinities ranging from 0–30 ppt. Fish held at 0 ppt salinity exhibited a statistically (P < 0.05) lower survival (20.0%) than those exposed to 5–30 ppt salinity concentrations. No differences were detected in survival (mean 99.1%) among fish held in the higher salinities. A second study examined the tolerance of older juveniles to lower salinities. Juvenile flounder (95.2 mm TL, 220-d-old) were acclimated to 0, 1,5 and 10 ppt salinities and reared for 2 wk. Results showed that fish could tolerate salinities of 0–10 ppt (100% survival). These data indicate that salinity tolerance of southern flounder increases with age. In addition to the short duration studies, a replicated 11-mo duration tank grow-out study was conducted at mean salinity 5.4 ppt and mean temperahue 22.6 C with an all male population. Flounder grew from a mean length of 100 mm to 213 mm TL and weight from 8.9 to 104.3 g. Growth of the cultured fish approximated that observed among male flounders in the wild.     

Supplementation of potassium, magnesium and sodium chloride in practical diets for the Pacific white shrimp, Litopenaeus vannamei, reared in low salinity waters

The culture of Litopenaeus vannamei in inland low salinity waters is currently being practiced in various countries around the world. These environments are often deficient in key ions essential for normal physiological function, including potassium (K⁺) and magnesium (Mg²⁺). Farmers have sometimes been able to counteract ionic deficiencies in the water profile by adding mineral salts containing sources of K⁺ and Mg²⁺. The purpose of this study was to explore the possibility of correcting deficiencies of K⁺ and Mg²⁺ in the water profile with dietary supplementation of these minerals. Two separate 7‐week experiments were conducted in 4.0 g⁻¹ artificial low salinity water to evaluate the effects of mineral supplements (K⁺, Mg²⁺ and NaCl) to diets of L. vannamei reared in low salinity waters. In trial 1 seven diets were formulated (10 g NaCl kg⁻¹, 20 g NaCl kg⁻¹, 150 mg kg⁻¹ Mg²⁺, 300 mg kg⁻¹ Mg²⁺, 5 g K⁺ kg⁻¹, 10 g K⁺ kg⁻¹, and a basal diet to serve as a control). Minerals were added in the form of purified potassium chloride (KCl), magnesium chloride (MgCl₂·6H₂O) and NaCl. Trial 2 evaluated the use of a coating agent for the Mg²⁺ and NaCl treatments, while a K⁺ amino acid complex was utilized in the K⁺ treatments to reduce mineral leaching. Trial 2 was performed using similar treatment levels as trial 1. Shrimp survival and growth were assessed in both experiments. Results from trial 1 indicated no significant differences in survival, growth or percent weight gain. Results from trial 2 revealed no significant differences in survival and growth in the NaCl and Mg²⁺ treatments. However, significant differences in growth (P < 0.05) were observed when using the 10 g K⁺ kg⁻¹ treatment, suggesting that dietary supplementation of a K⁺ amino acid complex may help improve growth of the species in low salinity waters.     

Quantitative genetic parameter estimates for size and growth rate traits in Pacific white shrimp, Penaeus vannamei (Boone 1931) when reared indoors

Heritabilities of size traits and growth rate traits, as well as genetic, phenotypic and environmental correlations were estimated at three ages for a captive population of Pacific white shrimp (Penaeus vannamei) grown indoors. A covariate, mean size or mean growth rate during early growth in individual tanks before tagging and mixing, was introduced in the analyses to reduce the error caused by unique previous growth conditions on variance components. Heritabilities of size traits increased with age, with the h² for TL, AL, TWt and Wi1AS being 0.15, 0.20, 0.20 and 0.22, respectively, at 17 weeks, increasing to 0.28, 0.33, 0.34 and 0.35 at 29 weeks of age. Heritabilities of growth rate traits estimated between consecutive growth periods decreased from the first (h² for ΔTL 0.65, ΔAL 0.71, ΔTWt 0.63 and ΔWi1AS 0.84) to the second period (h² for ΔTL 0.34, ΔAL 0.50, ΔTWt 0.54 and ΔWi1AS 0.52). Phenotypic correlations were always larger than genetic correlations for both, size and growth rate traits. Genetic correlations between size traits within age were high (r_G >0.95), but those between the same size trait at different ages decreased as the age difference increased in spite of a consistently high environmental correlation (r_E 0.80–0.85) between the same trait at different ages. Phenotypic and genetic correlation's between the same growth rate trait at the two different growth periods evaluated were negative or zero (r_G TL –0.26, AL –0.24, Wi1AS 0.00) with the exception of total weight (r_G TW 0.35) and the environmental correlations between growth periods were also low (r_E 0.13–0.32).