Molecular detection of Mycoplasma ovis in an outbreak of hemolytic anemia in sheep from Veracruz, Mexico.

Mycoplasma ovis is a small, pleiotropic bacterium, which parasitizes the external surface of erythrocytes of several species of artiodactyl mammals, especially sheep and goats. We here report an outbreak of ovine mycoplasmosis in a sheep flock of a private ranch (Universidad Veracruzana) in Veracruz, Mexico. For the identification of Mycoplasma and other hemoparasitic bacterial agents, we stained blood smears with the DiffQuick® technique and additionally amplified several fragments of 16S rDNA gene. We detected the presence of morulas in erythrocytes from 30 sick female adult sheep, and found Mycoplasma ovis DNA in all of them. Furthermore, three of these animals also tested positive for Anaplasma ovis. Our findings represent the first record of M. ovis and A. ovis in an outbreak of hemolytic anemia in a sheep flock, leading to severe livestock loss in a ranch of Mexico. This study highlights the importance of establishing an active surveillance of both pathogens in the country.

Recovery with a regular dose of antibiotics from bacillary hemoglobinuria in a Holstein cow.

One Holstein cow housed with 21 other cows exhibited clinical signs of pyrexia, anorexia and diarrhea along with severe hemoglobinuria. Hematological and biochemical analyses conducted before and after antibiotic therapy indicated severe hemolytic anemia and disruption of hepatic function. A general improvement in conditions was observed after an 11-day program of treatment comprising a regular dose of antibiotics and prescribed supportive therapies. A tentative diagnosis of bacillary hemoglobinuria was made based on the clinical and clinico-pathologic features on day 7. A molecular diagnosis was made by a PCR amplification of the flagellin gene of Clostridium haemolyticum using DNA extracted from the whole blood. The cow was diagnosed with the first recorded occurrence of bacillary hemoglobinuria of Holstein cattle in Japan.

Clostridium Perfringens Infection in a Febrile Patient with Severe Hemolytic Anemia.

BACKGROUND: Clostridium perfringens (C. perfringens) can cause various infections, including gas gangrene, crepitant cellulitis, and fasciitis. While C. perfringens sepsis is uncommon, it is often rapidly fatal because the alpha toxin of this bacterium induces massive intravascular hemolysis by disrupting red blood cell membranes. CASE REPORT: We present the case of a male patient with diabetes who developed a fatal liver abscess with massive intravascular hemolysis and septic shock caused by toxigenic C. perfringens. The peripheral blood smear showed loss of central pallor, with numerous spherocytes. Multiplex PCR only detected expression of the cpa gene, indicating that the pathogen was C. perfringens type A. CONCLUSIONS: C. perfringens infection should be considered in a febrile patient who has severe hemolytic anemia with a very low MCV, hemolyzed blood sample, and negative Coombs test. The characteristic peripheral blood smear findings may facilitate rapid diagnosis.

Red blood cell Thomsen-Friedenreich antigen expression and galectin-3 plasma concentrations in Streptococcus pneumoniae-associated hemolytic uremic syndrome and hemolytic anemia.

BACKGROUND: Pneumococcal hemolytic uremic syndrome (P-HUS) is a rare but severe complication of invasive pneumococcal disease (IPD) in young children. Consensual biologic diagnosis criteria are currently lacking. STUDY DESIGN AND METHODS: A prospective study was conducted on 10 children with culture-confirmed IPD. Five presented with full-blown P-HUS, three had an incomplete form with hemolytic anemia and mild or no uremia (P-HA), and two had neither HUS nor HA. Thomsen-Friedenreich (T), Th, and Tk cryptantigens and sialic acid expression were determined on red blood cells (RBCs) with peanut (PNA), Glycine soja (SBA), Bandeiraea simplicifolia II, and Maackia amurensis lectins. Plasma concentrations of the major endogenous T-antigen-binding protein, galectin-3 (Gal-3), were analyzed. RESULTS: We found that RBCs strongly reacted with PNA and SBA lectins in all P-HUS and P-HA patients. Three P-HUS and three P-HA patients showed also concomitant Tk activation. Direct antiglobulin test (DAT) was positive in three P-HUS (one with anti-C3d and two with anti-IgG) and two P-HA patients (one with anti-C3d and one with anti-IgG). RBCs derived from the two uncomplicated IPD patients reacted with PNA but not with SBA lectin. Gal-3 plasma concentrations were increased in all P-HUS patients. CONCLUSIONS: The results indicate high levels of neuraminidase activity and desialylation in both P-HUS and P-HA patients. T-antigen activation is more sensitive than DAT for P-HUS diagnosis. Combining PNA and SBA lectins is needed to improve the specificity of T-antigen activation. High concentrations of Gal-3 in P-HUS patients suggest that Gal-3 may contribute to the pathogenesis of P-HUS.

More complications occur in macrolide-resistant than in macrolide-sensitive Mycoplasma pneumoniae pneumonia.

We sought to understand the situation of macrolide-resistant genotypes of Mycoplasma pneumoniae, and analyze the relationship between macrolide-resistant genotypes and clinical manifestations of Mycoplasma pneumoniae pneumonia (MPP). Full-length sequencing of the 23S rRNA gene of M. pneumoniae was performed in 235 nasopharyngeal aspirates (NPAs) from children with MPP. We also retrospectively compared the clinical characteristics of macrolide-resistant (MR) M. pneumoniae infections and macrolide-sensitive (MS) M. pneumoniae infections. A total of 206 patients had point mutations in the M. pneumoniae 23S rRNA gene, and these patients are referred to as MR patients. The remaining 29 patients without point mutations are referred to as MS patients. Among 206 MR patients, 199 (96.6%) had A2063G mutations, 6 had A2063T mutations, and the remaining patients had an A2064G mutation. Among the clinical manifestations, we found that the median fever durations were 8 days (range, 0 to 42 days) and 6 days (0 to 14 days) (P < 0.01), the median hospitalization durations were 8 days (2 to 45 days) and 6 days (3 to 16 days) (P < 0.01), and the median fever durations after macrolide therapy were 5 days (0 to 42 days) and 3 days (0 to 10 days) (P < 0.01), respectively, in the MR and MS groups. We also found that the incidence of extrapulmonary complications in the MR group was significantly higher than that in the MS group (P < 0.05). Moreover, the radiological findings were more serious in the MR group than in the MS group (P < 0.05). The increasing prevalence of MR M. pneumoniae has become a significant clinical issue in the pediatric patients, which may lead to more extrapulmonary complications and severe clinical features and radiological manifestations.

Clostridial myonecrosis, haemolytic anaemia, hepatopathy, osteitis and transient hypertrophic cardiomyopathy after intramuscular injection in a Thoroughbred gelding.

A 9-year-old Thoroughbred gelding was presented for swelling over the left neck and inappetence. There was recent history of intramuscular administration of flunixin meglumine into the left neck. On examination, there was evidence of focal myositis, anaemia, haemolysis and pigmenturia. Culture of aspirated fluid from the left side of the neck produced a heavy growth of a Clostridium species. Complications of infection included haemolytic anaemia, hepatopathy, osteitis and transient hypertrophic cardiomyopathy. Treatment included intravenous fluid therapy, antibiotics, anti-inflammatory drugs, blood transfusion and surgical debridement. There was complete resolution of clinical, haematological, biochemical and echocardiographic abnormalities, and the horse returned to ridden work. This report highlights the complications that can arise from clostridial myonecrosis, including the effect on the myocardium.

Suppression of hepcidin expression and iron overload mediate Salmonella susceptibility in ankyrin 1 ENU-induced mutant.

Salmonella, a ubiquitous Gram-negative intracellular bacterium, is a food borne pathogen that infects a broad range of hosts. Infection with Salmonella Typhimurium in mice is a broadly recognized experimental model resembling typhoid fever in humans. Using a N-ethyl-N-nitrosurea (ENU) mutagenesis recessive screen, we report the identification of Ity16 (Immunity to Typhimurium locus 16), a locus responsible for increased susceptibility to infection. The position of Ity16 was refined on chromosome 8 and a nonsense mutation was identified in the ankyrin 1 (Ank1) gene. ANK1 plays an important role in the formation and stabilization of the red cell cytoskeleton. The Ank1(Ity16/Ity16) mutation causes severe hemolytic anemia in uninfected mice resulting in splenomegaly, hyperbilirubinemia, jaundice, extramedullary erythropoiesis and iron overload in liver and kidneys. Ank1(Ity16/Ity16) mutant mice demonstrated low levels of hepcidin (Hamp) expression and significant increases in the expression of the growth differentiation factor 15 (Gdf15), erythropoietin (Epo) and heme oxygenase 1 (Hmox1) exacerbating extramedullary erythropoiesis, tissue iron deposition and splenomegaly. As the infection progresses in Ank1(Ity16/Ity16), the anemia worsens and bacterial load were high in liver and kidneys compared to wild type mice. Heterozygous Ank1(+/Ity16) mice were also more susceptible to Salmonella infection although to a lesser extent than Ank1(Ity16/Ity16) and they did not inherently present anemia and splenomegaly. During infection, iron accumulated in the kidneys of Ank1(+/Ity16) mice where bacterial loads were high compared to littermate controls. The critical role of HAMP in the host response to Salmonella infection was validated by showing increased susceptibility to infection in Hamp-deficient mice and significant survival benefits in Ank1(+/Ity16) heterozygous mice treated with HAMP peptide. This study illustrates that the regulation of Hamp and iron balance are crucial in the host response to Salmonella infection in Ank1 mutants.

Sago haemolytic disease: towards understanding a novel food-borne toxicosis.

Sago haemolytic disease is a rare but sometimes fatal disease found primarily in the coastal regions of Papua New Guinea and among groups in which sago is a primary source of carbohydrate. It has been known since 1961 and fungi consistently have been suspected of being involved. Investigations carried out on stored sago and samples recovered from poisoning episodes have failed to indicate the consistent presence of mycotoxins. However, fungi (especially Aspergillus, Fusarium, Penicillium, Trichoderma) with strong haemolytic activity have been associated with sago, particularly when stored in open-weave baskets and sago-leaf-wrapped bundles. The haemolytic activity has been attributed to fatty acids (principally oleic, palmitic, linoleic) contained primarily in the fungal hyphae. It is hypothesized that when these acids are released through hyphal breakdown during digestion and are present in individuals with a low serum albumin level, free fatty acid excess occurs resulting in red cell membrane destruction and intravascular haemolysis. In extreme cases, blood transfusion is required. Methods of storage providing high levels of access to oxygen favour the development of fungi: eg, leaf-encased bundles and open-weave storage favour growth over that seen in starch stored under water, such as in earthen vessels. Ensuring storage does not exceed 3-4 weeks, encouraging anaerobic conditions of the starch and maintaining protein nutrition in communities where sago is relied upon should alleviate outbreak episodes.

Alterations in bone and erythropoiesis in hemolytic anemia: comparative study in bled, phenylhydrazine-treated and Plasmodium-infected mice.

Sustained erythropoiesis and concurrent bone marrow hyperplasia are proposed to be responsible for low bone mass density (BMD) in chronic hemolytic pathologies. As impaired erythropoiesis is also frequent in these conditions, we hypothesized that free heme may alter marrow and bone physiology in these disorders. Bone status and bone marrow erythropoiesis were studied in mice with hemolytic anemia (HA) induced by phenylhydrazine (PHZ) or Plasmodium infection and in bled mice. All treatments resulted in lower hemoglobin concentrations, enhanced erythropoiesis in the spleen and reticulocytosis. The anemia was severe in mice with acute hemolysis, which also had elevated levels of free heme and ROS. No major changes in cellularity and erythroid cell numbers occurred in the bone marrow of bled mice, which generated higher numbers of erythroid blast forming units (BFU-E) in response to erythropoietin. In contrast, low numbers of bone marrow erythroid precursors and BFU-E and low concentrations of bone remodelling markers were measured in mice with HA, which also had blunted osteoclastogenesis, in opposition to its enhancement in bled mice. The alterations in bone metabolism were accompanied by reduced trabecular bone volume, enhanced trabecular spacing and lower trabecular numbers in mice with HA. Taken together our data suggests that hemolysis exerts distinct effects to bleeding in the marrow and bone and may contribute to osteoporosis through a mechanism independent of the erythropoietic stress.

Treatment of severe hemolytic anemia caused by Clostridium perfringens sepsis in a liver transplant recipient.

BACKGROUND: Clostridium perfringens bacteremia accompanied by extensive intravascular hemolysis is an almost inescapably fatal infection. METHODS: Case report and literature review. RESULTS: A 52-year-old man with a recent history of liver transplantation developed sepsis and severe hemolytic anemia. The patient had multiple organ dysfunction syndrome and required aggressive transfusion, antibiotics, and continuous hemodialysis. Blood cultures grew C. perfringens. With appropriate resuscitation and antibiotic treatment, the patient had a complete, although complicated recovery. CONCLUSION: This is the first reported case of a liver transplant patient developing fulminant C. perfringens sepsis with hemolysis. This infection usually kills patients within hours of presentation. Early recognition and aggressive treatment is necessary to avoid this outcome.

[Porcine eperythrozoonosis: from Eperythrozoon suis to Mycoplasma suis]. / Porzine Eperythrozoonose: von Eperythrozoon suis zu Mycoplasma suis.

Mycoplasma suis (formerly known as Eperythrozoon suis ) is the most prevalent agent causing haemolytic anaemia in swine. The disease is also known as porcine eperythrozoonosis. M.suis is a small, pleomorphic bacteria parasitizing porcine erythrocytes. To date, no in vitro cultivation system for M.suis has been established and, therefore, our knowledge about the characteristics of M.suis and the pathogenesis of porcine eperythrozoonosis is rather limited. M.suis can cause acute disease, but the major significance of M.suis infections lies in the fact that M.suis can establish chronic and persistent infections leading to a higher susceptibility to other infections, especially of the respiratory and digestive tracts. The present article summarizes the current knowledge of the pathogen, the clinical signs and pathogenesis, diagnostic as well as therapy and prophylaxis.

Hemotrophic mycoplasmas induce programmed cell death in red blood cells.

Hemotrophic mycoplasmas (HM) are uncultivable bacteria found on and in the red blood cells (RBCs). The main clinical sign of HM infections is the hemolytic anemia. However, anemia-inducing pathogenesis has not been totally clarified. In this work we used the splenectomized pig as animal model and Mycoplasma suis as a representative for hemotrophic mycoplasmas to study anemia pathogenesis. Eryptosis, i.e. programmed cell death of RBCs, is characterized by cell shrinkage, microvesiculation and phosphatidylserine (PS) exposure on the outer membrane. The eryptosis occurrence and its influence on anemia pathogenesis was observed over the time-course of M. suis infections in pigs using 3 M. suis isolates of differing virulence. All 3 isolates induced eryptosis, but with different characteristics. The occurrence of eryptosis could as well be confirmed in vitro: serum and plasma of an acutely ill pig induced PS exposure on erythrocytes drawn from healthy pigs. Since M. suis is able to induce eryptotic processes it is concluded that eryptosis is one anemia-inducing factor during M. suis infections and, therefore, plays a significant role in the pathogenesis of infectious anemia due to HM infection.

Molecular identification of `Candidatus Mycoplasma haemovis' in sheep with hemolytic anemia.

We examined the presence of hemoplasmas, hemotropic mycoplasmas, among 11 sheep (Ovis aries) with regenerative and hemolytic anemia and found six of them were positive by real-time PCR. The positive samples were then subjected to conventional PCR for direct sequencing of the 16S rRNA gene. Nucleotide sequences of all the positive samples were identified as the 16S rRNA gene of `Candidatus Mycoplasma haemovis' by phylogenetic analysis, demonstrating the infections with this particular hemoplasma species in Japan.

Complete genome sequence of Mycoplasma haemofelis, a hemotropic mycoplasma.

Here, we present the genome sequence of Mycoplasma haemofelis strain Langford 1, representing the first hemotropic mycoplasma (hemoplasma) species to be completely sequenced and annotated. Originally isolated from a cat with hemolytic anemia, this strain induces severe hemolytic anemia when inoculated into specific-pathogen-free-derived cats. The genome sequence has provided insights into the biology of this uncultivatable hemoplasma and has identified potential molecular mechanisms underlying its pathogenicity.

Is Penicillium citrinum implicated in sago hemolytic disease?

Sago hemolytic disease (SHD) is an acute hemolytic syndrome affecting rural Papua New Guineans who depend on the starch of Metroxylon sagu as a staple carbohydrate. It is a suspected mycotoxicosis associated with fungal succession in stored and perhaps poorly fermented sago. Despite a mortality rate of approximately 25%, little is know about the disease. Recent studies have identified Penicillium citrinum as a possible candidate in the etiology of SHD. This is based on the frequency of isolation from sago starch and the hemolytic nature of the organism as demonstrated when cultured on sheep and human blood agar. A highly non-polar lipophilic P. citrinum fraction from C18 solid phase extraction demonstrated high hemolytic activity in a semi-quantitative assay using both mouse and human erythrocytes. When the red cell membrane proteins were subjected to sodium dodecyl-sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) separation, cleavage of protein band 3 and spectrin was demonstrated. This breach of major structural red cell proteins is consistent with the severe hemolysis found in vivo. Our findings warrant further investigation into the hemolytic activity of P. citrinum and its role as the etiological agent of SHD.

Anemia in cats with hemotropic mycoplasma infection: retrospective evaluation of 23 cases (1996-2005).

This study summarizes the diagnostic findings from all anemic cats diagnosed with hemotropic mycoplasma (HM) infections at the Western College of Veterinary Medicine-Veterinary Teaching Hospital between 1996 and 2005. The objectives were to determine the frequency of HM-induced anemia among all cats presented with anemia during this period, the clinical findings and risk factors associated with clinical HM infection, and factors affecting or predicting survival. Medical records were examined from 23 cats with HM-induced anemia from the total of 170 cats diagnosed with anemia during this period. The frequency of HM-induced anemia was 14% (23/170) among all anemic cats. Cats with HM-induced anemia were less likely to be purebred (P = 0.04) than other cats with anemia. Of the cats with HM-induced anemia, those with positive retroviral status (P = 0.01), concurrent illness (P < 0.01), or lack of erythroid regeneration (P = 0.01) were most likely to die. The 1-year survival of HM-infected cats was 65% (13/20).

Molecular characterization of two different strains of haemotropic mycoplasmas from a sheep flock with fatal haemolytic anaemia and concomitant Anaplasma ovis infection.

After the first outbreak of fatal Mycoplasma ovis infection (eperythrozoonosis) in a sheep flock in Hungary (1997), a second wave of the disease was noted in 2006, with different seasonal pattern and affected age group, as well as increased mortality (5.5%). The aim of the present study was to molecularly characterize the causative agent and to reveal underlying factors of the second wave of the disease. Remarkably, among the 33 sheep examined, 17 were infected with two strains of haemotropic mycoplasmas. Cloning and sequencing isolates of the latter showed that one of the strains was 99.4-99.8% identical to M. ovis (AF338268), while the second was only 96.8-97.9% identical and contained a 17-bp deletion. Different isolates of both strains were demonstrated in the same animal. When analyzing possible risk factors for fatal disease outcome, we found that among sheep born prior to the 1997 outbreak significantly more animals survived the second outbreak than succumbed to disease. In addition, locally born sheep were less frequently diseased than sheep introduced into the flock from other places. This suggests an immunoprotective effect in some animals. Concurrent infection with Anaplasma ovis was detected in 24 of the 33 evaluated sheep. In conclusion, this is the first study to demonstrate the existence of and characterize two genetically distinct ovine haemotropic mycoplasma strains in a sheep flock with fatal haemolytic anaemia.