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.

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.

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.

Swine influenza matrix 2 (M2) protein contributes to protection against infection with different H1 swine influenza virus (SIV) isolates.

A swine influenza virus (SIV) vaccine-challenge pig model was used to study the potential of a conserved matrix 2 (M2) protein vaccine alone or in combination with an inactivated H1N1-vaccine to protect against H1N1 and H1N2 viruses. The H1N1-vaccine and heterologous H1N2-challenge virus model has previously been shown to prolong fever and increase SIV-associated pneumonic lesions. The M2 vaccine in combination with the H1N1-vaccine reduced the H1N2 induced fever but not virus shedding. The M2 vaccine alone reduced respiratory signs and pneumonic lesions to levels similar to the negative control pigs following H1N2 infection. This study found that the M2 protein has potential as a vaccine for SIV-associated disease prevention. However, development of an immune response towards the major envelope HA protein was required to reduce SIV shedding.

Vaccine efficacy and immune response to swine influenza virus challenge in pigs infected with porcine reproductive and respiratory syndrome virus at the time of SIV vaccination.

The objective of this study was to assess the effect of concurrent infection with porcine reproductive and respiratory syndrome virus (PRRSV) on the efficacy of an inactivated swine influenza virus (SIV) vaccine. Eight groups of pigs were infected with a virulent PRRSV isolate either between the two SIV vaccines or at the time of SIV challenge. Control groups included SIV vaccination without PRRSV and pigs infected with SIV and/or PRRSV. Pigs infected with PRRSV during vaccination showed increased levels of macroscopic and microscopic lesions compared to pigs vaccinated against and challenged with only SIV indicating decreased SIV vaccine efficacy. In addition, pigs vaccinated in the presence of PRRSV showed increased clinical disease and shedding of SIV during the acute phase of SIV infection. No alterations in the systemic or local antibody response to either SIV vaccination or challenge were observed. These findings demonstrate that PRRSV infection has a significant impact on SIV vaccine efficacy that may be important for disease control.

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.

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.

Swine influenza virus: zoonotic potential and vaccination strategies for the control of avian and swine influenzas.

Influenza viruses are able to infect humans, swine, and avian species, and swine have long been considered a potential source of new influenza viruses that can infect humans. Swine have receptors to which both avian and mammalian influenza viruses bind, which increases the potential for viruses to exchange genetic sequences and produce new reassortant viruses in swine. A number of genetically diverse viruses are circulating in swine herds throughout the world and are a major cause of concern to the swine industry. Control of swine influenza is primarily through the vaccination of sows, to protect young pigs through maternally derived antibodies. However, influenza viruses continue to circulate in pigs after the decay of maternal antibodies, providing a continuing source of virus on a herd basis. Measures to control avian influenza in commercial poultry operations are dictated by the virulence of the virus. Detection of a highly pathogenic avian influenza (HPAI) virus results in immediate elimination of the flock. Low-pathogenic avian influenza viruses are controlled through vaccination, which is done primarily in turkey flocks. Maintenance of the current HPAI virus-free status of poultry in the United States is through constant surveillance of poultry flocks. Although current influenza vaccines for poultry and swine are inactivated and adjuvanted, ongoing research into the development of newer vaccines, such as DNA, live-virus, or vectored vaccines, is being done. Control of influenza virus infection in poultry and swine is critical to the reduction of potential cross-species adaptation and spread of influenza viruses, which will minimize the risk of animals being the source of the next pandemic.

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.

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.

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.

Lung inflammatory responses.

Inflammation is an important manifestation of respiratory disease in domestic animals. The respiratory system is mucosal in nature and has specific defense mechanisms used to control invasion by microbes and environmental elements. Inflammation can be beneficial or detrimental to the host. This article broadly discusses the primary mediators and mechanisms of inflammation within the respiratory tract of domestic animals. The role of cells, chemokines, cytokines and mediators in both acute and chronic inflammation are addressed. The pathogenesis of the initial insult determines the type of inflammation that will be induced, whether it is acute, chronic or allergic in origin. Maintenance of the microenvironment of cytokines and chemokines is critical for pulmonary homeostasis. Uncontrolled inflammation in the respiratory tract can be life threatening to the animal. The understanding of the mechanisms of inflammation, whether due to microbes or through inappropriate immune activation such as those occurring with allergies, is required to develop successful intervention strategies and control respiratory disease in animals.

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.

Transcriptional profiling of Mycoplasma hyopneumoniae during iron depletion using microarrays.

Mycoplasma hyopneumoniae, the causative agent of swine enzootic pneumonia and a major component of the porcine respiratory disease complex, continues to confound swine producers despite control programmes worldwide. The disease is chronic and self-limiting, but the host is subject to immunopathological changes that potentiate respiratory disease associated with other pathogens. The response of M. hyopneumoniae to environmental stress is of interest because of its relevance to virulence mechanisms in other bacterial pathogens. One of these stressors, iron deprivation, is a prominent feature of the host innate immune response, and most certainly impacts growth of mycoplasmas in vivo. To study this, microarray technology was applied to the transcriptome analysis of M. hyopneumoniae during iron deprivation. An array consisting of 632 of the 698 ORFs in the genome was used to compare the mRNA isolated from organisms grown under normal laboratory conditions with that from organisms subjected to iron deprivation with the chelator 2,2'-dipyridyl. This analysis identified 27 genes that were either up- or down-regulated in response to low-iron growth conditions (P<0.01), with an estimated false discovery rate below 10 %. These included genes encoding transport proteins, enzymes involved in energy metabolism, and components of the translation process. Ten of the 27 identified genes had no assigned function. These studies indicate that M. hyopneumoniae can respond to changes in environmental conditions, but the mechanism employed remains unknown.

Transcriptional profiling of Mycoplasma hyopneumoniae during heat shock using microarrays.

Bacterial pathogens undergo stress during host colonization and disease processes. These stresses result in changes in gene expression to compensate for potentially lethal environments developed in the host during disease. Mycoplasma hyopneumoniae colonizes the swine epithelium and causes a pneumonia that predisposes the host to enhanced disease from other pathogens. How M. hyopneumoniae responds to changing environments in the respiratory tract during disease progression is not known. In fact, little is known concerning the capabilities of mycoplasmas to respond to changing growth environments. With limited genes, mycoplasmas are thought to possess only a few mechanisms for gene regulation. A microarray consisting of 632 of the 698 open reading frames of M. hyopneumoniae was constructed and used to study gene expression differences during a temperature shift from 37 degrees C to 42 degrees C, a temperature swing that might be encountered during disease. To enhance sensitivity, a unique hexamer primer set was employed for generating cDNA from only mRNA species. Our analysis identified 91 genes that had significant transcriptional differences in response to heat shock conditions (P < 0.01) with an estimated false-discovery rate of 4 percent. Thirty-three genes had a change threshold of 1.5-fold or greater. Many of the heat shock proteins previously characterized in other bacteria were identified as significant in this study as well. A proportion of the identified genes (54 of 91) currently have no assigned function.

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.

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.