Armando E. Hoet

Reverse transcription-PCR assays for detection of bovine enteric caliciviruses (BEC) and analysis of the genetic relationships among BEC and human caliciviruses.

ABSTRACT Two genetically distinct bovine enteric caliciviruses (BECs) have been identified: the norovirus (NLV) Jena and Newbury Agent-2 (NA-2) BECs, which are genetically related to human noroviruses, and the Nebraska (NB) BECs, which is related to sapoviruses and lagoviruses but may also represent a new calicivirus genus. The prevalence of these two BEC genotypes in cattle is unknown. Although reverse transcription-PCR (RT-PCR) primers for human NLV recognize NLV-BECs, the genetic relationships between NLV from humans and the NLV-BECs commonly circulating in cattle is undefined. In the present study, veal calf fecal samples were assayed for enteric caliciviruses by using six RT-PCR primer sets designed for the detection of human NLVs or BECs. Caliciviruses genetically related to the NLV-BEC Jena and NA-2 strains or to the recently characterized NB BEC strain were identified in three of four and four of four sampled veal herds, respectively. Extended 3′-terminal genome sequences of two NLV-BECs, designated CV95-OH and CV186-OH, encoding the RNA-dependent RNA polymerase (RdRp; open reading frame 1 [ORF-1]), VP1 (ORF-2), and VP2 (ORF-3) genes were determined. Phylogenetic and sequence identity analyses of each genome region demonstrated these viruses to be most closely related to the NLV-BEC Jena and NA-2 strains. In initial testing, the human P289-P290 (P289/290) primer set was found to be the most sensitive for calicivirus detection. However, its failure to identify all positive fecal pools (as determined by other assays) led us to design two new primer sets, CBECU-F/R and NBU-F/R, for the sensitive and specific detection of NLV-BEC (NLV-BEC Jena and NA-2) and BEC-NB-like viruses, respectively. The RT-PCR assays with the new primers were compared against other primer sets, including P289/290. Composite results of the tests completed by using the new assays identified 72% (54 of 75) of veal calf fecal samples as positive, with 21 of 21 sequenced reaction products specific for the target RdRp gene. The same design strategy used for the new BEC assays may also be applicable to the design of similar assays for the detection of human caliciviruses (HuCVs). Our data support the genetic relationship between NLV-BECs and NLV-HuCVs but with the NLV-BECs comprising two clusters within a third NLV genogroup.

Detection of respiratory and enteric shedding of bovine coronaviruses in cattle in an Ohio feedlot

Recently, bovine coronavirus (BCV) has been isolated from new cattle arrivals to feedlots, but the association between respiratory and enteric infections with BCV in feedlot cattle remains uncertain. Fecal and nasal swab samples from 85 Ohio Agricultural Research and Development Center (OARDC) feedlot cattle averaging 7 months of age were collected at arrival (0) and at 4, 7, 14, and 21 days postarrival (DPA). An antigen capture enzyme-linked immunosorbent assay (ELISA) was used to detect concurrent shedding of BCV in fecal and nasal samples. All samples ELISA positive for BCV were matched with an equal number of BCV ELISA-negative samples and analyzed by reverse transcription-polymerase chain reaction (RT-PCR) of the N gene. Paired sera were collected at arrival and 21 DPA and tested for antibodies to BCV using an indirect ELISA. Information on clinical signs, treatments provided, and cattle weights were collected. The overall rates of BCV nasal and fecal shedding were 48% (41/85) and 53% (45/85) by ELISA and 84% (71/85) and 96% (82/85) by RT-PCR, respectively. The peak of BCV nasal and fecal shedding occurred at 4 DPA. Thirty-two cattle (38%) showed concurrent enteric and nasal shedding detected by both tests. Eleven percent of cattle had antibody titers against BCV at 0 DPA and 91% of cattle seroconverted to BCV by 21 DPA. The BCV fecal and nasal shedding detected by ELISA and RT-PCR were statistically correlated with ELISA antibody seroconversion (P < 0.0001); however, BCV fecal and nasal shedding were not significantly related to clinical signs. Seroconversion to BCV was inversely related to average daily weight gains (P < 0.06). Twenty-eight respiratory and 7 enteric BCV strains were isolated from nasal and fecal samples of 32 cattle in HRT-18 cell cultures. These findings confirm the presence of enteric and respiratory BCV infections in feedlot calves. Further studies are needed to elucidate the differences between enteric and respiratory strains of BCV and their role in the bovine respiratory disease complex of feedlot cattle.

Prevalence of Porcine Noroviruses, Molecular Characterization of Emerging Porcine Sapoviruses from Finisher Swine in the United States, and Unified Classification Scheme for Sapoviruses

ABSTRACT Noroviruses (NoVs) and sapoviruses (SaVs) are important human pathogens. Although the involvement of porcine NoVs in disease in pigs is unclear, they are genetically and antigenically closely related to human NoVs. Human NoV-like strains have been detected in pigs, raising public health concerns of potential interspecies transmission. Porcine SaVs are highly diverse and emerging in swine populations. Recently, at least three new genogroups of porcine SaVs have been proposed. In this study, we tested 413 pooled fecal samples collected from apparently healthy finisher pigs in North Carolina swine farms during 2009. Reverse transcription (RT)-PCR coupled hybridization assays were performed to detect known porcine NoVs. The overall prevalence of porcine NoVs determined was 18.9% based on this method. Samples were then tested by RT-PCR targeting the 5′ end of the capsid region for genogroup II (GII) NoVs, a group which includes human NoVs, followed by sequence analysis. All NoVs identified belonged to typical porcine NoV genotypes, and no human NoV-like strains were detected in specimens from these pigs. Porcine NoV-negative samples (n = 335) were subsequently screened using universal calicivirus primers, and 17 SaV strains were confirmed by sequencing. Based on the partial RNA-dependent RNA polymerase (RdRp) region, they clustered with GIII, GVII, and GVIII and with currently unclassified SaVs. According to analysis of the complete capsid sequences, 7 representative strains clustered with GVII, GVIII, and GIX? SaVs. We tentatively classified SaVs into 14 genogroups based on the complete capsid protein VP1. In summary, porcine NoVs and highly divergent SaVs were present in North Carolina finisher pigs.

The Global One Health Paradigm: Challenges and Opportunities for Tackling Infectious Diseases at the Human, Animal, and Environment Interface in Low- Resource Settings

Zoonotic infectious diseases have been an important concern to humankind for more than 10,000 years. Today, approximately 75% of newly emerging infectious diseases (EIDs) are zoonoses that result from various anthropogenic, genetic, ecologic, socioeconomic, and climatic factors. These interrelated driving forces make it difficult to predict and to prevent zoonotic EIDs. Although significant improvements in environmental and medical surveillance, clinical diagnostic methods, and medical practices have been achieved in the recent years, zoonotic EIDs remain a major global concern, and such threats are expanding, especially in less developed regions. The current Ebola epidemic in West Africa is an extreme stark reminder of the role animal reservoirs play in public health and reinforces the urgent need for globally operationalizing a One Health approach. The complex nature of zoonotic diseases and the limited resources in developing countries are a reminder that the need for implementation of Global One Health in low-resource settings is crucial. The Veterinary Public Health and Biotechnology (VPH-Biotec) Global Consortium launched the International Congress on Pathogens at the Human-Animal Interface (ICOPHAI) in order to address important challenges and needs for capacity building. The inaugural ICOPHAI (Addis Ababa, Ethiopia, 2011) and the second congress (Porto de Galinhas, Brazil, 2013) were unique opportunities to share and discuss issues related to zoonotic infectious diseases worldwide. In addition to strong scientific reports in eight thematic areas that necessitate One Health implementation, the congress identified four key capacity-building needs: (1) development of adequate science-based risk management policies, (2) skilled-personnel capacity building, (3) accredited veterinary and public health diagnostic laboratories with a shared database, and (4) improved use of existing natural resources and implementation. The aim of this review is to highlight advances in key zoonotic disease areas and the One Health capacity needs.

Detection of bovine torovirus and other enteric pathogens in feces from diarrhea cases in cattle

The objectives of this study were to determine the prevalence of bovine torovirus (BoTV) in bovine fecal samples from diarrhea cases submitted to the Ohio Animal Disease Diagnostic Laboratory (ADDL) and to assess if a relationship exists between BoTV and the other enteric pathogens detected. From November 1999 to May 2001, 259 specimens from 53 calves (≤6 months old), 27 young adults (≤2 years), 125 adults (≥2 years), and 54 animals of unknown age were examined by an antigen-capture enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase-polymerase chain reaction (RT-PCR) assay developed to detect BoTV Testing for other enteric pathogens was performed by ADDL, and the results were analyzed with the BoTV data. The BoTV was detected using ELISA or RT-PCR in 9.7% (25/259) of the clinical samples, 56% (14/25) of which were from calves (P < 0.001) representing 26.4% (14/53) of the calves tested. Of the BoTV-positive calves, 71% (10/14) were less than 3 weeks of age. In 11/25 positive specimens, BoTV was the only pathogen detected among those examined. Other enteric organisms detected alone or in combination with BoTV in calf samples were rotavirus, coronavirus, Salmonella spp., Cryptosporidium spp., and Giardia spp.; but no consistent association between BoTV and these organisms was observed. In summary, BoTV was detected in fecal samples from cattle with diarrhea, principally in young calves less than 3 weeks of age. Future studies of infectious diarrhea in cattle should also include assays for this etiologic agent.

Environmental Methicillin-Resistant Staphylococcus aureus in a Veterinary Teaching Hospital During a Nonoutbreak Period

Concurrent to reports of zoonotic and nosocomial transmission of methicillin-resistant Staphylococcus aureus (MRSA) in veterinary settings, recent evidence indicates that the environment in veterinary hospitals may be a potential source of MRSA. The present report is a cross-sectional study to determine the prevalence of MRSA on specific human and animal contact surfaces at a large veterinary hospital during a nonoutbreak period. A total of 156 samples were collected using Swiffers(®) or premoistened swabs from the small animal, equine, and food animal sections. MRSA was isolated and identified by pre-enrichment culture and standard microbiology procedures, including growth on Mueller-Hinton agar supplemented with NaCl and oxacillin, and by detection of the mecA gene. Staphylococcal chromosome cassette mec (SCCmec) typing and pulsed-field gel electrophoresis profile were also determined. MRSA was detected in 12% (19/157) of the hospital environments sampled. The prevalence of MRSA in the small animal, equine, and food animal areas were 16%, 4%, and 0%, respectively. Sixteen of the MRSA isolates from the small animal section were classified as USA100, SCCmec type II, two of which had pulsed-field gel electrophoresis pattern that does not conform to any known type. The one isolate obtained from the equine section was classified as USA500, SCCmec type IV. The molecular epidemiological analysis revealed a very diverse population of MRSA isolates circulating in the hospital; however, in some instances, multiple locations/surfaces, not directly associated, had the same MRSA clone. No significant difference was observed between animal and human contact surfaces in regard to prevalence and type of isolates. Surfaces touched by multiple people (doors) and patients (carts) were frequently contaminated with MRSA. The results from this study indicate that MRSA is present in the environment even during nonoutbreak periods. This study also identified specific surfaces in a veterinary environment that need to be targeted when designing and executing infection control programs.

Characterization of Multidrug-Resistant Salmonella enterica Serovar Heidelberg Isolated from Humans and Animals

Salmonella enterica serovar Heidelberg has been recognized as one of the most common serovar associated with foodborne infections in the United States. It is also frequently isolated from nonhuman sources and has increasingly shown resistance to various antimicrobial agents. The present study was undertaken to identify the predominant antimicrobial resistance phenotypes and genotypes of Salmonella Heidelberg (n = 95) isolates of human, swine, and turkey origin. Antimicrobial susceptibility was done using Kirby-Bauer disk diffusion method with a panel of 12 antimicrobials. Pulsed-field gel electrophoresis genotyping was used to determine the diversity of the isolates. The antimicrobial resistance genes and carriage of Class 1 and 2 integrons were determined by polymerase chain reaction. All Salmonella Heidelberg isolates from swine were resistant to one or more of the antimicrobials tested and the majority (73.3%) showed multidrug resistance to streptomycin, tetracycline, and kanamycin (R-type: StTeKm). About 80% of the Salmonella Heidelberg isolates of human origin were pan-susceptible, however, one isolate showed multidrug resistance to 10 of 12 antimicrobials tested. Among the multidrug-resistant (MDR) Salmonella Heidelberg isolates, Class 1 integrons with variable sizes of 1.2 to 1.5 kb were detected in six isolates (three each) from humans and swine. DNA sequencing revealed that Class 1 integrons of both human and swine origin carried a gene encoding aminoglycoside adenyltransferase (aadA). Resistance genes identified in other loci include aphA1-Iab, strA, bla(TEM), and tetA (B). Both human and swine MDR strains of Salmonella Heidelberg carried the resistance phenotypes on self-transferable plasmids. Dendrogram analysis of pulsotypes indicated possible clonality of Salmonella Heidelberg between isolates of human and swine origin. The findings in this study indicate the increasing significance of swine as reservoirs of emerging MDR serovars, such as MDR Salmonella Heidelberg, is of public health significance.

Identification of Escherichia coli and Salmonella enterica organisms with reduced susceptibility to ceftriaxone from fecal samples of cows in dairy herds.

OBJECTIVE To estimate the relationship between therapeutic use of ceftiofur and recovery of Escherichia coli and Salmonella spp with reduced susceptibility to ceftriaxone from feces of dairy cattle. ANIMALS 3,840 mature dairy cows on 50 dairy herds in Ohio. Procedures-Fecal samples were obtained from up to 100 mature dairy cows on each farm. Samples were screened for E coli and Salmonella spp with reduced susceptibility to ceftriaxone by use of selective media. RESULTS E coli with reduced susceptibility to ceftriaxone was recovered from 92% (46/50) of the herds and 60.9% (2,338/3,840) of cows. Salmonella spp were recovered from 44% (22/50) of the herds and 9.9% (382/3,840) of cows. No association was found between ceftiofur use and recovery of E coli with reduced susceptibility to ceftriaxone at the herd level. However, recovery of E coli with reduced susceptibility to ceftriaxone was more likely from cows in herds in which Salmonella spp were also recovered on the day of collection (odds ratio, 24.96; 95% confidence interval, 3.17 to 196.68) than from herds in which Salmonella spp were not recovered. Odds of recovery of E coli with reduced susceptibility to ceftriaxone from an individual cow increased 62% (odds ratio, 1.62; 95% confidence interval, 1.16 to 2.25) for every 454-kg increase in herd milk production. CONCLUSIONS AND CLINICAL RELEVANCE No evidence was found that the use of ceftiofur on dairy farms increases the prevalence or dissemination of Salmonella spp or E coli with reduced susceptibility to ceftriaxone.

Swab Type, Moistening, and Preenrichment for Staphylococcus aureus on Environmental Surfaces

ABSTRACT We compared five swabs, dry or premoistened and with or without preenrichment, to detect surface contamination with Staphylococcus aureus. Sensitivities varied based on swab type, as follows: 71.9% and 75% for rayon, 71.2% for cotton, 81.3% for polyester, and 53.2% for calcium alginate. Preenrichment improved sensitivity (80%, versus 61.3% for direct-plated specimens), as did premoistening (83.4%, versus 57.5% for dry swabs). All of the premoistened, preenriched swabs were positive.

Reduction of pathogen indicator organisms in dairy wastewater using an ecological treatment system.

Ecological treatment systems can provide a sustainable, plant-based alternative to traditional wastewater treatment. One factor essential to the success of these systems is ensuring their ability to reduce coliform concentrations in wastewater. Wastewater is the primary source of fecal contamination in aquatic ecosystems, containing total and fecal coliforms on the order of 10(8)-10(10) and 10(7)-10(9) CFU L(-1), respectively. This study assessed the ability of an ecological treatment system to reduce concentrations of total coliforms and Escherichia coli from dairy wastewater. Low strength wastewater was pumped into the system during July of 2005 and high strength in September 2005. Wastewater passes through a series of anaerobic, aerobic, and clarifier reactors and wetland cells before exiting the system. Regardless of wastewater strength, average total coliform and E. coli concentrations were consistently reduced by at least 99% from influent to effluent, with the majority of the reduction (76%) occurring in the first two reactors. Relationships between internal concentrations of solids and coliforms indicated that increased reduction of solids may further reduce coliform concentrations. Although U.S. Environmental Protection Agency discharge requirements for E. coli were not always met, the substantial reductions achieved indicate that ecological treatment systems have the potential to successfully reduce coliforms in wastewater to meet discharge limits. The results from this study will be used to guide design and management of future ecological treatment systems, so that larger and more consistent coliform reductions can be achieved.