Identity of the filterable hemolytic anemia agent of Sacks with Haemobartonella muris

Moore, D. H. (The Rockefeller University, New York, N. Y.), R. N. Arison, H. Tanaka, W. T. Hall, and M. Chanowitz. Identity of the filterable hemolytic anemia agent of Sacks with Haemobartonella muris. J. Bacteriol. 90:1669-1674. 1965.-In 1960 a new hemolytic agent in rats was reported. It was thought to be a filterable, nonsedimentable, replicating infectious agent, different from Haemobartonella muris. Rats which recovered from the infection developed a resistance to several kinds of transplantable tumors. It is here shown that this agent has the same properties as those reported in the literature and reconfirmed by us for H. muris. The size of the agent is approximately 500 mmu as determined by correlating bioactivity with Gradocol membrane filtrates and fractions from a diffusion cell, and its density is about midway between that of whole serum and distilled water (approximately 1.020) as determined by the sedimentation of bioactivity in an ultracentrifuge. Diffusion at 30 C indicated a lack of motility by the agent bodies. Rats were found to be protected against infection with the agent by daily administration of chlortetracycline and by prior infection with H. muris. The agent bodies were indistinguishable from H. muris in both the light and the electron microscope.     

Microbiological contamination of laboratory mice and rats in Korea from 1999 to 2003.

To survey the microbiological contamination of laboratory mice and rats in Korea during a 5-year period, we monitored animals housed in mouse and rat facilities with either barrier or conventional systems. At barrier and conventional mouse facilities, the most important pathogen identified was mouse hepatitis virus (MHV), while Mycoplasma pulmonis was the most important pathogen at conventional rat facilities. Interestingly, hantavirus was recovered from both barrier and conventional mouse facilities. The most common protozoon identified was Tritrichomonas muris in mouse facilities and Entamoeba muris in rat facilities. In addition, we found that the microbiological contamination of mice and rats in conventional facilities was severe. These results suggest that conventional facilities should be renovated and monitored regularly to decrease microbiological contamination. We also propose that hantavirus should be monitored in Korea as an important mouse pathogen.     

On molecular taxonomy: what is in a name?

Gene sequences of small portions of the genome are often used for premature detailed taxonomic changes, neglecting polyphasic taxonomy, which should also consider phenotypical characteristics. Three examples are given: (i) Recently, members of the genera Eperythrozoon and Haemobartonella have been moved, correctly so, from the Rickettsiales to the Mycoplasmatales, but were assigned to the genus Mycoplasma, mostly on the basis of 16S rRNA sequence analysis. Not only is the 16S rRNA sequence similarity between 'classical' Mycoplasma and these species of Eperythrozoon and Haemobartonella less than that between some other well-recognised bacterial genera, but their biological differences amply justify their classification in different genera of the Mycoplasmatales. Furthermore, the move creates considerable confusion, as it necessitates new names for some species, with more confusion likely to come when the 16S rRNA sequences of the type species of Eperythrozoon, a name which has priority over Mycoplasma, will be analysed. (ii) In the Rickettsiales, members of the genera Anaplasma, Ehrlichia, Cowdria, Neorickettsia and Wolhbachia are so closely related phylogenetically on the basis of 16S rRNA sequences, and for some also of groESL operon sequences, that they have recently been fused, correctly so, into one family, the Anaplasmataceae, while the tribes Ehrlichieae and Wolbachieae have been abolished. Sequence diversity within the 'classical' genus Ehrlichia has led to classifying E. phagocytophila (including E. equi and the agent of human granulocytic ehrlichiosis), E. platys and E. bovis in the genus Anaplasma, while others have been retained in Ehrlichia, which also includes Cowdria ruminantium. E. sennetsu and E. risticii have been transferred to the genus Neorickettsia. 16S rRNA and GroEL sequences of 'classical' Anaplasma and some members of 'classical' Ehrlichia do show a close relationship, but differences in citrate synthase gene sequences, the GC content of this gene, and sequences of the gene encoding the beta-subunit of RNA polymerase, not to speak of the phenotypical differences, do not justify the fusion into one genus. Because of the phylogenetical diversity in Ehrlichia it is recommended that a new genus name be created for the E. phagocytophila genogroup (and E. platys and E. bovis). (iii) One of the conclusions of studies on the phylogeny of ticks of the subfamilies Rhipicephalinae and Hyalomminae, based on nucleotide sequences from 12S rRNA, cytochrome c oxidase I, the internal transcribed spacer 2, 18S rRNA, as well as morphological characters, is that Boophilus should be considered as a subgenus of Rhipicephalus. While Boophilus and Rhipicephalus are undoubtedly close, the obviously important morphological and biological differences between the genera Rhipicephalus and Boophilus are thus overruled by similarities in the sequences of a number of genes and this leads to considerable confusion. Polyphasic taxonomy amply justifies maintaining Boophilus as a separate genus, phylogenetically near to Rhipicephalus. This note is a plea for a cautious and balanced approach to taxonomy, taking into account molecular genotypical information, as far as is possible from different genes, as well as phenotypical characteristics.     

Mycoplasma cavipharyngis and Mycoplasma fastidiosum, the closest relatives to Eperythrozoon spp. and Haemobartonella spp.

The 16S rRNA gene sequences of Mycoplasma cavipharyngis and Mycoplasma fastidiosum have been determined. Phylogenetic analysis showed that these species formed a new cluster within the so-called pneumoniae group of the mollicutes (class Mollicutes). This cluster will be referred to as the M. fastidiosum cluster. Interestingly, the M. fastidiosum cluster formed a sister lineage to the haemotrophic bacteria. Eperythrozoon spp. and Haemobartonella spp. The two latter genera, formerly classified as rickettsias, formed a stable phylogenetic entity in the tree as judged from branch lengths, bootstrap values and sequence signatures. Thus, the members of the M. fastidiosum cluster are the closest known relatives to the haemotrophic bacteria. Our data strongly support that the haemotrophic bacteria should be reclassified to reflect their actual phylogenetic affiliation.     

Relationship of hydrogen peroxide production by Mycoplasma pulmonis to virulence for catalase-deficient mice

Mycoplasma pulmonis, an etiological agent of murine pneumonia, produced about 0.065 mumoles of hydrogen peroxide (H(2)O(2)) per hr per 10(10) colony-forming units. When glucose was present at a concentration of 0.01 m, H(2)O(2) production was increased by 50%. To determine if H(2)O(2) production by M. pulmonis could be correlated with virulence, normal, acatalasemic, and acatalatic mice were infected with the organism. Three days after infection with M. pulmonis significantly more acatalatic mice had pneumonia than did normal or acatalasemic mice. The pneumonia in acatalatic mice was also more severe than in the other two groups. Five days after infection, pneumonia in the acatalatic mice was resolved, whereas normal mice were severely affected. The presence of pneumonia and the severity were correlated with the recovery of M. pulmonis from the lesions. In vitro studies of the effect of catalase on M. pulmonis showed that exogenously supplied catalase stimulated the growth of M. pulmonis at 37 C and prolonged its survival at 25 C. Hemolysis of sheep blood, guinea pig blood, rabbit blood, and normal and acatalasemic mouse blood by M. pulmonis was inversely related to the catalase activity of the erythrocytes. These findings suggest that H(2)O(2) secretion contributes to the virulence of M. pulmonis and to the death of the microorganism in the absence of host catalase.     

Identification of a haemomycoplasma species in anemic reindeer (Rangifer tarandus)

During an 18-mo period (May 2002-November 2003), 10 animals in a herd of 19 reindeer (Rangifer tarandus) at the National Animal Disease Center (NADC) experienced episodes of anemia. Affected animals had histories of weight loss, unthriftiness, occasionally edema of dependent parts and moderate anemia characterized by microcytosis or macrocytosis, hypochromasia, schistocytosis, keratocytosis, acanthocytosis, and dacryocytosis. Numerous basophilic punctate to ring-shaped bodies, measuring less than 1.0 microm, were found on the surface of red blood cells and were often observed encircling the outer margins of the cells. Based on cytologic findings, DNA preparations from selected affected animals in the NADC herd and one animal from a private herd experiencing similar episodes of anemia were assayed by polymerase chain reaction (PCR) for the presence of hemotropic bacteria using primers targeting the 16S rRNA genes of Mycoplasma (Eperythrozoon) suis, Mycoplasma (Haemobartonella) haemofelis, Anaplasma marginale, Anaplasma spp., and Ehrlichia spp. Amplification products were detected from four of the affected animals using primers specific for the 16S rRNA gene of M. haemofelis and Mycoplasma haemocanis. Product from one of the animals was sequenced and internal primers were designed from the resulting sequence to perform a nested PCR assay. Samples from 10 reindeer were positive using the nested PCR reaction and products from seven animals were sequenced; BLAST searches and phylogenetic analysis were performed on the resulting sequences. Sequence data from six animals revealed homology to an organism most closely related to Mycoplasma ovis, Mycoplasma wenyonii, and Mycoplasma haemolamae; sequence from a single animal was most closely related to M. haemofelis and M. haemocanis. This represents the first identification of a haemomycoplasma species in reindeer. Although several animals were also infected with abomasal nematodes, the presence of this newly described haemomycoplasma may have contributed to the anemic syndrome.     

Transformation of Mycoplasma pulmonis and Mycoplasma hyorhinis: transposition of Tn916 and formation of cointegrate structures

A procedure for transformation of the murine pathogen Mycoplasma pulmonis with plasmid pAM120 was developed. This plasmid replicates in Escherichia coli and contains the gram-positive transposon Tn916. The transformation protocol also proved effective for the swine pathogen Mycoplasma hyorhinis. The tetracycline resistance determinant of Tn916 was expressed in transformed myocoplasma cells, and Tn916 was found inserted into numerous sites in the recipient chromosomes of M. pulmonis and M. hyorhinis, indicating that transposition had occurred. Interestingly, some transformants of M. pulmonis and M. hyorhinis contained cointegrate structures which apparently had a complete copy of the entire donor plasmid (pAM120) inserted into the recipient chromosome. Subsequent transposition of inserted Tn916 was observed in passaged clones of transformed M. pulmonis.     

An ultrastructural comparison of the attachment sites between Gregarina steini and Cryptosporidium muris

Early developmental stages of Gregarina steini Berndt, 1902 from the intestine of Tenebrio molitor larvae were studied by transmission electron microscopy. The formation and structure of the eugregarine attachment site were compared with comparable features found on the feeder organelle of Cryptosporidium muris Tyzzer, 1907, from the stomach of experimentally infected rodents. The similarity of the attachment strategy between both organisms was revealed. The membrane fusion site in G. steini, formed by the trophozoite plasma membrane, host cell plasma membrane and a membrane-like structure limiting the cortical zone of the epimerite, resembles the Y-shaped membrane junction between the host cell plasma membrane, the trophozoite plasma membrane and membrane surrounding the anterior vacuole in C. muris. The anterior vacuole of C. muris appears to be the precursor of the feeder organelle and its structure is very similar to the epimeritic bud and the cortical zone of G. steini trophozoites. In both investigated organisms, the apical complex disappears early during cell invasion. The possibility of the epicellular location of Cryptosporidium on the surface of host cells is discussed.     

Arthropod transmission of Eperythrozoon coccoides in mice

The association of Eperythrozoon coccoides infection in laboratory mice, with commonly encountered external parasites, was examined. Transmission of E. coccoides was achieved by infesting recipient mice with as few as 10 live Polyplax serrata immediately after they were obtained from donor mice infected with E. coccoides. Polyplax serrata removed from such hosts and fasted for 24 hours also were able to initiate E. coccoides infections in recipient mice. Transovarial transmission of E. coccoides through P. serrata was not demonstrated. Transmission of E. coccoides by Myocoptes musculinus, Myobia musculi and Radfordia affinis was not observed.     

A phylogenetic analysis of the mycoplasmas: basis for their classification

Small-subunit rRNA sequences were determined for almost 50 species of mycoplasmas and their walled relatives, providing the basis for a phylogenetic systematic analysis of these organisms. Five groups of mycoplasmas per se were recognized (provisional names are given): the hominis group (which included species such as Mycoplasma hominis, Mycoplasma lipophilum, Mycoplasma pulmonis, and Mycoplasma neurolyticum), the pneumoniae group (which included species such as Mycoplasma pneumoniae and Mycoplasma muris), the spiroplasma group (which included species such as Mycoplasma mycoides, Spiroplasma citri, and Spiroplasma apis), the anaeroplasma group (which encompassed the anaeroplasmas and acholeplasmas), and a group known to contain only the isolated species Asteroleplasma anaerobium. In addition to these five mycoplasma groups, a sixth group of variously named gram-positive, walled organisms (which included lactobacilli, clostridia, and other organisms) was also included in the overall phylogenetic unit. In each of these six primary groups, subgroups were readily recognized and defined. Although the phylogenetic units identified by rRNA comparisons are difficult to recognize on the basis of mutually exclusive phenotypic characters alone, phenotypic justification can be given a posteriori for a number of them.     

Latent Haemobartonella muris infection: its transmission and decline in an inbred, ectoparasite-free strain of Wistar rat.

Ectoparasite-free, SPF Italian Wistar rats were consistently found to carry a latent infection with Haemobartonella muris, activable by splenectomy. In an inbred line this diminished and eventually ceased in six generations. Experimental infection from wild rats demonstrated that this was not apparently due to immunity.     

Cloning of mnuA, a membrane nuclease gene of Mycoplasma pulmonis, and analysis of its expression in Escherichia coli

Membrane nucleases of mycoplasmas are believed to play important roles in growth and pathogenesis, although no clear evidence for their importance has yet been obtained. As a first step in defining the function of this unusual membrane activity, studies were undertaken to clone and analyze one of the membrane nuclease genes from Mycoplasma pulmonis. A novel screening strategy was used to identify a recombinant lambda phage expressing nuclease activity, and its cloned fragment was analyzed. Transposon mutagenesis was used to identify an open reading frame of 1,410 bp, which coded for nuclease activity in Escherichia coli. This gene coded for a 470-amino-acid polypeptide of 53,739 Da and was designated mnuA (for "membrane nuclease"). The MnuA protein contained a prolipoprotein signal peptidase II recognition sequence along with an extensive hydrophobic region near the amino terminus, suggesting that the protein may be lipid modified or that it is anchored in the membrane by this membrane-spanning region. Antisera raised against two MnuA peptide sequences identified an M. pulmonis membrane protein of approximately 42 kDa by immunoblotting, which corresponded to a trypsin-sensitive nucleolytic band of the same size. Maxicell experiments with E. coli confirmed that mnuA coded for a nuclease of unknown specificity. Hybridization studies showed that mnuA sequences are found in few Mycoplasma species, suggesting that mycoplasma membrane nucleases display significant sequence variation within the genus Mycoplasma.     

Gene rearrangements in the vsa locus of Mycoplasma pulmonis

The vsa genes of Mycoplasma pulmonis encode the V-1 lipoproteins. Most V-1 proteins contain repetitive domains and are thought to be involved in mycoplasma-host cell interactions. Previously, we have reported the isolation and characterization of six vsa genes comprising a 10-kb region of the genome of M. pulmonis strain KD735-15. In the current study, vsa-specific probes were used to clone several fragments from a genomic library of KD735-15 DNA and assemble a single 20-kb contig containing 11 vsa genes. The middle region of the vsa locus contains a large open reading frame (ORF) that is not a vsa gene and has undergone an internal deletion in some strains. The ORF is predicted to encode a membrane protein that may have a role in disease pathogenesis. To examine vsa genes in a strain of M. pulmonis that is unrelated to KD735-15, strain CT was studied. Through Southern hybridization and genomic cloning analyses, CT was found to possess homologs of the KD735-15 vsaA, -C, -E, and -F genes and two unique genes (vsaG and vsaH) that were not found in KD735-15. High-frequency, site-specific DNA inversions serve to regulate the phase-variable production of individual V-1 proteins. As a result of the sequence analysis of vsa recombination products, a model in which DNA inversion arises from strand exchange involving at least six nucleotides of the vrs box is proposed.     

Male germ cell specific sulfogalactoglycerolipid is recognized and degraded by mycoplasmas associated with male infertility

Sulfogalactosylglycerolipid (SGG) is the major mammalian male germ cell glycolipid and has been implicated in sperm/egg binding. Mycoplasma pulmonis, a species of Mollicutes, is associated with male infertility in rodents. Purified SGG incubated in the presence of M. pulmonis was enzymatically degraded by both desulfation and deacylation. Desulfation occurred primarily at alkaline pH, and deacylation also increased with increased pH, indicating that these represent novel enzymatic activities. Digestion was facilitated, but not dependent on, the presence of detergent. Rat spermatozoa exposed to M. pulmonis showed a reduction in SGG content which was particularly marked for cauda (mature) spermatozoa. With the aid of tlc overlay binding procedure, intact M. pulmonis were found to bind specifically to sulfated glycolipids and thus SGG may provide the cell membrane receptor for this organism. The topology of mycoplasma binding to rat sperm was consistent with the known topology of sperm SGG. The reduced binding (and subsequent digestion) of caput spermatozoan SGG correlates with the membrane colocalization of SGG and its endogenous binding protein at this stage. Separation of SGG and its binding protein during epididymal sperm maturation appears to facilitate M. pulmonis binding to and digestion of cauda sperm SGG. The binding and degradation of the sperm SGG by M. pulmonis may play a role in the induction of infertility which follows infection with these organisms by interfering in sperm/egg receptor recognition.     

Genus- and species-specific identification of mycoplasmas by 16S rRNA amplification.

Systematic computer alignment of mycoplasmal 16S rRNA sequences allowed the identification of variable regions with both genus- and species-specific sequences. Species-specific sequences of Mycoplasma collis were elucidated by asymmetric amplification and dideoxynucleotide sequencing of variable regions, using primers complementary to conserved regions of 16S rRNA. Primers selected for Mycoplasma pneumoniae, M. hominis, M. fermentans, Ureaplasma urealyticum, M. pulmonis, M. arthritidis, M. neurolyticum, M. muris, and M. collis proved to be species specific in the polymerase chain reaction. The genus-specific primers reacted with all mycoplasmal species investigated as well as with members of the genera Ureaplasma, Spiroplasma, and Acholeplasma. No cross-reaction was observed with members of the closely related genera Streptococcus, Lactobacillus, Bacillus, and Clostridium or with any other microorganism tested. On the basis of the high copy number of rRNA, a highly sensitive polymerase chain reaction assay was developed in which the nucleic acid content equivalent to a single organism could be detected.     

Genus- and species-specific identification of mycoplasmas by 16S rRNA amplification.

Systematic computer alignment of mycoplasmal 16S rRNA sequences allowed the identification of variable regions with both genus- and species-specific sequences. Species-specific sequences of Mycoplasma collis were elucidated by asymmetric amplification and dideoxynucleotide sequencing of variable regions, using primers complementary to conserved regions of 16S rRNA. Primers selected for Mycoplasma pneumoniae, M. hominis, M. fermentans, Ureaplasma urealyticum, M. pulmonis, M. arthritidis, M. neurolyticum, M. muris, and M. collis proved to be species specific in the polymerase chain reaction. The genus-specific primers reacted with all mycoplasmal species investigated as well as with members of the genera Ureaplasma, Spiroplasma, and Acholeplasma. No cross-reaction was observed with members of the closely related genera Streptococcus, Lactobacillus, Bacillus, and Clostridium or with any other microorganism tested. On the basis of the high copy number of rRNA, a highly sensitive polymerase chain reaction assay was developed in which the nucleic acid content equivalent to a single organism could be detected.     

Deoxyribonucleic Acid Homology and Relative Genome Size in Mycoplasma

The deoxyribonucleic acid homologies of Mycoplasma laidlawii type A and type B, M. pulmonis (#47 and #63), and M. hominis were determined by membrane methodology. The homology data revealed a difference in genome size between M. laidlawii type A and type B. This difference also held with stringent conditions of annealing (high temperature). Little or negligible homology was shown to exist between the M. laidlawii strains type A and type B and M. pulmonis strains 47 and 63 and M. hominis. M. hominis showed less than 10% homology to the M. pulmonis and M. laidlawii strains. Neither of the M. laidlawii strains showed more than 2% annealing to the M. pulmonis strains. Reaction rate studies are suggested as a means of demonstrating the phylogenetic relationship between the Mycoplasma and other microorganisms.     

Experimental Arthritis in Mice with Mycoplasma pulmonis

A Mycoplasma pulmonis strain, recovered from the arthritic joints of mice employed in the serial passage of a chemically induced tumor, was found to be arthritogenic for mice under experimental conditions. Some joint involvement occurred in all mice challenged intravenously with this strain, and M. pulmonis was recovered frequently from the enlarged joints. The arthritis was migratory, appearing first in the radiocarpal joints and later in the tibiotarsal joints. There was little evidence of a generalized mycoplasmal infection as a consequence of the experimental challenge. Histopathologically, the early stages of the infection in the joints was characterized by an inflammatory response in the synovium and periarticular tissues. Exudate in the joint space contained about equal numbers of polymorphonuclear and mononuclear cells. The polyarthritis resolved slowly, but some residual joint enlargement was noted for as long as 4 months. Two other M. pulmonis strains were also observed to be arthritogenic for mice. Rats were not susceptible to M. pulmonis challenge. Characteristics of the nonsuppurative M. pulmonis arthritis in mice were compared to M. arthritidis joint infections in rats.     

Pneumonia due to mycoplasma in gnotobiotic mice. I. Pathogenicity of Mycoplasma pneumoniae, Mycoplasma salivarium, and Mycoplasma pulmonis for the lungs of conventional and gnotobiotic mice

Lutsky, Irving I. (Marquette University School of Medicine, Milwaukee, Wis.), and Avrum B. Organick. Pneumonia due to mycoplasma in gnotobiotic mice. I. Pathogenicity of Mycoplasma pneumoniae, Mycoplasma salivarium, and Mycoplasma pulmonis for the lungs of conventional and gnotobiotic mice. J. Bacteriol. 92:1154-1163. 1966.-Two species of mycoplasma of human origin, Mycoplasma pneumoniae and M. salivarium, were tested for their ability to produce respiratory disease in the Ha/ICR mouse when inoculated by the intranasal route. The mouse pathogen M. pulmonis was studied as a positive control. Conventional and gnotobiotic Ha/ICR mice were employed, the latter to provide a system free from indigenous mycoplasma and bacteria. Pneumonia from which mycoplasma were isolated was produced in all groups of the conventional Ha/ICR mice, including those inoculated with sterile broth. Only M. pulmonis produced disease when inoculated intranasally into the gnotobiotic mice, and the gross and microscopic lesions resembled those described in conventional mice. The gnotobiotic mouse provided a tool to study the pathogenicity of different mycoplasma species, and indicated marked differences in host specificity that could not be clearly seen when conventional mice were used.     

Mycoplasma pulmonis infection augments natural killer cell activity in mice

The goal of this study was to determine if experimental Mycoplasma pulmonis infection augmented splenic natural killer (NK) cell activity in mice. A 4 hour 51Cr-release in vitro assay using YAC-1 tumor target cells was employed to measure splenic NK cell activity in C57BL/6J mice infected intraperitoneally with M. pulmonis and in uninfected controls. Transient augmentation of the NK cells was observed, peaking at day 3 postinoculation (PI) and gradually returning to normal levels by day 10 PI. Selective depletion studies showed that the cells responsible for killing target cells were NK cells. They were nonadherent to nylon wool, not susceptible to Thy-1.2 antibody and susceptible to asialo GM1 ganglioside antibody. Inadvertent augmentation of the NK cell system due to M. pulmonis infection may complicate the interpretation of research data, especially in immunology and cancer studies.