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To the Editor: Streptococcus suis is a major swine pathogen and an emerging zoonotic agent that causes mainly meningitis and septic shock (1,2). Among the 35 described serotypes classified by differences in capsular antigens, serotype 2 is the most frequently isolated from humans worldwide, and serotype 14 cases are also increasing in some countries (1). In Southeast Asia, this pathogen affects not only workers in close contact with pig/pork by-products but also the general population, probably because of the widespread presence of backyard types of pig production, open meat markets, and some special dishes prepared with raw meat or blood (3). We report a case of peritonitis caused by an atypical S. suis serotype 21 strain in a patient in Argentina. A 62-year-old man from Santa Fe Province in Argentina, who had a history of tobacco and alcohol abuse, was hospitalized in 2013 as an emergency patient with symptoms of acute abdominal distress. Ten days before admission, abdominal distention, accompanied by intense upper abdominal pain, developed in the patient. The patient’s family reported that he had been having gastrointestinal bleeding 4 days before admission, and he was suspected of having diabetes. At admission, a physical examination indicated jaundice, hepatosplenomegaly, and ascites. A neurologic examination indicated that the patient was conscious, but disoriented, and that his vital signs were stable. The patient had a temperature of 38.9°C, a pulse rate of 130 beats/min, and blood pressure of 110/70 mm Hg. Other laboratory results were a leukocyte count of 2,900 cells/μL (70% neutrophils), a platelet count of 94,000/μL, a serum hemoglobin concentration of 13.20 g/dL, a glucose concentration of 195 mg/dL, a blood urea nitrogen level of 42 mg/dL, a creatinine level of 0.96 mg/dL; a serum bilirubin level of 3.01 mg/dL, an alanine aminotransferase level of 35 U/L, an aspartate aminotransferase level of 70 U/, a serum albumin level of 2.66 g/dL, and an increase in prothrombin time to 22 s. Spontaneous bacterial peritonitis was suspected. Abdominal paracentesis was performed and produced a turbid milky fluid, with a protein level of 1600 mg/dL; 1,340 cells/µL (90% neutrophils), a lactate dehydrogenase level of 221 U/L, and an amylase level of 34 U/L. Samples of blood and ascitic fluid were inoculated into aerobic and anaerobic blood culture bottles. Gram staining was performed and no organisms were observed. Treatment with intravenous ceftriaxone (2g/day) was started after a diagnosis of spontaneous bacterial peritonitis associated with liver cirrhosis was made. After 48 h of incubation, cultures of blood and ascetic fluid were plated onto sheep blood agar and chocolate agar and incubated at 35°C in an atmosphere of 5% CO2. After 24 h of incubation, cultures showed growth of α-hemolytic streptococci. An API Strep Test (bioMerieux, Marcy l’Etoile, France) identified the isolate as S. pneumoniae (probability 58.7%) or S. suis (probability 20.7%). However, these 2 probability values are unacceptable identification confidence levels. Therefore, the species and serotype were identified by sequence analysis of a 16S rRNA gene and a coagglutination test as described (4,5). The isolate was identified as S. suis serotype 21. The infection was considered resolved when all signs and symptoms of infection disappeared, a polymorphonuclear cell count in ascitic fluid decreased to

The knowledge about wine yeasts remains largely dominated by the extensive studies on Saccharomyces (S.) cerevisiae. Molecular methods, allowing discrimination of both species and strains in winemaking, can profitably be applied for characterization of the microflora occurring in winemaking and for monitoring the fermentation process. Recently, some novel yeast isolates have been described as hybrid between S. cerevisiae and Saccharomyces species, leaving the Saccharomyces strains containing non-Saccharomyces hybrids essentially unexplored. In this study, we have analyzed a yeast strain isolated from “Primitivo” grape ( http://www.ispa.cnr.it/index.php?page=collezioni&lang=en accession number 12998 ) and we found that, in addition to the S. cerevisiae genome, it has acquired genetic material from a non-Saccharomyces species. The study was focused on the analysis of chromosomal and mitochondrial gene sequences (ITS and 26S rRNA, SSU and COXII, ACTIN-1 and TEF), 2D-PAGE mitochondrial proteins, and spore viability. The results allowed us to formulate the hypothesis that in the MSH199 isolate a DNA containing an rDNA sequence from Hanseniaspora vineae, a non-Saccharomyces yeast, was incorporated through homologous recombination in the grape environment where yeast species are propagated. Moreover, physiological characterization showed that the MSH199 isolate possesses high technological quality traits (fermentation performance) and glycerol production, resistance to ethanol, SO2 and temperature) useful for industrial application.

To the Editor: The etiologic agent of the sixth pandemic of cholera was classical biotype of Vibrio cholerae O1. The ongoing seventh pandemic is caused by El Tor biotype. The biotypes are differentiated by phenotypic and genotypic characteristics. However, this differentiation blurred when V. cholerae O1 strains were detected in Matlab, Bangladesh, in 2006, in which characteristics were mixed. Genetically, the differences occurred in tcpA, which encodes the major adherence antigen rstR that regulates site-specific recombination of CTXϕ phage and ctxB that encodes the B subunit of cholera toxin. These genes had the characteristics of classical biotype in Matlab variants of El Tor strains. Later, various types of El Tor variants were reported in Southeast Asia, Africa, and Haiti. Differentiating features also occur in repeat toxin A gene (rtxA), chromosomal location of CTXϕ, the number of heptad repeats in ToxR binding region, and the occurrence of vibrio seventh pandemic islands I and II (1,2).

Melissococcus plutonius is the causative agent of an important honeybee disease, European foulbrood (EFB). In addition to M. plutonius strains with typical characteristics (typical M. plutonius), we recently reported the presence of atypical M. plutonius, which are phenotypically and genetically distinguished from typical M. plutonius. Because typical and atypical M. plutonius may have different pathogenic mechanisms, differentiation of these two types is very important for diagnosis and more effective control of EFB. In this study, therefore, a duplex PCR assay was developed to detect and differentiate typical and atypical M. plutonius rapidly and easily. On the basis of the results of comparative genomic analyses, we selected Na+/H+ antiporter gene and Fur family transcriptional regulator gene as targets for detection of typical and atypical strains, respectively, by PCR. Under optimized conditions, the duplex PCR system using the designed primers successfully detected and differentiated all typical and atypical M. plutonius strain/isolates tested, while no product was generated from any other bacterial strains/isolates used in this study, including those isolated from healthy honeybee larval guts. Detection limits of the PCR were 50 copies of chromosome/reaction for both types, and it could detect typical and atypical M. plutonius directly from diseased honeybee larvae. Moreover, the duplex PCR diagnosed mixed infections with both M. plutonius types more precisely than standard culture methods. These results indicate that the duplex PCR assay developed in this study is extremely useful for precise diagnosis and epidemiological study of EFB.

Experimental infection of chickens with an atypical * mycoplasma synoviae strain : comparaison of diagnosis methods. Fifteen chickens were inoculated with the atypical Mycoplasma synoviae (MS) K1415 strain. On different dates post inoculation, tracheal swab samples were collected for mycoplasma culture and blood samples were analyzed by slide agglutination test (SA) with commercial or homologous antigen, and commercial or experimental enzyme-linked immunosorbent assay (ELISA) tests. Results showed that MS isolation rate was low. SA with commercial antigen did not yield positive results, although birds were positive when tested with homologous antigen. With most ELISA tests, most sera remained negative. Only one experimental ELISA test gave positive results but this test is known to suffer from low specificity. These results illustrate the difficulty of diagnosis of infections with such MS variant strains., Quinze poulets sont infectés expérimentalement à l’aide de la souche variante Mycoplasma synoviae K1415. A différentes dates après inoculation, des écouvillonnages de trachée sont réalisés pour culture de mycoplasmes et des sérums sont prélevés et analysés par agglutination rapide sur lame (ARL) vis-à-vis d’un antigène commercial ou vis-à-vis d’un antigène homologue et par différents tests immuno-enzymatiques (ELISA) commerciaux ou expérimentaux. Les résultats montrent que le taux de réisolement de MS est faible. Les sérums testés par ARL vis-à-vis de l’antigène et par la plupart des tests ELISA paraissent négatifs. Ils sont positifs par la méthode d’ARL utilisant un antigène homologue ainsi que par l’une des méthodes immuno-enzymatiques, peu spécifique par ailleurs. Cette expérimentation démontre la difficulté de dépister, à l’aide des méthodes classiques, les infections dues à des souches variantes., Kempf Isabelle, Gesbert Fabienne, Czifra Georges. Infection expérimentale de poulets à l’aide d’une souche variante de Mycoplasma Synoviae : comparaison des méthodes de dépistage. In: Bulletin de l'Académie Vétérinaire de France tome 150 n°4, 1997. pp. 403-409.

The quarantine bacterium Clavibacter michiganensis subsp. michiganensis (Cmm) is the agent of tomato bacterial canker (TBC), one of the most destructive bacterial diseases of this crop, that causes severe economic losses worldwide. During a recent outbreak of TBC in Sicily (insular Italy), it was impossible to amplify the pat-1 gene by PCR with CMM5/CMM6 primers from affected greenhouse-grown plants from three farms, although Cmm-like colonies were consistently isolated. Microlog metabolic profile, 16S rDNA sequencing and the positive amplification of other Cmm DNA targets supported the identification of the strains as Cmm, suggesting the occurrence of a population lacking the virulence gene pat-1. A detailed phenotypic and molecular characterization of these Cmm strains and their virulence to tomatoes, as compared with Cmm reference strains is reported in this study. Pathogenicity tests revealed the strains under study were able to induce TBC, although delayed wilting symptoms were observed. Strains with or without pat-1 were undistinguishable, based on disease indexes.

To the Editor: Toxoplasma gondii parasites are obligate intracellular apicomplexans that can infect virtually all warm-blooded animals; felids are definitive hosts. The most common sources of human infection are ingestion of tissue cysts in undercooked meat or of food or water contaminated with oocysts shed by felids and transplacental transmission. Acquired toxoplasmosis in immunocompetent humans is frequently asymptomatic but is associated with cervical or occipital lymphadenopathy in ≈10% of patients. Severe or fatal outcomes for immunocompetent patients have been attributed to the virulence of specific T. gondii genotypes (1). We describe 3 cases of toxoplasmosis caused by atypical strains probably acquired by from ingestion of raw horse meat imported from Canada and Brazil. Patient 1, a 74-year-old man, was hospitalized locally (Antibes-Juan Les Pins, southern France) in March 2009 for asthenia and persistent febrile bronchitis. His medical history included severe smoking-related chronic obstructive pulmonary disease and coronary artery disease. He received broad-spectrum antimicrobial drugs and methylprednisolone. On day 23 after admission, he was transferred to our teaching hospital in Nice because of clinical deterioration and persistent fever. Disseminated toxoplasmosis was diagnosed on the basis of serologic evidence of recent primary T. gondii parasite infection and quantitative PCR detection of high Toxoplasma DNA levels in peripheral blood. Despite specific antitoxoplasma therapy with sulfadiazine and pyrimethamine, he remained febrile, his respiratory function worsened, and he died on day 27. Patient 2, a 24-year-old pregnant woman, was hospitalized in Draguignan, France, in December 2009 for full-term delivery. Three weeks earlier, routine serologic testing showed T. gondii parasite infection seroconversion. The newborn’s and mother’s ophthalmologic examinations were unremarkable. Congenital toxoplasmosis was diagnosed on the basis immunoglobulin M in the infant’s serum, positive quantitative PCR of samples from the placenta, and strain isolation after inoculation of mice with a placental preparation. Sulfadiazine and pyrimethamine were started. We performed a retrospective epidemiologic investigation of an unusual case of toxoplasmosis that occurred in March 1991. Patient 3, a 21-year-old pregnant woman living in the Nice area, was treated with spiramycine because routine serologic testing had shown T. gondii parasite infection seroconversion at 22 weeks’ gestation. Amniocentesis showed T. gondii tachyzoites in amniotic fluid by microscopic examination. At 26 weeks’ gestation, the woman underwent termination of pregnancy for ultrasonography-detected fetal severe abnormalities. Fetal necropsy showed numerous cerebral, cardiac, and hepatic abscesses with T. gondii tachyzoites. A few days after pregnancy termination, the woman experienced cervical lymphadenopathy, which lasted 3 years. She reported having eaten raw horse meat regularly during her pregnancy. Genetic analyses with microsatellite markers of the Toxoplasma spp. strains isolated from the 3 patients found 3 different atypical genotypes. Atypical strains are common in South America but unusual in France, where >95% of reported strains collected from human and animal toxoplasmosis cases belonged to the type II clonal lineage (2,3). Hence, isolation of an atypical Toxoplasma genotype from a patient in France strongly suggests contamination by a non-European strain, either during residence abroad or after ingestion of imported meat. Epidemiologic investigation of our case-patients that included questioning relatives, patients, and butchers found that eating raw horse meat imported from Canada (patient 1) or Brazil (patient 2) was the most likely source of the parasites. The geographic origin of the horse meat eaten by patient 3 is unknown. Moreover, an atypical T. gondii strain was isolated after mouse inoculation with horse meat from the first patient’s butcher. In all 3 cases, close relatives encouraged the patients to eat raw horse meat regularly because the practice is traditionally thought to reinforce health. Human toxoplasmosis cases associated with horse meat consumption are rarely reported but are probably underestimated (2). In the European Union, France and Italy account for more than two thirds of all horse meat eaten, predominantly raw, thereby increasing the likelihood of infection by various parasites, including Trichinella spp. and Toxoplasma spp (4). Under natural conditions, serologic prevalence of T. gondii parasites in horses worldwide may range from 0% to 80% (5). Many factors could account for this variation, including the sensitivity and specificity of the serologic test, ages of animals, geographic area and hygienic condition of farm management (5). The only prevalence survey of horses slaughtered for food that we are aware of was conducted in Canada and the United States and found antibodies to T. gondii parasites in 124 (6.9%) of 1,788 serum samples (6). T. gondii tissue cysts in meat are immediately killed by reaching an internal temperature of 67°C in all parts of meat during cooking (7). Deep freezing (