Comparative toxicity of Pfiesteria spp., prolonging toxicity of P. piscicida in culture and evaluation of toxin(s) stability

Toxicity of Pfiesteria piscicida (strain CAAE #2200) in the presence of fish (juvenile hybrid tilapia, Oreochromis sp., total length 3–6 cm) has been maintained in the laboratory for 19 months by serial transfer of toxic cells using a modified maintenance protocol. Toxicity was re-induced when toxin-producing P. piscicida cells were separated from fish and cultured on algal prey for 50 days and then re-introduced to new tanks containing fish. We confirmed toxicity in a strain of P. shumwayae (strain CAAE #101272). Toxicity to fish was demonstrated in culture filtrates (0.2 μm) derived from cultures of both Pfiesteria spp., however, it was markedly reduced in comparison to unfiltered water. Filtrates retained toxic activity when stored at −20 °C for up to 6 months. Toxicity to fish was retained when filtrates were held at room temperature for 48 h, at 70 °C for 30 min or at 88–92 °C for 2 h. P. piscicida killed all finfish species tested. Grass shrimp (Paleomonetes pugio; adult 2–3 cm), blue crab (Callinectes sapidus; juvenile 4–7 cm) and brine shrimp (Artemia sp.; 18–24 h post-hatch) were unaffected by concentrations of toxin(s) that killed juvenile tilapia in 4–24 h. Ichthyotoxic activity of filtrates from fish-killing cultures and stability of the toxic activity were similar among P. piscicida and P. shumwayae. These results confirm previously reported observations on toxicity of P. piscicidaand P. shumwayae to finfish. We have maintained toxicity in the laboratory for longer periods than have previously been routinely achieved, and we have demonstrated that the toxic activity is heat stable. In contrast to previous studies with other toxic P. piscicida strains, we did not observe toxic activity to blue crabs or other crustaceans.     

Invasion genetics from eDNA and thousands of larvae: A targeted metabarcoding assay that distinguishes species and population variation of zebra and quagga mussels

Identifying species and population genetic compositions of biological invasions at early life stages and/or from environmental (e)DNA using targeted high‐throughput sequencing (HTS) metabarcode assays offers powerful and cost‐effective means for early detection, analysis of spread patterns, and evaluating population changes. The present study develops, tests, and applies this method with a targeted sequence assay designed to simultaneously identify and distinguish between the closely related invasive Eurasian zebra and quagga mussels (Dreissena polymorpha and D. rostriformis) and their relatives and discern their respective population genetic patterns. Invasions of these dreissenid mussel species have markedly changed freshwater ecosystems throughout North America and Europe, exerting severe ecological and economic damage. Their planktonic early life stages (eggs and larvae) are morphologically indistinguishable, yet each species exerts differential ecological effects, with the quagga often outcompeting the zebra mussel as adults. Our targeted assay analyzes genetic variation from a diagnostic sequence region of the mitochondrial (mt)DNA cytochrome oxidase I (COI) gene, to assess temporal and spatial inter‐ and intra‐specific genetic variability. The assay facilitates analysis of environmental (e)DNA from water, early life stages from thousands of individuals, and simultaneous analysis of 50–100 tagged field‐collected samples. Experiments evaluated its accuracy and performance using: (a) mock laboratory communities containing known DNA quantities per taxon, (b) aquaria with mixed‐species/haplotype compositions of adults, and (c) field‐collected water and plankton versus traditional sampling of adult communities. Results delineated species compositions, relative abundances, and population‐level diversity differences among ecosystems, habitats, time series, and life stages from two allopatric concurrent invasions in the Great Lakes (Lake Erie) and the Hudson River, which had separate founding histories. Findings demonstrate application of this targeted assay and our approach to accurately and simultaneously discern species‐ and population‐level differences across spatial and temporal scales, facilitating early detection and ecological understanding of biological invasions.     

Waist Circumference,BMI, and Visceral Adipose Tissue in White Women and Women of African Descent

Although waist circumference (WC) is a marker of visceral adipose tissue (VAT), WC cut‐points are based on BMI category. We compared WC‐BMI and WC‐VAT relationships in blacks and whites. Combining data from five studies, BMI and WC were measured in 1,409 premenopausal women (148 white South Africans, 607 African‐Americans, 186 black South Africans, 445 West Africans, 23 black Africans living in United States). In three of five studies, participants had VAT measured by computerized tomography (n = 456). Compared to whites, blacks had higher BMI (29.6 ± 7.6 (mean ± s.d.) vs. 27.6 ± 6.6 kg/m², P = 0.001), similar WC (92 ± 16 vs. 90 ± 15 cm, P = 0.27) and lower VAT (64 ± 42 vs. 101 ± 59 cm², P < 0.001). The WC‐BMI relationship did not differ by race (blacks: β (s.e.) WC = 0.42 (.01), whites: β (s.e.) WC = 0.40 (0.01), P = 0.73). The WC‐VAT relationship was different in blacks and whites (blacks: β (s.e.) WC = 1.38 (0.11), whites: β (s.e.) WC = 3.18 (0.21), P < 0.001). Whites had a greater increase in VAT per unit increase in WC. WC‐BMI and WC‐VAT relationships did not differ among black populations. As WC‐BMI relationship did not differ by race, the same BMI‐based WC guidelines may be appropriate for black and white women. However, if WC is defined by VAT, race‐specific WC thresholds are required.     

Mortality,Morbidity and Health-Seeking Behaviour during the Ebola Epidemic 2014–2015 in Monrovia Results from a Mobile Phone Survey

Between March 2014 and July 2015 at least 10,500 Ebola cases including more than 4,800 deaths occurred in Liberia, the majority in Monrovia. However, official numbers may have underestimated the size of the outbreak. Closure of health facilities and mistrust in existing structures may have additionally impacted on all-cause morbidity and mortality. To quantify mortality and morbidity and describe health-seeking behaviour in Monrovia, Médecins sans Frontières (MSF) conducted a mobile phone survey from December 2014 to March 2015. We drew a random sample of households in Monrovia and conducted structured mobile phone interviews, covering morbidity, mortality and health-seeking behaviour from 14 May 2014 until the day of the survey. We defined an Ebola-related death as any death meeting the Liberian Ebola case definition. We calculated all-cause and Ebola-specific mortality rates. The sample consisted of 6,813 household members in 905 households. We estimated a crude mortality rate (CMR) of 0.33/10,000 persons/day (95%CI:0.25–0.43) and an Ebola-specific mortality rate of 0.06/10,000 persons/day (95%-CI:0.03–0.11). During the recall period, 17 Ebola cases were reported including those who died. In the 30 days prior to the survey 277 household members were reported sick; malaria accounted for 54% (150/277). Of the sick household members, 43% (122/276) did not visit any health care facility. The mobile phone-based survey was found to be a feasible and acceptable alternative method when data collection in the community is impossible. CMR was estimated well below the emergency threshold of 1/10,000 persons/day. Non-Ebola-related mortality in Monrovia was not higher than previous national estimates of mortality for Liberia. However, excess mortality directly resulting from Ebola did occur in the population. Importantly, the small proportion of sick household members presenting to official health facilities when sick might pose a challenge for future outbreak detection and mitigation. Substantial reported health-seeking behaviour outside of health facilities may also suggest the need for adapted health messaging and improved access to health care.     

Isolation and characterization of a trypsin fraction from the pyloric ceca of chinook salmon (Oncorhynchus tshawytscha)

A trypsin fraction was isolated from the pyloric ceca of New Zealand farmed chinook salmon (Oncorhynchus tshawytscha) by ammonium sulfate fractionation, acetone precipitation and affinity chromatography. The chinook salmon enzyme hydrolyzed the trypsin-specific synthetic substrate benzoyl-dl-arginine-p-nitroanilide (dl-BAPNA), and was inhibited by the general serine protease inhibitor phenyl methyl sulfonyl fluoride (PMSF), and also by the specific trypsin inhibitors — soybean trypsin inhibitor (SBTI) and benzamidine. The enzyme was active over a broad pH range (from 7.5 to at least pH 10.0) at 25 °C and was stable from pH 4.0 to pH 10.0 when incubated at 20 °C, with a maximum at pH 8.0. The optimum temperature for the hydrolysis of dl-BAPNA by the chinook salmon enzyme was 60 °C, however, the enzyme was unstable at temperatures above 40 °C. The molecular mass of the chinook salmon trypsin was estimated as 28 kDa by SDS–PAGE.     

Serological Comparison of the Three Morphological Phases of Coccidioides immitis by the Agar Gel Diffusion Method

Hyperimmune sera against spherules and against arthrospores of Coccidioides immitis were prepared by inoculation of rabbits. The antibody content of these sera was studied by the agar gel diffusion method. It was observed that antispherule pooled sera formed multiple precipitin bands with extracts of spherules and of arthrospores. The antiarthrospore pooled serum, however, failed to precipitate with the spherule extract, and formed a single band in the presence of an arthrospore solution. When the spherule and the arthrospore extracts were tested with a variety of different antisera, it was observed that the spherule preparation formed bands only in combination with anti-purified spherule pooled serum, whereas the arthrospore extract precipitated with anti-purified spherule, antiarthrospore, and anti-Histoplasma capsulatum pooled sera. It was also observed that a spherule culture supernatant solution formed five precipitin bands in combination with anti-spherule pooled sera, formed one band with pooled antiserum from rabbits with coccidioidomycosis, and did not precipitate in the presence of antiarthrospore pooled serum. Coccidioidin, however, formed two bands in the presence of any of these antisera. It was therefore concluded that extracts from the spherule phase of C. immitis differed from solutions obtained from the arthrospore and mycelial phases.     

THE IMPORTANCE OF MOSQUITO BEHAVIOURAL ADAPTATIONS TO MALARIA CONTROL IN AFRICA

Over the past decade the use of long‐lasting insecticidal nets (LLINs), in combination with improved drug therapies, indoor residual spraying (IRS), and better health infrastructure, has helped reduce malaria in many African countries for the first time in a generation. However, insecticide resistance in the vector is an evolving threat to these gains. We review emerging and historical data on behavioral resistance in response to LLINs and IRS. Overall the current literature suggests behavioral and species changes may be emerging, but the data are sparse and, at times unconvincing. However, preliminary modeling has demonstrated that behavioral resistance could have significant impacts on the effectiveness of malaria control. We propose seven recommendations to improve understanding of resistance in malaria vectors. Determining the public health impact of physiological and behavioral insecticide resistance is an urgent priority if we are to maintain the significant gains made in reducing malaria morbidity and mortality.     

Molecular systematics of the Canidae

Despite numerous systematic studies, the relationships among many species within the dog family, Canidae, remain unresolved. Two problems of broad evolutionary significance are the origins of the taxonomically rich canidae fauna of South America and the development in three species of the trenchant heel, a unique meat-cutting blade on the lower first molar. The first problem is of interest because the fossil record provides little evidence for the origins of divergent South American species such as the maned wolf and the bush dog. The second issue is problematic because the trenchant heel, although complex in form, may have evolved independently to assist in the processing of meat. We attempted to resolve these two issues and five other specific taxonomic controversies by phylogenetic analysis of 2,001 base pairs of mitochondrial DNA (mtDNA) sequence data from 23 canidae species. The mtDNA tree topology, coupled with data from the fossil record, and estimates of rates of DNA sequence divergence suggest at least three and possibly four North American invasions of South America. This result implies that an important chapter in the evolution of modern canids remains to be discovered in the fossil record and that the South American canidae endemism is as much the result of extinction outside of South America as it is due to speciation within South America. The origin of the trenchant heel is not well resolved by our data, although the maximum parsimony tree is weakly consistent with a single origin followed by multiple losses of the character in several extant species. A combined analysis of the mtDNA data and published morphological data provides unexpected support for a monophyletic South American canidae clade. However, the homogeneity partition tests indicate significant heterogeneity between the two data sets.     

Species-specific, nested PCR-restriction fragment length polymorphism detection of single Cryptosporidium parvum oocysts

Concurrent with recent advances seen with Cryptosporidium parvum detection in both treated and untreated water is the need to properly evaluate these advances. A micromanipulation method by which known numbers of C. parvum oocysts, even a single oocyst, can be delivered to a test matrix for detection sensitivity is presented. Using newly developed nested PCR-restriction fragment length polymorphism primers, PCR sensitivity was evaluated with 1, 2, 3, 4, 5, 7, or 10 oocysts. PCR detection rates (50 samples for each number of oocysts) ranged from 38% for single oocysts to 92% for 5 oocysts, while 10 oocysts were needed to achieve 100% detection. The nested PCR conditions amplified products from C. parvum, Cryptosporidium baileyi, and Cryptosporidium serpentis but no other Cryptosporidium sp. or protozoan tested. Restriction enzyme digestion with VspI distinguished between C. parvum genotypes 1 and 2. Restriction enzyme digestion with DraII distinguished C. parvum from C. baileyi and C. serpentis. Use of known numbers of whole oocysts encompasses the difficulty of liberating DNA from the oocyst and eliminates the standard deviation inherent within a dilution series. To our knowledge this is the first report in which singly isolated C. parvum oocysts were used to evaluate PCR sensitivity. This achievement illustrates that PCR amplification of a single oocyst is feasible, yet sensitivity remains an issue, thereby illustrating the difficulty of dealing with low oocyst numbers when working with environmental water samples.