The status of tularemia in Europe in a one-health context: a review.

The bacterium Francisella tularensis causes the vector-borne zoonotic disease tularemia, and may infect a wide range of hosts including invertebrates, mammals and birds. Transmission to humans occurs through contact with infected animals or contaminated environments, or through arthropod vectors. Tularemia has a broad geographical distribution, and there is evidence which suggests local emergence or re-emergence of this disease in Europe. This review was developed to provide an update on the geographical distribution of F. tularensis in humans, wildlife, domestic animals and vector species, to identify potential public health hazards, and to characterize the epidemiology of tularemia in Europe. Information was collated on cases in humans, domestic animals and wildlife, and on reports of detection of the bacterium in arthropod vectors, from 38 European countries for the period 1992-2012. Multiple international databases on human and animal health were consulted, as well as published reports in the literature. Tularemia is a disease of complex epidemiology that is challenging to understand and therefore to control. Many aspects of this disease remain poorly understood. Better understanding is needed of the epidemiological role of animal hosts, potential vectors, mechanisms of maintenance in the different ecosystems, and routes of transmission of the disease.

Molecular Detection of Persistent Francisella tularensis Subspecies holarctica in Natural Waters.

Tularemia, caused by the bacterium Francisella tularensis, where F. tularensis subspecies holarctica has long been the cause of endemic disease in parts of northern Sweden. Despite this, our understanding of the natural life-cycle of the organism is still limited. During three years, we collected surface water samples (n = 341) and sediment samples (n = 245) in two areas in Sweden with endemic tularemia. Real-time PCR screening demonstrated the presence of F. tularenis lpnA sequences in 108 (32%) and 48 (20%) of the samples, respectively. The 16S rRNA sequences from those samples all grouped to the species F. tularensis. Analysis of the FtM19InDel region of lpnA-positive samples from selected sampling points confirmed the presence of F. tularensis subspecies holarctica-specific sequences. These sequences were detected in water sampled during both outbreak and nonoutbreak years. Our results indicate that diverse F. tularensis-like organisms, including F. tularensis subsp. holarctica, persist in natural waters and sediments in the investigated areas with endemic tularemia.

The H2-Ab gene influences the severity of experimental allergic encephalomyelitis induced by proteolipoprotein peptide 103-116.

Immunization of H2(p) and H2(q) congenic C3H mouse strains with the PLP 103-116 peptide elicited two distinct experimental allergic encephalomyelitis (EAE) disease courses. C3H.Q (H2(q)) mice developed an acute-phase disease with classical ascending paralytic signs whereas C3H.NB (H2(p)) developed a highly variable disease course with symptoms originating from CNS above the spinal chord. C3H.Q lacks functional H2-E molecules and share H2-Aalpha with C3H.NB. To examine if the differences found at positions 85, 86, 88, and 89 in the Abeta-chains account for disease susceptibility, H2(q) mice were made transgenic with the Ab(p) gene. The Ab(p)-transgenic mice on the C3H.Q background developed a more severe disease course, demonstrating the importance of class II. However, the onset was not affected and the disease showed a classical ascending paralysis similar to the C3H.Q suggesting that the observed brain symptoms were related to nonclass II genes. Inhibition studies performed on affinity purified MHC class II molecules indicated that the PLP 103-116 peptide bound to A(p) with slightly higher affinity than to A(q). Both A(q) and A(p) formed long-lived stable complexes (t(1/2)>24 h) with the PLP 103-116 peptide, but a higher amount of the peptide was loaded on to A(p) compared with A(q). An F2 gene segregation experiment, in which the low PLP 103-116 binding A(r) molecule and the high binding A(p) molecule could be compared for the influence on the disease susceptibility, indicated a role for both peptide binding affinity and non-MHC genes. Based on our results, we conclude that the H2-Ab gene controls severity of EAE but not necessarily the onset or type of disease course and that affinity of the disease-promoting peptide for the class II molecule is a critical pathogenic factor.

The Th2 cytokines IL-4 and IL-10 are not crucial for the completion of allogeneic pregnancy in mice.

The physiological protection from fetal rejection is believed to be dependent on a Th2 type of inflammatory response at the maternal-fetal interface and the cytokines IL-4 and IL-10 have been suggested to play a critical role. We here present data from breeding experiments with IL-10 and IL-4 double-deficient mice indicating that neither maternal nor feto-placental deficiency of these cytokines are crucial for fetal or neonatal survival. The present study does not analyse possible developmental effects of maternal or fetal IL-10 and IL-4 double-deficiency in detail, but shows that an apparently normal breeding can be achieved in different crossings, providing that the mice are kept under very clean conditions.

Neonatal ingestion of IgG-containing milk increases the survival of adoptively transferred B-lineage cells in B cell-deficient mice.

This study shows that neonatal ingestion of immunoglobulin-containing milk increases the survival of adoptively transferred B-lineage cells in non-irradiated B cell-deficient (mu-/-) mice. Neonatal mu-/- mice were either transferred to lactating mu+/+ foster dams, allowing them to suckle IgG-containing milk, or were kept with their mu-) dams, without IgG in the milk. After adoptive transfer of spleen cells as adults, serum-IgG levels, numbers of plasma cells, T and B cells in spleen and bone marrow were determined. The results showed that the mice which had received milk-IgG had significantly higher levels of serum-IgG and splenic B cells, as well as a higher number of Ig-secreting cells in spleen and bone marrow. This indicates that the mice might have been tolerised against IgG as neonates, if allowed to ingest Ig-containing milk. There is, however, a possibility that B cells in the milk may also contribute to the observed tolerising effect. In summary, the results suggest that 'cross-fostering' could be a suitable method to facilitate the long-term reconstitution of B- and plasma cell numbers in non-irradiated B cell-deficient mice.

Immunoglobulin-secreting cells of maternal origin can be detected in B cell-deficient mice.

It is well known that the transfer of immunoglobulins (Igs) from mother to young via milk contributes to the offspring's immune defense. The present study suggests that not only is IgG transmitted to progeny, but that functional maternal Ig-secreting cells (or B cells) can also be transferred to the neonate. We have used B cell-deficient (micro(-/-)) mice and found that a high proportion of them obtain long-lasting, partial reconstitution of their serum Ig levels if born to micro(+/-) mothers. In some of these serum IgG-positive micro(-/-) mice, Ig-secreting cells were detected in spleen and bone marrow. To ensure that cells of maternal origin were present in the progeny, micro(-/-) offspring born to micro(+/-) dams transgenic for green fluorescent protein (GFP) were used. In spleens and bone marrow from some of these micro(-/-)GFP(-/-) offspring, GFP-positive cells were detected, which demonstrated that cells of maternal origin could infiltrate the progeny. In addition, splenic Ig-secreting cells were detected in micro(-/-) mice that were born to micro(-/-) dams and transferred to a lactating micro(+/+) foster dam at birth. This indicates that maternal Ig-secreting cells can be transferred postnatally via milk.

The 5' flank of mouse H19 in an unusual chromatin conformation unidirectionally blocks enhancer-promoter communication.

BACKGROUND: During mouse prenatal development, the neighbouring insulin-like growth factor II (Igf2) and H19 loci are expressed monoallelically from the paternal and maternal alleles, respectively. Identical spatiotemporal expression patterns and enhancer deletion experiments show that the Igf2 and H19 genes share a common set of enhancers. Deletion of a differentially methylated region in the 5' flank of the H19 gene partially relieves the repression of the maternal Igf2 and paternal H19 alleles in the soma. The mechanisms underlying the function of the 5' flank of the H19 gene are, however, unknown. RESULTS: Chromatin analysis showed that the 5' flank of the mouse H19 gene contains maternal-specific, multiple nuclease hypersensitive sites that map to linker regions between positioned nucleosomes. These features could be recapitulated in an episomal-based H19 minigene, which was propagated in human somatic cells. Although the 5' flank of the H19 promoter has no intrinsic silencer activity under these conditions, it unidirectionally extinguished promoter-enhancer communications in a position-dependent manner, without directly affecting the enhancer function. CONCLUSIONS: The unmethylated 5' flank of the H19 gene adopts an unusual and maternal-specific chromatin conformation in somatic cells and regulates enhancer-promoter communications, thereby providing an explanation for its role in manifesting the repressed state of the maternally inherited Igf2 allele.

Feral pigeons as carriers of Cryptococcus laurentii, Cryptococcus uniguttulatus and Debaryomyces hansenii.

We collected fresh droppings and cloaca samples from feral pigeons Columba livia in the southern Swedish city of Malmö, and isolated the following fungi: Debaryomyces hansenii var. hansenii, Cryptococcus laurentii and Cryptococcus uniguttulatus. The first two species are known to be pathogenic to humans. No strains of Cryptococcus neoformans var. neoformans were found. Our results indicate that feral pigeons can be carriers of medically significant fungi other than Cryptococcus neoformans var. neoformans.

IgG-mediated enhancement of antibody responses is low in Fc receptor gamma chain-deficient mice and increased in Fc gamma RII-deficient mice.

Immunization with IgG/Ag or IgE/Ag complexes leads to a higher production of specific Abs than immunization with Ag alone. The enhancing effect of IgE is exclusively dependent upon the low-affinity receptor for IgE, Fc epsilon RII, whereas the mechanism behind IgG-mediated enhancement is unknown. We have investigated whether receptors for the Fc part of IgG are required for responses to IgG/Ag. Mice lacking the gamma subunit of Fc receptors (FcRs) (FcR gamma-/-), Fc gamma RII (Fc gamma RII-/-), or Fc gamma RIII (Fc gamma RIII-/-) were immunized with BSA-2,4,6-trinitrophenyl (TNP) alone or BSA-TNP complexed to monoclonal TNP-specific IgG1, IgG2a, or IgG2b. As expected, all subclasses enhanced the Ab-response to BSA in wild-type mice. Enhancement was in the same order of magnitude in Fc gamma RIII-/- mice (

Adiaspiromycosis in a European beaver from Sweden.

An infection with the rare mycosis Chrysosporium parvum was diagnosed in a European beaver (Castor fiber) shot in northern Sweden. The animal was in normal body condition and no signs of disease were observed. In the lungs a large number of nodules, up to 5 mm diameter, were observed. A large number of adiaspores were observed in the interstitium of the lungs and in the mediastinal lymph node. A chronic inflammatory reaction dominated by mononuclear leukocytes and giant cells was observed around the spores. This is the first report of adiaspiromycosis (Chrysosporium parvum) in the European beaver.

Human choriocarcinoma-derived JEG-3 cells transfected with murine MHC class II Aq expression vectors present antigen to Aq-restricted murine T-cell hybridoma.

MHC class II molecules are not normally expressed on the cell surface of placental cells. This absence of class II molecules is assumed to be of importance for mammalian reproduction, since such expression is likely to increase the risk of harmful anti-placental immune responses. The present study was aimed to clarify whether post-transcriptional events prohibit proper cell surface expression of MHC class II molecules in cell lines of placental origin. The murine trophoblast cell line SM9-1 as well as the human choriocarcinoma-derived cell line JEG-3 were transiently co-transfected with MHC class II Aq a and b genes under the control of viral promoter systems. The transfected cells were stained for surface expression of MHC class II and assayed for antigen presentation in vitro. Only a small proportion of the transfected murine SM9-1 cells showed detectable class II cell surface expression, which made functional studies of this cell line difficult. The transfected JEG-3 cells, however, showed a high proportion of cells with distinct surface expression of murine class II Aq molecules and the antigen presentation assays revealed T cell activation upon addition of processed antigen, but not with unprocessed antigen. These results show that ectopic MHC class II gene transcription can result in cell surface expression of immunohistochemically detectable MHC class II on cells of placental origin. The fact that murine class II molecules could be expressed in a functional manner on human JEG-3 cells also strongly suggests that proper accessory gene activities are not essential for obtaining surface expression.

Hypervariable allelic expression patterns of the imprinted IGF2 gene in tumor cells.

The IGF2 gene, which encodes a growth factor, is subject to genomic imprinting. The frequently observed loss of IGF2 imprinting in a variety of tumors has been suggested to contribute to neoplasia. Since these reports have not documented the imprinting status of IGF2 at the cellular level, it cannot be excluded that the imprinting status might vary within the tumor. The possibility that loss of IGF2 imprinting in neoplastic cells reflects random imprinting patterns, was therefore addressed. We show here that individual cell populations of the JEG-3 choriocarcinoma cell line display heterogenous imprinting patterns of both IGF2 and H19. In addition, a lack of correlation between IGF2 and H19 imprinting status suggests that any regional parental imprint has been functionally lost. This notion is reinforced by the observation that JEG-3 cell subclones display a range of promoter-specific IGF2 allele usage. Moreover, we observed that the imprinting status of H19 and IGF2 were differentially modulated in JEG-3-derived tumors generated in nude mice. The results suggest that allele-specific expression of IGF2 operates in the absence of a parental imprint. Finally, our observations urge caution with respect to the general interpretation of biallelic expression as 'loss of imprinting'.

The paternal allele of the H19 gene is progressively silenced during early mouse development: the acetylation status of histones may be involved in the generation of variegated expression patterns.

Transcriptional silencing can reflect heritable, epigenetic inactivation of genes, either singly or in groups, during the life-time of an organism. This phenomenon is exemplified by parent-of-origin-specific inactivation events (genomic imprinting) for a subset of mammalian autosomal genes, such as H19. Very little is known, however, about the timing and mechanism(s) of silencing of the paternal H19 allele during mouse development. Using a novel in situ approach, we present evidence that the silencing of the paternal H19 allele is progressive in the trophectodermal lineage during early mouse development and generates variegated expression patterns. The silencing process apparently involves recruitment of histone deacetylases since the mosaic paternal-specific H19 expression reappears in trichostatin A-treated mouse conceptuses, undergoing in vitro organogenesis. Moreover, the paternal H19 alleles of PatDup.d7 placentas, in which a region encompassing the H19 locus of chromosome 7 is bipaternally derived, partially escape the silencing process and are expressed in a variegated manner. We suggest that allele-specific silencing of H19 share some common features with chromatin-mediated silencing in position-effect variegation.

Lack of detectable major histocompatibility complex class II a beta-chain messenger ribonucleic acid in placentas of interferon-gamma- and 5-azacytidine-treated mice.

Trophoblast cells do not normally express major histocompatibility complex (MHC) class II antigens during placental development in either mice or rats. We have previously observed that in vivo treatment of pregnant mice with interferon-gamma (IFN gamma) induces immunohistochemically detectable class II cell surface expression in many maternal cell types, but not on placental cells or other cells of extra-embryonic origin. Both IFN gamma- and 5-azacytidine-induced placental class II expression have been reported in mice by other scientists, however, which made it important to further clarify this issue. The present study was performed to analyze whether treatment of pregnant mice with recombinant IFN gamma or the drug 5-azacytidine in vivo can induce detectable MHC class II Ab mRNA expression. A strain of transgenic mice carrying a cytomegalovirus-regulated MHC class II Abq transgene, which was strongly expressed in the placenta, was used as a positive control in all in situ hybridizations and ribonuclease protection analyses. All mice were analyzed on gestation Days 12.5 and 17.5. Treatment of pregnant mice with IFN gamma did not induce detectable class II expression in the placental cells, whereas the maternal decidua showed expression both at the mRNA and protein level. Similarly, treatment with 5-azacytidine did not induce class II expression in the placenta, while a slight increase in mRNA expression was detected in the maternal decidual and uterine tissues. These results strengthen the opinion that MHC class II mRNA cannot normally be induced in murine placental cells after IFN gamma or 5-azacytidine treatments.

Maternal antibodies protect immunoglobulin deficient neonatal mice from mouse hepatitis virus (MHV)-associated wasting syndrome.

PROBLEM: Neonatal mice nursed by dams lacking immunoglobulins (Igs) may often suffer from lethal runting if raised under conventional conditions. The present study was performed in order to clarify a) the cause of the wasting syndrome and b) the protective role of antigen-specific milk antibodies. METHOD: Ig-deficient mouse embryos in a conventional environment were embryo-transferred to specified pathogen free (SPF) dams. Neonatal growth, mortality, and health status of mice from both environments was recorded. Suspected presence of mouse hepatitis virus (MHV) was tested by RT-PCR. Protective effects on neonatal mortality of milk containing different titers of anti-MHV antibodies were investigated in cross-fostering experiments. RESULTS: The SPF colony of Ig-deficient mice exhibited no breeding problems, whereas Ig-deficient neonates in the conventional environment suffered from lethal wasting syndrome. Serological screening of the mice kept in the two environments revealed that mice in the conventional room had high titers of antibodies against mouse hepatitis virus. Presence of MHV in runting neonates was confirmed by pathological examinations and RT-nested-PCR using MHV genome specific primers. Milk containing high titers of anti-MHV antibodies, when provided for 8 days or more, completely prevented Ig-deficient neonates from developing wasting syndrome in the conventional environment. CONCLUSION: These findings show that the neonatal wasting syndrome is associated with the presence of MHV and that neonates are efficiently protected by MHV-specific antibodies in the milk.

Systemic versus cartilage-specific expression of a type II collagen-specific T-cell epitope determines the level of tolerance and susceptibility to arthritis.

Immunization of mice with rat type II collagen (CII), a cartilage-specific protein, leads to development of collagen-induced arthritis (CIA), a model for rheumatoid arthritis. To define the interaction between the immune system and cartilage, we produced two sets of transgenic mice. In the first we point mutated the mouse CII gene to express an earlier defined T-cell epitope, CII-(256-270), present in rat CII. In the second we mutated the mouse type I collagen gene to express the same T-cell epitope. The mice with mutated type I collagen showed no T-cell reactivity to rat CII and were resistant to CIA. Thus, the CII-(256-270) epitope is immunodominant and critical for development of CIA. In contrast, the mice with mutated CII had an intact B-cell response and had T cells which could produce gamma interferon, but not proliferate, in response to CII. They developed CIA, albeit with a reduced incidence. Thus, we conclude that T cells recognize CII derived from endogenous cartilage and are partially tolerized but may still be capable of mediating CIA.

Expression of a novel member of estrogen response element-binding nuclear receptors is restricted to the early stages of chorion formation during mouse embryogenesis.

Members of the nuclear hormone receptor gene family of transcription factors have been shown to be expressed in characteristic patterns during mouse organogenesis and postnatal development. Using an RT-PCR based screening assay, we have identified nuclear receptors expressed in embryonal carcinoma stem cells. One of the cDNAs characterized, mERR-2, was found to be expressed exclusively during a narrow developmental window in trophoblast progenitor cells between days 6.5 and 7.5 post coitum (p.c.). From 8.5 days p.c. and onwards, the mERR-2 gene activity evaded detection as analysed by in situ hybridization. We also show that the mERR-2 gene product and the estrogen receptor share a common target DNA-sequence recognition specificity unique among members of the gene family. Furthermore, efficient homodimerization and DNA-binding of the orphan receptor mERR-2 was found to be dependent on interaction with the heat shock protein 90, a molecular chaperone hitherto recognized to interact only with the steroid hormone receptor subgroup of nuclear receptors. Based on our results we suggest that the mouse orphan receptor mERR-2 has the potential to regulate overlapping gene networks with the estrogen receptor and may participate in signal transduction pathways during a short developmental period coinciding with the formation of the chorion.

Pregnancy in B-cell-deficient mice: postpartum transfer of immunoglobulins prevents neonatal runting and death.

Mice lacking functional B cells because of a genetic deletion of the mu chain (IgM) gene were used to investigate the role of perinatal and postnatal transfer of maternal IgG in neonatal growth. Our results confirmed that immunoglobulin (Ig)-deficient mice successfully complete pregnancy and deliver healthy offspring. However, neonates nursed by Ig-deficient mothers showed growth retardation (runting) and high mortality during their first 10 days of life. This fatal course was seen whether or not the neonates were Ig-deficient. Cross-switching litters from phenotypically normal mothers to Ig-deficient mothers immediately after birth showed that perinatal Ig transfer normalized neonatal development for 10 days, but was not sufficient to sustain survival during the later part of the neonatal period. On the other hand, all Ig-deficient litters nursed by normal foster mothers showed normal development and 0% neonatal mortality. Administration of mouse IgG to an Ig-deficient mother or a neonate during the first critical week prevented runting. We assume that the growth- and health-promoting effects of IgG during early neonatal life are attributable mainly to the transfer of passive immunity to environmental pathogens. However, the finding that monoclonal IgG antibodies also enhanced neonatal growth and survival suggests that IgG-dependent growth-promoting mechanisms could be involved as well.

Expression of a transgenic class II Ab gene confers susceptibility to collagen-induced arthritis.

The major histocompatibility complex (MHC) class II region is assumed to influence autoimmune diseases such as rheumatoid arthritis. In the mouse, the H-2q haplotype is associated with susceptibility to collagen-induced arthritis, while the H-2p haplotype is not. The class II A molecules of these haplotypes differ by only four amino acids in the first domain of the beta chain. To test if this difference accounts for the MHC influence on susceptibility to collagen-induced arthritis, H-2p mice were made transgenic with an Abp gene altered to resemble the Abq gene. The transgenic A beta chain hybridized with the A alpha p chain and was shown to be physiologically expressed by testing antigen-presentation capacity to Aq-restricted T cell hybridomas and with FACS analyses. These transgenic mice developed an autoimmune response to type II collagen and also collagen-induced arthritis. The data unequivocally suggest the Ab gene as a major genetic susceptibility locus for autoimmune collagen-induced arthritis.