Carriage and Gene Content Variability of the pESI-Like Plasmid Associated with Salmonella Infantis Recently Established in United States Poultry Production.

Salmonella Infantis carrying extended spectrum ß-lactamase blaCTX-M-65 on a pESI-like megaplasmid has recently emerged in United States poultry. In order to determine the carriage rate and gene content variability of this plasmid in U.S. Salmonella Infantis, whole genome sequences of Salmonella isolates from humans and animals in the U.S. and internationally containing the pESI-like plasmid were analyzed. The U.S. Department of Agriculture Food Safety and Inspection Service (FSIS) identified 654 product sampling isolates containing pESI-like plasmids through hazard analysis and critical control point (HACCP) verification testing in 2017 and 2018. The Centers for Disease Control and Prevention identified 55 isolates with pESI-like plasmids in 2016-2018 through the National Antimicrobial Resistance Monitoring System. Approximately 49% of pESI-like plasmids from FSIS verification isolates and 71% from CDC NARMS contained blaCTX-M-65. Pan-plasmid genome analysis was also performed. All plasmids contained traN and more than 95% contained 172 other conserved genes; 61% contained blaCTX-M-65. In a hierarchical clustering analysis, some plasmids from U.S. animal sources clustered together and some plasmids from South America clustered together, possibly indicating multiple plasmid lineages. However, most plasmids contained similar genes regardless of origin. Carriage of the pESI-like plasmid in U.S. appears to be limited to Salmonella Infantis and carriage rates increased from 2017 to 2018.

Complete Genome Sequence of Multidrug-Resistant Salmonella enterica Serovar I 4,[5],12:i:- 2015 U.S. Pork Outbreak Isolate USDA15WA-1.

The genome of a multidrug-resistant (MDR) Salmonella enterica subsp. enterica serovar I 4,[5],12:i:- isolate from the 2015 U.S. pork outbreak was sequenced. The complete nucleotide sequence of USDA15WA-1 is 5,031,277 bp, including Salmonella genomic island 4 encoding tolerance to multiple metals and an MDR module inserted in the fljB region.

Validating the AMRFinder Tool and Resistance Gene Database by Using Antimicrobial Resistance Genotype-Phenotype Correlations in a Collection of Isolates.

Antimicrobial resistance (AMR) is a major public health problem that requires publicly available tools for rapid analysis. To identify AMR genes in whole-genome sequences, the National Center for Biotechnology Information (NCBI) has produced AMRFinder, a tool that identifies AMR genes using a high-quality curated AMR gene reference database. The Bacterial Antimicrobial Resistance Reference Gene Database consists of up-to-date gene nomenclature, a set of hidden Markov models (HMMs), and a curated protein family hierarchy. Currently, it contains 4,579 antimicrobial resistance proteins and more than 560 HMMs. Here, we describe AMRFinder and its associated database. To assess the predictive ability of AMRFinder, we measured the consistency between predicted AMR genotypes from AMRFinder and resistance phenotypes of 6,242 isolates from the National Antimicrobial Resistance Monitoring System (NARMS). This included 5,425 Salmonella enterica, 770 Campylobacter spp., and 47 Escherichia coli isolates phenotypically tested against various antimicrobial agents. Of 87,679 susceptibility tests performed, 98.4% were consistent with predictions. To assess the accuracy of AMRFinder, we compared its gene symbol output with that of a 2017 version of ResFinder, another publicly available resistance gene detection system. Most gene calls were identical, but there were 1,229 gene symbol differences (8.8%) between them, with differences due to both algorithmic differences and database composition. AMRFinder missed 16 loci that ResFinder found, while ResFinder missed 216 loci that AMRFinder identified. Based on these results, AMRFinder appears to be a highly accurate AMR gene detection system.

Antimicrobial Resistance Genes, Cassettes, and Plasmids Present in Salmonella enterica Associated With United States Food Animals.

The ability of antimicrobial resistance (AR) to transfer, on mobile genetic elements (MGEs) between bacteria, can cause the rapid establishment of multidrug resistance (MDR) in bacteria from animals, thus creating a foodborne risk to human health. To investigate MDR and its association with plasmids in Salmonella enterica, whole genome sequence (WGS) analysis was performed on 193 S. enterica isolated from sources associated with United States food animals between 1998 and 2011; 119 were resistant to at least one antibiotic tested. Isolates represented 86 serotypes and variants, as well as diverse phenotypic resistance profiles. A total of 923 AR genes and 212 plasmids were identified among the 193 strains. Every isolate contained at least one AR gene. At least one plasmid was detected in 157 isolates. Genes were identified for resistance to aminoglycosides (n = 472), ß-lactams (n = 84), tetracyclines (n = 171), sulfonamides (n = 91), phenicols (n = 42), trimethoprim (n = 8), macrolides (n = 5), fosfomycin (n = 48), and rifampicin (n = 2). Plasmid replicon types detected in the isolates were A/C (n = 32), ColE (n = 76), F (n = 43), HI1 (n = 4), HI2 (n = 20), I1 (n = 62), N (n = 4), Q (n = 7), and X (n = 35). Phenotypic resistance correlated with the AR genes identified in 95.4% of cases. Most AR genes were located on plasmids, with many plasmids harboring multiple AR genes. Six antibiotic resistance cassette structures (ARCs) and one pseudo-cassette were identified. ARCs contained between one and five resistance genes (ARC1: sul2, strAB, tetAR; ARC2: aac3-iid; ARC3: aph, sph; ARC4: cmy-2; ARC5: floR; ARC6: tetB; pseudo-ARC: aadA, aac3-VIa, sul1). These ARCs were present in multiple isolates and on plasmids of multiple replicon types. To determine the current distribution and frequency of these ARCs, the public NCBI database was analyzed, including WGS data on isolates collected by the USDA Food Safety and Inspection Service (FSIS) from 2014 to 2018. ARC1, ARC4, and ARC5 were significantly associated with cattle isolates, while ARC6 was significantly associated with chicken isolates. This study revealed that a diverse group of plasmids, carrying AR genes, are responsible for the phenotypic resistance seen in Salmonella isolated from United States food animals. It was also determined that many plasmids carry similar ARCs.

CTX-M-65 Extended-Spectrum ß-Lactamase-Producing Salmonella enterica Serotype Infantis, United States1.

Extended-spectrum ß-lactamases (ESBLs) confer resistance to clinically important third-generation cephalosporins, which are often used to treat invasive salmonellosis. In the United States, ESBLs are rarely found in Salmonella. However, in 2014, the US Food and Drug Administration found blaCTX-M-65 ESBL-producing Salmonella enterica serotype Infantis in retail chicken meat. The isolate had a rare pulsed-field gel electrophoresis pattern. To clarify the sources and potential effects on human health, we examined isolates with this pattern obtained from human surveillance and associated metadata. Using broth microdilution for antimicrobial susceptibility testing and whole-genome sequencing, we characterized the isolates. Of 34 isolates, 29 carried the blaCTX-M-65 gene with <9 additional resistance genes on 1 plasmid. Of 19 patients with travel information available, 12 (63%) reported recent travel to South America. Genetically, isolates from travelers, nontravelers, and retail chicken meat were similar. Expanded surveillance is needed to determine domestic sources and potentially prevent spread of this ESBL-containing plasmid.

Novel linezolid resistance plasmids in Enterococcus from food animals in the USA.

Objectives: To sequence the genomes and determine the genetic mechanisms for linezolid resistance identified in three strains of Enterococcus isolated from cattle and swine caecal contents as part of the US National Antimicrobial Resistance Monitoring System (NARMS) surveillance programme. Methods: Broth microdilution was used for in vitro antimicrobial susceptibility testing to assess linezolid resistance. Resistance mechanisms and plasmid types were identified from data generated by WGS on Illumina® and PacBio® platforms. Conjugation experiments were performed to determine whether identified mechanisms were transmissible. Results: Linezolid resistance plasmids containing optrA were identified in two Enterococcus faecalis isolates and one Enterococcus faecium. The E. faecium isolate also carried the linezolid resistance gene cfr on the same plasmid as optrA. The linezolid resistance plasmids had various combinations of additional resistance genes conferring resistance to phenicols (fexA), aminoglycosides [spc and aph(3')-III] and macrolides [erm(A) and erm(B)]. One of the plasmids was confirmed to be transmissible by conjugation, resulting in linezolid resistance in the transconjugant. Conclusions: To the best of our knowledge, this is the first identification of linezolid resistance in the USA in bacteria isolated from food animals. The oxazolidinone class of antibiotics is not used in food animals in the USA, but the genes responsible for resistance were identified on plasmids with other resistance markers, indicating that there may be co-selection for these plasmids due to the use of different antimicrobials. The transmissibility of one of the plasmids demonstrated the potential for linezolid resistance to spread horizontally. Additional surveillance is necessary to determine whether similar plasmids are present in human strains of Enterococcus.

Utility of Combining Whole Genome Sequencing with Traditional Investigational Methods To Solve Foodborne Outbreaks of Salmonella Infections Associated with Chicken: A New Tool for Tackling This Challenging Food Vehicle.

High consumption rates and a multitude of brands make multistate foodborne outbreaks of Salmonella infections associated with chicken challenging to investigate, but whole genome sequencing is a powerful tool that can be used to assist investigators. Whole genome sequencing of pathogens isolated from clinical, environmental, and food samples is increasingly being used in multistate foodborne outbreak investigations to determine with unprecedented resolution how closely related these isolates are to one another genetically. In 2014, federal and state health officials investigated an outbreak of 146 Salmonella Heidelberg infections in 24 states. A follow-up analysis was conducted after the conclusion of the investigation in which 27 clinical and 24 food isolates from the outbreak underwent whole genome sequencing. These isolates formed seven clades, the largest of which contained clinical isolates from a subcluster of case patients who attended a catered party. One isolate from a chicken processed by a large producer was closely related genetically (zero to three single-nucleotide polymorphism differences) to the clinical isolates from these subcluster case patients. Chicken from this large producer was also present in the kitchen of the caterer on the day before the event, thus providing additional evidence that the chicken from this producer was the outbreak source. This investigation highlights how whole genome sequencing can be used with epidemiologic and traceback evidence to identify chicken sources of foodborne outbreaks.

Implementation of Nationwide Real-time Whole-genome Sequencing to Enhance Listeriosis Outbreak Detection and Investigation.

Listeria monocytogenes (Lm) causes severe foodborne illness (listeriosis). Previous molecular subtyping methods, such as pulsed-field gel electrophoresis (PFGE), were critical in detecting outbreaks that led to food safety improvements and declining incidence, but PFGE provides limited genetic resolution. A multiagency collaboration began performing real-time, whole-genome sequencing (WGS) on all US Lm isolates from patients, food, and the environment in September 2013, posting sequencing data into a public repository. Compared with the year before the project began, WGS, combined with epidemiologic and product trace-back data, detected more listeriosis clusters and solved more outbreaks (2 outbreaks in pre-WGS year, 5 in WGS year 1, and 9 in year 2). Whole-genome multilocus sequence typing and single nucleotide polymorphism analyses provided equivalent phylogenetic relationships relevant to investigations; results were most useful when interpreted in context of epidemiological data. WGS has transformed listeriosis outbreak surveillance and is being implemented for other foodborne pathogens.

Detection of Shiga toxin-producing Escherichia coli (STEC) O157:H7, O26, O45, O103, O111, O121, and O145, and Salmonella in retail raw ground beef using the DuPont™ BAX® system.

Shiga toxin-producing Escherichia coli (STEC) and Salmonella are food-borne pathogens commonly associated with beef, and reliable methods are needed to determine their prevalence in beef and to ensure food safety. Retail ground beef was tested for the presence of E. coli O157:H7, STEC serogroups O26, O45, O103, O111, O121, and O145, and Salmonella using the DuPont™ BAX® system method. Ground beef (325 g) samples were enriched in 1.5 L of TSB with 2 mg/L novobiocin at 42°C for 18 h, and then evaluated using the BAX® System real-time PCR assays for E. coli O157:H7 and STEC suite, and the BAX® System standard PCR assays for E. coli O157:H7 MP and Salmonella. Samples positive for STEC target genes by the BAX® System assays were subjected to immunomagnetic separation (IMS) and plating onto modified Rainbow Agar O157. Enrichments that were PCR positive for Salmonella were inoculated into RV broth, incubated for 18 h at 42°C, and then plated onto XLT-4 agar. Presumptive positive STEC and Salmonella colonies were confirmed using the BAX® System assays. Results of the BAX® System STEC assays showed 20/308 (6.5%) of samples positive for both the Shiga toxin (stx) and intimin (eae) genes; 4 (1.3%) for stx, eae, and O26; 1 (0.3%) for stx, eae, and O45; 3 (1%) for stx, eae, and O103; and 1 (0.3%) for stx, eae, and O145. There were also 3 samples positive for stx, eae, and more than one STEC serogroup. Three (1.0%) of the samples were positive using the BAX® System real-time E. coli O157:H7 assay, and 28 (9.1%) were positive using the BAX® System Salmonella assay. STEC O103 and E. coli O157:H7 were isolated from 2/6 and 2/3 PCR positive samples, respectively. Salmonella isolates were recovered and confirmed from 27 of the 28 Salmonella PCR positive samples, and a portion of the isolates were serotyped and antibiotic resistance profiles determined. Results demonstrate that the BAX® System assays are effective for detecting STEC and Salmonella in beef.

Evaluation of a multiplex real-time PCR method for detecting shiga toxin-producing Escherichia coli in beef and comparison to the U.S. Department of Agriculture Food Safety and Inspection Service Microbiology laboratory guidebook method.

The "top-six" non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, and O145) most frequently associated with outbreaks and cases of foodborne illnesses have been declared as adulterants in beef by the U.S. Department of Agriculture Food Safety and Inspection Service (FSIS). Regulatory testing in beef began in June 2012. The purpose of this study was to evaluate the DuPont BAX System method for detecting these top six STEC strains and strains of E. coli O157:H7. For STEC, the BAX System real-time STEC suite was evaluated, including a screening assay for the stx and eae virulence genes and two panel assays to identify the target serogroups: panel 1 detects O26, O111, and O121, and panel 2 detects O45, O103, O145. For E. coli O157:H7, the BAX System real-time PCR assay for this specific serotype was used. Sensitivity of each assay for the PCR targets was ≥1.23 × 10(3) CFU/ml in pure culture. Each assay was 100% inclusive for the strains tested (20 to 50 per assay), and no cross-reactivity with closely related strains was observed in any of the assays. The performance of the BAX System methods was compared with that of the FSIS Microbiology Laboratory Guidebook (MLG) methods for detection of the top six STEC and E. coli O157:H7 strains in ground beef and beef trim. Generally, results of the BAX System method were similar to those of the MLG methods for detecting non-O157 STEC and E. coli O157:H7. Reducing or eliminating novobiocin in modified tryptic soy broth (mTSB) may improve the detection of STEC O111 strains; one beef trim sample inoculated with STEC O111 produced a negative result when enriched in mTSB with 8 mg/liter novobiocin but was positive when enriched in mTSB without novobiocin. The results of this study indicate the feasibility of deploying a panel of real-time PCR assay configurations for the detection and monitoring of the top six STEC and E. coli O157:H7 strains in beef. The approach could easily be adapted for additional multiplex assays should regulations expand to include other O serogroups or virulence genes.

Influence of primer sequences and DNA extraction method on detection of non-O157 Shiga toxin-producing Escherichia coli in ground beef by real-time PCR targeting the eae, stx, and serogroup-specific genes.

Non-O157 Shiga toxin-producing Escherichia coli (STEC) infections, particularly those caused by the "big six" or "top six" non-O157 serogroups (O26, O45, O103, O111, O121, and O145) can result in severe illness and complications. Because of their significant public health impact and the notable prevalence of STEC in cattle, methods for detection of the big six non-O157 STEC in ground beef have been established. Currently, the U.S. Department of Agriculture, Food Safety and Inspection Service detection methods for screening beef samples for non-O157 STEC target the stx(1), stx(2), and eae virulence genes, with the 16S rRNA gene as an internal control, in a real-time PCR multiplex assay. Further, the serogroup is determined by PCR targeting genes in the E. coli O-antigen gene clusters of the big six non-O157 serogroups. The method that we previously reported was improved so that additional stx variants, stx(1d), stx(2e), and stx(2g), are detected. Additionally, alignments of the primers targeting the eae gene were used to improve the detection assay so that eae subtypes that could potentially be of clinical significance would also be detected. Therefore, evaluation of alternative real-time PCR assay primers and probes for the stx and eae reactions was carried out in order to increase the stx and eae subtypes detected. Furthermore, a Tris-EDTA DNA extraction method was compared with a previously used procedure that was based on a commercially available reagent. The Tris-EDTA DNA extraction method significantly decreased the cycle threshold values for the stx assay (P < 0.0001) and eae assay (P < 0.0001), thereby increasing the ability to detect the targets. The use of different stx primers and probes increased the subtypes detected to include stx(1d), stx(2e), and stx(2g), and sequence data showed that modification of the eae primer should allow the known eae subtypes to be detected.

Differences in pathogenicity, response to vaccination, and innate immune responses in different types of ducks infected with a virulent H5N1 highly pathogenic avian influenza virus from Vietnam.

In a previous study, we found clear differences in pathogenicity and response to vaccination against H5N1 highly pathogenic avian influenza (HPAI; HA dade 2.3.4) between Pekin (Anas platyrhynchos var. domestica) and Muscovy (Cairina moschata) ducks vaccinated using a commercial inactivated vaccine (Re-1). The objective of the present study was to further investigate the pathogenicity of H5N1 HPAI viruses in different species of ducks by examining clinical signs and innate immune responses to infection with a different strain of H5N1 HPAI virus (HA clade 1) in two domestic ducks, Pekin and Muscovy, and one wild-type duck, mallard (Anas platyrhynchos). Protection conferred by vaccination using the Re-1 vaccine against infection with this virus was also compared between Pekin and Muscovy ducks. Differences in pathogenicity were observed among the virus-infected ducks, as the Muscovy ducks died 2 days earlier than did the Pekin and mallard ducks, and they presented more-severe neurologic signs. Conversely, the Pekin and mallard ducks had significantly higher body temperatures at 2 days postinfection (dpi) than did the Muscovy ducks, indicating possible differences in innate immune responses. However, similar expression of innate immune-related genes was found in the spleens of virus-infected ducks at this time point. In all three duck species, there was up-regulation of IFN-alpha, IFN-gamma, IL-6, CCL19, RIG-I, and MHC class I and down-regulation of MHC class II, but variable expression of IL-18 and TLR7. As in our previous study, vaccinated Muscovy ducks showed less protection against virus infection than did Pekin ducks, as evidenced by the higher mortality and higher number of Muscovy ducks shedding virus when compared to Pekin ducks. In conclusion, infection with an H5N1 HPAI virus produced a systemic infection with high mortality in all three duck species; however, the disease was more severe in Muscovy ducks, which also had a poor response to vaccination. The differences in response to virus infection could not be explained by differences in the innate immune responses between the different types of ducks when examined at 2 days dpi, and earlier time points need to be evaluated.

Isolation of Shiga toxin-producing Escherichia coli serogroups O26, O45, O103, O111, O121, and O145 from ground beef using modified rainbow agar and post-immunomagnetic separation acid treatment.

It is estimated that at least 70% of human illnesses due to non-O157 Shiga toxin-producing Escherichia coli (STEC) in the United States are caused by strains from the top six serogroups (O26, O45, O103, O111, O121, and O145). Procedures for isolating STEC from food products often use plating media that include antimicrobial supplements at concentrations that inhibit background microflora growth but can also inhibit target STEC growth. In this study, an agar medium with lower supplement concentrations, modified Rainbow agar (mRBA), was evaluated for recovery of STEC serogroups O26, O45, O103, O111, O121, and O145 from ground beef enrichments. A post-immunomagnetic separation (IMS) acid treatment step was additionally used to reduce background microflora and increase recovery of target STEC strains. Ground beef samples (325 g) were artificially contaminated with STEC and confounding organisms and enriched for 15 h. Recovery of the target STEC was attempted on the enrichments using IMS and plating onto mRBA and Rainbow agar (RBA). Additionally, acid treatment was performed on the post-IMS eluate followed by plating onto mRBA. Using the combination of mRBA and acid treatment, target STEC were isolated from 103 (85.8%) of 120 of the low-inoculated samples (1 to 5 CFU/325-g sample) compared with 68 (56.7%) of 120 using no acid treatment and plating onto RBA with higher levels of novobiocin and potassium tellurite. The combination of acid treatment and mRBA provides a significant improvement over the use of RBA for isolation of STEC serogroups O26, O45, O103, O111, O121, and O145 from raw ground beef.

Low pathogenicity avian influenza viruses infect chicken layers by different routes of inoculation.

In order to develop better control measures against avian influenza, it is necessary to understand how the virus transmits in poultry. In a previous study in which the infectivity and transmissibility of the pandemic H1N1 influenza virus was examined in different poultry species, we found that no or minimal infection occurred in chicken and turkeys intranasally (IN) inoculated with the virus. However, we demonstrated that the virus can infect laying turkey hens by the intracloacal (IC) and intraoviduct (IO) routes, possibly explaining the drops in egg production observed in turkey breeder farms affected by the virus. Such novel routes of exposure have not been previously examined in chickens and could also explain outbreaks of low pathogenicity avian influenza (LPAI) that cause a decrease in egg production in chicken layers and breeders. In the present study, 46-wk-old specific-pathogen-free chicken layers were infected by the IN, IC, or IO routes with one of two LPAI viruses: a poultry origin virus, A/chicken/CA/1255/02 (H6N2), and a live bird market isolate, A/chicken/NJ/12220/97 (H9N2). Only hens IN inoculated with the H6N2 virus presented mild clinical signs consisting of depression and anorexia. However, a decrease in number of eggs laid was observed in all virus-inoculated groups when compared to control hens. Evidence of infection was found in all chickens inoculated with the H6N2 virus by any of the three routes and the virus transmitted to contact hens. On the other hand, only one or two hens from each of the groups inoculated with the H9N2 virus shed detectable levels of virus, or seroconverted and did not transmit the virus to contacts, regardless of the route of inoculation. In conclusion, LPAI viruses can also infect chickens through other routes besides the IN route, which is considered the natural route of exposure. However, as seen with the H9N2 virus, the infectivity of the virus did not increase when given by these alternate routes.

Effect of age on the pathogenesis and innate immune responses in Pekin ducks infected with different H5N1 highly pathogenic avian influenza viruses.

The pathogenicity of H5N1 highly pathogenic avian influenza (HPAI) viruses in domestic ducks varies between different viruses and is affected by the age of the ducks, with younger ducks presenting a more severe disease. In order to better understand the pathobiology of H5N1 HPAI in ducks including the role of host responses, 2 and 5-week-old Pekin ducks were infected with three different H5N1 HPAI viruses. Virus-induced pathology ranged from no clinical signs to severe disease and mortality, with the 2-week-old ducks being more severely affected by the more virulent viruses. However, these more virulent viruses induced higher body temperatures in the 5-week-old ducks than in the 2-week-old ducks indicating possible differences in innate immune responses. To analyze the ducks host responses to H5N1 HPAI virus infection, expression of innate immune-related genes was measured in the spleens and lungs of infected ducks at the peak of virus infection. IFN-α, RIG-I, and IL-6 RNA levels were increased in spleens regardless of the virus given and the age of the ducks, however differences were observed in the levels of up-regulation of IFN-α and RIG-I between the 2 and the 5-week-old ducks with the more virulent virus. Differences in IL-2 gene expression were also observed. In the lungs, the levels of expression of innate immune-related genes were lower than in the spleen, with mostly up-regulation of RIG-I and IL-6 and down-regulation of IFN-α and IL-2; no significant difference in expression was found between the 2 and the 5-week-old ducks. The differences observed in the innate immune responses to infection with H5N1 HPAI viruses could explain in part the differences in pathogenicity found between the 2 and 5-week-old ducks, however earlier time points after infection and additional innate immune-related genes should be examined.

Characterization of H5N1 highly pathogenic avian influenza viruses isolated from poultry in Pakistan 2006-2008.

Nine avian influenza viruses (AIV), H5N1 subtype, were isolated from dead poultry in the Karachi region of Pakistan from 2006 to 2008. The intravenous pathogenicity indices and HA protein cleavage sites of all nine viruses were consistent with highly pathogenic AIV. Based on phylogenetic analysis of the HA genes, these isolates belong to clade 2.2 and both the HA and NA are closely related to each other (nucleotide identities above 99.0%) and to other Middle Eastern H5N1 AIV isolates (nucleotide identities above 98.0%). The phylogenetic data suggest that the virus in both epornitics of H5N1 HPAIV in commercial poultry in the Karachi region of Pakistan between 2006 and 2008 were from a very closely related source, however, there is inadequate epidemiological data to determine what the reservoir was for the virus between the 2006 and 2007 outbreaks other than that there was a single introduction into the region.

Pekin and Muscovy ducks respond differently to vaccination with a H5N1 highly pathogenic avian influenza (HPAI) commercial inactivated vaccine.

Domestic ducks are key intermediates in the transmission of H5N1 highly pathogenic avian influenza (HPAI) viruses, and therefore are included in vaccination programs to control H5N1 HPAI. Although vaccination has proven effective in protecting ducks against disease, different species of domestic ducks appear to respond differently to vaccination, and shedding of the virus may still occur in clinically healthy vaccinated populations. In this study we compared the response to vaccination between two common domestic duck species, Pekin (Anas platyrhynchos domesticus) and Muscovy (Cairina moschata), which were vaccinated with a commercial inactivated vaccine using one of three different schedules in order to elicit protection to H5N1 HPAI before one month of age. Clear differences in responses to vaccination were observed; the Muscovy ducks developed lower viral antibody titers induced by the same vaccination as Pekin ducks and presented with higher morbidity and mortality after challenge with an H5N1 HPAI virus. When comparing the response to infection in non-vaccinated ducks, differences were also observed, with infected Muscovy ducks presenting a lower mean death time and more severe neurological signs than Pekin ducks. However Pekin ducks had significantly higher body temperatures and higher levels of nitric oxide in the blood at 2 days post challenge than Muscovy ducks, indicating possible differences in innate immune responses. Comparison of the expression of innate immune related genes in spleens of the non-vaccinated infected ducks showed differences including significantly higher levels of expression of RIG-I in Pekin ducks and of IL-6 in Muscovy ducks. Both duck species showed an up-regulation of IFNα and MHC-I expression, and a down-regulation of MHC-II. In conclusion, differences in response to infection and vaccination were observed between the two domestic duck species. This information should be taken into account when developing effective vaccination programs for controlling H5N1 HPAI in different species of ducks.

In vivo and in vitro function of human UDP-galactose 4'-epimerase variants.

Type III galactosemia results from reduced activity of the enzyme UDP-galactose 4'-epimerase. Five disease-associated alleles (G90E, V94M, D103G, N34S and L183P) and three artificial alleles (Y105C, N268D, and M284K) were tested for their ability to alleviate galactose-induced growth arrest in a Saccharomyces cerevisiae strain which lacks endogenous UDP-galactose 4'-epimerase. For all of these alleles, except M284K, the ability to alleviate galactose sensitivity was correlated with the UDP-galactose 4'-epimerase activity detected in cell extracts. The M284K allele, however, was able to substantially alleviate galactose sensitivity, but demonstrated near-zero activity in cell extracts. Recombinant expression of the corresponding protein in Escherichia coli resulted in a protein with reduced enzymatic activity and reduced stability towards denaturants in vitro. This lack of stability may result from the introduction of an unpaired positive charge into a bundle of three α-helices near the surface of the protein. The disparities between the in vivo and in vitro data for M284K-hGALE further suggest that there are additional, stabilising factors present in the cell. Taken together, these results reinforce the need for care in the interpretation of in vitro, enzymatic diagnostic tests for type III galactosemia.

The effects of NS gene exchange on the pathogenicity of H5N1 HPAI viruses in ducks.

Until 2002, H5N1 highly pathogenic avian influenza (HPAI) viruses caused only mild respiratory infections in ducks. Since then, new viruses have emerged that cause systemic disease and high mortality in ducks and other waterfowl. Studies on HPAI virus pathogenicity in ducks have been limited, and there is no clear explanation of why the pathogenicity of some H5N1 HPAI viruses has increased. The nonstructural protein 1 (NS1 protein) is known to suppress immune responses in influenza virus-infected hosts affecting virus pathogenesis. In order to determine if the NS1 protein contributes to increased virulence in ducks, single-gene reassortant viruses were generated. Exchanging the NS genes from A/Ck/HK/220/97 (a virus that produces mild disease in ducks) and A/Dk/VN/201/05 (a very virulent virus for ducks) in the rEgret/02 background (a recombinant virus derived from A/Egret/HK/757.2/02, a highly pathogenic virus in ducks) resulted in decreased mean death times compared to infection with the rEgret/02 virus in ducks, but the change was not statistically significant. Infection with the reassortant viruses affected the expression of immune-related genes in spleens and lungs when compared to controls, but when compared among them, the expression of the duck genes was similar. Furthermore, virus titers in spleen, lung, and brain as well as antigen distribution in various tissues were similar in ducks infected with the reassortant viruses. All together these data show that, under these experimental conditions, exchanging the NS gene had minimal effect on the virus pathogenicity, and it suggests that other viral genes, or combination of genes, are most likely contributing to the increased virulence of H5N1 HPAI viruses in ducks.