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1.
J Am Assoc Lab Anim Sci ; 63(1): 89-98, 2024 Jan 01.
Article in English | MEDLINE | ID: mdl-37993117

ABSTRACT

In vitro fertilization (IVF), embryo cryopreservation, and embryo transfer (ET) are assisted reproductive technologies (ARTs) that are used extensively for the maintenance of mouse models in animal research. Inbred mouse strains with different genetic backgrounds vary in their reproductive performance. Cryopreservation can affect embryo quality and viability, and the genetic background of ET recipients can influence the ET result. In this retrospective study, we analyzed the out- comes of ETs performed in our facility during the last 6 y. We found that B6C3F1 mice with swollen ampullae show almost 3-fold higher pregnancy rates than mice with nonswollen ampullae when either freshly isolated or frozen-thawed embryos are implanted. Implantation of freshly collected embryos in recipients with swollen ampullae led to significantly higher pregnancy rates in comparison to implantation of frozen-thawed embryos, regardless of whether the latter were fertilized in vivo or in vitro. Moreover, we found a significant effect of genetic background on the birth rate; C57BL/6J mice and mice with a mixed genetic background had 34% higher birth rates than did C57BL/6N mice. Within the C57BL/6J group, the birth rates were significantly higher when using fresh in vivo-fertilized embryos, and cryopreservation negatively affected both in vivo- and in vitro-fertilized embryos. The success rate of obtaining one living pup was not significantly different between frozen-thawed and fresh embryos. Overall, a swollen ampulla is a strong indicator for a successful pregnancy, together with the embryo manipulation and genetic background. A better understanding of the factors that affect the reproductive outcome might lead to optimization of the ART protocols and contribute to a reduction in the number of mice used for these procedures.


Subject(s)
Embryo Transfer , Fertilization in Vitro , Pregnancy , Female , Mice , Animals , Retrospective Studies , Mice, Inbred C57BL , Embryo Transfer/veterinary , Embryo Transfer/methods , Fertilization in Vitro/veterinary , Embryo Implantation , Cryopreservation/veterinary , Mice, Inbred Strains
2.
Article in English | MEDLINE | ID: mdl-34379582

ABSTRACT

Nine strains of a Rodentibacter-related bacterium were isolated over a period of 38 years from a laboratory mouse (Mus musculus), seven laboratory rats (Rattus norvegicus) and a Syrian hamster (Mesocricetus auratus) in Düsseldorf and Heidelberg, Germany. The isolates are genotypically and phenotypically distinct from all previously described Rodentibacter species. Sequence analysis of 16S rRNA and rpoB gene sequences placed the isolates as a novel lineage within the genus Rodentibacter. In addition to the single-gene analysis, the whole genome sequence of the strain 1625/19T revealed distinct genome-to-genome distance values to the other Rodentibacter species. The genomic DNA G+C content of strain 1625/19T was 40.8 mol% within the range of Rodentibacter. At least six phenotypic characteristics separate the new isolates from the other Rodentibacter species, with Rodentibacter heylii being the most closely related. In contrast to the latter, the new strains display ß-haemolysis and are ß-glucuronidase, d-mannitol and sorbitol positive, but fail to produce lysine decarboxylase and trehalose. The genotypic and phenotypic differences between the novel strains and the other closely related strains of the genus Rodentibacter indicate that they represent a novel species within the genus Rodentibacter, family Pasteurellaceae, for which the name Rodentibacter haemolyticus sp. nov. is proposed. The type strain 1625/19T, (=DSM 111151T=CCM 9081T), was isolated in 2019 from the nose of a laboratory mouse (Mus musculus) in Düsseldorf, Germany.


Subject(s)
Mesocricetus/microbiology , Mice/microbiology , Pasteurellaceae , Phylogeny , Rats/microbiology , Animals , Animals, Laboratory/microbiology , Bacterial Typing Techniques , Base Composition , DNA, Bacterial/genetics , Fatty Acids/chemistry , Germany , Pasteurellaceae/classification , Pasteurellaceae/isolation & purification , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA
3.
J Microbiol Methods ; 182: 106150, 2021 03.
Article in English | MEDLINE | ID: mdl-33503485

ABSTRACT

Screening for the Rodentibacter species is part of the microbiologic quality assurance programs of laboratory rodents all over the world. Nevertheless, currently there are no PCR amplification techniques available for the diagnostic of R. ratti, R. heidelbergensis and of a Rodentibacter related ß-haemolytic taxon. The aim of this study was to utilize the differences in the sequence of the Internal Transcribed Spacer (ITS) regions of R. pneumotropicus, R. heylii, R. ratti, R. heidelbergensis and of the ß-haemolytic Rodentibacter taxon for the design of specific PCR assays for these species. The ITSile+ala sequence variations allowed the design of specific forward and reverse primers for each species included, that could be combined in different multiplex assays. The performance characteristics specificity and sensitivity registered for each primer pair against a diverse collection of Pasteurellaceae isolated from rats and mice and of further non-Pasteurellaceae strains was 100% for all five Rodentibacter species included. In addition, the PCR assays displayed high limits of detection and could be successfully used for detection of Rodentibacter spp. DNA in clinical swabs of laboratory mice and rats. Overall, the assays described here represent the first PCRs able to diagnose R. ratti, R. heidelbergensis and the ß-haemolytic Rodentibacter taxon, whose diagnostic to species level could further facilitate better understanding of their geographic distribution, prevalence, and biology in the future.


Subject(s)
Multiplex Polymerase Chain Reaction/methods , Pasteurellaceae Infections , Pasteurellaceae , RNA, Ribosomal, 16S/isolation & purification , Rodentia/microbiology , rRNA Operon , Animals , Mice , Pasteurellaceae/genetics , Pasteurellaceae/isolation & purification , Pasteurellaceae Infections/diagnosis , Pasteurellaceae Infections/microbiology , Pasteurellaceae Infections/veterinary , Rats
4.
Int J Syst Evol Microbiol ; 69(7): 2095-2100, 2019 Jul.
Article in English | MEDLINE | ID: mdl-31099739

ABSTRACT

A Gram-stain-positive, rod-shaped, aerobic, non-motile, white, opaque bacterial isolate, designated 924/12T, was isolated from the nose of a laboratory mouse in Düsseldorf, Germany. The 16S rRNA gene sequence analyses indicated the phylogenetic position of the strain within the genus Leucobacter. Similarity levels over 97 % were recorded between the 16S rRNA gene sequence of strain 924/12T and the type strains of the species Leucobacter chironomi DSM 19883T (99.5 %), followed by Leucobacter celersubsp. astrifaciens CBX151T (97.6 %), Leucobacter celersubsp. celer NAL101T (97.5 %), 'Leucobacter kyeonggiensis' F3-P9 (97.5 %), Leucobacter zeae CC-MF41T (97.3 %), Leucobacter chromiiresistens JG31T (97.1 %), Leucobacter triazinivorans JW-1T (97.1 %), Leucobacter corticis 2 C-7T (97.0 %) and Leucobacter aridicolis CIP108388T (97.0 %). DNA-DNA hybridization and whole genomic comparison, mandatory to taxonomically separate strain 924/12T from the type strain of L. chironomi, revealed similarity values of 40.4 and 30.8 %, respectively, thus below the threshold of 70 % recommended differentiating between species. The cell-wall amino acids of the novel isolate were diaminobutyric acid, alanine, glycine, threonine and glutamic acid. The major fatty acids were anteiso-C15 : 0, anteiso-C17 : 0 and iso-C16 : 0. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol, glycolipid and one unknown lipid, whereas the predominant menaquinones were MK-11 and MK-10. The genomic DNA G+C content of strain 924/12T was 70.6 mol%. Phylogenetic analyses based on the 16S rRNA gene sequences and the phenotypical differences between strain 924/12T and the other closely related type strains of the genus Leucobacter indicated that strain 924/12T represents a novel species within the genus Leucobacter, family Microbacteriaceae, for which the name Leucobacter muris sp. nov. is proposed. The type strain is 924/12T (=DSM 101948T=CCM 8761T).


Subject(s)
Actinobacteria/classification , Mice/microbiology , Nose/microbiology , Phylogeny , Actinobacteria/isolation & purification , Animals , Animals, Laboratory/microbiology , Bacterial Typing Techniques , Base Composition , Cell Wall/chemistry , DNA, Bacterial/genetics , Fatty Acids/chemistry , Germany , Glycolipids/chemistry , Nucleic Acid Hybridization , Phospholipids/chemistry , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA , Vitamin K 2/chemistry
5.
Reprod Biol Endocrinol ; 17(1): 28, 2019 Mar 02.
Article in English | MEDLINE | ID: mdl-30825879

ABSTRACT

BACKGROUND: Syndecan-1 is a heparan sulfate proteoglycan acting as a co-receptor for cytokines and growth factors mediating developmental, immunological and angiogenic processes. In human, the uteroplacental localization of Syndecan-1 and its reduced expression in pregnancy-associated pathologies, such as the intrauterine growth restriction, suggests an influence of Syndecan-1 in embryo-maternal interactions. The aim of the present study was to identify the effect of a reduced expression of Syndecan-1 on the reproductive phenotype of mice and their progenies. METHODS: Reproductive characteristics have been investigated using animals with reduced Syndecan-1 and their wildtype controls after normal mating and after vice versa embryo transfers. Female mice were used to measure the estrus cycle length and the weight gain during pregnancy, as well as for histological examination of ovaries. Male mice were examined for the concentration, motility, viability and morphology of spermatozoa. Organs like heart, lung, liver, kidney, spleen, brain and ovaries or testes and epididymis of 6-month-old animals were isolated and weighed. Statistical analyses were performed using two-tailed students t-test with P < .05 and P < .02, chi square test (P < .05) and Fisher's Exact Test (P < .05). A linear and a non-linear mixed-effects model were generated to analyze the weight gain of pregnant females and of the progenies. RESULTS: Focusing on the pregnancy outcome, the Syndecan-1 reduced females gave birth to larger litters. However, regarding the survival of the offspring, a higher percentage of pups with less Syndecan-1 died during the first postnatal days. Even though the ovaries and the testes of Syndecan-1 reduced mice showed no histological differences and the ovaries showed a similar number of primary and secondary follicles and corpora lutea, the spermatozoa of Syndecan-1 reduced males showed more tail and midpiece deficiencies. Concerning the postnatal and juvenile development the pups with reduced Syndecan-1 expression remained lighter and smaller regardless whether carried by mothers with reduced Syndecan-1 or wildtype foster mothers. With respect to anatomical differences kidneys of both genders as well as testes and epididymis of male mice with reduced syndecan-1 expression weighed less compared to controls. CONCLUSIONS: These data reveal that the effects of Syndecan-1 reduction are rather genotype- than parental-dependent.


Subject(s)
Estrus/physiology , Reproduction/physiology , Spermatozoa/physiology , Syndecan-1/metabolism , Animals , Animals, Newborn , Body Weight/genetics , Body Weight/physiology , Estrus/genetics , Female , Genotype , Humans , Litter Size/genetics , Male , Mice, 129 Strain , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Pregnancy , Reproduction/genetics , Spermatozoa/metabolism , Syndecan-1/genetics
6.
Lab Anim ; 53(5): 470-477, 2019 Oct.
Article in English | MEDLINE | ID: mdl-30567464

ABSTRACT

The extra-hospital epidemiology of Acinetobacter infections is a subject of debate. In recent years, the prevalence of animal multidrug-resistant Acinetobacter infections has increased considerably. The goal of the present study was to specify Acinetobacter species isolated from laboratory mice and to test them for their antimicrobial susceptibility. During routine microbiological monitoring of laboratory mice, 12 Acinetobacter spp. were isolated. By means of 16S rRNA and rpoB gene sequencing, seven of the isolates were identified as Acinetobacter radioresistens, three isolates belonged to Acinetobacter genomospecies 14BJ, one isolate was classified as Acinetobacter pitii and one as Acinetobacter sp. ANC 4051. The distribution of the minimal inhibitory concentration (MIC) values was uniform for 21 of the 23 antimicrobial agents tested, whereas a broad MIC distribution was recorded for tulathromycin and streptomycin. The MIC values recorded were low for the majority of the antibiotics tested. Nevertheless, very high MIC values, which will probably render a therapeutic approach using these substances unsuccessful, were recorded for florfenicol, tiamulin, tilmicosin and cephalothin in most of the isolates. In conclusion, we document colonization of laboratory mice with different Acinetobacter species, displaying similar antibiotic susceptibility profiles, with possible implications in the Acinetobacter epidemiology as well as in the husbandry and experimentation of the colonized animals.


Subject(s)
Acinetobacter/drug effects , Anti-Bacterial Agents/pharmacology , Drug Resistance, Bacterial , Mice/microbiology , Animals
7.
Lab Anim ; 51(4): 412-421, 2017 Aug.
Article in English | MEDLINE | ID: mdl-27932683

ABSTRACT

This study aims to determine the ability of laboratory animal bacteria to resist desiccation and inactivation by hydrogen peroxide vapour (HPV) on paper bedding pieces. Bedding pieces were saturated with bacterial suspensions in water or 2% (w/v) bovine serum albumin (BSA) in water, and held in a mouse facility. Viable counts showed variable survival rates over time for the bacterial species used ([ Pasteurella] pneumotropica, Muribacter muris, Pseudomonas aeruginosa, Acinetobacter redioresistens, Escherichia coli, Klebsiella oxytoca, Bordetella bronchiseptica, Bordetella hinzii, Enterococcus faecalis, ß-haemolytic Streptococcus spp., Staphylococcus aureus and Staphylococcus xylosus). Overall, BSA increased bacterial survival in the bedding pieces. The survival rates of Bacillus safensis were not influenced by BSA but depended on sporulation. When bedding pieces and Petri dishes inoculated with E. coli, P. aeruginosa and S. aureus were subjected to HPV disinfection, all bacterial species on the bedding pieces inoculated with bacterial suspensions in water were readily inactivated. By contrast, S. aureus and P. aeruginosa, but not E. coli cells survived HPV treatment in high numbers when inoculated on bedding pieces as a BSA suspension. Notably, all three bacterial species were readily inactivated by HPV even in the presence of BSA when smeared on smooth surfaces. In conclusion, the suspension medium and the carrier can influence the environmental survival and susceptibility of bacterial species to HPV. Our results may help to develop standard protocols that can be used to ensure the microbiological quality of experimental rodent housing.


Subject(s)
Bacteria/drug effects , Bacteria/growth & development , Bedding and Linens/microbiology , Disinfection/methods , Hydrogen Peroxide/pharmacology , Animals , Animals, Laboratory , Escherichia coli , Housing, Animal , Mice , Staphylococcus aureus
8.
PLoS One ; 10(10): e0138778, 2015.
Article in English | MEDLINE | ID: mdl-26430880

ABSTRACT

[Pasteurella] pneumotropica biotypes Jawetz and Heyl and [Actinobacillus] muris are the most prevalent Pasteurellaceae species isolated from laboratory mouse. However, mechanisms contributing to their high prevalence such as the ability to form biofilms have not been studied yet. In the present investigation we analyze if these bacterial species can produce biofilms in vitro and investigate whether proteins, extracellular DNA and polysaccharides are involved in the biofilm formation and structure by inhibition and dispersal assays using proteinase K, DNase I and sodium periodate. Finally, the capacity of the biofilms to confer resistance to antibiotics is examined. We demonstrate that both [P.] pneumotropica biotypes but not [A.] muris are able to form robust biofilms in vitro, a phenotype which is widely spread among the field isolates. The biofilm inhibition and dispersal assays by proteinase and DNase lead to a strong inhibition in biofilm formation when added at the initiation of the biofilm formation and dispersed pre-formed [P.] pneumotropica biofilms, revealing thus that proteins and extracellular DNA are essential in biofilm formation and structure. Sodium periodate inhibited the bacterial growth when added at the beginning of the biofilm formation assay, making difficult the assessment of the role of ß-1,6-linked polysaccharides in the biofilm formation, and had a biofilm stimulating effect when added on pre-established mature biofilms of [P.] pneumotropica biotype Heyl and a majority of [P.] pneumotropica biotype Jawetz strains, suggesting that the presence of ß-1,6-linked polysaccharides on the bacterial surface might attenuate the biofilm production. Conversely, no effect or a decrease in the biofilm quantity was observed by biofilm dispersal using sodium periodate on further biotype Jawetz isolates, suggesting that polysaccharides might be incorporated in the biofilm structure. We additionally show that [P.] pneumotropica cells enclosed in biofilms were less sensitive to treatment with amoxicillin and enrofloxacin than planktonic bacteria. Taken together, these findings provide a first step in understanding of the biofilm mechanisms in [P.] pneumotropica, which might contribute to elucidation of colonization and pathogenesis mechanisms for these obligate inhabitants of the mouse mucosa.


Subject(s)
Biofilms/drug effects , Pasteurella pneumotropica/metabolism , Animals , Anti-Bacterial Agents/pharmacology , Deoxyribonuclease I/pharmacology , Endopeptidase K/pharmacology , Mice , Microbial Sensitivity Tests , Microscopy, Confocal , Pasteurella pneumotropica/drug effects , Periodic Acid/pharmacology
9.
Berl Munch Tierarztl Wochenschr ; 128(7-8): 278-84, 2015.
Article in English | MEDLINE | ID: mdl-26281439

ABSTRACT

The impact of particular microbes on genetically engineered mice depends on the genotype and the environment. Infections resulting in clinical disease have an obvious impact on animal welfare and experimentation. In this study, we investigated the bacterial and fungal aetiology of spontaneous clinical disease of infectious origin among the genetically engineered mice from our institution in relation to their genotype. A total of 63 mice belonging to 33 different mice strains, from severe immunodeficient to wild-type, were found to display infections as the primary cause leading to their euthanasia. The necropsies revealed abscesses localized subcutaneously as well as in the kidney, preputial glands, seminal vesicles, in the uterus, umbilicus or in the lung. In addition, pneumonia, endometritis and septicaemia cases were recorded. Escherichia coli was involved in 21 of 44 (47.72%) of the lesions of bacterial origin, whereas [Pasteurella] pneumotropica was isolated from 19 of 44 (43.18%) cases. The infections with the two agents mentioned above included three cases of mixed infection with both pathogens. Staphylococcus aureus was considered responsible for five of 44 (11.36%) cases whereas Enterobacter cloacae was found to cause lesions in two of 44 (4.54%) mice. Overall, 16 of the 44 (36.36%) cases of bacterial aetiology affected genetically engineered mice without any explicit immunodeficiency or wild-type strains. The remaining 19 cases of interstitial pneumonia were caused by Pneumocystis murina. In conclusion, the susceptibility of genetically modified mice to opportunistic infections has to be regarded with precaution, regardless of the type of genetic modification performed. Beside the classical opportunists, such as [Pasteurella] pneumotropica and Staphylococcus aureus, Escherichia coli should as well be closely monitored to evaluate whether it represents an emerging pathogen in the laboratory mouse.


Subject(s)
Communicable Diseases, Emerging/veterinary , Escherichia coli Infections/veterinary , Mice, Transgenic/microbiology , Mycoses/veterinary , Rodent Diseases/genetics , Rodent Diseases/microbiology , Animals , Communicable Diseases, Emerging/genetics , Communicable Diseases, Emerging/microbiology , Escherichia coli Infections/genetics , Escherichia coli Infections/pathology , Immunocompromised Host , Mice , Mice, Inbred Strains , Mycoses/genetics , Mycoses/pathology , Opportunistic Infections/genetics , Opportunistic Infections/pathology , Opportunistic Infections/veterinary
10.
Lab Anim ; 48(4): 305-12, 2014 Oct.
Article in English | MEDLINE | ID: mdl-24876090

ABSTRACT

Correct identification of bacteria is crucial for the management of rodent colonies. Some bacteria are difficult to identify phenotypically outside reference laboratories. In this study, we evaluated the utility of 16S ribosomal DNA (rDNA) sequencing as a means of identifying a collection of 30 isolates of rodent origin which are conventionally difficult to identify. Sequence analysis of the first approximate 720 to 880 bp of the 5'- end of 16S rDNA identified 25 isolates (83.33%) with ≥ 99% similarity to a sequence of a type strain, whereas three isolates (10%) displayed a sequence similarity ≥ 97% but <99% to the type strain sequences. These similarity scores were used to define identification to species and genus levels, respectively. Two of the 30 isolates (6.67%) displayed a sequence similarity of ≥ 95 but <97% to the reference strains and were thus allocated to a family. This technique allowed us to document the association of mice with bacteria relevant for the colonies management such as Pasteurellaceae, Bordetella hinzii or Streptococcus danieliae. In addition, human potential pathogens such as Acinetobacter spp., Ochrobactrum anthropi and Paracoccus yeei or others not yet reported in mouse bacterial species such as Leucobacter chironomi, Neisseria perflava and Pantoea dispersa were observed. In conclusion, the sequence analysis of 16S rDNA proved to be a useful diagnostic tool, with higher performance characteristics than the classical phenotypic methods, for identification of laboratory animal bacteria. For the first time this method allowed us to document the association of certain bacterial species with the laboratory mouse.


Subject(s)
Animal Husbandry/methods , Animals, Laboratory/microbiology , Bacteria/genetics , Bacteria/isolation & purification , Mice/microbiology , Rats/microbiology , Animals , Bacteria/classification , Molecular Sequence Data , RNA, Ribosomal, 16S/genetics , Sequence Analysis, DNA
11.
J Microbiol Methods ; 95(2): 256-61, 2013 Nov.
Article in English | MEDLINE | ID: mdl-24055385

ABSTRACT

The rodents Pasteurellaceae have to be excluded from the specified pathogen free experimental animal facilities. Despite the biological and economic importance of Pasteurellaceae in relation to experimental animals just a few molecular based methods are available for their detection and identification. The aim of the present investigation was to develop a multiplex PCR assay allowing detection of all rodent Pasteurellaceae and identification of [Pasteurella] pneumotropica biotype Jawetz, [P.] pneumotropica biotype Heyl and [Actinobacillus] muris, as the most prevalent members of the group. For this, a Pasteurellaceae common forward primer located on the 16S rRNA gene was used in conjunction with four different reverse primers specific for [P.] pneumotropica biotype Jawetz, [P.] pneumotropica biotype Heyl, [A.] muris and a common reverse primer for all rodent Pasteurellaceae, all targeting the 16S-23S rRNA internal transcribed spacer sequences. The performance characteristics of the assay were tested against 125 Pasteurellaceae isolates belonging to eleven different species and including 34 strains of [P.] pneumotropica biotype Jawetz, 44 strains of [P.] pneumotropica biotype Heyl and 37 strains of [A.] muris. Additionally, eight other mouse associated bacterial species which could pose a diagnostic problem were included. The assay showed 100% sensitivity and specificity. Identification of the clinical isolates was validated by ITS profiling and when necessary by 16S rRNA gene sequencing. This multiplex PCR represents the first molecular tool able to detect and differentiate in a single assay among the Pasteurellaceae found in laboratory mouse and may become a reliable alternative to the present diagnostic methods.


Subject(s)
DNA Primers/genetics , Multiplex Polymerase Chain Reaction/methods , Pasteurellaceae/genetics , Pasteurellaceae/isolation & purification , Animals , Base Sequence , DNA, Bacterial/genetics , DNA, Ribosomal Spacer/genetics , Mice , Molecular Sequence Data , Pasteurellaceae/classification , Phenotype , RNA, Ribosomal, 16S/genetics , RNA, Ribosomal, 23S/genetics , Rodentia/microbiology , Sensitivity and Specificity , Sequence Analysis, DNA , Species Specificity
12.
J Microbiol Methods ; 94(2): 88-93, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23665068

ABSTRACT

[Actinobacillus] muris represents along with [Pasteurella] pneumotropica the most prevalent Pasteurellaceae species isolated from the laboratory mouse. Despite the biological and economic importance of Pasteurellaceae in relation to experimental animals, no molecular based methods for the identification of [A.] muris are available. The aim of the present investigation was to develop a PCR method allowing detection and identification of [A.] muris. In this assay, a Pasteurellaceae common forward primer based on a conserved region of the 16S rRNA gene was used in conjunction with two different reverse primers specific for [A.] muris, targeting the 16S-23S internal transcribed spacer sequences. The specificity of the assay was tested against 78 reference and clinical isolates of Pasteurellaceae, including 37 strains of [A.] muris. In addition, eight other mice associated bacterial species which could pose a diagnostic problem were included. The assay showed 100% sensitivity and 97.95% specificity. Identification of the clinical isolates was validated by ITS profiling and when necessary by 16S rRNA sequencing. This multiplex PCR represents the first molecular tool able to detect [A.] muris and may become a reliable alternative to the present diagnostic methods.


Subject(s)
Actinobacillus Infections/veterinary , Actinobacillus/isolation & purification , DNA Primers/genetics , DNA, Ribosomal Spacer/genetics , Polymerase Chain Reaction/methods , RNA, Ribosomal, 16S/genetics , RNA, Ribosomal, 23S/genetics , Rodent Diseases/microbiology , Actinobacillus/classification , Actinobacillus/genetics , Actinobacillus Infections/microbiology , Animals , Base Sequence , DNA, Bacterial/genetics , Mice , Molecular Sequence Data , Sensitivity and Specificity , Species Specificity
13.
J Microbiol Methods ; 90(3): 342-9, 2012 Sep.
Article in English | MEDLINE | ID: mdl-22771472

ABSTRACT

The internal transcribed spacer (ITS) regions of members of Pasteurellaceae isolated from rodents, including the [Pasteurella] pneumotropica biotypes Jawetz and Heyl, [Actinobacillus] muris, "Hemophilus influenzaemurium" and Bisgaard taxon 17 were studied and their feasibility to discriminate these species was analyzed. The reference strains of all species analyzed showed unique species-specific ITS patterns which were further present in 49 clinical isolates of [P.] pneumotropica biotypes Jawetz and Heyl and [A.] muris allowing their identification by comparison to the reference strains pattern. Sequence analysis of the amplified fragments revealed in all species, with exception of "H. influenzaemurium", a larger ITS(ile+ala) which contained the genes for tRNA(Ile(GAU)) and tRNA(Ala(UGC)) and a smaller ITS(glu) with the tRNA(Glu(UUC)) gene. "H. influenzaemurium" revealed two each of the larger and respectively the smaller ITS fragments. Both the length and the sequence of each ITS type were highly conserved within the [P.] pneumotropica biotypes Jawetz and Heyl and [A.] muris strains tested. On the contrary, ITS sequences revealed significant interspecies variations with identity levels ranging from 61.2 to 89.5% for ITS(ile+ala) and 56.5 to 86.8% for ITS(glu). Sequences regions with significant interspecies variation but highly conserved within the species were identified and might be used to design probes for the identification of rodent Pasteurellaceae to the species level.


Subject(s)
Animals, Laboratory/microbiology , DNA, Ribosomal Spacer/genetics , Pasteurella Infections/veterinary , Pasteurella pneumotropica/isolation & purification , Rodent Diseases/microbiology , Animals , Base Sequence , Conserved Sequence , Genetic Variation , Mice , Molecular Sequence Data , Molecular Typing/standards , Pasteurella Infections/microbiology , Pasteurella pneumotropica/genetics , Pasteurella pneumotropica/growth & development , RNA, Ribosomal, 16S/genetics , RNA, Ribosomal, 23S/genetics , Reference Standards , Sequence Alignment , Sequence Analysis, DNA
14.
Infect Immun ; 73(10): 6390-8, 2005 Oct.
Article in English | MEDLINE | ID: mdl-16177310

ABSTRACT

It is currently accepted that malaria-parasitized red blood cells (pRBC) are eliminated, like senescent erythrocytes, phagocytically by macrophages in the red pulp of the spleen. Here, however, we show that self-healing Plasmodium chabaudi malaria activates spleen closure in C57BL/6 mice. Confocal laser scanning microscopy revealed that spleen closing was manifested by elimination of entry into the red pulp of 3-microm polystyrol particles, pRBC, and nonparasitized red blood cells but not of bovine serum albumin. This spleen closure did not reflect a reduction in the number of phagocytic cells, as shown by flow cytometry, whereas marginal zone macrophages (MZM) were lost and red pulp macrophages entered the white pulp. Splenic trapping of pBRC was strongly reduced in the absence of MZM and marginal metallophilic macrophages (MMM), as it is in noninfected mice with a disrupted lymphotoxin beta receptor (LTbetaR(-/-)), and it was still significantly reduced when the number of MZM and MMM was diminished, as in tumor necrosis factor alpha-deficient (TNF-alpha(-/-)) mice. Moreover, mice deficient in TNF-alpha, tumor necrosis factor receptor I (TNFRI(-/-)), and LTbetaR exhibited progressive impairment in malaria-induced spleen closing. Treatment of C57BL/6 mice with TNF-alpha induced loss of MZM and spleen closing by about 20%. Our data indicate that TNF/TNFRI signaling is involved in regulating malaria-induced spleen closure, which is maximal during crisis, when parasitemia declines more than 100-fold. Consequently, the vast majority of pRBC cannot be destroyed by the spleen during crisis, suggesting that the known sophisticated sequestration system of Plasmodium parasites did not evolve to avoid splenic clearance.


Subject(s)
Erythrocytes/parasitology , Malaria/immunology , Parasitemia/immunology , Plasmodium chabaudi , Spleen/immunology , Animals , Lymphotoxin beta Receptor , Macrophages/immunology , Malaria/blood , Malaria/parasitology , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Parasitemia/parasitology , Parasitemia/pathology , Phagocytosis , Receptors, Tumor Necrosis Factor/genetics , Receptors, Tumor Necrosis Factor/physiology , Receptors, Tumor Necrosis Factor, Type I/genetics , Receptors, Tumor Necrosis Factor, Type I/physiology , Signal Transduction , Spleen/blood supply , Spleen/cytology , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/physiology
15.
J Cell Biochem ; 96(2): 339-48, 2005 Oct 01.
Article in English | MEDLINE | ID: mdl-16088918

ABSTRACT

The IMAP/IAN family of AIG1-like GTPases is conserved among vertebrates and angiosperm plants and has been postulated to regulate apoptosis, particularly in context with diseases such as cancer, diabetes, and infections. The human genes were recently renamed as gimap for GTPase of the immunity associated protein (GIMAP) family. Here we extend this new nomenclature to the murine gimap gene family. All gimap genes of the mouse are clustered on chromosome 6B with eight functional members and one pseudogene. The mGIMAP proteins contain one GTP-binding site and display molecular masses between 33 and 38 kDa except for the very unusual 77 kDa mGIMAP8 protein, which is the first characterized protein containing three GTP-binding domains. Northern blot analysis revealed expression of mgimap8 predominantly in the thymus. The low expression level observed in the spleen was further suppressed by Plasmodium chabaudi malaria. Confocal laser scanning microscopy demonstrated localization of mGIMAP8 at ER, Golgi, and mitochondria. Overexpression of mGIMAP8 could significantly impair anisomycin-induced activation of caspase 3. Our data support the view that mGIMAP8 exerts an anti-apoptotic effect in the immune system and is involved in responses to infections.


Subject(s)
Apoptosis , GTP Phosphohydrolases/metabolism , Gene Expression Regulation, Enzymologic , Malaria/enzymology , Malaria/pathology , Amino Acid Sequence , Animals , Apoptosis/genetics , Cell Line , Cricetinae , GTP Phosphohydrolases/chemistry , GTP Phosphohydrolases/genetics , Gene Expression Regulation, Enzymologic/genetics , Malaria/parasitology , Mice , Molecular Sequence Data , Plasmodium chabaudi/physiology , Sequence Alignment , Sequence Homology, Amino Acid
16.
Steroids ; 70(9): 604-14, 2005 Aug.
Article in English | MEDLINE | ID: mdl-15964042

ABSTRACT

Androgens can increase susceptibility toward numerous parasitic infections as well as modulate apoptosis of immune cells. According to the current view, androgens mediate immune cell activities not only through classical intracellular androgen receptors (AR), but also through membrane receptors on the cell surface. Here, using murine bone marrow-derived macrophages (BMMs), we examined the influence of testosterone on Leishmania donovani infection and cell viability in vitro as well as the possible mechanisms. Our data demonstrated that testosterone directly increased intramacrophage infection by L. donovani. In addition, testosterone decreased cell viability by way of apoptosis, accompanied by increased Fas, FasL, and Caspase-8 expression. However, these effects of testosterone could not be associated with the classical AR in BMMs since AR was not detectable using different experimental techniques. Instead, it was found that testosterone could bind to the surface of BMMs by the use of an impermeable testosterone-BSA-FITC in confocal laser scanning microscopy and flow cytometry. Collectively, our data indicated that the influence of testosterone on L. donovani infection and viability of BMMs was mediated through the binding sites of testosterone on cell surfaces, which provided a novel mode of direct action of testosterone on AR-free BMMs.


Subject(s)
Bone Marrow Cells/cytology , Leishmania donovani/microbiology , Macrophages/microbiology , Signal Transduction/physiology , Testosterone/pharmacology , Animals , Antigens, Differentiation/analysis , Apoptosis/drug effects , Binding Sites , Caspase 8 , Caspases/genetics , Caspases/metabolism , Cell Differentiation/drug effects , Cell Survival/drug effects , Estradiol/pharmacology , Fas Ligand Protein , Female , Gene Expression/drug effects , Macrophage Colony-Stimulating Factor/pharmacology , Macrophages/drug effects , Macrophages/metabolism , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred C57BL , Receptors, Androgen/analysis , Tumor Necrosis Factors/genetics , Tumor Necrosis Factors/metabolism , fas Receptor/genetics , fas Receptor/metabolism
17.
Microbes Infect ; 7(3): 399-409, 2005 Mar.
Article in English | MEDLINE | ID: mdl-15788153

ABSTRACT

Disrupted signaling through lymphotoxin beta receptor (LTbetaR) results in severe defects of the spleen and even loss of all other secondary lymphoid tissues, making mice susceptible to diverse infectious agents. Surprisingly, however, we find that female LTbetaR-deficient mice are even more resistant to blood stages of Plasmodium chabaudi malaria than wild-type C57BL/6 mice. Higher resistance of LTbetaR-deficient mice correlates with an earlier onset of reticulocytosis, and the period of anemia is shorter. After surviving fulminant parasitemias of about 35%, mice develop long-lasting protective immunity against homologous rechallenge, with both spleen and liver acting as anti-malaria effectors. Testosterone suppresses resistance, i.e. all mice succumb to infections during or shortly after peak parasitemia. At peak parasitemia, testosterone does not essentially affect cellularity and apoptosis in the spleen, but aggravates liver pathology in terms of increased cell swelling, numbers of apoptotic and binucleated cells and reduced serum alkaline phosphatase levels, and conversely, reduces inflammatory lymphocytic infiltrates in the liver. In the spleen, hybridization of cDNA arrays identified only a few testosterone-induced changes in gene expression, in particular upregulation of INFgamma and IFN-regulated genes. By contrast, a much larger number of testosterone-affectable genes was observed in the liver, including genes involved in regulation of the extracellular matrix, in chemokine and cytokine signaling, and in cell cycle control. Collectively, our data suggest that testosterone dysregulates the inflammatory response in spleen and liver during their differentiation to anti-malaria effectors in malaria-resistant female LTbetaR-deficient mice, thus contributing to the testosterone-induced lethal outcome of malaria.


Subject(s)
Liver/drug effects , Malaria/immunology , Receptors, Tumor Necrosis Factor/physiology , Spleen/drug effects , Testosterone/pharmacology , Animals , Female , Gene Expression/drug effects , Immunity, Innate/genetics , Lymphotoxin beta Receptor , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Parasitemia/immunology , Plasmodium chabaudi , Receptors, Tumor Necrosis Factor/genetics , Time Factors
18.
Gene ; 341: 291-304, 2004 Oct 27.
Article in English | MEDLINE | ID: mdl-15474311

ABSTRACT

There is a growing family of novel GTPases conserved among higher plants and vertebrates, abbreviated as AIG1, IAP, IMAP, and IAN, respectively. Here, we comparatively analyze the human gene family encoding GTPases of the immunity-associated protein family recently re-termed GIMAP. Chromosome 7q36.1 contains, within 300 kb, a gimap gene cluster with seven functional genes and one pseudogene (hgimap3). The six genes hgimap1, hgimap2, hgimap4, hgimap5, hgimap6, and hgimap7 encode 33-46 kDa proteins with one GTP-binding domain, whereas hgimap8 encodes a very unusual 75-kDa protein with three GTP-binding domains. All hgimap genes except hgimap2 have orthologs in the mouse. Major expression sites of hgimap mRNAs are the spleen and lymph nodes, but also other organs such as muscle, heart, placenta, and digestive tract display detectable hgimap mRNA levels. The proteins hGIMAP4 and hGIMAP7 can be localized at ER and Golgi apparatus, but not in mitochondria, lysosomes and nuclei. All hgimap genes were expressed at very low levels-if at all-in diverse cancer cell lines. Our data support the view that the GIMAP proteins are involved in the control of cell survival not only in cells of the immune system as commonly anticipated.


Subject(s)
GTP-Binding Proteins/genetics , Membrane Proteins/genetics , Multigene Family/genetics , Amino Acid Sequence , Animals , Blotting, Northern , CHO Cells , Cricetinae , Cricetulus , Exons , Female , GTP-Binding Proteins/metabolism , Gene Expression Profiling , Gene Order , Genes/genetics , Humans , Introns , Jurkat Cells , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Male , Membrane Proteins/metabolism , Microscopy, Confocal , Molecular Sequence Data , Phylogeny , Plasmids/genetics , Protein Isoforms/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Homology, Amino Acid , Tissue Array Analysis , Transfection
19.
Steroids ; 69(8-9): 585-90, 2004 Aug.
Article in English | MEDLINE | ID: mdl-15288774

ABSTRACT

We investigated the existence of membrane receptors for testosterone (mAR) in mouse macrophages of the cell lines IC-21 and RAW 264.7 as well as their roles in nongenomic pathways, gene expression and cell functioning. Both cell lines lack intracellular androgen receptors (iARs) and respond to testosterone with rapid rises in [Ca2+]i. These rises in [Ca2+]i can neither be inhibited by iAR- nor by iER blockers, but are rather mediated through mAR. Pharmacological approaches suggest that the mAR belongs to the class of membrane receptors which are coupled to phospholipase C via pertussis toxin (PTX) sensitive G-proteins. The mAR can be localized as specific surface binding sites for testosterone-BSA-FITC by confocal laser scanning microscopy (CLSM)and flow cytometry, and are characterized by their agonist-sequestrability. In order to examine a possible role of the testosterone-induced rise in [Ca2+]i on gene expression, a c-fos promoter reporter gene construct was transfected into RAW 264.7 macrophages. The increase in [Ca2+]i induced by testosterone cannot significantly activate the c-fos promoter directly. Also, no significant activation of ERK1/2, JNK/SAPK and p38 can be observed following testosterone-stimulation alone. However, testosterone-induced rises in [Ca2+]i do have specific effects on gene expression in context with lipopolysaccharide (LPS)-induced genotropic signaling: testosterone specifically down-regulates LPS-induced activation of c-fos promoter, p38 MAPK and NO production. In fetal calf serum (FCS)-induced genotropic signaling, the situation is reversed, i.e. testosterone augments the activation of c-fos promoter and ERK1/2. Our studies demonstrate a cross-talk between the testosterone-induced nongenomic Ca2+ signaling and the genotropic signaling induced by LPS and FCS in macrophages.


Subject(s)
Androgens/pharmacology , Macrophages/drug effects , Macrophages/metabolism , Androgens/metabolism , Animals , Calcium/metabolism , Cell Line , Gene Expression/drug effects , Gene Expression/physiology , Lipopolysaccharides/pharmacology , Mice , Nitric Oxide/biosynthesis , Oncogene Proteins v-fos/genetics , Oncogene Proteins v-fos/metabolism , Promoter Regions, Genetic , Receptors, Androgen/drug effects , Receptors, Androgen/metabolism , Signal Transduction/drug effects , Signal Transduction/physiology , Testosterone/metabolism , Testosterone/pharmacology , p38 Mitogen-Activated Protein Kinases/genetics , p38 Mitogen-Activated Protein Kinases/metabolism
20.
Steroids ; 67(11): 925-31, 2002 Oct.
Article in English | MEDLINE | ID: mdl-12234628

ABSTRACT

Increasing information indicates that testosterone actions on cells are mediated not only through the classical intracellular androgen receptor (iAR), but also through membrane androgen receptors (mAR) on cell surfaces. Here, we investigate the expression pattern of mAR and iAR in thymic T cells, which is compared with that of splenic T cells. Thymic T cells are testosterone-sensitive in vivo, i.e. treatment of female C57BL/10 mice with testosterone for 3 weeks decreased the total number of thymic T cells by approximately 90%. The percentage of CD4(-) CD8(-) T cells increased, whereas that of the subsequent CD4(+) CD8(+) T cells was diminished. Flow cytometry and confocal laser scanning microscopy (CLSM) with different anti-iAR antibodies localized iAR predominantly in the cytoplasm, but not on the surface of thymic T cells. The iAR are functionally active since the iAR are induced by testosterone to translocate from cytoplasm to nucleus, and they bind the testosterone analogue 3H-R1881 with high affinity (K(d) approximately 2.2 nM) and saturable capacity (approximately 10,000 binding sites per cell) as determined by Scatchard analysis. By contrast, the impeded ligand testosterone-BSA-FITC (T-BSA-FITC) did not bind to the surface of thymic T cells. In accordance, testosterone was unable to induce any rapid rise in the intracellular free Ca(2+) concentration of Fura-2 loaded thymocytes. This indicates that thymic T cells do not express any significant amounts of mAR. Conversely, splenic T cells express functionally active mAR, whereas their expressed iAR are not functional in the genomic pathway. Our results support the view of a delicately balanced developmental regulation of iAR and mAR in T cells.


Subject(s)
Gene Expression Regulation, Developmental , Receptors, Androgen/genetics , T-Lymphocytes/chemistry , Animals , Biological Transport/drug effects , CD4 Antigens/analysis , CD8 Antigens/analysis , Cell Membrane/chemistry , Cell Nucleus/metabolism , Cytoplasm/metabolism , Female , Flow Cytometry , Lymphocyte Count , Metribolone/metabolism , Mice , Mice, Inbred C57BL , Microscopy, Confocal , Receptors, Androgen/analysis , Receptors, Androgen/metabolism , Spleen/cytology , T-Lymphocytes/drug effects , T-Lymphocytes/ultrastructure , Testosterone/blood , Testosterone/pharmacology , Thymus Gland/cytology , Thymus Gland/drug effects , Thymus Gland/growth & development
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