Conference Scene: T-cell subset phenotype and function.

Organized by Euroscicon, this meeting focused on the complex and fast-paced research field of T-cell subset phenotype and function. During the past 20 years, this field has moved on from the simple Th1-Th2 paradigm to the discovery of a range of T-cell subsets, including Tregs and Th17 cells. The meeting brought together a variety of researchers currently exploring this field, to give insight into what we know, what we need to know and the potential implications of this research in the medical setting.

Identification of potential hosts and vectors of scrub typhus and tick-borne spotted fever group rickettsiae in eastern Taiwan.

Scrub typhus and tick-borne spotted fever group (SFG) rickettsioses are transmitted by chiggers (larval trombiculid mites) and hard ticks, respectively. We assessed exposure to these disease vectors by extensively sampling both chiggers and ticks and their small mammal hosts in eastern Taiwan during 2007 and 2008. The striped field mouse Apodemus agrarius Pallas (Rodentia: Muridae) was the most common of the small mammals (36.1% of 1393 captures) and presented the highest rate of infestation with both chiggers (47.8% of 110 760) and ticks (78.1% of 1431). Leptotrombidium imphalum Vercammen-Grandjean & Langston (Trombidiformes: Trombiculidae) and immature Rhipicephalus haemaphysaloides Supino (Ixodida: Ixodidae) were the most abundant chiggers (84.5%) and ticks (>99%) identified, respectively. Immunofluorescent antibody assay revealed high seropositive rates of rodents against Orientia tsutsugamushi Hyashi (Rickettsiales: Rickettsiaceae), the aetiological agent of scrub typhus (70.0% of 437 rodents), and tick-borne SFG rickettsiae (91.9% of 418 rodents). The current study represents a first step towards elucidating the potential hosts and vectors in the enzootic transmission of O. tsutsugamushi and tick-borne SFG rickettsiae in Taiwan. Further studies should focus on characterizing pathogens in L. imphalum and R. haemaphysaloides, as well as the proclivity of both vectors to humans. Uncovering the main hosts of adult ticks is also critical for the prevention of SFG rickettsial infections.

[The adaptive strategy of rodent populations living in conditions of radioactive and chemical environmental pollution].

The comparative analysis of demographic, morphological and physiological processes in mouselike rodents in pollution zones (90Sr + 90Y, 137Cs) on East-Ural radioactive track (EURT) and (Cu + Cd + Pb + Zn + SO2) on a site near copper-smelting factory is carried out. The direct (not mediated) defeat of animals by an irradiation leads to inherited adaptation (density preservation, tolerance increase to pollution, migration decrease and so forth). The mediated defeat of animals at pollution by metals influences animals as a result of degradation of a vegetative cover, reducing a forage reserve, shelters and reproduction places. Population is decreasing, migration is increasing. Hence, population reacts onto direct defeat of animals or on inhabitancy locuses degradation, id est unspecifically, without dependence from the physical and chemical nature of pollution.

Localization of Brugia malayi (sub-periodic) adults in different organs of Mastomys coucha and its influence on microfilaraemia and host antibody response.

Lymphatic filariasis caused by nematode parasites Wuchereria bancrofti or Brugia malayi is a spectral disease and produces wide range of immune responses and varying levels of microfilaraemia in infected individuals. The relationship between the immune response of host and the developmental stage of the parasite as well as the microfilariae (mf) density and specific location of the adult worms is yet to be understood. As an experimental model, B. malayi adapted in the experimental animal Mastomys coucha has been used widely for various studies in filariasis. The present study was to assess microfilaraemia as well as the humoral immune response of M. coucha during various stages of B. malayi development and their localization in different organs. The result showed that the density of mf in the circulating blood of the experimental animal depended upon the number of female worms as well as the location and co-existence of male and female worms. The mf density in the blood increased with the increase in the number of females. The clearance of inoculated infective stage (L3) or single sex infection or segregation of male and female to different organs of infected host resulted in a microfilaraemic condition. With respect to antibody response, those animals cleared L3 after inoculation and those with adult worm as well as mf showed low antibody levels. But those with developmental fourth stage and/or adult worms without mf showed significantly higher antibody levels.

Localization of Brugia malayi (sub-periodic) adults in different organs of Mastomys coucha and its influence on microfilaraemia and host antibody response

Lymphatic filariasis caused by nematode parasites Wuchereria bancrofti or Brugia malayi is a spectral disease and produces wide range of immune responses and varying levels ofmicrofilaraemia in infected individuals. The relationship between the immune response of host and the developmental stage of the parasite as well as the microfilariae (mf) density and specific location of the adult worms is yet to be understood. As an experimental model, B. malayi adapted in the experimental animal Mastomys coucha has been used widely for various studies in filariasis. The present study was to assess microfilaraemia as well as the humoral immune response of M. coucha during various stages of B. malayi development and their localization in different organs. The result showed that the density of mf in the circulating blood of the experimental animal depended upon the number of female worms as well as the location and co-existence of male and female worms. The mf density in the blood increased with the increase in the number of females. The clearance of inoculated infective stage (L3) or single sex infection or segregation of male and female to different organs of infected host resulted in amicrofilaraemic condition. With respect to antibody response, those animals cleared L3 after inoculation and those with adult worm as well as mf showed low antibody levels. But those with developmental fourth stage and/or adult worms without mf showed significantly higher antibody levels.

Immunotoxicology in wood mice along a heavy metal pollution gradient.

We carried out an immunotoxicological field study of wood mice in three populations along a heavy metal pollution gradient. Heavy metal concentrations in liver tissue indicated that exposure to silver, arsenic, cadmium, cobalt and lead decreased with increasing distance from a non-ferrous smelter. Host resistance to the endoparasite Heligmosomoides polygyrus decreased with increasing exposure, while the abundance of tick larvae and the nematode Syphacia stroma was unrelated to heavy metal exposure. Spleen mass was increased at the intermediate and the most polluted sites and was positively correlated with the number of H. polygyrus and tick larvae. Proportion of early apoptotic leukocytes increased towards the smelter and was positively related to cadmium exposure. Red and white blood cell counts and lysozyme activity showed no relationship with metal exposure. All together, our observations suggest negative effects of heavy metal exposure on the immune function of wood mice under field conditions.

Inflammatory antigens of Brugia malayi and their effect on rodent host Mastomys coucha.

The study was aimed at identifying pro- and anti-inflammatory cytokine releasing potential of Brugia malayi adult worm fractions and their role in filarial infection and pathogenesis. THP-1 cells were incubated with soluble somatic Brugia malayi adult worm extract (BmAS) and its Sephadex G-200 fractions BmAFI, BmAFII and BmAFIII and the effect of the fractions on parasitological, immunological and lymph node parameters was assessed in Mastomys coucha. BmAFII stimulated the pro-inflammatory TNF-alpha, IL-1beta and IL-6 release; IL-10 release was insignificant. Sensitization of animals with BmAFII and subsequent intraperitoneal implantation of worms enhanced CMI response. BmAFII also increased lymph node weight and cellularity, stimulated lymph node mast cells and eliminated intraperitoneally instilled worms. BmAFI stimulated several folds more release of IL-10, whereas TNF-alpha release was negligible. Sensitization with BmAFI elicited low CMI responses, moderately stimulated mast cells and facilitated survival of implanted adult parasites. Fifty percent of naive animals exposed to BmAFI showed oedematous lymph nodes and increased node weight. NCP-bound molecules corresponding to BmAFI and II showed cytokine-stimulating potential in vitro. It is concluded that BmAFII is protective and stimulates pro-inflammatory cytokines, whereas BmAFI facilitates parasite survival and stimulates IL-10.

Anti-Borrelia antibodies in rodents: important hosts in ecology of Lyme disease.

Data presented in this study focuses on the presence of anti-Borrelia antibodies in small mammals from Eastern Slovakia during 2000-2003. The total seropositivity observed was 18.78% in rodents. Amongst all species, the total seroprevalence in Apodemus flavicolis was the highest (20.87%), followed by Apodemus agrarius (19.58%) and Clethrionomys glareolus (11.11%). However, the prevalence in Apodemus flavicolis during the year 2000-2001 was higher (26.72%), which reduced to 10.60% in 2002-2003. To compare the year range of seroprevalence in other small mammals was not feasible due to the small sample number. Area-wise distribution of anti-Borrelia antibodies was even (18.75% to 20%) in this study, except in the Bot'any province (0%). This confirms the equal distribution of Borrelia spirochetes in the other 3 localities. Prevalence of anti-Borrelia antibodies during summer was significantly higher than during autumn and early spring. The overall study also reviews the importance of small mammals in Lyme disease ecology.

Evidence of the presence of Seoul virus in Cambodia.

A study was conducted in agricultural and urban areas in Cambodia to assess the presence of hantaviruses in rodent populations. In 1998, rodents were trapped in two villages and in Phnom Penh city around market places and a rubbish dump. IgG antibodies to Hantaan virus were detected in 54 (8.2%) rodents among 660 tested: 6.4% (13/203) among roof rats (Rattus rattus), 20.9% (39/187) among Norway rats (R. norvegicus), 16.7% (2/12) among unidentified Rattus species and none in 183 Polynesian rats (R. exulans) or in 75 bandicoot rats (Bandicota sp.). The presence of the viral genome was detected by a reverse transcription-PCR amplifying part of the sequence coding for the nucleoprotein in the S segment, in 87% of the seropositive rodents. Thirty-one representative cDNAs were sequenced. Phylogenetic studies of the sequences indicated a close relationship with Seoul virus. However, the Cambodian Seoul virus sequences clustered within two different phylogenetic lineages, one associated with R. rattus and the other with R. norvegicus.

Tests for positive selection on immune and reproductive genes in closely related species of the murine genus mus.

We examine variation among species of Mus in four genes involved in reproduction and the immune response for evidence of positive selection: the sperm recognition gene Zp-3, the testis-determining locus Sry, the testicular cell surface matrix protein Tcp-1, and the immune system protein beta(2) m. We use likelihood ratio tests in the context of a well-supported phylogeny to determine whether models that allow for positively selected sites fit the sequences better than models that assume purifying selection. We then apply a Bayesian approach to identify particular sites in each gene that have a high posterior probability of being under positive selection. We find no evidence of positive selection on the Tcp-1 gene, but for Zp-3, Sry, and beta(2) m, models that allow for positively selected sites fit the sequences better than alternatives. For each of these genes, we identify sites that have a high (> 95%) posterior probability of being positively selected. For Zp-3, two of these sites occur near the sperm-binding region, while one occurs in a region whose functional role remains unstudied but where the pattern of change predicts functional importance. A single site in Sry shows an elevated rate of replacement substitution but occurs in a region of apparently little functional importance; therefore, relaxation of functional constraints may better explain the rapid evolution of this site. Three sites in beta(2) m have a posterior probability > 50% of being under positive selection. While the functional role for two of these sites is unknown, the third is known to influence the ability of MHC class I molecules to present antigens to the immune system; therefore, the elevated rate of replacement substitutions at this site is consistent with selection acting to promote variability in immune system proteins.

Testosterone depresses innate and acquired resistance to ticks in natural rodent hosts: a force for aggregated distributions of parasites.

The effects of testosterone on acquired resistance to ticks, Ixodes ricinus, in their natural rodent hosts (voles, Clethrionomys glareolus, and wood-mice, Apodemus sylvaticus) were investigated by manipulating testosterone levels and exposing the hosts to repeated tick infestations. Testosterone reduced both innate and acquired resistance to tick feeding. During primary infestations, attachment rates were higher on rodents with high testosterone levels than on oil-implanted controls. Successive infestations on voles were accompanied by a decrease in tick feeding success and survival, but this decrease was significantly greater in ticks fed on control voles than in those fed on voles implanted with testosterone. When reduced feeding success had been induced, either by vaccination with tick salivary gland extract or by 4 successive infestations, implantation with testosterone partially reversed the acquired resistance. These effects of testosterone will generate heterogeneities within the rodent population with respect to tick distribution and microparasite transmission. The lowest innate and acquired resistance to tick feeding occurs in that fraction of the host population, i.e., sexually active males, most actively involved in the transmission of both Babesia microti and Borrelia burgdorferi s.l.

Nomenclature and overview of the mouse (Mus musculus and Mus sp.) immunoglobulin kappa (IGK) genes.

'Nomenclature and overview of the mouse (Mus musculus and Mus sp.) immunoglobulin kappa (IGK) Genes', the 19th report of the 'IMGT Locus in Focus' section, provides the first complete list of all the mouse (M. musculus) IGK genes. The mouse (M. musculus) locus spans 3,200 kb. The total number of mouse (M. musculus) IGK genes per haploid genome is 164 (174 if the orphons are included). The functional genomic repertoire comprises 93 IGKV belonging to 18 subgroups, 5 IGKJ and 1 IGKC gene. IMGT gene names and definitions of the mouse (M. musculus) IGK genes on chromosome 6 and IGK orphons are provided with the gene functionality and the number of alleles, according to the concepts of IMGT-ONTOLOGY and to rules of the IMGT Scientific chart, with the accession numbers of the IMGT reference sequences. These tables and figures are available at the IMGT Marie-Paule page of IMGT, the international ImMunoGeneTics database (http://imgt.cines.fr) created by Marie-Paule Lefranc, Université Montpellier II, CNRS, France.

Preliminary study on potential circulation of arenaviruses in the rodent population of Nakhon Pathom Province, Thailand and their medical importance in an evoluting environment.

Preliminary serological investigations were prefered to detect evidence of arenavirus infection in rodents. The study examined virus antibody in 367 rodents trapped in 6 different geographical areas of Nakhon Pathom Province, Thailand from February-March, 1998. The overall seroprevalence among rodents was 13.3%, mostly in Bandicota savilei (35.7%) and Rattus norvegicus (31.5%). Between ecology, behavior and sex of the rodents, seroprevalence was not significantly different (p>0.05), however the seroprevalence found among different geographical areas of Nakhon Pathom Province were significantly different (p<0.0001).

Immune system cell markers in the northern brown bandicoot, Isoodon macrourus.

This is the first study to report the presence of T and B lymphocyte markers and antigen presenting-like molecules in a marsupial bandicoot. Intra-cytoplasmic markers for CD3 and CD5, as well as surface Thy-1.1 and CD1a molecules were located in lymphocytes of T dependent regions of immuno-lymphoid tissue in the northern brown bandicoot using immunohistochemical techniques. Similarly, intra-cytoplasmic domains of CD79a, CD79b molecules and surface IgG molecules enabled characterisation of B lymphocytes and plasma cells. The phenotypic expression of these molecules parallels findings in eutherians, suggesting firstly the conservation of lineage epitopes for T and B subsets and secondly, the potential for similar functional properties of immune system cells between marsupials and eutherians. In addition, the presence of MHC class II and CD1a molecules on dendritic-like cells may indicate similar mechanisms for antigen processing and presentation as reported in eutherians. The use of such immune system cell markers will enable functional studies to characterise the marsupial immune system as well as ontogeny studies of immune competence.

Immunomodulatory effects in Litomosoides sigmodontis-infected Mastomys coucha.

The levels of parasite-specific IgG1 and IgG2 antibodies and mitogen-induced and parasite-specific proliferative T-cell responses were determined in Litomosoides sigmodontis-infected Mastomys coucha throughout an observation period of 400 days post infection (p.i.). These were compared with the respective reactions in animals that had been immunized with intrauterine stages/microfilariae of the parasite and in animals that had been challenged after immunization as determined at up to 60 days after challenge. IgG1 antibodies to adult antigen developed early and reached a plateau at 120 days p.i., whereas IgG2 antibodies were not found before day 60 p.i., increased with rising parasitemia, reached a plateau at 200 days p.i., and, in some animals, even became the predominant IgG subclass. Proliferative responses of spleen lymphocytes to concanavalin A (Con A) and lipopolysaccharides (LPS), but not Con-A-induced interleukin 2 (IL-2) production, were found to be suppressed in infected animals during patency as compared with uninfected controls. Spleen cells of infected animals showed a weak proliferative reaction to male antigen but were unresponsive to female and microfilarial antigen during prepatency and early patency. Subsequently, when microfilaremia decreased (200 days p.i.), continuously increasing responses to all antigens were observed. Immunized M. coucha developed specific IgG1 and IgG2 antibodies, and their spleen cells showed strong proliferative responses to the three L. sigmodontis antigens. Challenge infections down-regulated the proliferative responses of spleen cells to filarial antigens as early as during the prepatent phase of the challenge infection but supported existing IgG1 and IgG2 responses.

Prevalence of IgG antibodies response to Borrelia burgdorferi s.l. in populations of wild rodents from Mazury lakes district region of Poland.

Three rodent species: Clethrionomys glareolus, Apodemus flavicollis and Microtus arvalis from Mazury Lakes District of Poland were examined for antibodies to Borrelia burgdorferi by enzyme-labelled protein G assay (ELGA). C. glareolus had an exceptionally high prevalence of B. burgdorferi antibodies - 58%, but A. flavicollis and M. arvalis also showed significant prevalence of 16.6% and 10.5%, respectively. The ELGA method is highly specific with good reproducibility. Nevertheless, some differences of sensitivity of assessed samples were season dependent. However, high seroprevalence did not coincide with infestation rates of examined rodents by I. ricinus ticks. The results indicated that in Mazury Lakes District, naturally infected rodents play an important role as an animal reservoir host for B. burgdorferi, and these animals may increase the risk of human infections in some habitats used as recreation areas. Also, this study shows that ELGA method based on the affinity of protein G for IgG of wild animals may be widely used to determine the competent zoonotic reservoir of B. burgdorferi.

Isolation and genetic characterization of a hantavirus (Bunyaviridae: Hantavirus) from a rodent, Oligoryzomys microtis (Muridae), collected in northeastern Peru.

This paper describes the isolation and partial genetic characterization of a hantavirus from a pygmy rice rat, Oligoryzomys microtis, collected within the urban area of Iquitos, Loreto Department, Peru. The virus, designated HTN-007, exhibited the highest degree of genetic similarity to Rio Mamore virus, which was originally described from the same rodent species in eastern Bolivia. Comparison of small and medium segment nucleotide sequence data from HTN-007 and Rio Mamore virus revealed 87% and 85% sequence identity, respectively. Based on these analyses, HTN-007 appears to be a variant of Rio Mamore virus. As such, it represents the first successful isolation of Rio Mamore virus and the first evidence for the existence of a hantavirus in Peru. Serologic studies done by immunofluorescence on blood samples of 56 O. microtis trapped at the collection site indicated that 21.4% had antibodies to hantavirus. In view of the proximity of this rodent species to humans and the close phylogenetic relationship of Rio Mamore virus to hantaviruses that have been associated with human disease, Rio Mamore virus may be a hantavirus of some public health importance in tropical South America.

Allelic diversity at the Mhc-DQA locus in cotton rats (Sigmodon hispidus) and a comparison of DQA sequences within the family muridae (Mammalia: Rodentia).

The cotton rat (Sigmodon hispidus) is a common murid rodent of the southern United States, Mexico, and Central America. Using single-stranded conformation polymorphism analysis and DNA sequencing techniques, 11 DQA exon 2 alleles were detected among 180 S. hispidus from Caddo County, Oklahoma, USA. The alleles represent a single locus exhibiting a high level of polymorphism. Nucleotide and amino acid distance values among DQA alleles of S. hispidus were higher than those within Mus musculus and species of Rattus. Although the distribution of polymorphic amino acid residues among alleles of S. hispidus was similiar to that of Mus and Rattus, some residues of the alpha-helix region were more variable in S. hispidus. Comparisons of nonsynonymous and synonymous substitutions indicated a trend toward higher numbers of nonsynonymous substitutions; however, this difference was not significant statistically among S. hispidus alleles. To examine evolution of DQA alleles within Muridae, we performed a phylogenetic analysis that included DQA alleles from S. hispidus, Peromyscus leucopus, M. musculus, R. norvegicus, and six Australian species of Rattus. Results depicted monophyly for each genus, and this concordance between species and gene trees represents a lack of evidence for trans-species persistence of alleles among these genera.

Receptor-mediated interference mechanism responsible for resistance to polytropic leukemia viruses in Mus castaneus.

The Asian mouse Mus castaneus is resistant to infection by the polytropic mink cell focus-inducing (MCF) subgroup of murine leukemia viruses (MuLVs). Genetic crosses showed this recessive resistance to be governed by a single gene that maps at or near the gene encoding the polytropic viral receptor, Rmc1. To investigate this resistance, we mated M. castaneus with mice carrying the wild mouse Sxv variant of the Rmc1 receptor that allows infection by xenotropic as well as polytropic virus. Unlike other F1 hybrids of M. castaneus, these F1 mice were resistant to both xenotropic and polytropic classes of MuLVs. Analysis of backcrossed progeny of the F1 hybrids mated to Sxv mice indicates that resistance is due to inheritance of two M. castaneus genes. Cells from individual backcross mice were also examined for cell surface antigen by fluorescence-activated cell sorter analysis with monoclonal antibodies reactive with xenotropic or MCF virus env glycoproteins. A correlation was observed between virus resistance and antigen, suggesting that virus resistance is due to expression of endogenous viral envelope genes that interfere with infection by exogenous virus. Since the inbred strain Rmc1 receptor remains functional in the presence of these M. castaneus genes, and since M. castaneus contains multiple copies of xenotropic MuLV env genes, we suggest that these resistance genes control expression of xenotropic env glycoprotein that interferes with exogenous virus in cells containing the Sxv variant of Rmc1.

Comparison of Ehrlichia muris strains isolated from wild mice and ticks and serologic survey of humans and animals with E. muris as antigen.

In metropolitan Tokyo, the Ehrlichia muris seropositivity rate of 24 wild mice was 63% in Hinohara Village, but in the surrounding areas, it was 0 to 5%. This finding suggests that the reservoir of E. muris is focal. Among the 15 seropositive mice, ehrlichiae were isolated from 9 Apodemus speciosus mice and 1 A. argenteus mouse, respectively. Five ehrlichial isolates were obtained from 10 ticks (Haemaphysalis flava) collected in Asuke Town, Aichi Prefecture, where the E. muris type strain had been isolated. These new isolates were compared with the E. muris type strain. The mouse virulence and ultrastructure of the new isolates were similar to those of the type strain, and all of them were cross-reactive with each other, as well as with the type strain, by indirect immunofluorescent-antibody test. The levels of similarity of the base sequences of the 16S rRNA gene of one of the A. speciosus isolates and one of the tick isolates to that of the E. muris type strain were 99.79 and 99.93%, respectively. We suggest that all of these isolates are E. muris; that E. muris is not limited to Eothenomys kageus but infects other species of mice; and that E. muris is present at locations other than Aichi Prefecture. It appears that H. flava is a potential vector of E. muris. Twenty (1%) of 1803 humans from metropolitan Tokyo were found to be seropositive for E. muris antibodies. A serological survey revealed that exposure to E. muris or organisms antigenically cross-reactive to E. muris occurred among dogs, wild mice, monkeys, bears, deer, and wild boars in Gifu Prefecture, nearby prefectures, and Nagoya City, central Japan. However, human beings and Rattus norvegicus rats in this area were seronegative. These results indicate broader geographic distribution of and human and animal species exposure to E. muris or related Ehrlichia spp. in Japan.