RT-PCR Assays for Seven Serotypes of Epizootic Haemorrhagic Disease Virus & Their Use to Type Strains from the Mediterranean Region and North America

Epizootic haemorrhagic disease virus (EHDV) infects wild ruminants, causing a frequently fatal haemorrhagic disease. However, it can also cause bluetongue-like disease in cattle, involving significant levels of morbidity and mortality, highlighting a need for more rapid and reliable diagnostic assays. EHDV outer-capsid protein VP2 (encoded by genome-segment 2 [Seg-2]) is highly variable and represents the primary target for neutralising antibodies generated by the mammalian host. Consequently VP2 is also the primary determinant of virus “serotype”, as identified in virus neutralisation tests (VNT). Although previous reports have indicated eight to ten EHDV serotypes, recent serological comparisons and molecular analyses of Seg-2 indicate only seven EHDV “types”. Oligonucleotide primers were developed targeting Seg-2, for use in conventional RT-PCR assays to detect and identify these seven types. These assays, which are more rapid and sensitive, still show complete agreement with VNT and were used to identify recent EHDV isolates from the Mediterranean region and North America.     

Calcium signatures and signaling in cytosol and organelles of tobacco cells induced by plant defense elicitors

Calcium signatures induced by two elicitors of plant defense reactions, namely cryptogein and oligogalacturonides, were monitored at the subcellular level, using apoaequorin-transformed Nicotiana tabacum var Xanthi cells, in which the apoaequorin calcium sensor was targeted either to cytosol, mitochondria or chloroplasts. Our study showed that both elicitors induced specific Ca(2+) signatures in each compartment, with the most striking difference relying on duration. Common properties also emerged from the analysis of Ca(2+) signatures: both elicitors induced a biphasic cytosolic [Ca(2+)] elevation together with a single mitochondrial [Ca(2+)] elevation concomitant with the first cytosolic [Ca(2+)] peak. In addition, both elicitors induced a chloroplastic [Ca(2+)] elevation peaking later in comparison to cytosolic [Ca(2+)] elevation. In cryptogein-treated cells, pharmacological studies indicated that IP(3) should play an important role in Ca(2+) signaling contrarily to cADPR or nitric oxide, which have limited or no effect on [Ca(2+)] variations. Our data also showed that, depending on [Ca(2+)] fluxes at the plasma membrane, cryptogein triggered a mitochondrial respiration increase and affected excess energy dissipation mechanisms in chloroplasts. Altogether the results indicate that cryptogein profoundly impacted cell functions at many levels, including organelles.     

Assessment of digestive enzymes activity during the fry development of the endangered Caspian brown trout Salmo caspius

The study of digestive enzymes activity at Salmo caspius fry showed that enzymes were available at the moment of mouth opening on the first day post hatching (dph) and the activity of enzymes showed no significant difference from the hatching day 28 dph. An increased activity was seen between 32 and 43 dph and this activity was significantly higher than the activity during the first 28 days. In the primary stages after yolk sac resorption (43–58 dph), enzymes activity showed an increased profile, however none of them showed a significant difference between 43 and 58 dph.     

Molecular Characterization of Cyst Nematode Species (Heterodera spp.) from the Mediterranean Basin using RFLPs and Sequences of ITS-rDNA

Fifteen populations of cyst‐forming nematodes belonging to 11 known and one unidentified species collected in countries bordering the Mediterranean Sea were studied using polymerase chain reaction restriction fragment length polymorphism (PCR–RFLP) and internal transcribed spacer (ITS)‐rDNA sequences. RFLP profiles generated by the restriction enzymes AluI, AvaI, Bsh1236I, HaeIII, Hin6I, MvaI, PstI and RsaI are presented for Heterodera carotae, H. ciceri, H. fici, H. filipjevi, H. goettingiana, H. hordecalis, H. humuli, H. mediterranea, H. ripae and H. schachtii. Molecular data support the first detection of H. filipjevi from wheat in Italy and H. ripae from nettle in Greece. A relative high level of sequence divergence between populations of H. hordecalis was observed. This suggests that two species might presently be grouped under this taxon. The phylogenetic relationship between the Mediterranean cyst‐forming nematode species is analysed based on the ITS‐rDNA sequences.     

Serum lipoprotein composition, lecithin cholesterol acyltransferase and tissue lipase activities in pregnant diabetic rats and their offspring receiving enriched n-3 PUFA diet

The effects of dietary n-3 polyunsaturated fatty acids on lipoprotein concentrations and on lipoprotein lipase (LPL), hepatic triglyceride lipase (HTGL) and lecithin cholesterol acyltransferase (LCAT) activities were studied in streptozotocin-induced diabetic rats during pregnancy and in their macrosomic offspring from birth to adulthood. Pregnant diabetic and control rats were fed Isio-4 diet (vegetable oil) or EPAX diet (concentrated marine omega-3 EPA/DHA oil), the same diets were consumed by pups at weaning. Compared with control rats, diabetic rats showed, during pregnancy, a significant elevation in very low density lipoprotein (VLDL) and low and high density lipoprotein (LDL-HDL(1))-triglyceride, cholesterol and apoprotein B100 concentrations and a reduction in apoprotein A-I levels. HTGL activity was high while LPL and LCAT activities were low in these rats. The macrosomic pups of Isio-4-fed diabetic rats showed a significant enhancement in triglyceride and cholesterol levels at birth and during adulthood with a concomitant increase in lipase and LCAT activities. EPAX diet induces a significant diminution of VLDL and LDL-HDL(1) in mothers and in their macrosomic pups, accompanied by an increase in cholesterol and apoprotein A-I levels in HDL(2-3) fraction. It also restores LPL, HTGL and LCAT activities to normal range. EPAX diet ameliorates considerably lipoprotein disorders in diabetic mothers and in their macrosomic offspring.     

Implication of the lymphocyte-specific nuclear body protein Sp140 in an innate response to human immunodeficiency virus type 1

The viral infectivity factor (Vif) of human immunodeficiency virus type 1 (HIV-1) neutralizes an unidentified antiviral pathway that occurs only in nonpermissive (NP) cells. Using a yeast two-hybrid screen of a human lymphocyte cDNA library, we identified several potential Vif partners. One, the nuclear body protein Sp140, was found specifically in all NP cells (n = 12 cell lines tested; P < or = 0.001), and HIV-1 infection induced its partial dispersal from nuclear bodies into cytosolic colocalization with Vif. Our results implicate Sp140 in a response to HIV-1 that may be related to or coordinated with the pathway that inactivates HIV-1 lacking vif.     

Ethnicity, infection and sudden infant death syndrome

Epidemiological studies found the incidence of SIDS among Indigenous groups such as Aboriginal Australians, New Zealand Maoris and Native Americans were significantly higher than those for non-Indigenous groups within the same countries. Among other groups such as Asian families in Britain, the incidence of SIDS has been lower than among groups of European origin. Cultural and childrearing practices as well as socio-economic factors have been proposed to explain the greater risk of SIDS among Indigenous peoples; however, there are no definitive data to account for the differences observed. We addressed the differences among ethnic groups in relation to susceptibility to infection because there is evidence from studies of populations of European origin that infectious agents, particularly toxigenic bacteria might trigger the events leading to SIDS. The risk factors for SIDS parallel those for susceptibility to infections in infants, particularly respiratory tract infections which are also major health problems among Indigenous groups. Many of the risk factors identified in epidemiological studies of SIDS could affect three stages in the infectious process: (1) frequency or density of colonisation by the toxigenic species implicated in SIDS; (2) induction of temperature-sensitive toxins; (3) modulation of the inflammatory responses to infection or toxins. In this review we compare genetic, developmental and environmental risk factors for SIDS in ethnic groups with different incidences of SIDS: low (Asians in Britain); moderate (European/Caucasian); high (Aboriginal Australian). Our findings indicate: (1) the major difference was high levels of exposure to cigarette smoke among infants in the high risk groups; (2) cigarette smoke significantly reduced the anti-inflammatory cytokine interleukin-10 responses which control pro-inflammatory responses implicated in SIDS; (3) the most significant effect of cigarette smoke on reduction of IL-10 responses was observed for donors with a single nucleotide polymorphism for the IL-10 gene that is predominant among both Asian and Aboriginal populations. If genetic makeup were a major factor for susceptibility to SIDS, the incidence of these deaths should be similar for both populations. They are, however, significantly different and most likely reflect differences in maternal smoking which could affect frequency and density of colonisation of infants by potentially pathogenic bacteria and induction and control of inflammatory responses.     

Arginine 447 plays a pivotal role in substrate interactions in a neuronal glutamate transporter

Glutamate transporters from the central nervous system play a crucial role in the clearance of the transmitter from the synaptic cleft. Glutamate is cotransported with sodium ions, and the electrogenic translocation cycle is completed by countertransport of potassium. Mutants that cannot interact with potassium are only capable of catalyzing electroneutral exchange. Here we identify a residue involved in controlling substrate recognition in the neuronal transporter EAAC-1 that transports acidic amino acids as well as cysteine. When arginine 447, a residue conserved in all glutamate transporters, is replaced by cysteine, transport of glutamate or aspartate is abolished, but sodium-dependent cysteine transport is left intact. Analysis of other substitution mutants shows that the replacement of arginine rather than the introduced cysteine is responsible for the observed phenotype. In further contrast to wild type, acidic amino acids are unable to inhibit cysteine transport in R447C-EAAC-1, indicating that the selectivity change is manifested at the binding step. Electrophysiological analysis shows that in the mutant cysteine, transport has become electroneutral, and its interaction with the countertransported potassium is impaired. Thus arginine 447 plays a pivotal role in the sequential interaction of acidic amino acids and potassium with the transporter and, thereby, constitutes one of the molecular determinants of coupling their fluxes.     

Development of 2 types of competitive enzyme-linked immunosorbent assay for detecting antibodies to the rinderpest virus using a monoclonal antibody for a specific region of the hemagglutinin protein

A competitive enzyme-linked immunosorbent assay (C-ELISA) has been developed and standardized for the detection of antibodies to the rinderpest virus (RPV) in sera from cattle, sheep, and goats. The test is specific for rinderpest because it does not detect antibodies to peste-des-petits-ruminants virus (PPRV). The test depends on the ability of the monoclonal antibody (MAb) directed against the hemagglutinin (H) protein of RPV to compete with the binding of RPV antibodies in the positive serum to the H protein of this virus. This MAb recognized a region from amino acids 575 to 583 on the H protein of RPV that is unique to the RPV H protein and is not present on the hemagglutinin-neuraminidase protein of PPRV. Another C-ELISA (peptide C-ELISA) was set up using this specific region as an antigen. A threshold value of 64.4% inhibition was established for the RPV C-ELISA, with 90 known RPV-negative and 30 RPV-positive serum samples. Using common serum samples, a cutoff value of 43.0% inhibition for the peptide C-ELISA was established. Based on statistical analysis, the overall sensitivity and specificity of the RPV C-ELISA, relative to those of a commercial kit, were found to be 90.00% and 103.33%, respectively. However, the sensitivity and specificity of the peptide C-ELISA were found to be 180.00% and 73.33%, respectively. Although a common MAb in 2 new C-ELISA systems was used, variation in their percent inhibition, due to the use of different antigens, was observed. Taking into consideration the difference in percent inhibition of the 2 described assays and the commercial kit (50%), it was found that the RPV C-ELISA and the peptide C-ELISA are more specific and sensitive tools than the commercial kit for assessing herd immune status and for epidemiologic surveillance.     

A Chronic Autochthonous Fifth Clade Case of Candida auris Otomycosis in Iran

Candida auris, a multidrug-resistant nosocomial pathogen, has emerged globally with high morbidity and mortality among immunocompromised individuals and COVID19 hospitalized patients. Five major clades of C. auris have been previously described. The fifth clade is exclusively found in Iran where C. auris isolates are genetically distinct from other clades by?>?200,000 single-nucleotide polymorphisms. The origin of C. auris remains unclear, and limited clinical data are available at present regarding clade V infection or colonization. Herein, another case of otomycosis in Iran caused by an isolate of C. auris belonging to the fifth clade is reported. Genotyping revealed that the obtained C. auris isolate from Isfahan clustered with earlier clade V isolates from Babol, cities around 600 km separated, which indicates that C. auris clade V is established in Iran. C. auris is thought to exist more commonly in Iran, given that limited diagnostic capacity in the country has probably curbed the identification of more C. auris cases. Therefore, surveillance of the environment, patients and healthcare facilities in different geographical regions in Iran is urgently required.