Joanne B. Messick

'Candidatus Mycoplasma haemodidelphidis' sp. nov., 'Candidatus Mycoplasma haemolamae' sp. nov. and Mycoplasma haemocanis comb. nov., haemotrophic parasites from a naturally infected opossum (Didelphis virginiana), alpaca (Lama pacos) and dog (canis familiaris) : Phylogenetic and secondary structural relatedness of their 16S rRNA genes to other mycoplasmas

The 16S rRNA sequence of newly characterized haemotrophic bacteria in an opossum (Didelphis virginiana) and alpaca (Lama pacos) was determined. In addition, the 16S rRNA sequence of a haemotrophic parasite in the dog (Canis familiaris) was determined. Sequence alignment and evolutionary analysis as well as secondary structural similarity and signature nucleotide sequence motifs of their 16S rRNA genes, positioned these organisms in the genus Mycoplasma. The highest scoring sequence similarities were 16S rRNA genes from haemotrophic mycoplasma species (Haemobartonella and Eperythrozoon spp.). However, the lack of several higher-order structural idiosyncrasies used to define the pneumoniae group, suggests that these organisms and related haemotrophic mycoplasmas represent a new group of mycoplasmas. It is recommended that the organisms be named Candidatus Mycoplasma haemodidelphidis, Candidatus Mycoplasma haemolamae and Mycoplasma haemocanis comb. nov., to provide some indication of the target cell and host species of these parasites, and to reflect their phylogenetic affiliation.

Combined deletion of mouse dematin-headpiece and β-adducin exerts a novel effect on the spectrin-actin junctions leading to erythrocyte fragility and hemolytic anemia

Dematin and adducin are actin-binding proteins of the erythrocyte “junctional complex.” Individually, they exert modest effects on erythrocyte shape and membrane stability, and their homologues are expressed widely in non-erythroid cells. Here we report generation and characterization of double knock-out mice lacking β-adducin and the headpiece domain of dematin. The combined mutations result in altered erythrocyte morphology, increased membrane instability, and severe hemolysis. Peripheral blood analysis shows evidence of severe hemolytic anemia with reduced number of erythrocytes/hematocrit/hemoglobin and an ∼12-fold increase in the number of circulating reticulocytes. The presence of a variety of misshapen and fragmented erythrocytes correlates with increased osmotic fragility and reduced in vivo life span. Despite the apparently normal protein composition of the mutant erythrocyte membrane, the retention of the spectrin-actin complex in the membrane under low ionic strength conditions is significantly reduced by the double mutation. Atomic force microscopy reveals an increase in grain size and a decrease in filament number of the mutant membrane cytoskeleton, although the volume parameter is similar to wild type erythrocytes. Aggregated, disassembled, and irregular features are visualized in the mutant membrane, consistent with the presence of large protein aggregates. Importantly, purified dematin binds to the stripped inside-out vesicles in a saturable manner, and dematin-membrane binding is abolished upon pretreatment of membrane vesicles with trypsin. Together, these results reveal an essential role of dematin and adducin in the maintenance of erythrocyte shape and membrane stability, and they suggest that the dematin-membrane interaction could link the junctional complex to the plasma membrane in erythroid cells.

Clinical and clinicopathologic features of dogs that consumed foodborne hepatotoxic aflatoxins: 72 cases (2005–2006)

OBJECTIVEnTo characterize clinical signs, clinicopathologic features, treatments, and survival in dogs with naturally acquired foodborne aflatoxicosis.nnnDESIGNnRetrospective case series.nnnANIMALSn72 dogs that consumed aflatoxin-contaminated commercial dog food.nnnPROCEDURESnMedical records of affected dogs were reviewed. Between December 2005 and March 2006, dogs were identified as having foodborne aflatoxin hepatotoxicosis on the basis of the history of consumption of contaminated food or characteristic histopathologic lesions (subject dog or a recently deceased dog in the same household or kennel). Recorded information included signalment, clinical features, clinicopathologic test results, treatments, and survival. Data were analyzed by survival status.nnnRESULTSnMost dogs were of large breeds from breeding kennels. No significant differences were found in age or weight between 26 (36%) survivor dogs and 46 (64%) nonsurvivor dogs. Severity of clinical signs varied widely; 7 dogs died abruptly. In order of onset, clinical features included anorexia, lethargy, vomiting, jaundice, diarrhea (melena, hematochezia), abdominal effusion, peripheral edema, and terminal encephalopathy and hemorrhagic diathesis. Common clinicopathologic features included coagulopathic and electrolyte disturbances, hypoproteinemia, increased serum liver enzyme activities, hyperbilirubinemia, and hypocholesterolemia. Cytologic hepatocellular lipid vacuolation was confirmed in 11 dogs examined. In comparisons of clinicopathologic test results between survivor and nonsurvivor dogs, only granular cylindruria (7/21 dogs) consistently predicted death. Best early markers of aflatoxicosis were low plasma activities of anticoagulant proteins (protein C, antithrombin) and hypocholesterolemia. Despite aggressive treatment, many but not all severely affected dogs died.nnnCONCLUSIONS AND CLINICAL RELEVANCEnSerum liver enzyme activities and bilirubin concentration were unreliable early markers of aflatoxin hepatotoxicosis in dogs. Hypocholesterolemia and decreased plasma protein C and antithrombin activities may function as exposure biomarkers.

Can we continue research in splenectomized dogs? Mycoplasma haemocanis: old problem--new insight.

We report the appearance of a Mycoplasma haemocanis infection in laboratory dogs, which has been reported previously, yet, never before in Europe. Outbreak of the disease was triggered by a splenectomy intended to prepare the dogs for a hemorrhagic shock study. The clinical course of the dogs was dramatic including anorexia and hemolytic anemia. Treatment included allogeneic transfusion, prednisone, and oxytetracycline. Systematic follow-up (n = 12, blood smears, antibody testing and specific polymerase chain reaction) gives clear evidence that persistent eradication of M. haemocanis is unlikely. We, therefore, had to abandon the intended shock study. In the absence of effective surveillance and screening for M. haemocanis, the question arises whether it is prudent to continue shock research in splenectomized dogs.

Physical Map and Genome Sequencing Survey of Mycoplasma haemofelis (Haemobartonella felis)

ABSTRACT Mycoplasma haemofelis is an uncultivable red-cell pathogen of cats. Isolated M. haemofelis DNA was used to create a bacterial artificial chromosome library and physical map. Random sequencing of this material revealed 75 genes that had not been previously reported for M. haemofelis or any other hemotrophic mycoplasma.

Competitive, quantitative PCR analysis of Haemobartonella felis in the blood of experimentally infected cats

The DNA from the organism, Haemobartonella felis, was extracted from the leukocyte-poor peripheral blood of four experimentally infected cats. Since these organisms are currently unculturable, a competitive, quantitative PCR method developed by Zachar et al. [Nucleic Acid Res. 21 (1993) 2017–2018] was used to estimate the numbers of H. felis organisms in the blood of these cats. This estimation was based on the assumption that there is only one copy of the 16S rRNA gene in the genome of H. felis. It was also based on the efficiency of the DNA extraction, lysing efficiency, as well as the difference in amplification rates between a cloned version of the 16S rRNA gene and genomic DNA from the organism. The number of organisms in the peripheral blood of the cats at peak bacteremia was estimated to be between 3.0·105 and 1.1·108 per microliter of blood. Using this method, the sensitivity of the PCR was determined by estimating the lowest limits of detection. It was determined that as few as 52 organisms were detectable by PCR.

LIGHT AND ELECTRON MICROSCOPIC FEATURES OF EPERYTHROZOON-LIKE PARASITES IN A NORTH AMERICAN OPOSSUM (DIDELPHIS VIRGINIANA)

Abstract Epierythrocytic parasites associated with a severe anemic episode have not been previously reported in the opossum. A Wright-Giemsa–stained peripheral blood smear from an anemic North American opossum (Didelphis virginiana) revealed numerous organisms attached to red blood cells either singularly or in chains. Ring forms of the organism were common and could be found free in the plasma. Electron microscopy revealed that these organisms were attached to the intact plasma membrane in depressions on the surface of red blood cells. Delicate fibrils between the organism and adjacent membrane were observed. The organisms were round to oval with a diameter of 300–750 nm and were enclosed by a single limiting membrane. The light and electron microscopic features of these epierythrocytic organisms are similar to those reported for Eperythrozoon and Haemobartonella species.

The splenectomized dog: do we have to say farewell to an established hemorrhagic shock model?

To the Editor:Braz et al. report the effects of fluid resuscitation in acutely splenectomized dogs (1). We would like to address an issue that we believe is dramatically underestimated. Recently, we reported the endemic occurrence of Mycoplasma haemocanis infection in laboratory dogs (2)—an incident