Molecular profiles of<Emphasis Type="Italic"> Trypanosoma brucei</Emphasis>,<Emphasis Type="Italic"> T. evansi</Emphasis> and<Emphasis Type="Italic"> T. equiperdum</Emphasis> stocks revealed by the random amplified polymorphic DNA method

A total of 20 random primers (10-mers) were used to amplify RAPD markers from the genomic DNA of four Trypanosoma brucei stocks from East and West Africa, four T. evansi stocks from Africa, Asia and South America and one T. equiperdum stock from Asia. Between 65 and 88 reproducible fragments ranging from 0.25 to 2.15 kb were generated from these stocks depending on the stock/primer combination. The similarity coefficient (SC) among the stocks of T. brucei from Kenya, Nigeria, Tanzania and Zambia ranged from 62.9% to 74.0% (average: 67.6%). The SC among the stocks of T. evansi from Kenya, China and Brazil was 76.4%–95.5% (average: 86.4%), while the SC between T. evansi stock from China and Brazil was 95.5%. For T. evansi and T. equiperdum, the SC among the stocks ranged from 81.2% to 94.4% (average: 87.6%). As for the SC among the stocks of T. brucei and T. evansi, it was found to be from 54.7% to 80.3% (average: 68.0%) and the SC among stocks of T. brucei and T. equiperdum was from 59.4% to 76.9% (average: 68.1%). Our results indicate that the stocks of T. evansi from China and from Brazil are more closely related to the stock of T. equiperdum from China than to the stocks of T. evansi isolated from Kenya and to the stocks of T. brucei. In addition, our results further support the hypothesis that T. evansi stocks from China and Brazil could have arisen from a single lineage. The possible evolution of T. evansi and T. equiperdum is also discussed.     

Persistent Colonization of Nine Cystic Fibrosis Patients with an<Emphasis Type="Italic"> Achromobacter</Emphasis> (<Emphasis Type="Italic">Alcaligenes</Emphasis>)<Emphasis Type="Italic"> xylosoxidans</Emphasis> Clone

The present study was conducted to investigate the increasing incidence of Achromobacter (previously Alcaligenes) xylosoxidans isolates being recovered from sputum samples of cystic fibrosis patients at a cystic fibrosis department for adults in Athens, Greece. During the 1-year study period, a total of 34 isolates were detected persistently in 9 of 71 cystic fibrosis patients. The isolates exhibited resistance to multiple antimicrobial agents. Isolates that were recovered repeatedly from each patient exhibited identical macrorestriction profiles with pulsed-field gel electrophoresis, indicating that the same strain persisted in the lungs of these patients. Isolates from five of the patients were genetically related, suggesting a common-source outbreak of Achromobacter xylosoxidans colonization or infection.     

Host specificity ofGiardia muris isolates from mouse and golden hamster

One isolate ofGiardia muris from a naturally infected laboratory mouse (Mus musculus) and one from a naturally infected golden hamster (Mesocricetus auratus) were passaged three times by the inoculation of ten cysts (the minimal infectious dose) into barrier-maintained homologous hosts. Both of the resultant isolates were tested for infectivity by intragastric inoculation of 3–5×10⁵ cysts into 40 mice (2 inbred strains), 40 rats (2 inbred strains), and 19 golden hamsters (1 outbred strain). Rats were not susceptible to infection with either isolate. Mice and golden hamsters did develop infections following their inoculation with the heterologous isolates. The mean intensity of heterologous infections with the hamster isolates was significantly lower than that of homologous infections. The mouse isolate induced a higher mean intensity of infection in hamsters as compared with homologous recipients. The mean intensity of infections induced by both isolates was greater in male hamsters than in females.     

Towards a New Reference Test for Surra in Camels

Current serological diagnosis of Trypanosoma evansi infection in camels is based on the native variable antigen type RoTat 1.2. The goal of this study was to develop a novel serological diagnostic test based on a nonvariable protein and freed from the use of rats or mice for its production. An enzyme-linked immunosorbent assay using a recombinant extracellular domain of invariant surface glycoprotein 75 (ELISA/rISG75) was developed and tested on a collection of 184 camel sera. The results were compared to those obtained from three established antibody detection tests based on variable surface glycoprotein RoTat 1.2: an ELISA for T. evansi (ELISA/T. evansi), a card agglutination test for trypanosomiasis (CATT/T. evansi), and an immune trypanolysis (TL) assay. The ELISA/rISG75 and the ELISA/T. evansi showed a sensitivity of 94.6% (95% confidence interval [CI], 87.8 to 98.2%, at 19% positivity cutoff value) and 98.9% (95% CI, 94.1 to 99.8, at 12% positivity cutoff value), respectively. The ELISA/rISG75 had 100% specificity (CI, 95.9 to 100%), while the ELISA/T. evansi showed 98.9% specificity (CI, 95.9 to 100%). The ELISA/rISG75 demonstrated an almost perfect agreement with the TL assay, the CATT/T. evansi, and the ELISA/T. evansi, with kappa scores of at least 0.94. The ELISA/rISG75, having a performance comparable to that of the gold standard (the TL assay) and being independent of antigenic variation, may become a new reference test for surra in camels. It opens avenues for the diagnosis of T. evansi infections in other hosts as well as for the development of a pan-Trypanozoon test for detection of Trypanosoma brucei brucei, T. b. gambiense, T. b. rhodesiense, T. evansi, and T. equiperdum.Trypanosoma evansi is the causative agent of surra in domestic animals such as camels, equines, cattle, buffaloes, small ruminants, and dogs. Wild animals such as capybaras can act as reservoir hosts. Although T. evansi is noninfective for healthy humans, a case of human infection by T. evansi in India in 2004 has been reported; however, this infection was due to a genetic mutation in the host''s APOL1 gene (20). Camels and horses are very sensitive to T. evansi, and death can occur within 3 months without treatment. Cattle and other ruminants infected by T. evansi suffer from immunosuppression, resulting in increased susceptibility to other diseases or vaccination failure (5, 17). T. evansi is mechanically transmitted by bloodsucking flies, such as Tabanidae and Stomoxys species. The disease occurs in Africa, Asia, and South and Central America and causes important economic losses (16).Control of surra still relies mainly on the observation of clinical signs and subsequent treatment of sick animals, which is inefficient and results in high morbidity and mortality of undiagnosed animals that in the meantime act as reservoirs. Definitive diagnosis of T. evansi infection is achieved by microscopic demonstration of the parasite, which method, however, suffers from limited sensitivity. The most sensitive parasite detection test is the mini-anion-exchange centrifugation method, but it is seldom used (4, 9). Indirect diagnosis is possible through detection of specific antibodies in the mammalian hosts. All currently available antibody detection tests, including an immune trypanolysis (TL) assay (21), an enzyme-linked immunosorbent assay for T. evansi (ELISA/T. evansi) (22), a card agglutination test for trypanosomiasis for T. evansi (CATT/T. evansi) (1), and a latex agglutination test for T. evansi (24), are based on the native variant surface glycoprotein (VSG) of the predominant variable antigen type (VAT) RoTat 1.2 of T. evansi. Production of these tests requires the mass culture of T. evansi homogeneously expressing RoTat 1.2 VSG in rats or mice. In addition, the results of all these serological tests may remain negative in animals infected with T. evansi type B in Kenya, as this type has been reported not to express the RoTat 1.2 VAT (13-15).On the cell surface of a bloodstream form trypanosome, among the VSGs and nonvariable surface proteins, there are an estimated 5 × 10⁴ invariant surface glycoprotein (ISG75) molecules (28). The ISG75 gene family is present and transcribed in the bloodstream form of all species and subspecies of the Trypanozoon subgenus, including Trypanosoma brucei brucei, T. b. gambiense, T. b. rhodesiense, T. evansi, and T. equiperdum. This multicopy gene family consists of two main groups that share at least 75% similarity among their cDNA and genomic DNA sequences (19). Within each group, there is at least 92% similarity among the cDNA sequences. A putative ISG75, regardless of its belonging to group I or group II, has a conserved topology: a large N-terminal extracellular domain, a single α-helix transmembrane domain, and a small cytoplasmic domain at the C terminus (19, 27). Since ISG75 is not subject to antigenic variation, its corresponding antibodies circulate during the course of infections in mice (29), and its extracellular domain can be produced in Escherichia coli by a standardized protocol (18), it is considered a highly relevant antigen for diagnosis of Trypanozoon infections.The purpose of this study was to develop an antibody detection ELISA for T. evansi infection in camels by using recombinant ISG75 (rISG75). This ELISA/rISG75 was tested against a panel of 184 camel sera in parallel with the currently used serological diagnostic tests, including the TL assay, the CATT/T. evansi, and the ELISA/T. evansi.     

Vertical transmission of<Emphasis Type="Italic"> Toxocara cati</Emphasis> Schrank 1788 (Anisakidae) in the cat

In eight cats and their offspring the mode of transmission of Toxocara cati following natural and experimental infection was investigated in three experiments. In experiments 1 and 2 the kittens of four cats with a chronic natural infection and of four cats with an acute experimental infection, respectively, were examined. In experiment 3 two queens of experiment 2 were mated again to examine whether in the adult cat dormant larvae exist in the tissue, that can be reactivated during pregnancy or lactation to infect the offspring. Additionally, the muscle tissue and organs of two adult cats, one with chronic one with acute infection, were examined for hypobiotic larvae. Pre-natal infections with T. cati did not occur in experiments 1 or 2. In none of the kittens that were examined directly after birth were larvae found. In the offspring of experiment 1 one single larva of T. cati was found 28 days post-partum. Whereas in the kittens of experiment 2 up to 333 larvae were found in one animal. Lactogenic transmission of larvae occurs after acute infection of the queen during late pregnancy but not during chronic natural infection. There is no evidence for the existence of arrested somatic larvae in the adult cat as an important host-finding strategy in the life cycle of T. cati. Following milk-borne infections, the majority of larvae seem to undergo direct development in the intestine without tracheal migration. Only a small number of larvae was found in other organs.     

Characterization of coagulase-negative Staphylococci causing nosocomial infections in preterm infants

The species spectrum, antibiotic susceptibility, and genomic profile of coagulasenegative staphylococci (CNS) isolated from infected preterm infants were compared with those obtained in CNS from nursery personnel.Staphylococcus epidermidis was the predominant species in the 66 investigated preterm infants (171 isolates), accounting for 64 % of all isolates. A high proportion ofStaphylococcus haemolyticus (32 %) could be detected. In contrast to the results in patients, the spectrum in nursery personnel was broad and included more species of CNS. All isolates of CNS from preterm infants demonstrated a low rate of susceptibility to the -lactam antibiotics (2 % sensitivity to penicillin and 6 % sensitivity to oxacillin). Sensitivity to gentamicin (9 %) was also rare. An unexpected observation was susceptibility to teicoplanin in only 70 % of all CNS isolated from patients due to the high proportion ofStaphylococcus haemolyticus. Analysis of the genomic profile of 33 isolates ofStaphylococcus haemolyticus by pulsed-field gel electrophoresis revealed a relationship between the strains. An outbreak of one particular strain ofStaphylococcus haemolyticus in the neonatal intensive care unit investigated can therefore not be excluded.     

The typing of Trypanosoma evansi isolates using mobile genetic element (MGE) PCR

The mobile genetic element PCR (MGE-PCR) is a simple and sensitive technique that can be used to detect genetic variability in Trypanosoma brucei ssp. To investigate the reliability of MGE-PCR in genotyping Trypanosoma evansi, stocks that were isolated directly from camels and after their respective passage in mice were analyzed. Construction of a dendrogram using the MGE-PCR banding profiles revealed a clear distinction between T. evansi and T. brucei, as well as discriminating the T. evansi strains (T. evansi with minicircle types B and A). A minor host-dependent clustering shows a genetic difference of <15%. Changes in the banding profiles were observed after serial passage of T. evansi type B in mice, while those of T. evansi type A were identical. It is apparent that significant random insertion mobile element positional variation occurs when T. evansi isolates are introduced into a new host, a factor that needs to be considered when MGE-PCR is used to determine genetic variation in T. evansi isolates that have different host origins.     

The efficacy of inhibitors involved in spermidine metabolism in<Emphasis Type="Italic"> Plasmodium falciparum</Emphasis>,<Emphasis Type="Italic"> Anopheles stephensi</Emphasis> and<Emphasis Type="Italic"> Trypanosoma evansi</Emphasis>

In the present study, we have tested the effect of different polyamine inhibitors of the spermidine metabolizing enzymes deoxyhypusine synthase and homospermidine synthase in different chloroquine resistant Plasmodium falciparum strains, in the mosquito Anopheles stephensi (Diptera: Culicidae) and in a Trypanosoma evansi clone I from strain STIB 806 K China. Recent experiments have shown that agmatine is a growth inhibitor of the malaria parasite P. falciparum (Kaiser et al. 2001) in vitro. A comparison of agmatine efficacy with the new antimalarials artemisinin, triclosan and conventional chloroquine showed similar or even better results on the basis of growth inhibition and the reduction of developmental forms. However, no effect of triclosan or agmatine was observed at the ribonucleic acid level. In a second set of experiments, we tested the effect of 1,7-diaminoheptane and agmatine on oocyst formation in A. stephensi after infection with Plasmodium yoelii. Agmatine had an antisporozoite effect since 1,000 M led to a 59.5% inhibition of oocysts. A much weaker inhibitor of oocyst formation was 1,7-diaminoheptane. The most effective in in vitro inhibition of T. evansi was dicyclohexylamine, an inhibitor of spermidine biosynthesis with an IC₅₀ value of 47.44 M and the deoxyhypusine inhibitor 1,7-diaminoheptane with an IC₅₀ value of 47.80 M. However, both drugs were ineffective in in vivo experiments in a Trypanosoma mouse model. Two different spermidine analogues, 1,8-diaminooctane and 1,3-diaminopropane with IC₅₀ values of 171 M and 181.37 M, respectively, were moderate inhibitors in vitro and ineffective in vivo.     

Identification of highly conserved loci by genome painting

Fluorescencein situ hybridization was used to identify patterns of DNA similarity among the genomes of several rodent taxa. Total genomic or Cot-1 DNAs were used as hybridization probes against metaphase preparations across different taxonomic levels, including three species ofMicrotus (suborder Sciurognathi),Mus musculus (suborder Sciurognathi) andCtenomys steinbachi (suborder Hystricognathi). The hybridization patterns ofMus orPeromyscus (sciurognath) DNA toMus metaphases, which were consistent with what is known of the satellite sequences in these species, demonstrated the efficacy of this approach for molecular cytogenetics and evolutionary biology. Additional hybridizations to chromosomes ofCtenomys orMicrotus identified loci consisting of highly conserved DNA sequences. This approach has proved useful in investigating genome homologies across divergent rodent lineages. Chromosome microdissection can be used to characterize these regions further.     

Molecular characterization of Trypanosoma evansi isolates from water buffaloes (Bubalus bubalis) in the Philippines

Trypanosoma evansi infection in the Philippines is frequently reported to affect the country’s livestock, particularly, the buffaloes. To assess the prevalence and intraspecific diversity of T. evansi in the country, blood samples from water buffaloes in different geographical regions were collected during an outbreak. T. evansi was detected in all 79 animals tested using PCR targeting the RoTat 1.2 VSG gene. Sequencing of the rDNA complete internal transcribed spacer (ITS) region including the 5.8S subunit showed high similarity (99–100%) between Philippine isolates and known T. evansi isolates in Genbank. Tree construction based on the same region confirmed the close relationship between Philippine and reported Thai isolates as compared to Egyptian isolates separated by relatively small genetic distances, 47 polymorphisms, despite the clustering in four branches. Overall, the results of this study prove genetic diversity within T. evansi species despite previous reports on limited heterogeneity among isolates worldwide.     

Postpartum pathology in Yankasa ewes experimentally infected with <Emphasis Type="Italic">Trypanosoma evansi</Emphasis> during pregnancy

The study was designed to determine the pathological changes in postpartum Yankasa ewes experimentally infected with Trypanosoma evansi (T. evansi) during pregnancy. Thirteen pregnant Yankasa ewes were divided into two groups comprising six ewes in group A and seven ewes in group B. Ewes in group A served as the uninfected control while ewes in group B were infected with approximately 1.0?×?10⁶ of T. evansi per ewe through the jugular vein at the second trimester of pregnancy. They were closely monitored for trypanosomosis from the time of infection up till parturition. One ewe from each group was humanely sacrificed 10 days postpartum and examined for pathological changes. At postmortem, no gross lesion was seen in the infected and control ewes and their placenta. Histologically, there were no changes on the placenta, reproductive tract (ovary, oviduct, uterus, cervix), and viscera organs (liver, lungs, heart, kidney, and spleen) of both infected and control ewes. However, the infected ewe had histopathological changes in the hypothalamus characterized by neuronal degeneration and microglial infiltration, while in the pituitary gland, there was mononuclear cell infiltration. The thyroid gland was infiltrated by inflammatory macrophages and lost its glandular secretions. All these histopathological changes were absent in the uninfected ewe, suggesting that T. evansi is capable of disrupting the endocrine function of reproduction in Yankasa ewes.     

Interferon-gamma levels during the course ofTrypanosoma cruzi infection ofCalomys callosus (Rodentia-Cricetidae) and Swiss mice

Serum levels of interferon-gamma (IFN-) were evaluated inCalomys callosus, and Swiss mice during the course of infection by four strains ofTrypanosoma cruzi. All strains stimulated the production of this interleukine; however, the timing of its onset and permanence When chronically infected animals with no detectable serum IFN- were challenged with the homologous strain, they produced quantities comparable with those obtained during the acute phase of infection. InC. callosus there was a correlation between H₂O₂ liberation by peritoneal macrophages and serum IFN- levels, whereas no such correlation was found in mice.C. callosus had a higher capacity to heal histopathological lesions, whereas lesions in mice were progressive. The results obtained suggest thatC. callosus develops welladapted immune mechanisms that may be important for its role as a reservoir ofT. cruzi.     

Chromatin preferences of the perichromosomal layer constituent pKi-67

The proliferation-associated nuclear protein pKi-67 relocates from the nucleolus to the chromosome surface during the G2/M transition of the cell cycle and contributes to the formation of the perichromosomal layer. We investigated the in-vivo binding preferences of pKi-67 for various chromatin blocks of the mitotic chromosomes from the human and two mouse species, Mus musculus and M. caroli. All chromosomes were decorated with pKi-67 but displayed a gap of pKi-67 decoration in the centromere and NOR regions. pKi-67 distribution in a rearranged mouse chromosome showed that the formation of the centromeric gap was controlled by the specific chromatin in that region. While most chromatin served as a substrate for direct or indirect binding of pKi-67, we identified three types of chromatin that bound less or no pKi-67. These were: (1) the centromeric heterochromatin defined by the alpha satellite DNA in the human, by the mouse minor satellite in M. musculus and the 60- and 79-bp satellites in M. caroli; (2) the pericentromeric heterochromatin in M. musculus defined by the mouse major satellite, and (3) NORs in the human and in M. musculus defined by rDNA repeats. In contrast, the conspicuous blocks of pericentromeric heterochromatin in human chromosomes 1, 9 and 16 containing the 5-bp satellite showed intense pKi-67 decoration. The centromeric gap may have a biological significance for the proper attachment of the chromosomes to the mitotic spindle. In this context, our results suggest a new role for centromeric heterochromatin: the control of the centromeric gap in the perichromosomal layer.     

Intraspecific variation ofTaenia taeniaeformis as determined by various criteria

The intraspecific variation of four laboratory-reared isolates ofTaenia taeniaformis the SRN and KRN isolates from Norwa rats,Rattus norvegicus, captured in Japan and malaysia, respectively; the BMM isolate from a house mouse,Mus musculus, captured in Belgium; and the ACR isolate from a gray red-backed vole,Clethrionomys rufocanus bedfordiae, captured in Japan was examined by various criteria. Eggs of each of the four isolates were orally inoculated into several species of intermediate host. They were most infective to the rodent species from which the original metacestode of each isolate had been isolated in the field, and only the ACR isolate was infective to the gray red-backed vole. Although little difference was found between the SRN, KRN, and BMM isolates by the other criteria, including the morphology of rostellar hooks, the protein composition of the metacestode, and restriction endonuclease analysis of DNA, the ACR isolate was clearly different from the others. It was considered that the ACR isolate was independent as a strain distinct from the other three isolates.     

Repeated epidemics caused by extended-spectrum beta-lactamase-producingSerratia marcescens strains

An outbreak ofSerratia marcescens involving 42 patients admitted to the general intensive care unit of the Hospital of Varese, Italy, occurred from March 1994 to August 1995. The causative strains were resistant to oxyimino-cephalosporins and monobactams due to their production of an extended-spectrum-lactamase. Another outbreak caused bySerratia marcescens strains had occurred in the same unit a few months earlier, from February to October 1993, with the strains involved producing a novel TEM-derived extended-spectrum-lactamase. In order to verify whether there were any relationships between isolates from the two epidemics, the strains and their enzymes were characterized. Biochemical data and gene amplification experiments showed that the isolates of the second outbreak harbored a non-conjugative plasmid of approximately 48 kb, codifying for the production of an SHV-derived extended-spectrum-lactamase with pI 8.2. Restriction fragment length polymorphism analysis of total genomic DNA by pulsed-field gel electrophoresis ofSerratia marcescens isolates unambiguously identified two different bacterial clones responsible for the two epidemics. Epidemiological and microbiological investigations demonstrated the long persistence ofSerratia marcescens strains and their circulation in other hospital wards, thus suggesting their possible role as a long-term reservoir for further epidemic spread.     

Tandem repeat protein as potential diagnostic antigen for <Emphasis Type="Italic">Trypanosoma evansi</Emphasis> infection

Trypanosoma evansi infection (surra) causes significant losses in livestock production in tropical and sub-tropical areas. The current ELISA recommended by OIE for diagnosis of the disease is based on trypanosome lysate antigen. However, antigenic variation and unstable nature of cell lysate antigen make it difficult to standardize the assay. Thus, there are needs to develop recombinant antigen-based ELISA that improve stability, sensitivity, and specificity of the test. Since tandem repeat (TR) proteins of trypanosomatid parasites generally possess high antigenicity, they have been considered to be the promising antigens for trypanosomosis and leishmaniosis. In this study, IgG responses against 14 recombinant TR proteins of trypanosomes were examined by ELISA. Serum samples were obtained from three water buffaloes experimentally infected with T. evansi. Since Trypanosoma congolense GM6 (TcoGM6) elicited highest IgG responses to all water buffaloes, we further bioinformatically and molecular biologically identified Trypanosoma brucei brucei GM6 (TbbGM6) and T. evansi GM6 (TeGM6) TR genes, respectively. As expected, predicted amino acid sequences of TbbGM6 and TeGM6 were identical while the nucleic acid sequence homology between TbbGM6 and TcoGM6 was 63.8%. All buffaloes became clearly positive in recombinant TbbGM6 (rTbbGM6)-based ELISA at 48 days post-infection, suggesting that rTbbGM6 is usable as a serodiagnostic antigen for chronic T. evansi infection.     

The identification of free-living environmental isolates of amoebae from Bulgaria

A survey was carried out in Bulgaria to determine the presence of free-living amoebae (FLA) from environmental sources. In 171 (61.1%) of 280 samples, isolates of Acanthamoeba with group II or III morphology, as well as Hartmannella spp. were recovered. Five isolates named 6 (artificial lake), Ep (lake), G2 (soil), R4* (river) and PK (spring water)—all exhibiting a highly efficient proliferation in axenic cultures—were subsequently cloned and subjected to molecular analyses for identification and genotyping In accordance with morphological findings, PCR-based analyses identified four isolates (6, Ep, G2, R4*) belonging to the genus Acanthamoeba. Confirmation of these findings was obtained by phylogenetic analysis using partial sequencing of the 18S rDNA (ASA.S1) Acanthamoeba-gene. Comparison of these sequences with corresponding regions from other Acanthamoeba strains available from GenBank sorted all four isolates into the sequence type group T4 that contains most of the pathogenic Acanthamoeba strains already identified. The fifth isolate (PK) exhibited morphological characteristics matching those of Hartmannella, and scored negative in the Naegleria fowleri and Acanthamoeba PCRs.     

Phylogenetic analysis of<Emphasis Type="Italic"> Theileria</Emphasis> species transmitted by<Emphasis Type="Italic"> Haemaphysalis qinghaiensis</Emphasis>

The phylogenetic relationships between six isolates of Theileria spp. infective to small ruminants, and two isolates of Theileria spp. infective to yak, all transmitted by Haemaphysalis qinghaiensis, together with the Theileria orientalis/sergenti/buffeli group and T. sinensis, were analyzed using the 18S ssrRNA gene sequence. The target DNA segment was amplified by polymerase chain reaction (PCR). The PCR product was used either for direct sequencing or was ligated to the PCR II vector for sequencing. The length of the 18S ssrRNA gene of all Theileria spp. involved in this study was around 1,740 bp. Two phylogenetic trees were inferred based on the 18S ssrRNA gene sequence of the Chinese isolates only, and Chinese isolates and other species of Theileria available in GenBank. In the first tree, the Theileria sp. infective to yaks was found to be T. sinensis. The Theileria sp. infective to small ruminants was found to be composed of two separate species of Theileria. Theileria sp. from Qinghai, Madang, Ningxian and Lintan, which was identical to the unidentified Theileria sp. described previously, is designated Theileria sp (China 1). The Theileria sp. from Longde, Zhangjiachuan and Lintan, which has not been described previously, is designated Theileria sp. (China 2) in order to avoid confusion. In the second tree, Theileria sp. (China 1) was closely related to benign Theileria, such as T. buffeli and T. sergenti, while Theileria sp. (China 2) was separated from other Theileria spp. The results indicate that H. qinghaiensis transmit at least three species of Theileria, two which are infective to sheep and goats, but not yak and one which is infective to yaks and cattle, but not to sheep and goats.     

Variable number tandem repeat loci providing discrimination within widespread genotypes of <Emphasis Type="Italic">Acinetobacter baumannii</Emphasis>

Some genotypes of Acinetobacter baumannii, defined by pulsed-field gel electrophoresis (PFGE), have been found in many hospitals. Our aim was to find variable number tandem repeat (VNTR) loci capable of providing discrimination among isolates with highly similar or identical PFGE profiles, to gain insights into the epidemiology. Thirteen loci identified in A. baumannii ATCC 17978 were tested using a panel of isolates that included multiple representatives of genotypes belonging to the three European clonal lineages. Two loci, with repeat units of 9 and 6 bp respectively were selected. Repeat numbers varied between 3 and 29, and 9 and 26 respectively at the two loci. The repeat numbers of representatives of each genotype often differed between hospitals, providing a means of tracking patient transfers and possible transmissions between patients. The results suggest that this analysis accurately reflects the known epidemiological information, and provides a valuable tool for cross-infection studies.     

Detection of antibody for the serodiagnosis of hantavirus infection in different rodent species

Summary Peroxidase-labeled staphylococcal protein A, streptococcal protein G, and antibodies directed against Mus musculus (mouse), Rattus norvegicus (rat), Mesocretus auratus (hamster), and Peromyscus leucopus were examined for their reactivity with immunoglobulin G (IgG) from various rodent species. The purpose of this study was to identify the optimal secondary antibodies or reagents for specific serodiagnosis of hantavirus infection in various rodent species. Using ELISA, a total of 65 sera from 29 rodent species of the family Muridae and one serum sample from family Octodontidae were compared for IgG reactivity with the six different reagents. The results demonstrate that the reactivities of the secondary antibodies and reagents to the sera varied, even among sera from rodents of the same genus. Hantavirus-specific antibody ELISA revealed that hantavirus-infected rodent sera obtained from M. musculus, R. norvegicus, Apodemus agrarius, A. peninsulae, and Bandicota indica bound to the six different conjugates in a similar pattern as that detected in IgG ELISA. These results indicate that the applicability of secondary antibodies and protein A and G should be carefully evaluated before use for serodiagnosis in different rodent species.Received January 23, 2003; accepted May 26, 2003