Prevalence of mcr-1 in the Cecal Contents of Food Animals in the United States.

A survey of 2,003 cecal content samples from chickens, turkeys, cattle, and swine at slaughter facilities in the United States was conducted to estimate the prevalence of the mcr-1 gene conferring resistance to colistin in Enterobacteriaceae Two cecal samples from swine had Escherichia coli with IncI2 plasmids bearing the mcr-1 gene.

National Antimicrobial Resistance Monitoring System: Two Decades of Advancing Public Health Through Integrated Surveillance of Antimicrobial Resistance.

Drug-resistant bacterial infections pose a serious and growing public health threat globally. In this review, we describe the role of the National Antimicrobial Resistance Monitoring System (NARMS) in providing data that help address the resistance problem and show how such a program can have broad positive impacts on public health. NARMS was formed two decades ago to help assess the consequences to human health arising from the use of antimicrobial drugs in food animal production in the United States. A collaboration among the Centers for Disease Control and Prevention, the U.S. Food and Drug Administration, the United States Department of Agriculture, and state and local health departments, NARMS uses an integrated "One Health" approach to monitor antimicrobial resistance in enteric bacteria from humans, retail meat, and food animals. NARMS has adapted to changing needs and threats by expanding surveillance catchment areas, examining new isolate sources, adding bacteria, adjusting sampling schemes, and modifying antimicrobial agents tested. NARMS data are not only essential for ensuring that antimicrobial drugs approved for food animals are used in ways that are safe for human health but they also help address broader food safety priorities. NARMS surveillance, applied research studies, and outbreak isolate testing provide data on the emergence of drug-resistant enteric bacteria; genetic mechanisms underlying resistance; movement of bacterial populations among humans, food, and food animals; and sources and outcomes of resistant and susceptible infections. These data can be used to guide and evaluate the impact of science-based policies, regulatory actions, antimicrobial stewardship initiatives, and other public health efforts aimed at preserving drug effectiveness, improving patient outcomes, and preventing infections. Many improvements have been made to NARMS over time and the program will continue to adapt to address emerging resistance threats, changes in clinical diagnostic practices, and new technologies, such as whole genome sequencing.

Transcriptome changes in Mycoplasma hyopneumoniae during infection.

Mycoplasma hyopneumoniae causes swine pneumonia and contributes significantly to the porcine respiratory disease complex. The mechanisms of pathogenesis are difficult to address, since there is a lack of genetic tools, but microarrays are available and can be used to study transcriptional changes that occur during disease as a way to identify important virulence-related genes. Mycoplasmas were collected from bronchial alveolar lavage samples and compared to broth-grown cells using microarrays. Bronchial alveolar lavage was performed on pigs 28 days postinfection, and mycoplasmas were isolated by differential centrifugation. Mycoplasma RNA-enriched preparations were then obtained from total RNA by subtracting eucaryotic ribosomal and messenger RNAs. Labeled cDNAs were generated with mycoplasma open reading frame-specific primers. Nine biological replicates were analyzed. During lung infection, our analysis indicated that 79 M. hyopneumoniae genes were differentially expressed (P < 0.01), at a false-discovery rate of <2.7%. Of the down-regulated genes, 28 of 46 (61%) lacked an assigned function, in comparison to 21 of 33 (63%) of up-regulated genes. Four down-regulated genes and two up-regulated genes encoded putative lipoproteins. secA (mhp295) (P = 0.003) and two glycerol transport permease genes (potA [mhp380; P = 0.006] and ugpA [mhp381; P = 0.003]) were up-regulated in vivo. Elongation factor EF-G (fusA [mhp083]) (P = 0.002), RNA polymerase beta chain (rpoC [mhp635]) (P = 0.003), adenylate kinase (adk [mhp208]) (P = 0.001), prolyl aminoacyl tRNA synthetase (proS [mhp397]) (P = 0.009), and cysteinyl-tRNA synthetase (cysS [mhp661]) (P < 0.001) were down-regulated in vivo.

Real-time PCR assays to address genetic diversity among strains of Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae is an important cause of pneumonia in pigs around the world, but confirming its presence in (or absence from) pigs can be difficult. Culture for diagnosis is impractical, and seroconversion is often delayed after natural infection, limiting the use of serology. Numerous PCR assays for the detection of M. hyopneumoniae have been developed, targeting several different genes. Recently, genetic diversity among strains of M. hyopneumoniae was demonstrated. The effect of this diversity on the accuracy and sensitivity of the M. hyopneumoniae PCR assays could result in false-negative results in current PCR tests. In this study, a panel of isolates of M. hyopneumoniae, M. flocculare, M. hyorhinis, and M. hyosynoviae were tested with a number of M. hyopneumoniae-specific PCR assays. Some M. hyopneumoniae PCR assays tested did not detect all isolates of M. hyopneumoniae. To increase the efficiency of PCR testing, two new real-time PCR assays that are specific and capable of detecting all of the M. hyopneumoniae isolates used in this study were developed.

Comparison of three serological assays to determine the cross-reactivity of antibodies from eight genetically diverse U.S. swine influenza viruses.

Swine influenza virus is an economically important pathogen to the U.S. swine industry. New influenza subtypes and isolates within subtypes with different genetic and antigenic makeup have recently emerged in U.S. swineherds. As a result of the emergence of these new viruses, diagnosticians' ability to accurately diagnose influenza infection in pigs and develop appropriate vaccine strategies has become increasingly difficult. The current study compares the ability of subtype-specific commercial enzyme-linked immunosorbent assays (ELISA), hemagglutination inhibition (HI), and serum neutralization (SN) assays to detect antibodies elicited by multiple isolates within different subtypes of influenza virus. Pigs were infected with genetically and antigenically different isolates of the 3 major circulating subtypes within populations of swine (H1N1, H1N2, and H3N2). Serum was collected when all pigs within a group collectively reached HI reciprocal titers >or=160 against that group's homologous challenge virus. The antibody cross-reactivity of the sera between isolates was determined using ELISA, HI, and SN assays. In addition, the correlation between the 3 assays was determined. The assays differed in their ability to detect antibodies produced by the viruses used in the study. The results provide important information to diagnostic laboratories, veterinarians, and swine producers on the ability of 3 common serological assays used in identifying infection with influenza in pigs.

Array-based genomic comparative hybridization analysis of field strains of Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae is the causative agent of porcine enzootic pneumonia and a major factor in the porcine respiratory disease complex. A clear understanding of the mechanisms of pathogenesis does not exist, although it is clear that M. hyopneumoniae adheres to porcine ciliated epithelium by action of a protein called P97. Previous studies have shown variation in the gene encoding the P97 cilium adhesin in different strains of M. hyopneumoniae, but the extent of genetic variation among field strains across the genome is not known. Since M. hyopneumoniae is a worldwide problem, it is reasonable to expect that a wide range of genetic variability may exist given all of the different breeds and housing conditions. This variation may impact the overall virulence of a single strain. Using microarray technology, this study examined the potential variation of 14 field strains compared to strain 232, on which the array was based. Genomic DNA was obtained, amplified with TempliPhi, and labeled indirectly with Alexa dyes. After genomic hybridization, the arrays were scanned and data were analyzed using a linear statistical model. The results indicated that genetic variation could be detected in all 14 field strains but across different loci, suggesting that variation occurs throughout the genome. Fifty-nine percent of the variable loci were hypothetical genes. Twenty-two percent of the lipoprotein genes showed variation in at least one field strain. A permutation test identified a location in the M. hyopneumoniae genome where there is spatial clustering of variability between the field strains and strain 232.

Are swine workers in the United States at increased risk of infection with zoonotic influenza virus?

BACKGROUND: Pandemic influenza strains originate in nonhuman species. Pigs have an important role in interspecies transmission of the virus. We examined multiple swine-exposed human populations in the nation's number 1 swine-producing state for evidence of previous swine influenza virus infection. METHODS: We performed controlled, cross-sectional seroprevalence studies among 111 farmers, 97 meat processing workers, 65 veterinarians, and 79 control subjects using serum samples collected during the period of 2002-2004. Serum samples were tested using a hemagglutination inhibition assay against the following 6 influenza A virus isolates collected recently from pigs and humans: A/Swine/WI/238/97 (H1N1), A/Swine/WI/R33F/01 (H1N2), A/Swine/Minnesota/593/99 (H3N2), A/New Caledonia/20/99 (H1N1), A/Panama/2007/99 (H3N2), and A/Nanchang/933/95 (H3N2). RESULTS: Using multivariable proportional odds modeling, all 3 exposed study groups demonstrated markedly elevated titers against the H1N1 and H1N2 swine influenza virus isolates, compared with control subjects. Farmers had the strongest indication of exposure to swine H1N1 virus infection (odds ratio [OR], 35.3; 95% confidence interval [CI], 7.7-161.8), followed by veterinarians (OR, 17.8; 95% CI, 3.8-82.7), and meat processing workers (OR, 6.5; 95% CI, 1.4-29.5). Similarly, farmers had the highest odds for exposure to swine H1N2 virus (OR, 13.8; 95% CI, 5.4-35.4), followed by veterinarians (OR, 9.5; 95% CI, 3.6-24.6) and meat processing workers (OR, 2.7; 95% CI, 1.1-6.7). CONCLUSIONS: Occupational exposure to pigs greatly increases workers' risk of swine influenza virus infection. Swine workers should be included in pandemic surveillance and in antiviral and immunization strategies.

The immune response and maternal antibody interference to a heterologous H1N1 swine influenza virus infection following vaccination.

This study investigated the efficacy of a bivalent swine influenza virus (SIV) vaccine in piglets challenged with a heterologous H1N1 SIV isolate. The ability of maternally derived antibodies (MDA) to provide protection against a heterologous challenge and the impact MDA have on vaccine efficacy were also evaluated. Forty-eight MDA(+) pigs and 48 MDA(-) pigs were assigned to 8 different groups. Vaccinated pigs received two doses of a bivalent SIV vaccine at 3 and 5 weeks of age. The infected pigs were challenged at 7 weeks of age with an H1N1 SIV strain heterologous to the H1N1 vaccine strain. Clinical signs, rectal temperature, macroscopic and microscopic lesions, virus excretion, serum and local antibody responses, and influenza-specific T-cell responses were measured. The bivalent SIV vaccine induced a high serum hemagglutination-inhibition (HI) antibody titer against the vaccine virus, but antibodies cross-reacted at a lower level to the challenge virus. This study determined that low serum HI antibodies to a challenge virus induced by vaccination with a heterologous virus provided protection demonstrated by clinical protection and reduced pneumonia and viral excretion. The vaccine was able to prime the local SIV-specific antibody response in the lower respiratory tract as well as inducing a systemic SIV-specific memory T-cell response. MDA alone were capable of suppressing fever subsequent to infection, but other parameters showed reduced protection against infection compared to vaccination. The presence of MDA at vaccination negatively impacted vaccine efficacy as fever and clinical signs were prolonged, and unexpectedly, SIV-induced pneumonia was increased compared to pigs vaccinated in the absence of MDA. MDA also suppressed the serum antibody response and the induction of SIV-specific memory T-cells following vaccination. The results of this study question the effectiveness of the current practice of generating increased MDA levels through sow vaccination in protecting piglets against disease.

2006 AAHA canine vaccine guidelines.

In 2005, AAHA's Canine Vaccine Task Force met to reexamine and revise guidelines on the use of vaccines in dogs. The results of the Task Force's work are summarized and tabulated in this article and are published in their entirety on the AAHA website (www.aahanet.org). The 2006 AAHA Canine Vaccine Guidelines contain information on new technological developments in vaccines, an introduction to conditionally licensed vaccines, and detailed recommendations on the use of available vaccines. Perhaps the most noteworthy addition to the guidelines is a separate set of recommendations created for shelter facilities. Vaccines are classified as core (universally recommended), noncore (optional), or not recommended. The Task Force recognizes that vaccination decisions must always be made on an individual basis, based on risk and lifestyle factors.

Development of a reverse transcription-PCR assay to detect porcine circovirus type 2 transcription as a measure of replication.

Porcine circovirus type 2 (PCV2) is a non-enveloped, single-stranded, circular DNA virus. In situ hybridization and PCR assays have detected PCV2 DNA in multiple organs and cell types from infected pigs; however, it is not clear if this represents replicating virus or virion DNA. We describe the development of a single-tube RT-PCR assay to differentiate PCV2 replication products and virus DNA. Primers targeted to the open-reading frame 2 (ORF2) of PCV2 were designed to amplify both virus DNA (984 bp) and the spliced Cap mRNA (594 bp). The 984 bp fragment, but not the 594 bp fragment, was amplified from PCV2 stock, confirming that the spliced Cap mRNA was not present in the PCV2 stock. The 594 bp fragment was amplified from DNase-treated RNA extracted from PCV2-infected PK-15 cells, and was detected as early as 14 h post-infection. No products were amplified from either the PCV1 stock or PCV1-infected PK-15 cells, or from cells infected with UV-inactivated PCV2. Therefore, the presence of the 594 bp fragment is specific for PCV2 replication. This assay will be useful in assessing cell populations that support PCV2 replication in vivo or in vitro and advance the understanding of PCV2 replication and pathogenesis.

Interleukin-10, interleukin-12, and interferon-gamma levels in the respiratory tract following mycoplasma hyopneumoniae and PRRSV infection in pigs.

The cytokine profile associated with either a T helper 1 (Th1) or Th2 response in a porcine respiratory disease model was assessed by measuring IL-12, IL-10 and IFN-gamma using RT-PCR and ELISA, respectively. IL-10, IL-12, and IFN-gamma levels in pulmonary alveolar macrophages and bronchial lavage fluid were increased in pigs infected with porcine reproductive and respiratory syndrome virus (PRRSV), Mycoplasma hyopneumoniae, or both pathogens. At 10 days post-infection (DPI), both IL-10 and IL-12 mRNA levels were increased in both groups infected with PRRSV. The IL-12 levels were increased in pigs infected with both pathogens and IFN-gamma protein levels were increased in pigs infected with PRRSV alone and only numerically increased in the dual infection. At 28 DPI, IL-12 mRNA levels and IL-10 protein levels were increased in all infected groups. The mRNA level of IL-12 remained elevated in the group infected with both pathogens at 42 DPI. Production of IFN-gamma did not appear to be closely correlated with elimination of virus from the respiratory tract. However, when the virus existed in the lung, the local IFN-gamma production appeared to increase. Although IL-12 mRNA levels were significantly elevated in the pigs infected with both pathogens, the increased protein levels of IL-12 may compromise the immune system's ability to clear PRRSV from the lung. This could explain the prolonged presence of PRRSV, IFN-gamma production and the increased pneumonia observed in the lungs of dual-infected pigs. The increased levels of cytokines associated with both Th1 and Th2 responses in the respiratory tract of pigs infected with PRRSV and M. hyopneumoniae provides valuable information on the pathogenesis of these diseases.

Impact of immunizations with porcine reproductive and respiratory syndrome virus on lymphoproliferative recall responses of CD8+ T cells.

The objective of this study was to investigate the immune responses elicited by either a modified-live (MLV) or a killed virus (KV) porcine reproductive and respiratory syndrome virus (PRRSV) vaccine. Specifically, we investigated the effects of multiple vaccinations on antigen-specific cellular and antibody responses against PRRSV. Twelve sows were obtained from herds with either a history of repeated MLV or KV PRRSV vaccination and a non-vaccinated, PRRSV-negative herd. Within herd, sows were divided into three groups and vaccinated with MLV, KV, or injected with saline. On day 0, 27, and 38, recall responses of peripheral blood mononuclear cells (PBMC) to the parent strains of the vaccines (e.g., MLV-VR2332 or KV-ISUP) were examined. The concentrations of total PRRSV-specific and virus-neutralizing serum antibodies were determined by ELISA and serum neutralization assays. Following immunization, the antigen-specific proliferation of CD8alphabeta(+), CD4(+)CD8alphaalpha(+) T cells in the naive sows was greater than in sows repeatedly vaccinated with KV or MLV. This diminished lymphoproliferative responses of CD8alphabeta(+) and CD4(+)CD8alphaalpha(+) T cells could be partially overcome by heterologous immunization. However, B cell proliferation, PRRSV antibody concentrations and virus neutralizing antibody titers were not enhanced by heterologous immunization and only KV vaccination increased antibody levels in previously immunized (MLV or KV) sows.

Diagnosis of Mycoplasma hyopneumoniae.

Mycoplasma hyopneumoniae, the cause of enzootic pneumonia, remains an important pathogen in the swine industry. This small, complex organism colonizes the ciliated cells of the respiratory tract, resulting in little exposure to the immune system. Confirming the presence of M. hyopneumoniae, as well as identifying its role in respiratory disease and pneumonia, remains challenging to the veterinary profession. While culture of the organism remains the gold standard for identification, the use of serology, the polymerase chain reaction and various assays to detect the presence of M. hyopneumoniae in tissue is common in diagnostic laboratories. Because of the role M. hyopneumoniae plays in increasing the severity of pneumonia associated with concurrent bacterial and viral infections, understanding the pathogenesis and diagnostic assays available is critical for developing effective intervention strategies to control respiratory disease on a herd basis.

Use of a Mycoplasma hyopneumoniae nested polymerase chain reaction test to determine the optimal sampling sites in swine.

A number of polymerase chain reaction (PCR)-based diagnostic tests have been developed for Mycoplasma hyopneumoniae, including one from this research group. This report presents further development, optimization, and standardization of a nested PCR test. Detection sensitivity was 1 fg of M. hyopneumoniae chromosomal DNA (approximately 1 organism). This exceeded the sensitivity of or compared favorably with other published PCR tests. Polymerase chain reaction primers to porcine beta2-microglobulin were included as internal controls for amplifiable chromosomal DNA from porcine samples. To standardize the test, a number of samples from experimentally infected pigs, including nasal, tonsil, tracheobronchial swabs, lung tissue, bronchial alveolar lavage (BAL) fluid, and tracheobronchial brush samples, were examined by PCR. Samples obtained from BAL fluid and tracheobronchial sites were most predictive of infection, whereas nasal swabs and lung tissue were not reliable indicators of experimentally induced infection. In conclusion, the nested PCR developed for this study was found to be a highly sensitive and specific diagnostic tool for M. hyopneumoniae, but the enhanced sensitivity may be unnecessary if the proper sites are sampled.

Effectiveness of transdermal, needle-free injections for reducing pork carcass defects.

A needle-free, transdermal injection device was evaluated for effectiveness of vaccine delivery and for injection site lesions in swine. A total of 130 pigs were vaccinated for pseudorabies virus (PRV) and Mycoplasma hyopneumoniae (M. hyopneumoniae). Pigs were divided into three groups; one group served as unvaccinated controls, the second group was vaccinated with conventional hypodermic needles and the third group was vaccinated with a needle-free, airpowered transdermal injection device. Blood samples collected for up to 36 days post-injection showed that both injection methods produced similar serological responses that were significantly greater than for unvaccinated controls. Injection sites, collected at slaughter from each carcass, showed minimal development of lesions and no carcass defects. The results show the needle-free, transdermal injection system to be effective and safe. Elimination of needles will prevent residual needle fragments in carcasses and associated carcass defects that develop from needle-induced injection-site lesions.

Issues encountered in development of enzyme-linked immunosorbent assay for use in detecting Influenza A virus subtype H5N1 exposure in swine.

A potential mechanism by which highly pathogenic avian Influenza A virus subtype H5N1 could more readily infect human beings is through the infection of and adaptation in pigs. To detect the occurrence of such infection, monitoring of pig populations through serological screening would be highly desirable. In the current study, hemagglutination inhibition assays were able to detect antibodies against H5N1 developed in pigs, but because of antigenic variation between clades, the use of multiple virus strains were required. Whole recombinant virus and recombinant hemagglutinin antigen enzyme-linked immunosorbent assays (ELISAs) were generated that could detect antibody against multiple H5N1 strains, but which also detected antibody against endemic swine influenza viruses. A recombinant hemagglutinin antigen-based ELISA was as effective as the whole virus antigen ELISAs in detecting antibody against the H5N1 virus strains used and eliminated nearly all of the cross-reactivity with non-H5N1 virus antibody. The current study also highlighted the difficulty in establishing a decision (cutoff) value that would effectively counterbalance nonspecific reactivity against sensitivity. The results provide important information and considerations for the development of serological screening assays for highly pathogenic avian H5N1 viruses.

Antibody responses following vaccination versus infection in a porcine circovirus-type 2 (PCV2) disease model show distinct differences in virus neutralization and epitope recognition.

Porcine circovirus associated disease (PCVAD) encompasses a group of syndromes linked to infection with porcine circovirus type 2 (PCV2). Based on the hypothesis that the immune responses to vaccination versus infection are quantitatively and qualitatively different, the objective of this study was to evaluate immunity, virus replication and disease protection in pigs vaccinated with PCV2 capsid protein (CP) and during infection. The disease model included dual infection with PCV2 and porcine reproductive and respiratory syndrome virus (PRRSV), a virus known to enhance disease progression and severity. The principal effect of PRRSV infection was to increase peak PCV2 viremia by almost 40-fold; however, PCV2 failed to show a reciprocal effect on PRRSV. In vaccinated pigs, there was no evidence of disease or PCV2 replication following dual virus challenge. Immunity following vaccination favored PCV2 neutralizing activity; whereas, PCV2 infection and disease produced high levels of non-neutralizing antibody, primarily directed against a polypeptide in the C-terminal region of CP. These results support the notion that the magnitude of the total antibody response cannot be used as a measure of protective immunity. Furthermore, protection versus disease lies in the immunodominance of specific epitopes. Epitope specificity should be taken into consideration when designing PCV2 vaccines.

Immunomodulators, immunostimulants, and immunotherapies in small animal veterinary medicine.

Immunomodulators, immunostimulants, and immunotherapies are important tools used by veterinary practitioners and researchers to control and direct the immune system of small animals. This article is an overview and summary of some of the most common immunomodulatory agents used in companion animals emphasizing steroidal and nonsteroidal agents, T-cell inhibitors, cytotoxic drugs, immunostimulators and biologic response modifying agents, and neoplasia chemotherapeutic agents.

One Health approach to identify research needs in bovine and human babesioses: workshop report.

BACKGROUND: Babesia are emerging health threats to humans and animals in the United States. A collaborative effort of multiple disciplines to attain optimal health for people, animals and our environment, otherwise known as the One Health concept, was taken during a research workshop held in April 2009 to identify gaps in scientific knowledge regarding babesioses. The impetus for this analysis was the increased risk for outbreaks of bovine babesiosis, also known as Texas cattle fever, associated with the re-infestation of the U.S. by cattle fever ticks. RESULTS: The involvement of wildlife in the ecology of cattle fever ticks jeopardizes the ability of state and federal agencies to keep the national herd free of Texas cattle fever. Similarly, there has been a progressive increase in the number of cases of human babesiosis over the past 25 years due to an increase in the white-tailed deer population. Human babesiosis due to cattle-associated Babesia divergens and Babesia divergens-like organisms have begun to appear in residents of the United States. Research needs for human and bovine babesioses were identified and are presented herein. CONCLUSIONS: The translation of this research is expected to provide veterinary and public health systems with the tools to mitigate the impact of bovine and human babesioses. However, economic, political, and social commitments are urgently required, including increased national funding for animal and human Babesia research, to prevent the re-establishment of cattle fever ticks and the increasing problem of human babesiosis in the United States.