Detection and genetic characterization of porcine pegivirus in pigs in the United States.

Using next-generation sequencing on vesicular swab and serum from swine from the USA exhibiting lameness and vesicles, porcine pegivirus (PPgV) was first identified and genetically characterized in the United States. Further screening using RT-PCR revealed that 24 of 159 (15.1%) serum samples were positive for PPgV. Future studies are needed to understand clinical impacts of the virus.

Risk of exposure to Coxiella burnetii from ruminant livestock exhibited at Iowa agricultural fairs.

Coxiella burnetii is a zoonotic pathogen typically associated with clinical and asymptomatic infection in ruminant livestock. A re-emerging pathogen of significant public health importance, C. burnetii has caused recent epidemics in the United States and Europe, and public livestock exhibitions are increasingly scrutinized as a potential source of C. burnetii exposure. Although C. burnetii prevalence data among North American domestic ruminants are extremely limited, contemporary studies suggest that this pathogen is both geographically widespread and highly prevalent on a herd basis, especially in dairy cattle and goat populations. We utilized a real-time PCR assay to detect C. burnetii faecal shedding by clinically normal, non-periparturient beef cattle, meat goats and sheep exhibited at Iowa agricultural fairs. Individual faecal samples were collected from beef cattle, meat goats and sheep exhibited at twelve Iowa county fairs during the summer of 2009. The sample pool was blocked by species and fair, and ten samples from each block were randomly selected for the diagnostic assay; this test pool is considered sufficient to identify with 95% confidence a shedding animal in a population prevalence of 2.85% (cattle and sheep) and 6.25% (goats). Detection of C. burnetii DNA was determined through use of a real-time PCR assay validated for use in bovine, ovine and caprine faeces; threshold of detection is one DNA copy per PCR (sensitivity 95.8%, specificity 100%). All tested samples were negative for C. burnetii DNA. We conclude that non-dairy, non-periparturient ruminants exhibited at Iowa fairs are unlikely to shed C. burnetii in their faeces and that this population should not be considered to be a significant exposure risk to other livestock or fair attendees.

Pathogenesis of porcine epidemic diarrhea virus isolate (US/Iowa/18984/2013) in 3-week-old weaned pigs.

Porcine epidemic diarrhea virus (PEDV) is associated with clinical diarrhea in naïve swine of all ages. This report describes timing of antibody generation and disease progression following infection with a US PEDV isolate by assessing fecal viral shedding, morphometric analysis of intestinal lesions, and magnitude of immunohistochemical staining. Sixty-three, 3-week-old pigs were randomly allocated into control (n=27) and challenged (n=36) groups. Challenged pigs were administered 1 mL of 1 × 10(3) PFU/mL of US/Iowa/18984/2013 PEDV isolate by oro-gastric gavage. Three control and four challenged pigs were necropsied on days post-inoculation (dpi) 1, 2, 3, 4, 7, and weekly thereafter, until study termination on dpi 35. Clinical disease, fecal shedding, body weight, and temperature were monitored during the study period. Diarrhea was observed in challenged pigs beginning for some on dpi 2, affecting a majority of pigs by dpi 6 and subsiding by dpi 10. Average daily gain was significantly lower (P<0.001) for one week post-infection in challenged pigs. PEDV was detected in feces by PCR on dpi 1 and continued in a subset of pigs until dpi 24. PEDV-specific antigen was detected in villous enterocytes of challenged pigs by immunohistochemistry (IHC) on dpi 1, 2, 3, 4, 7, and 14. Microscopic lesions included severe diffuse atrophic enteritis with significantly reduced (P<0.001) villous length observed on dpi 3, 4, and 7. Under the conditions of this study, fecal shedding of PEDV and IHC staining can precede and continue beyond the observation of clinical signs, thus increasing the risk of viral transmission.

Selective breeding for 50 kHz ultrasonic vocalization emission produces alterations in the ontogeny and regulation of rough-and-tumble play.

Ultrasonic vocalizations (USVs) are emitted by rodents and can signal either negative or positive affective states in social and nonsocial contexts. Our recent work has utilized selective breeding based upon the emission of 50 kHz USVs in response to standard cross species hand play-namely experimenters 'tickling' rats. Previous work has shown that high-tickle responsive animals (i.e., rats emitting abundant 50 kHz USVs) are gregarious and express enhanced positive emotional behaviors relative to animals exhibiting low 50 kHz USVs. The present study extends this work by examining the developmental profile of play behavior and the suppression of play behavior by predator (cat) odor in juvenile high-line and low-line animals. Results support dissociations in key play measures between these groups, with high-line animals emitting more dorsal contacts during play and low-line animals emitting more pinning behavior. For cat-odor induced play suppression, we found that high-line animals exhibit elevated suppression of play for a prolonged period compared to low-line rats. In contrast, low-line animals returned to normal levels of play just 1 day post-predator odor experience. These findings support the idea that emotional arousal may differ between these selectively bred groups, and extends previous work by demonstrating a possible influence of altered emotional learning and conditioning in these phenotypically different animals. One possibility is that high-line animals exhibit enhanced associative learning abilities leading to stronger negative contextual conditioning. These findings suggest that selection for positive or negative social-emotional phenotypes may also segregate genes that control emotional learning abilities in unanticipated ways.

Rats selectively bred for low levels of 50 kHz ultrasonic vocalizations exhibit alterations in early social motivation.

In rats, the rates of 50 kHz ultrasonic vocalizations (USVs) can be used as a selective breeding phenotype and variations in this phenotype can be an indicator of affective states. The 50 kHz USV is elicited by rewarding stimuli (e.g., food, sexual behavior) and therefore can express a positive affective state. Conversely, the 22 kHz USV is elicited by aversive stimuli (e.g., presence of a predator, social defeat) indicating a negative affective state. In the present study, we tested the effect of selectively breeding for 50 kHz USVs on a variety of maternal social/emotional behaviors in young rat pups (PND 10-12). These measures consisted of an assessment of isolation calls and conditioned odor preference paradigm. Results indicate that animals selected for low levels of 50 kHz USVs show the greatest alterations in social behaviors compared to the control animals. The low line animals had an increase in isolation calls tested during place preference conditioning and a decrease in 50 kHz ultrasonic calls in all conditions. These same low line animals failed to show a typical preference for a maternally-associated odor during the place preference test. The different social behaviors of the high line animals did not consistently vary from those of the control group. These results have important implications for the study of genetic and epigenetic mechanisms underlying emotional states, and possibly contribute to the research underlying the emotional changes in developmental disorders such as autistic spectrum disorder by providing a novel animal model that displays communication deficits that are interdependent with significant social behavioral impairments.

Comparison of molecular and biological characteristics of a modified live porcine reproductive and respiratory syndrome virus (PRRSV) vaccine (ingelvac PRRS MLV), the parent strain of the vaccine (ATCC VR2332), ATCC VR2385, and two recent field isolates of PRRSV.

The objectives of this study were to compare the molecular and biological characteristics of recent porcine reproductive and respiratory syndrome virus (PRRSV) field isolates to those of a modified live virus (MLV) PRRS vaccine and its parent strain. One hundred seventeen, 4-week-old pigs were randomly assigned to six groups. Group 1 (n = 20) served as sham-inoculated negative controls, group 2 (n = 19) was inoculated with Ingelvac PRRS MLV vaccine, group 3 (n = 20) was inoculated with the parent strain of the vaccine (ATCC VR2332), group 4 (n = 19) was inoculated with vaccine-like PRRSV field isolate 98-38803, group 5 (n = 19) was inoculated with PRRSV field isolate 98-37120, and group 6 (n = 20) was inoculated with known high-virulence PRRSV isolate ATCC VR2385. The levels of severity of gross lung lesions (0 to 100%) among the groups were significantly different at both 10 (P < 0.0001) and 28 days postinoculation (p.i.) (P = 0.002). At 10 days p.i., VR2332 (26.5% +/- 4.64%) and VR2385 (36.4% +/- 6.51%) induced gross lesions of significantly greater severity than 98-38803 (0.0% +/- 0.0%), 98-37120 (0.8% +/- 0.42%), Ingelvac PRRS MLV (0.9% +/- 0.46%), and negative controls (2.3% +/- 1.26%). At 28 days p.i., 98-37120 (17.2% +/- 6.51%) induced gross lesions of significantly greater severity than any of the other viruses. Analyses of the microscopic-interstitial-pneumonia-lesion scores (0 to 6) revealed that VR2332 (2.9 +/- 0.23) and VR2385 (3.1 +/- 0.35) induced significantly more severe lesions at 10 days p.i. At 28 days p.i., VR2385 (2.5 +/- 0.27), VR2332 (2.3 +/- 0.21), 98-38803 (2.6 +/- 0.29), and 98-37120 (3.0 +/- 0.41) induced significantly more severe lesions than Ingelvac PRRS MLV (0.7 +/- 0.17) and controls (0.7 +/- 0.15). The molecular analyses and biological characterizations suggest that the vaccine-like isolate 98-38803 (99.5% amino acid homology based on the ORF5 gene) induces microscopic pneumonia lesions similar in type to, but different in severity and time of onset from, those observed with virulent strains VR2385 and the parent strain of the vaccine. Our data strongly suggest that isolate 98-38803 is a derivative of Ingelvac PRRS MLV and that the isolate is pneumovirulent.

Evolution of porcine reproductive and respiratory syndrome virus during sequential passages in pigs.

Porcine reproductive and respiratory syndrome (PRRS) viruses are recognized as possessing a high degree of genetic and antigenic variability. Viral diversity has led to questions regarding the association of virus mutation and persistent infection in the host and has raised concerns vis-à-vis protective immunity, the ability of diagnostic assays to detect novel variants, and the possible emergence of virulent strains. The purpose of this study was to describe ongoing changes in PRRS virus during replication in pigs under experimental conditions. Animals were inoculated with a plaque-cloned virus derived from VR-2332, the North American PRRS virus prototype. Three independent lines of in vivo replication were maintained for 367 days by pig-to-pig passage of virus at 60-day intervals. A total of 315 plaque-cloned viruses were recovered from 21 pigs over the 367-day observation period and compared to the original plaque-cloned virus by virus neutralization assay, monoclonal antibody analysis, and sequencing of open reading frames (ORFs) 1b (replicase), 5 (major envelope protein), and 7 (nucleocapsid) of the genome. Variants were detected by day 7 postinoculation, and multiple variants were present concurrently in every pig sampled over the observation period. Sequence analysis showed ORFs 1b and 7 to be highly conserved. In contrast, sequencing of ORF 5 disclosed 48 nucleotide variants which corresponded to 22 amino acid variants. Although no epitopic changes were detected under the conditions of this experiment, PRRS virus was shown to evolve continuously in infected pigs, with different genes of the viral genome undergoing various degrees of change.

Fecal shedding of Campylobacter and Arcobacter spp. in dairy cattle.

Campylobacter jejuni, Campylobacter coli, and Arcobacter spp. were detected in feces of healthy dairy cows by highly specific multiplex-PCR assays. For C. jejuni, at this one-time sampling, cows from 80.6% of farm operations (n = 31) and 37.7% of individual dairy cattle fecal samples (n = 2,085) were positive. Farm management factors were correlated with prevalence in herds in which >25% of cows were positive for C. jejuni. Statistical significance was set at a P of 0.20. Using these criteria, application of manure with broadcast spreaders (P = 0.17), feeding of whole cottonseed or hulls (P = 0.17) or alfalfa (P = 0.15), and accessibility of feed to birds (P = 0.17) were identified as possible risk factors for C. jejuni infection. C. coli was detected in at least one animal in 19.4% of operations and 1.8% of individual cows (n = 2,085). At the herd level, use of broadcaster spreaders was not a risk factor for C. coli infection. For Arcobacter, cows from 71% of dairy operations (n = 31) and 14.3% of individual dairy cattle fecal samples (n = 1,682) were positive. At the herd level, for Arcobacter spp., feeding of alfalfa (P = 0.11) and use of individual waterers (P = 0.19) were protective. This is the first description of Arcobacter spp. in clinically healthy dairy cattle and the first attempt to correlate their presence with C. jejuni.

Effect of challenge dose and route on porcine reproductive and respiratory syndrome virus (PRRSV) infection in young swine.

Porcine reproductive and respiratory syndrome virus (PRRSV) is perceived to be highly infectious because of the rapid spread of the virus through populations of domestic swine throughout the world. However, no information has been published on the minimum infectious dose of PRRSV and the effect of challenge dose on clinical response. In this experiment, ten groups of pigs (n = 3 per group) were inoculated with one of five different quantities (10(1)-10(5) fluorescent foci units per millilitre) of PRRSV (isolate ISU-P) by either intramuscular or intranasal routes. Clinical signs and body temperature were monitored for 21 days. Serum was collected periodically throughout the study period to monitor the presence of virus in serum and the early immune response of pigs. A 2-mL inoculum containing 10(1) fluorescent foci units of virus per millilitre was found sufficient to achieve infection by either route. Time to onset of clinical signs was highly associated with challenge dose (P < 0.01), regardless of route of exposure. However, no dose- or route-dependent differences in the severity of clinical manifestation were observed. No significant differences in the time of onset or degree of humoural immune response to PRRSV infection were observed between different treatment groups. However, intramuscular exposure appeared to induce a more uniform antibody response compared to intranasal exposure. These results confirmed that PRRSV is highly infectious; a fact that should be taken into consideration when designing strategies for the prevention and control of PRRSV.

Antigenic and genetic variations of the 15 kD nucleocapsid protein of porcine reproductive and respiratory syndrome virus isolates.

The antigenic variability of the 15 kD nucleocapsid protein of porcine reproductive and respiratory syndrome (PRRS) virus was characterized with a panel of 24 monoclonal antibodies (MAbs) raised against the American PRRS virus isolate ISU-P. Five continuous epitopes designated EpORF7-A through E were revealed by the reactivity pattern of these MAbs with 67 American field isolates, two modified-live vaccine viruses, and the European Lelystad virus as determined by the indirect immnofluorescence assay and Western immunoblotting and confirmed by additivity and blocking enzyme-linked immunosorbent assays. The reactivity pattern of isolates in the IFA permitted their subdivision into 4 American antigenic groups which represented 84.1, 11.6, 2.9 and 1.4% of viruses tested. The antigenic variation among isolates was correlated to single, group specific nucleotide substitutions and mediated by a combination of at least 4 of the 5 epitopes. EpORF7-A was conserved in all American isolates and the Lelystad virus which constituted a separate antigenic group. Consequently, monoclonal antibodies specific for EpORF7-A may prove useful as the antigenic basis for a universal diagnostic test for the PRRS virus. EpORF7-C, D and E were only present in the American isolates tested.

Prevalence and genetic variability of Arcobacter species in mechanically separated turkey.

A survey for Arcobacter spp. and Arcobacter butzleri in mechanically separated turkey was conducted during the winter of 1995 and summer and fall of 1996. Arcobacter spp. and A. butzleri were identified by polymerase chain reaction and species-specific oligonucleotide probes. Arcobacter spp. were isolated from 77% (303 out of 395) of the mechanically separated turkey samples with 74% (223 out of 303) of these samples positive for A. butzleri. Of the 121 A. butzleri isolates tested, 86 different patterns were evident following amplification of enterobacterial repetitive intergenic consensus sequences. The extent of genetic polymorphism indicated multiple sources of contamination.

Application of multiplex polymerase chain reaction for rapid identification of Campylobacter jejuni and C coli associated with reproductive failure.

OBJECTIVE: To evaluate a multiplex polymerase chain reaction (PCR) to distinguish Campylobacter jejuni from C coli as causes of reproductive failure. PROCEDURE: Review of clinical cases of reproductive failure attributed to C jejuni or C coli. RESULTS: A case of swine abortion was attributable to infection with C coli. The porcine abortion isolates were verified as C coli by restriction fragment length polymorphism and multiplex PCR. Cases of endometritis in a fox and in mink caused by C jejuni were reviewed, and isolates were confirmed as C jejuni by results of the multiplex PCR. CONCLUSION: Multiplex PCR was useful in identifying C coli and C jejuni recovered from atypical cases of reproductive failure. Multiplex PCR in conjunction with conventional assays may be useful for verifying other unusual instances of campylobacteriosis.

Differentiation of Campylobacter jejuni and Campylobacter coli by polymerase chain reaction.

A multiplex PCR assay was developed using two primer sets for the identification and differentiation of Campylobacter coli and Campylobacter jejuni. Primer Set I amplifies a 460-bp fragment present in C. coli and C. jejuni. Set II amplifies a 160-bp target unique to C. jejuni. When the assay was performed on reference strains, amplification of C. coli yielded only the 460-bp fragment. Amplification of C. jejuni generated both the 160- and 460-bp fragments. Campylobacter field strains (n = 85) isolated from raw poultry were identified by PCR and by conventional biochemical methods. Species determination by the two methods agreed for 83 of the 85 isolates examined. By PCR, 23 were identified as C. coli and 62 as C. jejuni. One isolate was unidentifiable by biochemical testing. The PCR assay identified this isolated as C. coli. In addition, one strain which was identified as C. coli by biochemical testing was determined to be C. jejuni by PCR. The PCR assay offers an alternative to traditional biochemical typing methods for the identification and differentiation of C. coli and C. jejuni isolated from poultry. It is accurate, simple to perform, and can be completed within 8 h.

Specific identification of Campylobacter fetus by PCR targeting variable regions of the 16S rDNA.

Campylobacter fetus is recognized as a human and animal pathogen. The isolation and differentiation of C. fetus in diagnostic laboratories is hindered by its relatively slow growth and lack of distinguishing biochemical characteristics. We developed a fast, reliable PCR assay that specifically amplifies a 554-bp segment of the 16S rDNA from C. fetus. Fifty-two ATCC reference strains and 255 bacterial field isolates comprising the genera Campylobacter, Arcobacter, Helicobacter, Escherichia, Listeria, Salmonella, and Wolinella were evaluated using this PCR protocol. Only C. fetus strains were amplified. Sequence analysis of amplicons from ATCC and field strains of C. fetus confirmed the presence of the target DNA fragment. The detection limit of the technique was 5.9 x 10(3) CFU/ml. This PCR assay can yield reliable detection of C. fetus within 3 h after isolation of presumptive colonies on agar plates.

Multiplex PCR for the identification of Arcobacter and differentiation of Arcobacter butzleri from other arcobacters.

A multiplex polymerase chain reaction (PCR) assay to identify Arcobacter isolates and to distinguish A. butzleri from other arcobacters is described. The test uses two primer sets. Set I targets a section of the 16S rRNA genes of Arcobacter spp. Set II amplifies a portion of the 23S rRNA genes unique to A. butzleri. Specificity of the primer sets was evaluated using ATCC reference strains of A. butzleri, A. cryaerophilus, A. skirrowii, Bacteroides spp., Campylobacter spp., Helicobacter spp. and Wolinella succinogenes. Upon PCR amplification, all of the Arcobacter isolates yielded a 1223 bp product, whereas A. butzleri ATCC 49616 exhibited both a 1223 bp and a 686 bp product. No PCR product was observed for other closely related ATCC strains (n = 37). We next analyzed by multiplex PCR field strains of Arcobacter spp. (n = 108) which had been previously characterized to the species level by either DNA-DNA hybridization, dot blot hybridization, ribotyping or by serology. The 1223 bp multiplex PCR product identified all of the isolates as Arcobacter. The presence of both the 1223 and 686 bp amplicons identified 66 strains as A. butzleri. Speciation by multiplex PCR agreed with results obtained by the other methods. The multiplex PCR assay is specific, rapid and easy to interpret and, thus, will aid in elucidating the prevalence, epidemiology and zoonotic potential of Arcobacter.

Cumulative trauma disorders among hand therapists.

OBJECTIVES: This study examined the prevalence of cumulative trauma disorders (CTDs) among hand therapists. Factors such as hand therapy tasks and number of years spent performing these tasks were examined in their contribution to CTD symptoms of the upper extremity. STUDY DESIGN: Of the 356 questionnaires distributed to registrants at the 1996 Hand Conference, 195 (55%) were returned. A t-test was used to determine whether the number of years spent practicing hand therapy was a significant factor in the development of CTD symptoms among hand therapists. RESULTS: Of the 195 respondents, 73% reported they had previously experienced CTD symptoms, and 46% reported they were currently experiencing CTD symptoms. A significant difference was found (P < 0.05) in the development of CTD symptoms with regard to number of years practiced. CONCLUSION: The work practices of hand therapists place them at risk for developing CTDs. Hand therapists who spend more years practicing are more likely to incur CTDs.

Classification of Arcobacter species isolated from aborted pig fetuses and sows with reproductive problems in Brazil.

Seventeen field isolates of Arcobacter species were recovered in Brazil from aborted porcine fetal livers (n = 3), kidneys (n = 2), and thoracic fluid (n = 1). Arcobacter species were also recovered from uterine and oviductal tissues (n = 5) and a placenta from sows with reproductive problems. These isolates were initially presumed to be Arcobacter cryaerophilus on the basis of aerobic growth at 30 degree C, indoxyl acetate hydrolysis, catalase and oxidase reactions, growth on MacConkey agar, sensitivity to 3.5% sodium chloride, and susceptibility to nalidixic acid (40 mg/ml). The isolates were confirmed as Arcobacter using polymerase chain reaction, and were classified as A. cryaerophilus 1A (24%), A. cryaerophilus 1B (71%), and A. butzleri (6%) using restriction fragment length polymorphism.

Identification of Arcobacter isolates by PCR.

A polymerase chain reaction (PCR) assay was developed for the identification of the three species of Arcobacter which have been recovered from clinically ill or healthy humans and/or livestock, namely Arcobacter butzleri, Arcobacter skirrowii and Arcobacter cryaerophilus. The assay utilizes primers targeted to the genes encoding 16S rRNA of Arcobacter spp. The assay reduces the amount of time required to positively identify strains of Arcobacter.