Municipal wastewater treatment plants as removal systems and environmental sources of human-virulent microsporidian spores

Municipal wastewater treatment plants play a vital role in reducing the microbial load of sewage before the end-products are discharged to surface waters (final effluent) or local environments (biosolids). This study was to investigate the presence of human-virulent microsporidian spores (Enterocytozoon bieneusi, Encephalitozoon intestinalis, and Encephalitozoon hellem) and enterococci during treatment processes at four Irish municipal secondary wastewater treatment plants (plants A–D). Microsporidian abundance was significantly related to seasonal increase in water temperature. Plant A had the least efficient removal of E. intestinalis spores (32%) in wastewater, with almost 100% removal at other plants both in April and July. Some negative removal efficiencies were obtained for E. bieneusi (at plants C and D, −100%) and for E. hellem (at plants A and D, −90% and −50%). In addition, a positive correlation was found between the levels of enterococci and E. bieneusi in July (r _s = 0.72, P < 0.05). In terms of the dewatered biosolids, a median concentration as high as 32,000 spores/Kg of E. hellem was observed at plant D in July. Plant C sewage sludge contained the lowest microsporidian loadings (E. bieneusi; 450 spores/L and 1,000 spores/L in April and July, respectively). This study highlights the seasonal variation in concentrations of microsporidian spores in the incoming sewage. Spores in final effluents and dewatered biosolids can be the source of human-virulent microsporidian contamination to the local environment. This emphasizes a considerably high public health risk when sewage-derived biosolids are spread during summer months. This study also suggested enterococci as a potential indicator of the presence of microsporidian spores in wastewater, especially for E. bieneusi.     

Relationships among bather density, levels of human waterborne pathogens, and fecal coliform counts in marine recreational beach water

During summer months, samples of marine beach water were tested weekly for human waterborne pathogens in association with high and low bather numbers during weekends and weekdays, respectively. The numbers of bathers on weekends were significantly higher than on weekdays (P < 0.001), and this was associated with a significant (P < 0.04) increase in water turbidity. The proportion of water samples containing Cryptosporidium parvum, Giardia duodenalis, and Enterocytozoon bieneusi was significantly higher (P < 0.03) on weekends than on weekdays, and significantly (P < 0.01) correlated with enterococci counts. The concentration of all three waterborne pathogens was significantly correlated with bather density (P < 0.01). The study demonstrated that: (a) human pathogens were present in beach water on days deemed acceptable for bathing according to fecal bacterial standards; (b) enterococci count was a good indicator for the presence of Cryptosporidium, Giardia, and microsporidian spores in recreational marine beach water; (c) water should be tested for enterococci during times when bather numbers are high; (d) re-suspension of bottom sediments by bathers caused elevated levels of enterococci and waterborne parasites, thus bathers themselves can create a non-point source for water contamination; and (e) exposure to recreational bathing waters can play a role in epidemiology of microsporidiosis. In order to protect public health, it is recommended to: (a) prevent diapered children from entering beach water; (b) introduce bather number limits to recreational areas; (c) advise people with gastroenteritis to avoid bathing; and (d) use showers prior to and after bathing.     

Prevalence and molecular characteristics of <Emphasis Type="Italic">Enterocytozoon bieneusi</Emphasis> in cattle in Korea

Enterocytozoon bieneusi is the most common microsporidium associated with AIDS patients. Moreover, its detection in increasing numbers of immunocompetent patients has made it an emerging pathogen. This organism was also identified in a wide range of animals, and the zoonotic potential of human infections is of particular interest. In this study, 538 fecal samples from cattle in Korea were analyzed for the presence of E. bieneusi by PCR. Approximately 15% were found to be positive, with higher rates being detected over the summer months. The internal transcribed spacer (ITS) regions of the rRNA gene of ten E. bieneusi positive samples were amplified using nested PCR and sequenced. Genetic polymorphisms, which were represented by six distinct genotypes (CEbA–CEbF), were found among the E. bieneusi isolates. Five isolates from this study had identical ribosomal ITS to the previously known E. bieneusi genotype ITSs in cattle and other animals. Four isolates were previously unreported but were quite similar to the previously known genotypes of E. bieneusi from cattle and other animals. One isolate was identical to the human E. bieneusi type D, which indicated some E. bieneusi isolates from cattle in the country may be of public health importance. To the best of my knowledge, this is the first report of E. bieneusi study in cattle in Asia.     

New record of <Emphasis Type="Italic">Afrimenopon waar</Emphasis> (Eichler) (Phthiraptera: Menoponidae) from budgerigar <Emphasis Type="Italic">Melopsittacus undulatus</Emphasis> (Psittaciformes: Psittacidae) from Karachi,Pakistan

Human microsporidiosis, a serious disease of immunocompetent and immunosuppressed people, can be due to zoonotic transmission of microsporidian spores. A survey utilizing chromotrope 2R stain and fluorescent in situ hybridization techniques for testing feces from 193 captive mammals demonstrated that 3 animals (1.6%) shed Encephalitozoon bieneusi spores. These include two critically endangered species (i.e., black lemurs, Eulemur macaco flavifrons; and Visayan warty pig, Sus cebifrons negrinus) and a threatened species (mongoose lemur, Eulemur mongoz). The concentration of spores varied from 2.7 × 10⁵ to 5.7 × 10⁵/g of feces, and all infections were asymptomatic. The study demonstrates that E. bieneusi spores can originate from captive animals, which is of particular epidemiologic importance because the close containment of zoological gardens can facilitate pathogen spread to other animals and also to people such as zoo personnel and visitors.     

Strongyloides stercoralis: identification of antigens in natural human infections from endemic areas of the United States

Using Western-blot analysis, we identified eight immunodominant antigens (apparent molecular weights 96, 86, 75, 56, 41, 32, 28, and 26 kDa) of Strongyloides stercoralis in natural human infections. For this study, 78 individual serum samples were obtained from S.␣stercoralis-infected patients residing in endemic areas of the United States. Poly A⁺ RNA was translated in vitro in the rabbit-reticulocyte lysate system. The newly synthesized translation products were immuno-precipitated with S. stercoralis human infection sera. All eight of the identified antigens were detected in the immunoprecipitates. The potential of these antigens as targets for immunodiagnosis is also discussed. Received: 28 January 1997 / Accepted: 25 April 1997     

Bacterial diversity of periodontal and implant-related sites detected by the DNA Checkerboard method

The aim of this study was to compare the microbial composition of the subgingival biofilm from teeth and implant sulci in relation to contents originating from internal parts of the implant, abutment and implant prosthesis. Twenty subgingival biofilm samples from the mesial and distal aspects of each tooth/implant and 29 samples from the internal parts of titanium implants, abutments and implant prostheses were evaluated for the presence of 18 bacterial species using DNA Checkerboard and the differences between samples from teeth and implants were assessed with Pearson’s correlation analysis. The periodontal and peri-implantar sulci presented significantly higher bacterial counts than the implant-related sites (p < 0.05 and p < 0.01, respectively). The highest counts were observed for Capnocytophaga gingivalis, Prevotella intermedia, P. nigrescens and P. micra. The correlation between the counts in the periodontal and peri-implantar sulci was r = 0.66 (p < 0.001). Weaker correlations between samples from the internal parts of the implants and periodontal sulcus (r = 0.49; p < 0.001) or peri-implant sulcus (r = 0.42; p < 0.001) were found. All 18 bacterial species were detected to be colonising the subgingival sulcus of teeth and implants, and implant components in the evaluated patients. Significant correlations between the microbiota were found, the strongest being between the periodontal and peri-implantar sulci.     

<Emphasis Type="Italic">Streptococcus pneumoniae</Emphasis> skin and soft tissue infections: characterization of causative strains and clinical illness

Streptococcus pneumoniae is an uncommon cause of skin and soft tissue infections, yet the incidence and clinical significance of its isolation in samples of skin or soft tissues in unselected hospital samples is poorly understood. In the present study, a review was conducted of the records of all patients with skin and soft tissue infections due to S. pneumoniae at a university hospital between January 1994 and December 2005. The isolates were identified by standard methods and were serotyped, and susceptibility testing was performed by the broth microdilution method following the guidelines of the Clinical and Laboratory Standards Institute. During the study period, 3,201 isolates of S. pneumoniae were recovered from several sources. Of these, 69 (2.2%) were from skin and soft tissue samples (69 patients). Complete information could not be obtained for 13 patients. Of the 56 patients remaining, 36 (64.3%) were infected and fulfilled the inclusion criteria. The following types of infections were observed: surgical wound infection (n = 11), burn infection (n = 7), pyomyositis (n = 6), cellulitis (n = 4), perineal or scrotal abscess (n = 3), and other (n = 5). Thirty-one (86%) patients had a favorable outcome, and 5 (13.8%) patients died. Mortality was directly attributable to S. pneumoniae infection in three of the five fatal cases. Of the 39 S. pneumoniae isolates obtained (36 from skin and soft tissues, three from blood cultures), 58.9% were penicillin nonsusceptible, 7.7% were cefotaxime nonsusceptible, and 20.5% were erythromycin resistant. The most frequent serotypes were 3, 19, 11, and 23. Of the overall number of isolates of S. pneumoniae recovered in a general institution, 2.2% involved skin and soft tissues (of which 64% were clinically significant). Mortality due to pneumococcal skin and soft tissue infections was low. R. Pallarés, coordinator (e-mail: rpallares@bell.ub.es)     

A monoclonal antibody against Schistosoma haematobium soluble egg antigen: efficacy for diagnosis and monitoring of cure of S. haematobium infection

A monoclonal antibody (mAb), 2F/11F, raised against Schistosoma haematobium soluble egg antigen (SEA) was found to be nonreactive with S. mansoni SEA or other parasite antigens (Fasciola hepatica, Echinococcus granulosus). This IgG₁ mAb recognized a repetitive epitope on S. haematobium SEA in the molecular-weight regions of 70, 42, and 35 kDa. It was employed as both an antigen-capture and a biotinylated detection antibody in a sandwich enzyme-linked immunosorbent assay (ELISA) for the detection of circulating schistosome antigen (CSA) and had a detection limit of <1 ng S. haematobium SEA/ml. CSA levels were measured in serum and urine samples from 116 S. haematobium-infected rural students before therapy and at 4, 8, and 12 weeks after praziquantel treatment. Serum and urine samples from 50 S. mansoni -infected patients, 15 patients harboring other parasites, and 30 noninfected individuals were also assessed. CSA was detected in 90.5% of serum samples and 94% of urine samples from S. haematobium-infected patients. CSA was undetectable in serum from the 15 patients harboring other parasites and in 94% of serum samples and 84% of urine samples from S. mansoni-infected patients. In the S. haematobium-infected group a positive correlation was detected between CSA levels in serum and urine samples and the egg load per 10 ml urine. A significant reduction in CSA levels was detected in serum and urine samples after praziquantel therapy. CSA was undetectable in 87% of serum samples and 81.5% of urine samples from schistosomiasis haematobium patients at 12 weeks post-treatment. These data demonstrate that the use of mAb 2F/11F for detection of CSA provides a sensitive method for the immunodiagnosis and monitoring of cure of schistosomiasis haematobium. Received: 5 December 1998 / Accepted: 26 June 1999     

Prepatent periods of different Oesophagostomum spp. isolates in experimentally infected pigs

To define prepatent periods of different Oesophagostomum spp. isolates we carried out two separate experiments, one using two monospecific laboratory isolates and another using laboratory isolates as well as isolates obtained from pig herds having different management systems and with different anthelmintic treatment histories. Pigs were inoculated with 1,000–2,000 infective larvae. Fecal samples were collected daily beginning on days 15 and 16 postinoculation (p.i.). Fecal cultures were set up at different times to yield larvae that could be identified by DNA analyses. All pigs started to excrete eggs on days 18–24 p.i. The mean prepatent period was 20.2 ± 1.4 days, with no significant difference being observed between species and isolates. Prepatent periods of 17–19 days were found for the monospecific laboratory isolates of O. dentatum and O. quadrispinulatum. These findings conflict with parasitology textbooks; therefore, suggestions as to the possible reasons for the observed short prepatent periods are given. Received: 13 November 1996 / Accepted: 15 January 1997     

Prevalence rates of Borrelia burgdorferi sensu lato in host-seeking Ixodes ricinus ticks in Europe

Borrelia burgdorferi sensu lato spirochetes have been found in all examined Ixodes ricinus (L.) populations in Europe. The overall mean proportions of unfed I. ricinus infected with B. burgdorferi s.l. were 1.9% (range 0–11%), 10.8% (2–43%) and 17.4% (3–58%) for larvae (n = 5699), nymphs (n = 48 804) and adults (n = 41 666), respectively. However, the results varied according to the method used. Cultivation in BSK medium is the least sensitive technique (an average of 11% adult ticks found infected), whereas polymerase chain reaction detecting spirochetal DNA is probably the most sensitive method (29% adults found infected). Microscopic methods (dark field, phase contrast, direct or indirect fluorescence) are generally comparable to each other (17–20% adults found infected) and should be regarded as standard procedures because they also make possible a quantitative estimation of spirochetes in the vector. Some technical problems of these methods are discussed. Received: 18 August 1997 / Accepted: 12 September 1997     

Presumed pseudobacteremia outbreak resulting from contamination of proportional disinfectant dispenser

Reported here are the microbiological and epidemiological details of a presumed outbreak of aerobic gram-negative bacilli infections affecting 19 hematological patients, which was traced to contaminated disinfectant. Over a 5-month period, the following organisms were isolated from the blood cultures of 19 neutropenic patients: Pseudomonas fluorescens (n = 13), Achromobacter xylosoxidans (n = 12), Comamonas testosteroni (n = 2) or Stenotrophomonas maltophilia (n = 1). The affected patients were all treated with an expensive regimen of broad-spectrum antibiotic therapy. The same bacteria were recovered from environmental samples as well as from the water pipes of an apparatus for dispensing disinfectant (didecyldimethylammonium chloride). Genotyping results indicated that many of the clinical strains were identical to strains isolated from the apparatus. It was eventually discovered that the night staff was in the habit of disinfecting the blood-culture bottles before use, thereby contaminating the bottles with bacteria contained in the disinfectant. Contamination of the apparatus resulted from faulty maintenance.     

Evaluation of a serum-free transport medium supplemented with cyanobacterial extract, for the optimal survival of Helicobacter pylori from biopsy samples and strains

The purpose of this work was to evaluate the use of an alternative transport medium supplemented with a cyanobacterial extract (CE), free of animal derivatives, to preserve the viability of Helicobacter pylori strains during long-term transportation and allow its recovery from biopsy samples. The transport media evaluated were Mueller–Hinton broth 0.3% agar (MH) and 0.3% of CE (MH-CE). MH broth 5% fetal calf serum (FCS) was used as the reference medium (MH-FCS). Biopsy samples from 134 patients, H. pylori NCTC 11638 and six clinical isolates were studied. A higher recovery (p ≤ 0.001) at 4°C was obtained in MH-CE than in MH-FCS after 96 h of storage. Only MH-CE allowed recovery after 120 h. The H. pylori recovery at room temperature after 96 h was higher (p ≤ 0.005) in MH-CE than in MH-FCS. Similar survival rates were observed in biopsy samples conserved in MH-CE and MH-FCS at 4°C. The recovery after 48 h at room temperature in MH-CE was higher (p ≤ 0.05) than MH-FCS and was the only medium allowing recovery after 72 h. The MH-CE medium is a simple, inexpensive and animal derivatives-free transport medium that can be used to preserve H. pylori viability and its recovery from biopsy samples.     

Occurrence and genotypic characteristics of <Emphasis Type="Italic">Enterocytozoon bieneusi</Emphasis> in pigs with diarrhea

There is limited information available on the association between Enterocytozoon bieneusi and diseases in animals or on the characteristics of the strains involved. This study examined the occurrence of E. bieneusi in piglets with and without diarrhea to determine its involvement. Among 472 fecal samples from 472 piglets (237 with diarrhea and 235 without) up to 7 weeks of age, 67 (approximately 14%) were polymerase chain reaction (PCR) positive for E. bieneusi. Of the 237 piglets with diarrhea, 38 (approximately 16%) tested positive for E. bieneusi. Of the 235 healthy piglets, 29 (approximately 12%) tested positive for E. bieneusi. This species was detected only in the younger group of piglets with diarrhea, particularly those aged less than 1 week and between 1 and 2 weeks. This suggests that E. bieneusi is a possible cause of diarrhea in piglets. This organism, however, produced asymptomatic infections in the older piglets, as there was no significant difference in the rates of occurrence between the diarrheic and nondiarrheic older piglets (aged older than 4 weeks). The internal transcribed spacer (ITS) region of the ribosomal ribonucleic acid gene of the ten E. bieneusi-positive samples was amplified using nested PCR and subsequently sequenced. Genetic polymorphisms, which were represented by five distinct genotypes (PEbA–PEbE), were found among the E. bieneusi isolates. The five genotypes identified in this study differed from each other by two to six single-nucleotide polymorphisms. Nine isolates from four genotypes (PEbA–PEbD) were homologous to previously known types that had originally been isolated from pigs. However, one isolate from the PEbE genotype was identical to type CAF1, which was originally isolated from humans. In addition, the phylogenetic relationships determined by the neighbor-joining analysis of the ITS sequences indicated this genotype to be more distant from the other pig-specific genotypes. Thus, this isolate from pigs may be distantly related to the pig-specific genotypes and may be capable of infecting humans.     

Haematology, morphology and blood cells characteristics of male and female Siamese fighting fish (Betta splendens)

The aim of the present study was to obtain baseline data on blood cell size, morphology and haematological parameters in Siamese fighting fish (Betta splendens) since there is limited information in the published literature. Blood samples from the caudal vein of apparently healthy Siamese fighting fish (male: n = 40 and female: n = 36) were collected. Haematological values of the blood samples were determined using standard techniques. The morphological features of blood cells were described according to observations made by light microscopy. The various types of blood cells measurement were carried out with the help of a stage and an ocular micrometre at a magnification of ×1,000. Erythrocytes, thrombocytes and four types of leucocytes: lymphocytes, monocytes, heterophils and eosinophils, were distinguished and characterised. The average size of the erythrocyte cell and nucleus was 97.33 and 16.28 μm², respectively. Results showed a positive correlation between erythrocyte size and nucleus size for Siamese fighting fish (r = 0.470, p < 0.01). We also found sex-dependent differences for total white blood cell count, lymphocytes and heterophils in Siamese fighting fish (p < 0.05). Statistical analysis revealed that differences in other haematological parameters and blood cell morphology, between male and female fish were not statistically significant (p > 0.05).     

High-density fecal Enterococcus faecium colonization in hospitalized patients is associated with the presence of the polyclonal subcluster CC17

Enterococcus faecium belonging to the polyclonal subcluster CC17, with a typical ampicillin-resistant E. faecium (AREfm) phenotype, have become prevalent among nosocomial infections around the world. High-density intestinal AREfm colonization could be one of the factors contributing to the successful spread of these pathogens. We aimed to quantify the enterococcal intestinal colonization densities in stool samples from AREfm-colonized and non-colonized patients using fluorescent in situ hybridization (FISH). Stool samples were collected from AREfm-colonized (n = 8) and non-colonized (n = 8) patients. The relative number of Enterococcus faecalis and E. faecium was determined by FISH using specific 16S rRNA probes, while the total amount of bacterial cells was counted by staining the sample with 4′,6-diamidino-2-phenylindole (DAPI). The median bacterial cell numbers in fecal samples, counted by DAPI staining, were 7.7 × 10⁹ and 4.8 × 10⁹ cells/g for AREfm-colonized and non-colonized patients, respectively (p = 0.34). The E. faecium densities in AREfm-colonized patients, accounting for 0.5–7% of all fecal bacterial cells, exceeded E. faecalis levels by over ten-fold. E. faecium was not detected in non-colonized patients. This study demonstrated high E. faecium cell densities in stool samples from patients colonized with AREfm. Increased cell densities may contribute to host-to-host transmission and environmental contamination, facilitating the spread of AREfm in the hospital setting.     

Simultaneous detection of human bocavirus and adenovirus by multiplex real-time PCR in a Belgian paediatric population

Since the discovery of human bocavirus (hBoV), the virus has been detected worldwide in respiratory tract samples from young children by various polymerase chain reaction (PCR) assays and real-time PCRs (Q-PCR). Until now, no data have been reported on the presence of hBoV in Belgium and the detection of hBoV in a multiplex Q-PCR setting has not been described. The aim of this study was to develop a fast and reliable multiplex Q-PCR for the simultaneous detection of hBoV DNA and adenovirus (AdV) DNA. During the winter of 2004–2005, 445 nasopharyngeal aspirates (NPAs) were analysed from 404 Belgian children up to 5 years old with acute respiratory tract infections (ARTIs). (Co)infections with hBoV, AdV, respiratory syncytial virus (RSV), human metapneumovirus (hMPV) and influenza A virus were investigated. A viral agent was detected in 61% (n = 272/445) of the NPAs. Multiplex Q-PCR found a prevalence of 11% (n = 51/445) hBoV and 13% (n = 58/445) AdV. Coinfections were more frequently found with AdV (62%; n = 36/58) than with hBoV (49%; n = 25/51). Follow-up samples were available from 22 patients with ARTIs. In three patients, hBoV DNA persisted for one month. Multiplex Q-PCR may help in closing the diagnostic gap by addressing a broader range of potential respiratory pathogens.     

Risk factors for multidrug-resistant tuberculosis in a tuberculosis unit in Madrid,Spain

The setting for this retrospective cohort study was a specialised tuberculosis unit in Madrid, Spain. The objective was to describe the risk factors for multidrug-resistant tuberculosis (MDR-TB). The medical records of all patients admitted to the unit were reviewed retrospectively to identify factors associated with multidrug resistance. Patients with positive culture for M. tuberculosis and with available drug-susceptibility tests were included. The variables assessed were age, gender, country of origin, homelessness, alcohol consumption, intravenous drug use, methadone substitution therapy, contact with a tuberculosis patient, sputum smear, site of disease, previous tuberculosis treatment, HIV infection, history of imprisonment, diabetes mellitus and chronic obstructive pulmonary disease. Thirty patients with MDR-TB and 666 patients with non-MDR-TB were included from the years 1997 to 2006. The only factors associated with MDR-TB in multivariate analysis were previous tuberculosis treatment (OR: 3.44; 95% CI: 1.58–7.50; p = 0.003), age group 45–64 years (OR: 3.24; 95% CI: 1.34–7.81; p = 0.009) and alcohol abuse (OR: 0.12; 95% CI: 0.03 to 0.55; p = 0.003). In our study, patients who had had previous treatment for tuberculosis, who were 45–64 years of age or who had no history of alcohol abuse were more likely to have MDR-TB.     

Sequence and immunogenicity of the Taenia saginata homologue of the major surface antigen of Echinococcus spp.

A clone (R-Tso18) was isolated from a Taenia saginata oncosphere cDNA library by screening with sera from rabbits immunised with oncosphere extract. It contained a full-length cDNA sequence of 1893 bp with an open reading frame of 1680 bp, corresponding to 559 amino acids with a deduced molecular mass of 65.173 kDa and an isoelectric point of 6.08. The R-Tso18 protein showed 80–84% nucleotide identity with the major protoscolex surface antigens of Echinococcus multilocularis (EM10) and E. granulosus (EG10). Preliminary immunogenicity studies employing the radio-labeled R-Tso18 protein in immune co-precipitation assays indicated sero-positivity for T. saginata-infected calf sera (6/13), T. solium cysticercosis human (7/22) and pig (2/2) sera and E. multilocularis (6/10)- and E. granulosus (1/12)-infected human sera, whereas other helminth-infection sera were negative. As immuno-precipitation is a relatively insensitive assay, it was concluded that further studies on the diagnostic potential of the purified recombinant R-Tso18 antigen, or its peptides, are merited. Received: 9 July 1997 / Accepted: 3 November 1997     

Prevalence and genotypes of Enterocytozoon bieneusi in weaned beef calves on cow-calf operations in the USA

To determine the prevalence and genotype distribution of Enterocytozoon bieneusi in weaned beef calves in the USA, fecal samples were collected from 819 calves (6–18 months of age) from 49 operations. Feces were sieved and subjected to density gradient centrifugation to remove fecal debris and to concentrate spores. DNA extracted from each sample was subjected to the polymerase chain reaction (PCR) to amplify the complete internal transcriber spacer (ITS). All PCR-positive specimens were sequenced to determine the genotype(s) present. Overall, E. bieneusi was detected in 34.8% of the 819 fecal samples. The highest prevalence was found in the Midwest region (42.7%) followed by the South (35.8%) and the West (23.2%). The prevalence of E. bieneusi varied considerably from operation to operation (0–100%). A prevalence of 100% was observed in three operations, one in the Midwest and two in the South; E. bieneusi was not found in six operations, three in the South and three in the West. Sequence analysis revealed the presence of six genotypes, four previously reported (I, J, BEB4, and type IV) and two novel genotypes (BEB8 and BEB9). Mixed infections were identified in five specimens, three contained I and BEB4 and two contained J and BEB4. Most of the positive calves (238 of 285) harbored genotypes with zoonotic potential including I (59), J (108), BEB4 (65), type IV (1), mixed I/BEB4 (3), and mixed J/BEB4 (2).