Molecular epidemiology of Theileria annulata and identification of 18S rRNA gene and ITS regions sequences variants in apparently healthy buffaloes and cattle in Pakistan

A molecular epidemiological survey was conducted to determine the prevalence of piroplasms in buffaloes and cattle from Sheikhupura and Okara districts of Punjab, Pakistan using reverse line blot (RLB) hybridization assay. The genetic diversity within 18S rRNA gene and ITS regions sequences of various obtained Theileria species (spp.) was also investigated. Briefly, 102 blood samples from buffaloes and cattle in the study districts were collected on blood collection cards and brought to the laboratory. DNA was extracted; the V4 hypervariable region of 18S rRNA was amplified and analyzed using RLB. Out of total samples analyzed, 61 (59.8%) were hybridized with Babesia/Theileria (B/T) genus-specific probe. Only one species of piroplasm was detected in buffaloes and cattle in study districts, i.e. Theileria (T.) annulata. Six samples only hybridized with B/T genus-specific and Theileria genus-specific probes but not with any species-specific probe indicating the presence of novel species or variants. The sequences of 18S rRNA gene and ITS regions of these six samples revealed the presence of T. annulata variants as confirmed through sequence identity estimation and phylogenetic analyses. Meanwhile, an unexpected sequence variation was observed within the 18S rRNA gene and ITS regions sequences of T. annulata identified in the present study. This is the first report on the simultaneous detection of species of piroplasms infecting buffaloes and cattle in Pakistan and molecular characterization of T. annulata 18S rRNA gene and ITS regions. The present study may address the new insights into the epidemiology of theileriosis which will help researches in designing control strategies and developing various molecular diagnostic tools at national level.     

Seroprevalence of Toxoplasma gondii in beef cattle and dairy cattle in northeast China

The seroprevalence of Toxoplasma gondii in beef cattle and dairy cattle in Heilongjiang Province, northeast China, was surveyed between April 2009 and May 2011. A total of 1803 (693 beef cattle and 1110 dairy cattle) serum samples were collected from 10 administrative regions rearing beef cattle and dairy cattle, and antibodies to T. gondii were examined by indirect hemagglutination (IHA) test. The overall seroprevalence of T. gondii in beef cattle and dairy cattle was 2.6% (46/1803), and the prevalence in beef cattle (3.0%) was slightly higher than that in dairy cattle (2.3%). The prevalence of antibodies in adult animals was higher than that in calves, but the differences among the age groups were not significant (p>0.05). The seroprevalence in female (3.4%) and male (2.5%) beef cattle was not statistically significant (p>0.05). Though the prevalence in intensively reared beef cattle and dairy cattle was lower than that in semi-intensively reared animals, the difference was not statistically significant (p>0.05). The results of this survey indicated the presence of T. gondii infection in beef cattle and dairy cattle in Heilongjiang Province, the coldest province in China, which may cause economic losses to the local livestock industry, and may be a source of T. gondii infection for humans in this region.     

Seroepidemiological survey of trypanozoon infection in horses in the suspected dourine-infected Bale highlands of the Oromia region, Ethiopia

This paper presents the results of a seroepidemiological survey of trypanozoon infection in horses carried out between September 2007 and June 2008. The survey was conducted to determine the seroprevalence of anti-trypanozoon antibodies in 880 serum samples collected randomly from selected horse-breeding districts of the Bale highlands of Ethiopia. The seroprevalence of trypanozoon infection was found to be 173 (19.66%) and 140 (15.91%) for the CATT/T. evansi and LATEX/T. evansi tests, respectively. The high seroprevalence of trypanozoon infection strongly indicates that the infection is endemic. Neither test can differentiate between anti-trypanozoon antibodies caused by infection with T. equiperdum (the causative agent of dourine) and those of T. evansi (the causative agent of surra). The findings of the present study suggest that field-applicable screening serological tests such as the CATT/T. evansi and LATEX/T. evansi could be useful for epidemiological studies and the control of trypanozoon infection.     

Detection of Chagas infections using Trypanosoma evansi crude antigen demonstrates high cross-reactions with Trypanosoma cruzi.

Antigenic similarities between salivarian trypanosomes are known for a long time, but similarities between salivarian and stercorarian trypanosomes have been very little investigated. Phylogenetically, these genus and species appear to be far. However, in a preliminary work we had shown strong reactions of chagasic human sera using T. evansi antigens in Western-blotting and ELISA. In the current work an ELISA test using T. evansi crude antigens was probed with one hundred and two sera of chagasic Bolivian patients previously diagnosed which presented different pathologies. The sensitivity of the ELISA T. evansi was 92.6% similar to that of ELISA T. cruzi. The specificity evaluated using 20 sera of patients infected by Leishmania sp. reaches a comparable value of that obtained with the T. cruzi immunofluorescent assay. Finally, the sensitivity and the specificity of the ELISA T. evansi were not really different from conventional serology of Chagas. In spite of their taxonomic position in various sections and their old divergence, these observations prove a strong antigenic community between T. cruzi and T. evansi. Consequently, the common antigens which remain to be characterized, could be an alternative source of antigen for the detection of antibodies against T. cruzi. Given that T. evansi seems to have strong antigenic communities with the majority of the pathogenic current trypanosomoses of mammals, it is very attractive to identify and characterize these highly conserved antigens which could be suitable targets to develop tools for diagnosis, prophylaxy and chemotherapy against several human and animal trypanosomoses.     

Detection of Trypanosoma lewisi from wild rats in Southern China and its genetic diversity based on the ITS1 and ITS2 sequences

Trypanosoma lewisi has widely been considered as a non-pathogenic rat trypanosome. However, more and more cases of humans infected with T. lewisi have been reported around the world, indicating that it can infect humans in some undetermined circumstances. Quick and sensitive diagnosis of infection by T. lewisi is important for both treatment of patients and epidemiological studies of this parasite. In this paper, three methods i.e. wet blood smear (diagnosis by microscopy), PCR and LAMP were used to detect T. lewisi from 238 wild rats (Rattus norvegicus) collected from the field in Huadu, Guangdong province, China. Infection rates of these samples detected by the 3 methods was 6.7% (16/238), 12.6% (30/238), and 18.9% (45/238), respectively. LAMP could detect all samples shown positive by microscopical observation of wet smear and by single PCR indicating good potential for application in the detection of T. lewisi. So far as we know, this is the first report of the LAMP method being used to detect T. lewisi in wild rats. The specific T. lewisi LAMP primers were able to amplify the target fragment from the genomic DNA of 19 T. lewisi strains isolated from Huadu, Guangdong province (n=16), Changchun, Jilin province of China (n=1) and from Thailand (n=2). Based on the analyses of ITS1 (internal transcribed spacer 1) and ITS2 sequences, these 19 strains show a very close genetic relationship with over 96-97% similarity to the other corresponding sequences of T. lewisi published in Genbank. Phylogenetic trees of the species in the subgenus Herpetosoma were constructed, based on the ITS1 and ITS2 sequences, and these results also indicate that they are closely related and in the same clade.     

Microsatellite and minisatellite genotyping of Theileria parva population from southern Africa reveals possible discriminatory allele profiles with parasites from eastern Africa

The control of Theileria parva, a protozoan parasite that threatens almost 50% of the cattle population in Africa, is still a challenge in many affected countries. Theileria parva field parasites from eastern Africa, and parasites comprising the current live T. parva vaccine widely deployed in the same region have been reported to be genotypically diverse. However, similar reports on T. parva parasites from southern Africa are limited, especially in Corridor disease designated areas. Establishing the extent of genetic exchange in T. parva populations is necessary for effective control of the parasite infection.Twelve polymorphic microsatellite and minisatellite loci were targeted for genotypic and population genetics analysis of T. parva parasites from South Africa, Mozambique, Kenya and Uganda using genomic DNA prepared from cattle and buffalo blood samples. The results revealed genotypic similarities among parasites from the two regions of Africa, with possible distinguishing allelic profiles on three loci (MS8, MS19 and MS33) for parasites associated with Corridor disease in South Africa, and East Coast fever in eastern Africa. Individual populations were in linkage equilibrium (V_D<L), but when considered as one combined population, linkage disequilibrium (V_D>L) was observed. Genetic divergence was observed to be more within (AMOVA = 74%) than between (AMOVA = 26%) populations. Principal coordinate analysis showed clustering that separated buffalo-derived from cattle-derived T. parva parasites, although parasites from cattle showed a close genetic relationship. The results also demonstrated geographic sub-structuring of T. parva parasites based on the disease syndromes caused in cattle in the two regions of Africa.These findings provide additional information on the genotypic diversity of T. parva parasites from South Africa, and reveal possible differences based on three loci (MS8, MS19 and MS33) and similarities between buffalo-derived T. parva parasites from southern and eastern Africa.     

Genetic characterization of Trypanosoma evansi isolated from a patient in India.

The first human case of trypanosomiasis caused by Trypanosoma evansi was recently discovered in India. We have focused on the parasite to investigate whether this atypical infection was due to a particular genotype of T. evansi. The SRA gene was not detected by PCR in the Indian human T. evansi (TEVH) DNA sample. TEVH appears to be closely related to Vietnam WH, with identical alleles for TRBPA and MT30-33 AC/TC microsatellites. Furthermore, T. evansi has homogeneous kDNA minicircles and the minicircles of isolate TEVH were shown to be of Type A. Thus, the T. evansi isolated from an Indian patient appears to be a typical T. evansi as far as we can judge, suggesting that the explanation for this unusual infection may lie with the patient.     

High genetic diversity and superinfection by Anaplasma marginale strains in naturally infected Angus beef cattle during a clinical anaplasmosis outbreak in southeastern Brazil

Anaplasma marginale is an obligate intracellular Gram-negative bacterium that is parasitic to erythrocytes and is the main agent of bovine anaplasmosis. This disease causes severe anemia and reduces weight gain and milk production, thus giving rise to major economic losses relating to livestock worldwide. The genetic diversity of this bacterium has been characterized based on sequences of major surface proteins (MSPs), especially MSP1α. This has enabled identification of several geographical strains, according to different amino acid sequences. The aim of this study was to investigate the genetic diversity of A. marginale in naturally infected Angus beef cattle during a disease outbreak in southeastern Brazil. Four blood samples were collected over a four-month period from each of 20 animals on a cattle farm in Itú, São Paulo, Brazil. Serum samples were subjected to indirect ELISA to detect anti-A. marginale IgG antibodies. The 80 whole-blood samples obtained were subjected to DNA extraction, quantitative real-time PCR (qPCR) for the msp1β gene, semi-nested PCR (snPCR) for the msp1α gene, cloning of the target fragment and sequencing using the Sanger method. The sequences obtained were analyzed for genetic diversity using the RepeatAnalyzer software. Both iELISA tests, using recombinant MSP5 and the Anaplasma antibody test kit (VMRD), revealed high seroprevalence: 91.25% and 97.5%, respectively. In qPCR, 100% of the samples were positive, with between 10³ and 10⁷ DNA copies/μL. In the snPCR based on the msp1α gene, 57.5% (46/80) of the samples were positive. Microsatellite analysis on the 36 sequences obtained showed the presence of genotypes H (58.3%), F (25%), E (19.4%), C (2.7%) and G (2.7%). The RepeatAnalyzer software identified 36 strains in the study region, among which some had not previously been described in the literature (13 27 13 27 13 F; 16 FF; τ 27; 63 29 104 29; LJ1 13 LJ1 13; 16 F 17; 16 F 91). High genetic diversity of A. marginale bacteria was found on this farm in Itú, São Paulo.     

Further evidence from SSCP and ITS DNA sequencing support Trypanosoma evansi and Trypanosoma equiperdum as subspecies or even strains of Trypanosoma brucei

The subgenus Trypanozoon includes three species Trypanosoma brucei, Trypanosoma evansi and Trypanosoma equiperdum, which are morphologically identical and indistinguishable even using some molecular methods. In this study, PCR-based single strand conformation polymorphism (PCR-SSCP) was used to analyze the ribosomal DNA of the Trypanozoon species. Data indicate different patterns of ITS2 fragments between T. brucei, T. evansi and T. equiperdum by SSCP. Furthermore, analysis of total ITS sequences within these three members of the subgenus Trypanozoon showed a high degree of homology using phylogenetic analysis but were polyphyletic in haplotype networks. These data provide novel nuclear evidence to further support the notion that T. evansi and T. equiperdum should be subspecies or even strains of T. brucei.     

Characterization of Trypanosoma evansi type B.

A distinctive feature of Trypanosoma evansi is the possession of a kinetoplast that contains homogeneous DNA minicircles, but lacks DNA maxicircles. Two major sequence variants of the minicircle have been described and here we have sequenced the type B variant and designed a specific PCR test to distinguish it from type A. Further a test based on maxicircles to distinguish T. brucei brucei from T. evansi was designed and evaluated. Using the designed PCR tests, we detected three type B isolates from camel blood samples collected in northern Kenya, more than 20 years after the first isolation of type B. Comparison of minicircle sequences from all four type B isolates shows >96% identity within the group, and 50-60% identity to type A minicircles. Phylogenetic analysis based on minicircle sequences reveals two clusters, one comprising isolates of type A and one of type B, while random amplification of polymorphic DNA show slight polymorphic bands within type B. Most T. evansi isolates analysed were heterozygous at a repetitive coding locus (MORF2). All type B isolates had one genotype designated 3/5 based on the alleles present. Three camel isolates, which had homogenous type A minicircles, lacked the RoTat 1.2 gene, while another five isolates were T. b. brucei, based on the heterogeneity of their minicircles and presence of maxicircles as demonstrated by PCR amplification of the gene for cytochrome oxidase subunit 1. Our results confirm the existence of T. evansi type B isolates, T. b. brucei and existence of T. evansi type A without RoTat 1.2 gene in Kenyan isolates.     

Sequence Analysis of the Second Internal Transcribed Spacer (ITS2) Region of rDNA for Species Identification of Trichostrongylus Nematodes Isolated From Domestic Livestock in Iran

Background

Infectivity of herbivores with Trichostrongylus nematodes is widespread in many countries, having a major economic impact on breeding, survivability, and productivity of domestic livestock. This study was carried out on Trichostrongylus species isolated from domestic livestock in order to develop an easy-to-perform method for species identification.

Methods

Trichostrongylus isolates were collected from sheep, goat, cattle, and buffaloes in Khuzestan Province, southwest Iran. Primary species identification was carried out based on morphological characterization of male worms. PCR amplification of ITS2-rDNA region was performed on genomic DNA and the products were sequenced. Phylogenetic analysis of the nucleotide sequence data was conducted employing Bayesian Inference approach. Consequently, a restriction fragment length polymorphism (RFLP) profile was designed to differentiate Trichostrongylus species.

Results

A consensus sequence of 238 nucleotides was deposited in the GenBank for Iranian isolates of Trichostrongylus species including T. colubriformis, T. capricola, T. probolurus and T. vitrinus. The designated RFLP using restriction enzyme TasI could readily differentiate among species having different ITS2 sequence. The molecular analysis was in concordance with morphological findings.

Conclusion

Phylogenetic analysis indicated a close relationship among the sequences obtained in this study and reference sequence of relevant species. ITS2-RFLP with TasI is recommended for molecular differentiation of common Trichostrongylus species.     

Genetic characterization of a diverse Escherichia coli O157:H7 population from a variety of farm environments

Many of the current studies on the genetic diversity of Escherichia coli O157:H7 have focused on pathogenic clinical, veterinary, or food isolates. These studies did not explore the diversity of the larger population in the farm environment. Research on selected farm isolates address this wider diversity but have typically been limited to a specific geographic locale or farm type, thus giving limited insight into the greater diversity across geographic regions and varied environments. The objective of this study was to evaluate a diverse population of E. coli O157:H7 collected from a variety of locations and farm environments. Eighty-eight isolates were collected from four farm types (swine, dairy, beef, and poultry) across the southeastern and western United States. Eighteen farms were sampled every 3 months over a period of 24 months. Isolates were analyzed by ribotyping and pulsed field gel electrophoresis (PFGE). Real-time PCR was used to determine the presence or absence of key pathogenic genes (stx1, stx2, and eae). The data indicate a significant amount of genetic diversity, however, ribotype analysis revealed meaningful clusters within the larger population. These groupings were consistent with PFGE analysis. Most of these isolates were clustered by location (i.e. from the same state or region) or farm type. Of the isolates in these clusters, most did not contain pathogenic genes. Of notable interest is a single group in which the majority of isolates, collected from four of the five states sampled, contained at least one stx gene and the eae gene suggesting the existence of a specific pathogenic cluster. These data suggest that, while there is notable diversity within the broader E. coli O157:H7 population, pathogenic isolates may be limited to a subset of strains within the population.     

Eleventh international meeting on Trypanosoma evansi: report of the Working Group. Paris, 17 May 1990.

Three objectives have been achieved by the Working Group since its creation in 1983: more detailed information (and consequently better awareness) of zones infected by T. evansi; refinement of diagnostic techniques and the development of test kits suitable for field use; development of a new synthetic trypanocide. Exchange of strains between specialist laboratories should be encouraged in order to compare isolates from Africa, Asia and South America by using current techniques of biotechnology, and to open the way to better knowledge of the pathogenicity of T. evansi and to the discovery of effective prophylactic measures. Research reported to the meeting was concerned with the taxonomy and genetics of T. evansi, the cloning and sequencing of nuclear and kinetoplastic DNA (kDNA), chromosomal polymorphism in relation to antigenic variation, the detection of lymphocytic interleukin 2 and its receptors in infected ponies, the use of monoclonal group antibodies to detect T. evansi, and the importance of natural receptivity of the host. A text concerning the diagnosis of surra (T. evansi infection) was drafted and forwarded to the Office International des Epizooties (OIE) for incorporation in the OIE Manual of recommended diagnostic techniques and requirements for biological products. A concise dossier was presented on the pharmacology and pharmacodynamics of the new trypanocide, melarsomine (proprietary name Cymelarsan). The recommended active dosage was 0.25 mg/kg body weight, given as a single intramuscular or subcutaneous injection. Laboratory tests were also reported with ronidazole, demonstrating its trypanocidal activity in rats.     

Type-specific PCR assays for Babesia bovis msa-1 genotypes in Asia: Revisiting the genetic diversity in Sri Lanka,Mongolia, and Vietnam

Babesia bovis is the most virulent Babesia organism, resulting in a high mortality rate in cattle. The genetic diversity of B. bovis merozoite surface antigens (MSAs), such as MSA-1, MSA-2b, and MSA-2c, might be linked to altered immune profiles in the host animals. The present study aimed to develop type-specific PCR assays for Asian msa-1 genotypes, thereby re-analyzing the genetic diversity of msa-1 in Sri Lanka, Mongolia, and Vietnam. Specific primers were designed for nine Asian msa-1 genotypes, which had been detected based on the phylogeny constructed using msa-1 gene sequences retrieved from the GenBank database. Specificity of the type-specific PCR assays was confirmed using plasmids containing the inserts of msa-1 gene fragments that represent Asian genotypes. Furthermore, no amplicons were observed by these PCR assays when DNA samples of Babesia bigemina, Babesia ovata, Theileria annulata, Theileria orientalis, Trypanosoma evansi, Trypanosoma theileri, Anaplasma marginale, and Anaplasma bovis, and non-infected bovine blood were analyzed. In total, 109 B. bovis-positive blood DNA samples sourced from Sri Lanka (44 cattle), Mongolia (26 cattle), and Vietnam (23 cattle and 16 water buffaloes) were then screened by the type-specific PCR assays. The sequences derived from all of the PCR amplicons were phylogenetically analyzed. Out of 109 DNA samples, 23 (20 from cattle and 3 from water buffaloes) were positive for at least one genotype. In agreement with previous studies, five and four different genotypes were detected among the DNA samples from Sri Lanka and Vietnam, respectively. In contrast, four genotypes, including three novel genotypes, were detected from Mongolia. Five DNA samples were found to be co-infected with multiple genotypes. The sequences of the PCR amplicons clustered phylogenetically within the corresponding clades. These findings indicated that the type-specific PCR assays described herein are useful for the determination of genotypic diversity of the B. bovis msa-1 gene in Asia.     

Epidemiology and genetic diversity of Anaplasma marginale in Zamora-Chinchipe,Ecuador

Bovine anaplasmosis, caused by the tick-borne pathogen Anaplasma marginale, is a hemolytic disease that constitutes a major constraint to cattle production in tropical and subtropical regions including Ecuador. However, the epidemiological situation of this hemoparasitosis in Ecuador is poorly characterized. The present study was aimed to determine the prevalence and genetic diversity of A. marginale in cattle of Ecuador. A cross-sectional study was carried out covering several farms from six out nine cantons of the Zamora-Chinchipe province. A total of 185 cattle were randomly selected and blood samples were collected from the animals. The studied group of animals included six breeds, three age groups, and both sexes. The molecular diagnostic was performed based on a nPCR assay targeting the A. marginale msp5 gene. Anaplasma marginale prevalence was 63.8 % and the bacteria were detected in all the cantons studied. Thirteen representative strains were selected and genetically characterized based on the msp1α gene. Genetic diversity analysis revealed that different strains circulate in the bovine herds studied. The results suggest that cattle movement may contribute to the circulation of common strains in the area. The results demonstrate a high prevalence of A. marginale in the region which should be considered by the sanitary authorities. The epidemiological surveillance for this disease should increase to anticipate acute disease outbreaks with high mortality. Bovine anaplasmosis outbreaks can cause economic losses and the death of several animals; therefore, measures for the prevention and control of this disease are required.     

Tenth international meeting on Trypanosoma evansi: report of the working group. Paris, 24 May 1989

Some epidemiological surveys have provided information on the incidence of Trypanosoma evansi infection among dromedaries in Mali, and among buffaloes in Java and Indonesia. The disease among camels has been reported again from Kazakhstan in the USSR, with the coexistence of T. evansi and Cephalopina titillator in animals which developed acute infection. The disease has been studied among horses in Venezuela and among buffaloes in Vietnam and Indonesia, and suspected among horses in Brazil. Diagnostic kits for rapid and reliable detection of T. evansi are being made available free of charge, upon request, by the institutes which have developed these new techniques, namely: detecting the parasite by agglutination-lysis; detecting antibody (by a modification of CATT); detecting antigen (by using monoclonal antibodies). Once these various diagnostic procedures developed by competent institutes have been evaluated and used widely, the next step will be to standardise the techniques and the antigens. Differential diagnosis of T. evansi and T. equiperdum is still difficult in the case of akinetoplastic strains. For improved evaluation of T. evansi isolates, a proposal has been made to form collections of complementary DNA (cDNA) with a view to exchanging these copies and the original strains. The advice of the International Commission on Zoological Nomenclature has been requested for definitive adoption of a binomial designation for the species T. evansi. With more extensive data on the pharmacology and pharmacokinetics of Cymelarsan and laboratory testing of a new trypanocide called "IMOL 881", research on trypanocides continues.     

Characterisation of a monoclonal antibody against Trypanosoma evansi and its application for detecting circulating antibodies

Monoclonal antibodies were obtained against Trypanosoma evansi. The 2-4F6 IgM monoclonal antibody (Mab) was chosen for the study because of its ability to detect antigens and its specificity (as it did not recognise T. cruzi, T. equiperdum, Babesia equi or B. caballi). The immunoblot test revealed that the 2-4F6 IgM Mab recognises epitopes in two antigenic bands, one measuring 85 kDa and the other 122 kDa. An immunoassay for antigen detection in serum using polyclonal antibodies for capture, the Mab 2-4F6 as primary antibody and an antimouse IgM as secondary antibody gave positive results in 10 of the 11 equidae infected with T. evansi, whereas 20 controls gave negative results. These research results show that the Mab 2-4F6 and the antigen it recognises are useful in identifying equidae infected with T. evansi.     

Phylogenetic analyses of typical bovine rotavirus genotypes G6, G10, P[5] and P[11] circulating in Argentinean beef and dairy herds

Group A rotavirus (RVA) is one of the main causes of neonatal calf diarrhea worldwide. RVA strains affecting Argentinean cattle mainly possess combinations of the G6, G10, P[5] and P[11] genotypes. To determine RVA diversity among Argentinean cattle, representative bovine RVA strains detected in diarrheic calves were selected from a survey conducted during 1997–2009. The survey covered the main livestock regions of the country from dairy and beef herds. Different phylogenetic approaches were used to investigate the genetic evolution of RVA strains belonging to the prevalent genotypes. The nucleotide phylogenetic tree showed that all genotypes studied could be divided into several lineages. Argentinean bovine RVA strains were distributed across multiple lineages and most of them were distinct from the lineage containing the vaccine strains. Only the aminoacid phylogenetic tree of G6 RVA strains maintained the same lineages as observed at the nucleotide level, whereas a different clustering pattern was observed for the aminoacid phylogenetic trees of G10, P[5] and P[11] suggesting that the strains are more closely related at the aminoacid level than G6 strains. Association between P[5] and G6(IV), prevalent in beef herd, and between P[11] and G6(III) or G10 (VI and V), prevalent in dairy herds, were found. In addition, Argentinean G6(III), G10, P[5] and P[11] bovine RVA strains grouped together with human strains, highlighting their potential for zoonotic transmission. Phylogenetic studies of RVA circulating in animals raised for consumption and in close contact with humans, such as cattle, contribute to a better understanding of the epidemiology of the RVA infection and evolution.     

Genetic similarity between cysticerci of Taenia solium isolated from human brain and from pigs.

Mitochondrial (mt) cox1 and ribosomal ITS1 DNA sequences from Taenia solium cysticercus isolates from pigs and cysticerci (racemose and cellulose types) from patients with neurocysticercosis were amplified by the polymerase chain reaction (PCR). The amplicons were sequenced in order to determine the genetic relationship between these types of cysticerci. Phylogenetic trees were constructed and evolutionary distances were calculated. ITS1 and mt cox1 cysticerci sequence data were compared with previously published Taenia spp. sequences. The variation in the ITS1 and cox1 sequences of samples collected from Mexico was minimal, regardless of geographical origin, size or colour of cysticerci from either pigs or human brain. These results suggest that the racemose and cellulose types represent genetically identical metacestodes of T. solium. Alignment of the mt cox1 sequences of the Mexican samples with sequences of other Taenia taxa showed that most were very similar to T. solium from Mexico and T. solium from Colombia; one T. solium Mexican isolate and Taenia hydatigena were placed in the same group close to Taenia crassiceps. The ITS1 sequences for the Mexican T. solium samples indicated the majority were in the same group as the Latin American T. solium. Two Mexican T. solium samples and T. solium from Philippines were placed together in a different group.     

Prevalence and genetic profiling of virulence determinants of non-O157 Shiga toxin-producing Escherichia coli isolated from cattle, beef, and humans, Calcutta, India.

We investigated the prevalence of Shiga toxin-producing Escherichia coli (STEC) in hospitalized diarrhea patients in Calcutta, India, as well as in healthy domestic cattle and raw beef samples collected from the city's abattoir. Multiplex polymerase chain reaction using primers specific for stx1 and stx2 detected STEC in 18% of cow stool samples, 50% of raw beef samples, and 1.4% and 0.6% of bloody and watery stool samples, respectively, from hospitalized diarrhea patients. Various virulence genes in the STEC isolates indicated that stx1 allele predominated. Plasmid-borne markers, namely, hlyA, katP, espP, and etpD, were also identified. Bead enzyme-linked immunosorbent assay and Vero cell assay were performed to detect and evaluate the cytotoxic effect of the Shiga toxins produced by the strains. STEC is not an important cause of diarrhea in India; however, its presence in domestic cattle and beef samples suggests that this enteropathogen may become a major public health problem in the future.