Monitoring Taiwanese bovine arboviruses and non-arboviruses using a vector-based approach.

In current sampling approaches, there exists a divergence between the surveillance of arthropod-borne and that of non-arthropod-borne viruses. It is commonly held that the collection of vector specimens applies only to arbovirus surveillance and that the surveillance of non-arboviruses must rely on traditional methods that involve the sampling of blood, faeces or saliva, or other examinations. The vector-based approach is a sampling method that has the ability to survey both arboviruses and non-arboviruses by distinguishing engorged vector specimens from entire vector samples. Accordingly, five arboviruses and three non-arboviruses were detected in a study using a vector-based approach conducted during 2012-2015. Hence, this report provides the first description of the Taiwanese vector species for the bovine arboviruses detected. The present investigations demonstrate that the vector-based approach applies not only to the surveillance of arboviruses, but also has potential as a possible tool for monitoring non-arboviruses on livestock farms in the future.

Docosahexaenoic acid synthesis from alpha-linolenic acid is inhibited by diets high in polyunsaturated fatty acids.

The conversion of the plant-derived omega-3 (n-3) α-linolenic acid (ALA, 18:3n-3) to the long-chain eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) can be increased by ALA sufficient diets compared to ALA deficient diets. Diets containing ALA above an optimal level result in no further increase in DHA levels in animals and humans. The present study evaluates means of maximizing plasma DHA accumulation by systematically varying both linoleic acid (LA, 18:2n-6) and ALA dietary level. Weanling rats were fed one of 54 diets for three weeks. The diets varied in the percentage of energy (en%) of LA (0.07-17.1 en%) and ALA (0.02-12.1 en%) by manipulating both the fat content and the balance of vegetable oils. The peak of plasma phospholipid DHA (>8% total fatty acids) was attained as a result of feeding a narrow dietary range of 1-3 en% ALA and 1-2 en% LA but was suppressed to basal levels (∼2% total fatty acids) at dietary intakes of total polyunsaturated fatty acids (PUFA) above 3 en%. We conclude it is possible to enhance the DHA status of rats fed diets containing ALA as the only source of n-3 fatty acids but only when the level of dietary PUFA is low (<3 en%).

Correlations between blood and tissue omega-3 LCPUFA status following dietary ALA intervention in rats.

The aim of this study was to assess relationships between the fatty acid contents of plasma and erythrocyte phospholipids and those in liver, heart, brain, kidney and quadriceps muscle in rats. To obtain a wide range of tissue omega-3 (n-3) long chain polyunsaturated fatty acids (LCPUFA) we subjected weanling rats to dietary treatment with the n-3 LCPUFA precursor, alpha linolenic acid (ALA, 18:3 n-3) for 3 weeks. With the exception of the brain, we found strong and consistent correlations between the total n-3 LCPUFA fatty acid content of both plasma and erythrocyte phospholipids with fatty acid levels in all tissues. The relationships between eicosapentaenoic acid (EPA, 20:5 n-3) and docosapentaenoic acid (DPA, 22:5 n-3) content in both blood fractions with levels in liver, kidney, heart and quadriceps muscle phospholipids were stronger than those for docosahexaenoic acid (DHA, 22:6 n-3). The strong correlations between the EPA+DHA (the Omega-3 Index), total n-3 LCPUFA and total n-3 PUFA contents in both plasma and erythrocyte phospholipids and tissues investigated in this study suggest that, under a wide range of n-3 LCPUFA values, plasma and erythrocyte n-3 fatty acid content reflect not only dietary PUFA intakes but also accumulation of endogenously synthesised n-3 LCPUFA, and thus can be used as a reliable surrogate for assessing n-3 status in key peripheral tissues.

Omega-3 long chain fatty acid synthesis is regulated more by substrate levels than gene expression.

The conversion of linoleic acid (LA) and alpha-linolenic acid (ALA) to long chain polyunsaturated fatty acids (LCPUFA) is known to involve desaturation and elongation steps. Although there is evidence that genes for these steps can be regulated by extremes of dietary PUFA, the degree to which there is meaningful regulation of LCPUFA levels in tissues by diet as a result of changes in expression of desaturase and elongase genes is unclear. In this study, we tested the effect of increasing ALA levels in diets of rats from 0.2% to 2.9% energy (en) against a constant LA level (1%en) on plasma and liver phospholipid LCPUFA content together with the expression of hepatic genes involved in PUFA metabolism, the desaturases FADS1 and FADS2, the elongases ELOV2 and ELOV5, and the transcription factors sterol regulatory element-binding protein-1c (SREBP-1c) and peroxisome proliferator-activated receptor alpha (PPARalpha). The levels of plasma and liver eicosapentaenoic acid (EPA) and docosapentaenoic acid (DPA) increased in proportion to dietary ALA whereas docosahexaenoic acid (DHA) increased only up to 1%en ALA. A low PUFA (0.4%en) reference diet stimulated the expression of delta 6 desaturase (FADS2) and elongase 2 (ELOVL2) when compared to higher PUFA diets. There was, however, no difference in the expression of any of the genes in rats, which were fed diets containing between 0.2%en and 2.9%en ALA and mRNA expression was unrelated to tissue/plasma LCPUFA content. These data suggest that the endogenous synthesis of n-3 LCPUFA from the precursor ALA is regulated independently of changes in the expression of the synthetic enzymes or regulatory transcription factor, and provides evidence that n-3 LCPUFA synthesis is regulated more by substrate competition for existing enzymes than by an increase in their mRNA expression.

Molecular differentiation and diversity of Forcipomyia taiwana (Diptera: Ceratopogonidae) based on the mitochondrial cytochrome oxidase II sequence.

Forcipomyia taiwana (Shiraki), a biting midge, is one of the most annoying blood-sucking pests in Taiwan. In this study, partial DNA sequences of cytochrome c oxidase II from 113 individuals collected from 11 locations around the island were analyzed to delineate the differentiation pattern and possible dispersal processes of F. taiwana in Taiwan. The uncorrected nucleotide divergences, composed of mostly transition substitutions, were high (up to 2.7%) among the samples. Average comparable variations (approximately equal to 0.7%) were found within and between populations. Phylogenetic analysis suggested that several distinct lineages exist and some can be found simultaneously in some populations. A relationship between sequence divergences among populations and their relative geographical distances was observed. Moreover, haplotype diversity was high in all populations, and low to middle levels (Fst = 0.004-0.288) of genetic differentiation were found among populations. Linearized calibration from sequence divergences and phylogenetic analysis showed that different ancestral lineages of F. taiwana possibly emerged as early as 0.6 million years ago. Taken together, genetic exchanges among these divergently ancestral lineages, likely caused by recent artificial events, have possibly led to the similarly diversified compositions of F. taiwana populations all around Taiwan nowadays.

Study on the species-specificity of Isospora michaelbakeri by experimental infection.

Isospora michaelbakeri is one of the Isospora species most commonly found in the wild field, which can cause severe infection and mortality in young sparrows. In this study, we selected I. michaelbakeri (Chung Hsing strain) as a pathogen to orally inoculate russet sparrows (Passer rutilans), spotted munia (Lonchura punctulata), canary (Serinus canaria), Java sparrows (Padda oryzivora), chicken (Gallus domesticus), ducks (Anas platyrhynchos) and BALB/c mice. The results indicated that I. michaelbakeri infected only russet sparrows. Infected sparrows displayed lethargy, muscular weakness and fluffy feathers, followed by rapid death. Liver and spleen enlargement was seen in the infected birds. Schizonts were identified in thin smears from the venous blood, enlarged livers and spleens. Histopathological examination revealed schizonts and merozoites from the liver and spleen of infected russet sparrows, but not from other species experimentally inoculated with I. michaelbakeri in the present study.

Molecular cloning of the precursor polypeptide of mastoparan B and its putative processing enzyme, dipeptidyl peptidase IV, from the black-bellied hornet, Vespa basalis.

Mastoparan B, a cationic toxin, is the major peptide component in the venom of Vespa basalis. Molecular cloning of its cDNA fragment revealed that this toxin was initially synthesized as a precursor polypeptide, containing an N-terminal signal sequence, a prosequence, the mature toxin, and an appendix glycine at C-terminus. Sequence alignment between precursors of mastoparan B and melittin from honeybee venom showed a significant conservation in prosequence. Alternate positions existing in both prosequences were either proline or alanine known as the potential cleaving sites for dipeptidyl peptidase IV. Subsequently, a putative dipeptidyl peptidase IV cDNA fragment was cloned from Vespa basalis venom gland. The prosequence may possibly be removed via sequential liberation of dipeptides during the processing of mastoparan B.

Induction of tumour necrosis factor, interleukin-1beta and matrix metalloproteinases in pulmonary fibrosis of rats infected with Angiostrongylus cantonensis.

In angiostrongyliasis, chronic parasite-induced granuloma formation can lead to tissue destruction and fibrosis. Here, the histomorphology of granulomatous fibrosis and proteinase production in the lungs of Angiostrongylus cantonensis-infected Sprague-Dawley rats were investigated. The relationship between metalloproteinases and granulomatous fibrosis was investigated following infection of each rat with 60 infective larvae. Granulomata and fibrosis were marked in the lungs of rats on day 180 post-inoculation. Reverse transcriptase polymerase chain reaction of lung mRNA showed an up-expression of proinflammatory cytokine including tumour necrosis factor alpha (TNF-alpha) and interleukin-1 beta (IL-1beta). According to Western blot analysis, matrix metalloproteinase-2 (MMP-2) proenzyme was presented in the lungs of uninfected and infected rats, and partial conversion of 72 kDa proenzyme to the 64 kDa active form occurred in infected rats. In addition, increased protein levels of MMP-9 and MMP-13 were detected in infected lungs, but were undetectable in controls. The results suggest that TNF-alpha, IL-1beta, MMP-2, -9, and -13 may be associated with the granulomatous fibrosis.

Ultrastructural localization of matrix metalloproteinase-9 in eosinophils from the cerebrospinal fluid of mice with eosinophilic meningitis caused by Angiostrongylus cantonensis.

Matrix metalloproteinase-9 (MMP-9) has been implicated in the pathogenesis of eosinophilic meningitis caused by Angiostrongylus cantonensis. In the present study, such meningitis in mice was found to be associated with elevated expression of MMP-9 mRNA, elevated MMP-9 concentrations and enhanced MMP-9 activity in the cerebrospinal fluid (CSF). Immunocytochemistry showed that an anti-MMP-9 antibody reacted with macrophages, neutrophils and eosinophils from the CSF. As eosinophils are generally considered to be effector cells in host defence against A. cantonensis infection, high-resolution immuno-electron microscopy was then used to confirm the localization of MMP-9 in the eosinophils from the CSF. The method used, which was based on immunogold, indicated that the eosinophilic MMP-9 was mostly localized in the 'small' granules in the cytoplasm and along the cell membrane, and not in the crystalloid-containing secretory granules observed. It therefore appears that MMP-9 is synthesised and/or stored in the small granules of the eosinophils, and is released into the subarachnoid space of the host's brain by secretion or cell rupture.

Ultrastructural studies on the reproductive system of male Aedes aegypti (Diptera: Culicidae) infected with dengue 2 virus.

Dengue 2 virus was found by transmission electron microscopy to be present in the reproductive tissues of male Aedes aegypti (L) 14 d after intrathoracic inoculation. Dengue 2 particles were detected in the matrix, epithelial cells, and the peripheral fat body of the testes; secretory droplets of columnar cells of the accessory glands; and the epithelial and muscle cells of the seminal vesicles. However, none was found in the germ cells (i.e., spermatogonia, spermatocytes, spermatid, or spermatozoa). These observations indicate that fluid transfer may be the mechanism of venereal transmission of dengue 2 virus by Ae. aegypti.

Human IgE and IgG antibodies to mosquito proteins detected by the immunoblot technique.

Immunoblot technique was used in this study to detect IgE and IgG antibodies in human sera against mosquito antigens. Mosquito proteins were separated on SDS-polyacrylamide gels and transferred electrophoretically to nitrocellulose papers. After incubation with sera from different individuals, the precipitated bands were analyzed with enzyme-labeled goat anti-human immunoglobulins. Distinct patterns of antigen were recognized by IgE and IgG antibodies.

Persistence of hepatitis B viral antigens in Culex quinquefasciatus.

Culex quinquefasciatus mosquitoes were fed on or inoculated with blood or serum positive for hepatitis B viral antigens and pools of mosquitoes were tested by radioimmunoassay daily for 3 weeks after exposure to detect the viral antigens. Hepatitis B surface antigen (HBsAg) was detectable up to 3 weeks, while hepatitis B e antigen (HBeAg) persisted only for 3 days in mosquitoes after feeding on hepatitis B viral antigens-positive blood. Mosquitoes inoculated with serum were HBsAg-positive for 3 weeks and HBeAg positive for 4 days after inoculation. These results suggest that biological multiplication of hepatitis B virus did not occur in these mosquitoes. The possibility of mechanical transmission of hepatitis B antigens by mosquitoes is discussed.