To pool or not to pool? Guidelines for pooling samples for use in surveillance testing of infectious diseases in aquatic animals

Samples from multiple animals may be pooled and tested to reduce costs of surveillance for infectious agents in aquatic animal populations. The primary advantage of pooling is increased population‐level coverage when prevalence is low (<10%) and the number of tests is fixed, because of increased likelihood of including target analyte from at least one infected animal in a tested pool. Important questions and a priori design considerations need to be addressed. Unfortunately, pooling recommendations in disease‐specific chapters of the 2018 OIE Aquatic Manual are incomplete and, except for amphibian chytrid fungus, are not supported by peer‐reviewed research. A systematic review identified only 12 peer‐reviewed aquatic diagnostic accuracy and surveillance studies using pooled samples. No clear patterns for pooling methods and characteristics were evident across reviewed studies, although most authors agreed there is a negative effect on detection. Therefore, our purpose was to review pooling procedures used in published aquatic infectious disease research, present evidence‐based guidelines, and provide simulated data examples for white spot syndrome virus in shrimp. A decision tree of pooling guidelines was developed for use by peer‐reviewed journals and research institutions for the design, statistical analysis and reporting of comparative accuracy studies of individual and pooled tests for surveillance purposes.     

Impact of weather variables and season on sporulation of Phytophthora pluvialis and Phytophthora kernoviae

Phytophthora pluvialis and Phytophthora kernoviae are the causal agents of important needle diseases on Pinus radiata in New Zealand. Little is known about the epidemiology of the diseases, making the development of control strategies challenging. To investigate the seasonality and climatic drivers of sporulation, inoculum traps, consisting of pine fascicles floating on water in plastic containers, were exchanged fortnightly at five sites in P. radiata plantations between February 2012 and December 2014. Sections of needle baits were plated onto selective media and growth of Phytophthora pluvialis and P. kernoviae recorded. To explore the generalizability of these data, they were compared to detection data for both pathogens from the New Zealand Forest Health Database (NZFHDB). Further, equivalent analyses on infection of Rhododendron ponticum by P. kernoviae in Cornwall, UK allowed the comparison of the epidemiology of P. kernoviae across different host systems and environments. In New Zealand, inoculum of P. pluvialis and P. kernoviae was detected between January–December and March–November, respectively. Inoculum of both species peaked in abundance in late winter. The probability of detecting P. pluvialis and P. kernoviae was greater at lower temperatures, while the probability of detecting P. pluvialis also increased during periods of wet weather. Similar patterns were observed in NZFHDB data. However, the seasonal pattern of infection by P. kernoviae in the UK was the opposite of that seen for sporulation in New Zealand. Phytophthora kernoviae was likely limited by warmer and drier summers in New Zealand, but by colder winter weather in the UK. These results emphasize the importance of considering both environmental drivers and thresholds in improving our understanding of pathogen epidemiology.     

A historical perspective on measurement of soil and plant water status and plant water status

Summary Surrounded by many sophisticated instruments, data acquisition systems and connecting computers, contemporary soil and plant scientists may not fully appreciate what really was known in earlier times and how the science has developed. A short review and a few examples of the growth of measurements and ideas are presented here.     

Kinetic and stoichiometric assessment of the antioxidant activity of flavonoids by electron spin resonance spectroscopy

There is current interest in the use of naturally occurring flavonoids as antioxidants for the preservation of foods and the prevention of diseases such as atherosclerosis and cancers. To establish the molecular characteristics required for maximum antioxidant activity, electron spin resonance (ESR) spectroscopy has been used to determine the stoichiometry and kinetics of the hydrogen-donating ability of 15 flavonoids and d-alpha-tocopherol to galvinoxyl, a resonance-stabilized, sterically protected aryloxyl radical. The second-order reaction rates, which will be governed by O-H bond dissociation energies, were myricetin > morin > quercetin > fisetin approximately catechin > kaempferol approximately luteolin > rutin > d-alpha-tocopherol > taxifolin > tamarixetin > myricetin 3',4',5'-trimethyl ether > datiscetin > galangin > hesperitin approximately apigenin. Reactivity is highly dependant on the configuration of OH groups on the flavonoid B and C rings, there being little contribution from the A ring to antioxidant effectiveness. Highest reaction rates and stoichiometries were observed with flavonols capable of being oxidized to orthoquinones or extended paraquinones. However, rates and stoichiometries did not always correlate and the data suggest that kinetic factors may be of greater importance within a biological context.     

Preparation of tetrahydroagathic acid: a serum metabolite of isocupressic acid, a cattle abortifacient in ponderosa pine

Isocupressic acid (1) was used to synthetically prepare a mixture of (8S,13R,S)-labda-15,19-dioic acid (tetrahydroagathic acid) (5) via a two-step oxidation procedure followed by hydrogenation of the double bonds at C13 and C8. Reduction of the C8,17 double bond was stereospecific producing only the 8S isomer and confirmed by the nOe interaction between the resulting C17 and C20 methyl groups. The 13R and 13S isomers of 5 were separated and analyzed by HPLC/MS, and (13S)-tetrahydroagathic acid was isolated and identified by comparison to a standard prepared by hydrogenation of naturally occurring (13S)-dihydroagathic acid (4). (13R,S)-dihydroagathic acid was prepared by selective sodium metal-catalyzed hydrogenation of the C13,14 allylic double bond of agathic acid (3). The prepared compounds were then used as standards to confirm the presence of 4 and 5 and their respective 13R and 13S isomers in bovine serum samples. Tetrahydroagathic acid was shown to be the only metabolite detected in serum samples taken from a suspected cattle abortion case submitted for diagnosis; and, thus, 5 could be a valuable diagnostic marker for pine needle-induced abortions.     

Lupine induced "crooked calf disease" in Washington and Oregon: identification of the alkaloid profiles in Lupinus sulfureus, Lupinus leucophyllus, and Lupinus sericeus

Several lupines (Lupinus spp.) present on western U.S. rangelands contain alkaloids that are teratogenic to livestock and cause congenital birth defects in calves (crooked calf disease). Periodically, large losses of calves due to lupine-induced "crooked calf disease" occur in northern Oregon and eastern Washington state. Five lupine populations from this area representing three species (L. leucophyllus, L. sulfureus, and L. sericeus) were evaluated taxonomically and by gas chromatography/mass spectrometry, and the major alkaloids in each lupine species were identified. The teratogenic alkaloid anagyrine was present in both of the lupine species responsible for the high outbreaks in east-central Washington and northeastern Oregon. However, the alkaloid profiles of the two lupines identified as L. leucophyllus were dissimilar, as were the alkaloid profiles of the two lupines identified as L. sulfureus. Botanical classification is not sufficient to determine potential teratogenicity, and it must be followed by chemical characterization to determine risk to livestock.     

Degradation of selected polycyclic aromatic hydrocarbons in coastal sediments: Importance of microbes and polychaete worms

Rates of degradation of polycyclic aromatic hydrocarbons (PAH) were examined after addition of crude oil enriched with anthracene, fluoranthene, benz(a)anthracene, and benzo(a)pyrene to coastal sediments in a laboratory (20°) flowing seawater system. Three types of sediment (fine sand, medium sand, and marsh sediment) with and without the benthic polychaete worm, Capitella capitata, were used. After extraction from the sediment, PAH concentrations were measured by liquid chromatography with fluorescent detection. Microbial degradation was studied by incubating sediments with radioactive PAH and measuring subsequent production of ¹⁴C0₂. Concentrations of the four PAH decreased significantly with time in fine and medium sized sands. PAH levels also decreased in marsh sediment but trends were not significant (P < 0.05). C. capitata stimulated PAH degradation. Microbial degradation was more rapid in upper surfaces than in lower layers of the sediments.     

Element mass balances for South Carolina Coastal Plain watersheds

Element mass balance estimates for South Carolina Coastal Plain watersheds indicate that fertilizers and liming materials are the major sources for inputs of Ca, Mg, K, Cl, and HCO₃ whereas precipitation is the major input for Na and SO₄. Stream flow is the chief mode of output for all of these elements. A balance between input and output is evident only for Cl. Retentions of 50% or more are shown by Ca, Mg, K, HCO₃ and SO₄ whereas Na shows an apparent net loss. The retention of Ca, Mg and HCO₃ suggests that less than 25% of the dolomitic liming materials applied to the landscape actually dissolve and that the carbonate chemistry of Lower Coastal Plain streams is therefore probably largely controlled by seepage of groundwaters from underlying calcareous aquifers. The retention of K and the loss of Na may be due to cation exchange reactions on soil clays whereas the apparent retention of SO₄ is probably due to reduction to H₂S in floodplain environments and soil adsorption.     

Winter flocking behaviour of speckled warblers and the Allee effect

The aggregation of individuals into foraging flocks is one behavioural trait that, if disrupted, can cause the Allee effect, which is a slowing in population growth at low density or small population size, and this can greatly increase the risk of extinction. Here, I describe intraspecific flocking behaviour of a colour-banded population of speckled warblers, Chthonicola sagittata, a species that has declined across a large part of its range in the fragmented temperate woodlands of Australia. I make predictions about the context in which the Allee effect might be expressed and the consequences for the viability of populations living in small habitat remnants. Speckled warblers lived in discrete, stable social groups throughout winter, the nucleus of which was the residents from one or more adjacent breeding territories. The timing and mode of flock formation and the size of flocks varied between two winters, apparently in response to the severity of conditions; thus flocking probably facilitates increased foraging efficiency and predator detection, potentially leading to increased survival in harsh conditions. Because flock territories were up to 30 ha each, and larger territories are likely, birds living in remnants smaller than 40 ha may suffer increased mortality if there are too few birds available to form flocks of an appropriate size to facilitate the benefits of grouping when conditions are most extreme. Further, in small remnants where survival is reduced, dominance behaviour and male-male competition may act to compound the Allee effect by reducing reproductive success. Regardless of these predictions, speckled warbler populations may only be viable in remnants that are large enough to support multiple flocks, to enable rapid recruitment to breeding vacancies and thus provide adequate numbers of birds for flocking.