Genomic-Bioinformatic Analysis of Transcripts Enriched in the Third-Stage Larva of the Parasitic Nematode Ascaris suum

Differential transcription in Ascaris suum was investigated using a genomic-bioinformatic approach. A cDNA archive enriched for molecules in the infective third-stage larva (L3) of A. suum was constructed by suppressive-subtractive hybridization (SSH), and a subset of cDNAs from 3075 clones subjected to microarray analysis using cDNA probes derived from RNA from different developmental stages of A. suum. The cDNAs (n = 498) shown by microarray analysis to be enriched in the L3 were sequenced and subjected to bioinformatic analyses using a semi-automated pipeline (ESTExplorer). Using gene ontology (GO), 235 of these molecules were assigned to ‘biological process’ (n = 68), ‘cellular component’ (n = 50), or ‘molecular function’ (n = 117). Of the 91 clusters assembled, 56 molecules (61.5%) had homologues/orthologues in the free-living nematodes Caenorhabditis elegans and C. briggsae and/or other organisms, whereas 35 (38.5%) had no significant similarity to any sequences available in current gene databases. Transcripts encoding protein kinases, protein phosphatases (and their precursors), and enolases were abundantly represented in the L3 of A. suum, as were molecules involved in cellular processes, such as ubiquitination and proteasome function, gene transcription, protein–protein interactions, and function. In silico analyses inferred the C. elegans orthologues/homologues (n = 50) to be involved in apoptosis and insulin signaling (2%), ATP synthesis (2%), carbon metabolism (6%), fatty acid biosynthesis (2%), gap junction (2%), glucose metabolism (6%), or porphyrin metabolism (2%), although 34 (68%) of them could not be mapped to a specific metabolic pathway. Small numbers of these 50 molecules were predicted to be secreted (10%), anchored (2%), and/or transmembrane (12%) proteins. Functionally, 17 (34%) of them were predicted to be associated with (non-wild-type) RNAi phenotypes in C. elegans, the majority being embryonic lethality (Emb) (13 types; 58.8%), larval arrest (Lva) (23.5%) and larval lethality (Lvl) (47%). A genetic interaction network was predicted for these 17 C. elegans orthologues, revealing highly significant interactions for nine molecules associated with embryonic and larval development (66.9%), information storage and processing (5.1%), cellular processing and signaling (15.2%), metabolism (6.1%), and unknown function (6.7%). The potential roles of these molecules in development are discussed in relation to the known roles of their homologues/orthologues in C. elegans and some other nematodes. The results of the present study provide a basis for future functional genomic studies to elucidate molecular aspects governing larval developmental processes in A. suum and/or the transition to parasitism

Seasonal changes in plankton respiration and bacterial metabolism in a temperate shelf sea

The seasonal variability of plankton metabolism indicates how much carbon is cycling within a system, as well as its capacity to store carbon or export organic matter and CO2 to the deep ocean. Seasonal variability between November 2014, April 2015 and July 2015 in plankton respiration and bacterial (Bacteria+Archaea) metabolism is reported for the upper and bottom mixing layers at two stations in the Celtic Sea, UK. Upper mixing layer (UML, >75 m in November, 41 - 70 m in April and ~50 m in July) depth-integrated plankton metabolism showed strong seasonal changes with a maximum in April for plankton respiration (1.2- to 2-fold greater compared to November and July, respectively) and in July for bacterial production (2-fold greater compared to November and April). However UML depth-integrated bacterial respiration was similar in November and April and 2-fold lower in July. The greater variability in bacterial production compared to bacterial respiration drove seasonal changes in bacterial growth efficiencies, which had maximum values of 89 % in July and minimum values of 5 % in November. Rates of respiration and gross primary production (14C-PP) also showed different seasonal patterns, resulting in seasonal changes in 14C-PP:CRO2 ratios. In April, the system was net autotrophic (14C-PP:CRO2 > 1), with a surplus of organic matter available for higher trophic levels and export, while in July balanced metabolism occurred (14C-PP:CRO2 = 1) due to an increase in plankton respiration and a decrease in gross primary production. Comparison of the UML and bottom mixing layer indicated that plankton respiration and bacterial production were higher (between 4 and 8-fold and 4 and 7-fold, respectively) in the UML than below. However, the rates of bacterial respiration were not statistically different (p > 0.05) between the two mixing layers in any of the three sampled seasons. These results highlight that, contrary to previous data from shelf seas, the production of CO2 by the plankton community in the UML, which is then available to degas to the atmosphere, is greater than the respiratory production of dissolved inorganic carbon in deeper waters, which may contribute to offshore export

Direct Counting of Bacteria Preserved with Lugol Iodine Solution

Lugol iodine solution was compared with glutaraldehyde as a preservative for marine bacteria. Direct counts with the fluorochrome 4′,6-diamidino-2-phenylindole show no significant difference between the preservatives, but the use of Lugol solution has several advantages over glutaraldehyde, especially in the handling and storage of samples. Bacteria were counted in water samples that were preserved with Lugol iodine solution and stored at room temperature for 4 years. Microprotozoa were also counted in samples preserved with Lugol iodine solution by using the fluorochrome fluorescein isothiocyanate

Genetic Diversity and Zoonotic Potential of Cryptosporidium parvum Causing Foal Diarrhea▿

Cryptosporidium isolates from diarrheic foals in New Zealand (n = 9) were identified as C. parvum, subtyped at two polymorphic loci, and compared with human (n = 45) and bovine (n = 8) isolates. Foal C. parvum isolates were genetically diverse, markedly similar to human and bovine isolates, and carried GP60 IIaA18G3R1 alleles, indicating a zoonotic potential

A survey of gastrointestinal nematode species in red deer (Cervus elaphus) farms in New Zealand using PCR

Gastrointestinal nematodes are recognised as an animal health issue for farmed red deer. The aim of this study was to explore the range of species infecting farmed deer herds and their farm-level prevalence in New Zealand. Faecal samples were collected from 12-24-month-old deer (n = 6-26; mean 19) on 59 farms located in the North (n = 25) and South (n = 34) Islands. Sub-samples of faeces were pooled by farm and cultured to recover third stage larvae. Twenty four larvae were randomly selected and identified to species using a multiplex PCR (total = 1217 larvae). At farm-level the most prevalent nematodes were Oesophagostomum venulosum 83% (n = 49) and the deer-specific nematodes in the subfamily Ostertagiinae (= Ostertagia-type) including, Spiculoptera asymmetrica 73% (n = 43), Ostertagia leptospicularis 47% (n = 28), Spiculoptera spiculoptera 47% (n = 28). The recently identified Trichostrongylus askivali was present on 32% (n = 19) of the farms and Oesophagostomum sikae on 17% (n = 10). In the analysis of the total number of larvae identified, the proportion was in similar order, 45% (n = 548) were O. venulosum, 14% (n = 173) S. asymmetrica, 10% (n = 124) S. spiculoptera, 9% (n = 114) O. leptospicularis, T. askivali, 3% (n = 40) and only 2% were O. sikae (n = 20). This study is the first to show the farm-level prevalence of nematode species in deer in New Zealand and the first to use PCR as a diagnostic tool. It provides data consistent with cross-infection from sheep/cattle to deer, and provided tentative insights into the proportions of the main GIN species across the deer population including O. sikae and T. askivali which have only recently been identified in New Zealand

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