Detection of Cryptosporidium parvum oocysts in environmental water in Hokkaido, Japan

Control of cryptosporidiosis is important in public health. Rivers that are polluted with Cryptosporidium and drinking water that is treated for drinking water production from polluted rivers could result in the waterborne disease of cryptosporidiosis. We carried out an epidemiological study of natural water supplies in Hokkaido, one of the largest dairy prefectures in Japan. To detect Cryptosporidium oocysts in environmental water, the filtration method was used for 28 samples, which were collected from 10 rivers. A method adapted from the United States Environmental Protection Agency (U.S. EPA) filtration method using a cartridge filter has been used for the collection of samples. Oocysts were separated from a pellet by discontinuous sucrose gradient method. Twelve samples were collected from 10 rivers and parasites were purified by iron (III) flocculation method. Cryptosporidium parvum oocysts were identified with the immunofluorescence antibody technique using DIF kit (Cellabs Pty. Ltd., Sydney/Australia). We detected Cryptosporidium oocysts in 6 out of 10 rivers sampled. Fifty percentage (14/28) of the samples were Cryptosporidium-positive. The average number of Cryptosporidium oocysts was 16.73/100 L (max. 80/100 L).     

Detection of natural infection of Boophilus microplus with Babesia equi and Babesia caballi in Brazilian horses using nested polymerase chain reaction

The potential role of Boophilus microplus as a natural tick vector of Babesia equi and Babesia caballi in Brazilian horses was assessed using nested polymerase chain reaction (PCR)-based marker assay. B. equi merozoite-specific 218bp gene fragment was detected in almost 96% of horse blood samples, and 45.3-62.5% of females, eggs, larvae, and nymphs of B. microplus collected from 47 horses at Campo Grande in the State of Matto Grosso, Brazil. Except for the partially-fed female ticks, the B. caballi-specific 430bp gene fragment was amplified from horse blood samples, and all developmental stages. Parasite DNA from both species was detected in horse blood samples and B. microplus, with the preponderance of B. equi DNA. No DNA samples were positive solely for B. caballi parasite. Only 32% of the Giemsa-stained thin blood smears were positive for Babesia parasites, as against detection of B. equi parasite DNA in 95.7% of the blood samples by nested PCR. We have obtained molecular evidence that strengthens earlier experimental and ultrastructural studies in Brazil incriminating B. microplus as a natural vector of B. equi, and possibly of B. caballi. The detection of B. equi and B. caballi DNA in eggs and larvae of B. microplus is likewise suggestive of the possibility of both transovarial and transstadial parasite transmission in this tick vector.     

Sex differences in metabolic rate and swimming performance in pink salmon (Oncorhynchus gorbuscha): the effect of male secondary sexual traits

Do secondary sexual traits, such as large dorsal hump and hooked snout, decrease the swimming efficiency of male pink salmon during freshwater migration? This is the first study to address the effects of secondary sexual traits in pink salmon on oxygen uptake and swimming capacity. We conducted a laboratory experiment using a swimming respirometer and a field study using electromyogram (EMG) telemetry in the Shibetsu River, Hokkaido, Japan. We compared the relationship between MO₂ (mg O₂·kg^?1·h^?1) and swimming velocity U (m·s^?1) in male and female fish, and also investigated the effects of morphological traits (secondary sexual characters) on the relationship between MO₂ (mg O₂·kg^?1·h^?1) and swimming velocity U (m·s^?1). Additionally, we compared energy costs and swimming behaviour during upstream migration between male and female pink salmon. The laboratory experiment revealed that MO₂ exponentially increased with increasing U; this increase was described by MO₂ = 167.9e^1.23U for males and 144.9e^1.14U for females. Linear mixed models found that hump height and the upper jaw length in males significantly and positively affected the relationship between MO₂ and U; no effect was found in females. The field study found that swimming velocity for both sexes estimated from EMG calibration was lower than optimal swimming velocity (U_opt) calculated from the laboratory experiment. We suggest that pink salmon in the Shibetsu River do not swim at the optimal swimming velocity because of the short migration distance involved (20 km).     

P-55 Febrile neutrophilic dermatosis in a miniature schnauzer resembling Sweet''s syndrome in humans

The purpose of this study was to evaluate the histopathologic changes seen in feline plasmacytic pododermatitis in an attempt to correlate clinical response with any specific histopathologic feature, and to evaluate these specimens for possible infectious organisms. Fourteen skin biopsies from cats with plasmacytic pododermatitis and a clinical follow-up of 12–36 months were included in the study. Sections were stained with H&E as well as with a polyclonal anti- Mycobacterium bovis [Bacille Calmette–Guerin (BCG)] antibody cross-reactive to a broad spectrum of fungi and bacteria. Sections stained with the anti-BCG antibody were graded as positive or negative. Inflammatory cell types and density, overall pattern, and the presence or involvement of adnexal structures were assessed in sections stained with H&E. All sections were negative for organisms within the actual footpad tissue with the anti-BCG antibody stains. Two samples revealed a few foci of cocci on the outer surface of the ulcers.
Funding: Self-funded.     

Spatial distribution analysis of the North Pacific spiny dogfish,Squalus suckleyi,in the North Pacific using generalized additive models

The North Pacific spiny dogfish (SPD), Squalus suckleyi, is a commercially exploited shark species that plays an important role in the ecosystem. To elucidate the distribution of the SPD in the North Pacific and to evaluate the effects of sea surface temperature (SST) and prey availability on its distribution, we estimated the probability of SPD presence using a generalized additive model with a binomial error distribution from SPD presence/absence data on 14,824 operations in fishery‐independent gillnet surveys between 1972 and 2011. The habitat model was structured in the east and west to reflect differences in the North Pacific oceanic environments. In the east, a higher probability of SPD presence was identified along the coast from the eastern Gulf of Alaska to Queen Charlotte Sound. In the west, it was identified around northern Japan. The estimated distribution was continuous between the two areas, whereas the probability of SPD presence was relatively low. Although the probability of SPD presence was higher at SSTs between 6°C and 12°C, the SST at the peak probability of SPD presence differed between the west and east. The prey species, Japanese sardine, Sardinops melanostictus, and walleye pollock, Gadus chalcogrammus, in the west and boreal clubhook squid, Onychoteuthis borealijaponica, in the east significantly affected the probability of SPD presence, which was higher if the prey species co‐existed with SPD. Therefore, SPD might adapt their distribution to that of available prey species. SPD stock assessment and management in these two important areas are required for its sustainable utilization.     

Neospora caninum NcSRS2 is a transmembrane protein that contains a glycosylphosphatidylinositol anchor in insect cells

We investigated the terminal location of NcSRS2, a surface antigen of Neospora caninum that has potential use for diagnosis, and demonstrated its importance as a vaccine component against neosporosis, in an insect-baculovirus expression system. To examine the role of the hydrophobic C-terminal tail in NcSRS2, four types of recombinant baculoviruses were constructed. Immunoblotting and N-terminal amino acid analysis revealed cleavage of a 6 kDa of the N-terminal signal peptide in the mature NcSRS2 protein. The recombinant NcSRS2 (rNcSRS2) lacking 25, and 62 amino acids from the termination codon were detected in supernatants from recombinant virus-infected cells, but not in recombinants with truncated 147 amino acids from the termination codon, and intact NcSRS2 gene (401 amino acids). By flow cytometric and confocal laser scanning microscopic analyses, the truncation of the hydrophobic C-terminal tail in NcSRS2 was shown to result in the reduction of protein expression on the cell surface relative to intact rNcSRS2. Except for the recombinant lacking the 147 C-terminal residues, three other rNcSRS2 were detected in the supernatants after treatment with phosphatidylinositol-specific phospholipase C. Our results demonstrate that the N. caninum NcSRS2 is a transmembrane protein that contains a glycosylphosphatidylinositol-anchor molecule in insect cells, and that the hydrophobic C-terminal domain is an essential component for GPI-membrane attachment. We have likewise shown the usefulness of the insect-recombinant baculovirus system in the expression of rNcSRS2.     

Transgene manipulation in rainbow trout using Cre recombinase

Cre/loxP-mediated cell targeting is considered to be a powerful tool for biotechnology in farmed fish. As a first step toward establishing cell targeting in salmonids, we analyzed the functionality of the Cre/loxP system in rainbow trout. We first established stable transgenic strains carrying the DsRed gene, which was flanked by loxP sites and further spliced with the EGFP gene. By microinjecting Cre complementary RNA (cRNA) into fertilized eggs of the transgenic trout, the functionality of the Cre/loxP system was evaluated. The results showed that all of the embryos exhibited green fluorescence in at least some of their cells. While 19 out of 20 embryos comprised cells showing both green and red fluorescence, the remaining embryo showed only green fluorescence. Polymerase chain reaction (PCR) using primers designed to recognize sequences outside of the two loxP sites revealed that, in addition to long intact fragments, the 19 individuals carried short fragments that were equivalent in length to the loxP-excised fragments. The remaining green embryo carried only this short fragment. DNA sequencing of the short fragment revealed that it lacked the DNA fragments flanking the loxP sites and the spliced fragments did not contain any sequence rearrangements. These results suggest that the Cre/loxP system is functional in rainbow trout.     

Characterization of Antioxidant Activity of Heated Mycosporine-like Amino Acids from Red Alga Dulse Palmaria palmata in Japan

We recently demonstrated the monthly variation and antioxidant activity of mycosporine-like amino acids (MAAs) from red alga dulse in Japan. The antioxidant activity of MAAs in acidic conditions was low compared to that in neutral and alkali conditions, but we found strong antioxidant activity from the heated crude MAA fraction in acidic conditions. In this study, we identified and characterized the key compounds involved in the antioxidant activity of this fraction. We first isolated two MAAs, palythine, and porphyra-334, from the fraction and evaluated the activities of the two MAAs when heated. MAAs possess absorption maxima at around 330 nm, while the heated MAAs lost this absorption. The heated MAAs showed a high ABTS radical scavenging activity at pH 5.8–8.0. We then determined the structure of heated palythine via ESI-MS and NMR analyses and speculated about the putative antioxidant mechanism. Finally, a suitable production condition of the heated compounds was determined at 120 °C for 30 min at pH 8.0. We revealed compounds from red algae with antioxidant activities at a wide range of pH values, and this information will be useful for the functional processing of food.