Topic: Bacterial Disease Presenter: Anderson, Alyssa Abstract #: 1 Title: Improvement of Mycoplasma hyopneumoniae Culture and Isolation Focusing on Sample Selection and Handling Authors: Anderson, Alyssa,1 Dalquist, L.,2 Leuwerke, B.,2 Sponheim, A.,3 Fano, E.,3 Pieters, M.1 1Veterinary Population Medicine Department, College of Veterinary Medicine, University of Minnesota, St. Paul, MN. 2Swine Veterinary Center, St. Peter, MN. 3Boehringer Ingelheim Vetmedica, Inc., St. Joseph, MO, USA
Introduction Historically, Mycoplasma hyopneumoniae (Mhp) is known to be challenging to culture and to isolate from tissue samples of infected pigs. Successful culture and isolation of Mhp is influenced by many factors, such as specialized media, sample bacterial load, and presence of other bacteria in the sample. The most recent reported success rate for Mhp culture was 8%. Despite the bacterium’s slow and complex growth, there has been an increased interest towards obtaining isolates currently circulating in the field for Mhp control measures, including antibiotic sensitivity testing. Therefore, the objective of the study was to develop a protocol for sample selection and handling for successful Mhp culture and isolation from field samples.
Materials and Methods Herds for the study were chosen based on a criterion, which consisted of having a history of Mhp clinical activity, observation of clinical signs suggestive of Mhp infection at the time of sampling, and documentation of Mhp positive lung tissues by qPCR. Four continuous flow farms with growing pigs or gilts were enrolled in the study. A pre-screening protocol was implemented that consisted of two stages: 1) Identification of Mhp infected pigs and 2) Final pig selection for sample collection. The first stage consisted of collecting laryngeal swabs from 10 gilts/growing pigs per age group (16 to 29 weeks; 2-3 ages; cross sectional) 3 to 5 weeks after the onset of Mhp clinical signs. The second stage consisted of selecting 2 to 5 Mhp positive pigs expressing clinical signs suggestive of infection, such as labored breathing, a dry cough, and having the lowest qPCR Cq value from the previous laryngeal sampling. Selected pigs were humanely euthanized and necropsied. Macroscopic lesions suggestive of enzootic pneumonia, such as cranio-ventral consolidation on the apical and cardiac lung lobes, were assessed. Entire plucks were collected from pigs with characteristic lesions and immediately stored on ice until transported to a -20oC freezer. Within 24 hours of collection, the frozen lungs were transported to the Mycoplasma Laboratory, University of Minnesota Veterinary Diagnostic Laboratory to be processed for Mhp culture and isolation. Upon arrival at the laboratory, frozen lungs were thawed at 4oC. Mhp culture using modified Friis broth was attempted and growth was observed by the presence of an acid shift in pH with no turbidity. After various passages, culture was verified Mhp positive using qPCR prior to single colony cloning. Mhp isolation was confirmed using 16s sequencing in cloned colonies.
Results Pigs chosen to be necropsied for Mhp culture and isolation presented laryngeal swab Cq values ranging from 28.48 to 35.91, averaging a 31.25 Cq value. Of the lung samples collected from each farm, Mhp culture success rate ranged from 25 to 100%. Average pig age (weeks) and Cq values for laryngeal and bronchial swabs in which a Mhp culture was obtained was 19 weeks, 30.97, and 21.33 respectively. At least one Mhp isolate was obtained from each of the four farms, resulting in an overall isolation success rate of 100%.
Under the conditions of the investigation, Mhp culture and isolation success rate was 100% among all farms from the first sample collection attempt. The improvement of Mhp culture and isolation was accomplished through the use of diagnostic sampling and assessing clinical signs in naturally infected swine, combined with sample freezing and thawing prior to processing. In the future, this pre-screening protocol can be used as a guideline in order to obtain tissue samples with an essential bacterial load for successful Mhp culture and isolation.
Topic: Bacterial Disease Presenter: Fano, Eduardo Abstract #: 2 Title: Mycoplasma hyopneumoniae Molecular Characterization and Analysis Tools to Understand Transmission Patterns Within and Between Endeminc Swine Populations Authors: Laura Dalquist,1 Amanda Sponheim,2,3 Eduardo Fano,2 Alyssa Anderson,3 Maria Pieters3; 1Swine Vet Center, MN, USA; 2Boehringer Ingelheim Vetmedica, Inc., MO, USA; 3University of Minnesota, MN, USA
Introduction In the midst of increasing interest in control and elimination of Mycoplasma hyopneumoniae (Mhp) in the field, a need to better understand the epidemiology of this pathogen was recognized. Epidemiologic questions of interest included evidence of Infection Chain™ versus lateral transmission in within flow and identification of number variants within one site, flow and system. Several new tools have become readily available for molecular characterization and analysis of Mycoplasma hyopneumoniae (Mhp) that have assisted in answering these questions.
Materials and Methods To begin documentation of Mhp infection and exposure between production stages, samples from known Mhp positive gilts, sows, nursery pigs and finishing pigs within the same flow were targeted for laryngeal swab sampling. Samples from surrounding sites, with no flow similarity to the target sites, were requested to be included in the project. Ten pigs were sampled per production stage, targeting clinical signs associated with Mhp, including dry coughing and labored breathing. Pigs recently treated for Mhp infection were not sampled. In order to document lateral spread, a minimum of 20 production flows were planned to be targeted with at least 100 samples analyzed. Mhp PCR positive laryngeal swab samples with a Ct ≤ 32 were sent to the University of Minnesota VDL for P146 full sequence anlaysis and to the Mycoplasma Lab, University of Minnesota for MLVA analysis. P146 full sequences and MLVA typing for each variant were uploaded into the Disease BioPortal (CADMS, UC-Davis), along with the corresponding site information for temporal-spatial genomic analysis. MLVA typing analysis is currently in development in the Disease BioPortal.
Results Thus far, molecular characterization has been successfully completed on six flows, including 39 sites and 51 Mhp P146 full sequence and MLVA typing events. The Disease BioPortal temporal- spatio-genomic visualizer has been utilized for analysis of Mhp P146 full sequences. To date, the authors have not identified lateral transmission of Mhp, but rather transmission has appeared to occur vertically within the Infection Chain™, as suggested by the similarity of sequences and types of variants from vertically related sites.
Conclusions Under the conditions of this project, transmission of Mhp has occured within the Infection Chain™. Lateral transmission of Mhp has not been documented in this project to date. By beginning the process of documentation and comparison of variants within sites, flows, and systems, more informed decisions can be made on control and elimination of Mhp in the field. Tools for molecular characterization and analysis of Mhp, including Mhp P146 full sequencing, MLVA typing, and the Disease BioPortal are readily available for use in the field and allow for a better understanding of the transmission of Mhp. A database has been created for variants classified by site, flow, and system to assist in comparison to new variants that may be identified in the project.
Topic: Bacterial Disease Presenter: Fano, Eduardo Abstract #: 3 Title: Field Application of Mycoplasma hyopneumoniae Molecular Characterization and Analysis Tools in Outbreak Investigations after Elimination Efforts Authors: Fano, Eduardo; Sponheim, Amanda, Boehringer Ingelheim Vetmedica, Inc., St. Joseph, MO; Dalquist, Laura; Swine Vet Center, Anderson, Alyssa, Pieters, Maria; University of Minnesota, St. Paul, MN, USA
Introduction Elimination of Mycoplasma hyopneumoniae (Mhp) from the breeding herd is a considerable effort and financial investment and steps should be taken to evaluate the long term success of the program. Since Mhp can be transmitted “vertically” from sow to piglet as well as laterally via new animal introductions or aerosol, and/or fomites, it is necessary to identify methods to determine origin of variant(s) when detected. This may be accomplished utilizing new tools that have become readily available for molecular characterization and analysis of Mhp. With the aid of these tools, variant(s) within sow herds and downstream flows may be identified prior to Mhp herd elimination programs to create a baseline for comparison in the event of identification of Mhp within flows post-elimination.
Materials and Methods In order to begin documentation and identification of Mhp variants within sow herds prior to the beginning of Mhp elimination programs, known Mhp positive gilts and sows were targeted for laryngeal swab sampling at GDUs and/or sow farms. Ten females were sampled at each facility, targeting clinical signs associated with Mhp, including dry coughing and labored breathing. Gilts or sows recently treated for Mhp infection were not sampled. Mhp PCR positive laryngeal swab samples with a Ct ≤ 32 were submitted to the University of Minnesota VDL for P146 full sequence analysis and to the Mycoplasma Research Lab, University of Minnesota for MLVA analysis. P146 full sequences for each variant were uploaded into the Disease BioPortal (CADMS, UC-Davis), along with the corresponding site information for temporal-spatial genomic analysis. In the event of clinical signs or Mhp detection via diagnostic monitoring (outbreak confirmation) during the post-eliminaiton phase, 10 females were sampled per sow herd, again targeting Mhp associated clinical signs. Laryngeal swab samples were submitted as described above and P146 full sequences for each variant were uploaded to Disease BioPortal for comparison to original variant(s). MLVA typing analysis is currently in development in the Disease BioPortal.
Results In this study, molecular characterization tools have been utilized post-Mhp elimination to identify the original and a new variant in one sow herd, and a new variant in five sow herds and one GDU (Figure 1). The tools are also being utilized to investigate the origin of the new variant.
Conclusions The use of these tools for molecular characterization and analysis of Mhp has allowed a comprehensive understanding of variant origin and potential routes of transmision and has avoided production system and veterinarians assuming that elimination programs had not eradicated the original variant in all six sow herds. Molecular characterization of Mhp variants within GDUs and sow herds prior to elimination is an important component of the program, and is essential for launching an investigation in the event of Mhp identification post-elimination. Tools for molecular characterization and analysis of Mhp are available for use in the field. These tools, including P146 full sequences, MLVA typing, and phylogenetic tree and site location analysis in the Disease BioPortal allow for a better understanding of the transmission of Mhp. A database has been created and utilized along with Disease BioPortal to assist in determination of Mhp variant origin after identification of Mhp within flows post-elimination. The information presented in this study supports better preparation of Mhp elimination programs and enables accurate decision making in the field.
Topic: Bacterial Disease Presenter: Hayer, Shivdeep Abstract #: 4 Title: Antimicrobial Susceptibility and Virulence Profiles of Non-Typhoidal Salmonella enterica Isolates Obtained from Porcine Clinical Samples Received at the Minnesota Veterinary Diagnostic Laboratory Authors: Hayer, Shivdeep; Hong, Samuel; Perez, Andres; Alvarez, Julio; Alshalchi, Sahar; Rovira, Albert; Olsen, Karen; Vidovic, Sinis; College of Veterinary Medicine, University of Minnesota, St. Paul, MN USA
Animal reservoirs have been identified as a source of emergence and spread of antimicrobial resistant non-typhoidal Salmonella enterica clones among human populations. Moreover, recently emerging serovars, such as Salmonella enterica serovar 4,5,12:i:-, are being isolated with an increasing frequency in swine. However, such emerging serovars have not been fully characterized. The present study aims at describing and contrasting the antibiotic resistance and virulence potential of 40 isolates each of two serovars (Salmonella Typhimurium var 5- and Salmonella 4,5,12:i:-) isolated from clinical samples of swine origin in 2015 at the Minnesota Veterinary Diagnostic Laboratory. Minimum inhibitory concentrations for nine antibiotics were estimated using Sensititre automated dilution system. Isolates were classified as resistant/non-resistant using cut-off values provided by CLSI. Hundred, 97.5 and 75-92.5% of Salmonella Typhimurium var 5- and Salmonella 4,5,12:i:- isolates were resistant against oxytetracycline, sulphadimethoxine and ampicillin, respectively. Between 12.5-20% of Salmonella 4,5,12:i:- and 7.5 to 32.5% of Salmonella Typhimurium var 5- were resistant against ceftiofur, enrofloxacin, gentamicin, and trimethoprim-sulphamethoxazole, respectively. Proportion of Salmonella Typhimurium var 5- isolates resistant to florfenicol and neomycin (62.5 and 45%, respectively) was significantly higher (Fisher’s exact test, p<0.05) as compared to Salmonella 4,5,12:i:- isolates (7.5 and 20%, respectively). Prevalence of virulence genes coding for invasion protein E (invE), bovine colonization factor (bcfC), magnesium transporter C (mgtC) and a chaperone protein (ssaE), was high (75-100%) in both serovars as estimated using PCR. Future work will aim at characterization of more virulence genes and to study the population structure of these isolates using molecular epidemiological techniques.
Topic: Bacterial Disease Presenter: Kanankege, Kaushi Abstract #: 5 Title: Space-Time Analysis of Porcine Reproductive and Respiratory Syndrome (PRRS) in Midwestern Sow Farms During 2011 -2015 Authors: Kanankege, K.S.T.1, VanderWaal, K.L.1, Morrison B.M.1, Perez, A.M1; ; 1Department of Population Medicine, College of Veterinary Medicine, University of Minnesota, USA
Porcine Reproductive and Respiratory Syndrome (PRRS) has caused considerable losses to the swine industry since it was initially reported in the USA, in 1987. The Swine Health Monitoring Project (SHMP) routinely gathers a variety of swine health data including PRRS status, and vaccination status in sow farms of participating systems.
The objective of the study here was to recognize spatiotemporal trends of PRRS status at selected SHMP-monitored sow farms in the Midwestern area, using data from 2011-2015. A total of 358 sow farms from 10 management systems were assessed. A linear mixed effect model confirmed a predictable seasonality of PRRS outbreaks over time with a decline in May-June period, the smallest number of outbreaks reported in October, and a dramatic increase in November – December. However, in 2014 and 2015 a deviation from the temporal pattern observed during 2011 -2013 was detected, with a larger number of outbreaks reported throughout the year. There were statistically significant differences between management systems over time. Farm capacity was not statistically associated with the probability of having a PRRS outbreak. Compared to non- filtered farms, farms with air filtration were significantly less likely to suffer PRRS outbreaks.
In subsequent versions of the model here, new variables will be incorporated to fit a prediction model of the risk of PRRS outbreaks in sow farms, which will contribute to the evaluation of preventive and control interventions for the disease in the US.
Topic: Bacterial Disease Presenter: Kaptur Jr, Ronald Abstract #: 6 Title: Evaluation of Aivlosin® (Tylvalosin) Type A Medicated Article for 14 Days and Tylan® (Tylosin) Medicated Feed for 21 Days, in the Control of an Artificial Infection of Lawsonia intracellularis in Pigs Authors: Ronald Kaptur Jr,1 DVM; Dan Rosener,1 DVM; Elizabeth Abbott2, BVMS, PhD, MRCVS, Rickie Domangue3, PhD, Kelly Lechtenberg4, DVM, PhD, Nathan Winkelman5, DVM, Kathleen Rooney6, MS, DVM 1Pharmgate Animal Health, Omaha, Nebraska; 2ECO Animal Health, London, United Kingdom; 3Statistical Consulting Services, Broadway, Virginia; 4Central States Research Centre, Inc., Oakland, Nebraska, 5Swine Services Unlimited Inc., Rice, Minnesota, 6The Veterinary Consultancy, LLC., Kalamazoo, MI
Introduction Porcine proliferative enteropathy (PPE, or ileitis) is an important enteric disease of pigs caused by the obligate intracellular bacterium Lawsonia intracellularis. PPE remains endemic on the majority of swine farms despite various interventions. The chronic form of PPE typically occurs in growing pigs at 6 to 20 weeks of age, causing reduced rates of weight gain and increased variability in body size. The economic impact of this endemic production disease can be very significant due to a longer fattening period and late finishing mortality. AIVLOSIN®17% Tylvalosin Medicated Premix, from Pharmgate Animal Health, is a new formulation of tylvalosin, a novel macrolide antibiotic with potent activity against L. intracellularis. AIVLOSIN 17% is a granulated premix containing 17% tylvalosin (TVN), indicated for control of PPE in groups of swine in buildings experiencing an outbreak of this disease. The premix is intended for inclusion in complete feed at the rate of 42.5 ppm (38.6 g/ton) which is administered continuously as the sole ration for 14 consecutive days.
Materials and Methods The comparative challenge study was conducted at 2 independent sites in the U.S. with each site following a common study protocol. The study involved 288 weaned commercial hybrid barrows and gilts averaging 6-8 weeks of age at the time of arrival acquired from high-health-status farms. At each site, 144 pigs were assigned to 6 blocks containing 18 total pens based on gender and body weight (8 pigs per pen) that were randomly assigned to either: non-medicated control (0 ppm tylvalosin, 0 ppm tylosin), AIVLOSIN® 17% (42.5 ppm tylvalosin fed continuously as the sole ration for 14 consecutive days); and Tylan® 40 (110 ppm tylosin fed continuously for 21 consecutive days). Following acclimation, all pigs were orally challenged with an intestinal mucosal homogenate prepared from a recent field case of acute hemorrhagic PPE containing 3.84 × 109 to 5.2 × 109 L. intracellularis organisms. All treatments were initiated when at least 15% of pigs were observed to be clinically affected with PPE (6 days post challenge). PPE clinical assessments were comprised of abnormal fecal, abdominal, and demeanor scores and were recorded for two time periods; 8-14 days and 15-21 days after the initiation of treatment. Other parameters assessed were average daily gain (ADG) and feed efficiency (feed/gain) over three different time points: study days -1-14 and 15-27 (bracketing the treatment phase), and study days -1-84 that encompass the entire study period.
Results and Discussion Judicious use of antibiotics will be paramount as the swine industry transitions to the new VFD rules that will be in effect on January 1, 2017. The study results show that Aivlosin 17% is the judicious antibiotic choice, by effectively treating acute ileitis in 14 days compared to 21 days for Tylan and by using fewer grams of antibiotic per ton of feed, thereby reducing the overall amount of antibiotic used while also being more cost efficient for practitioners. For both time periods, pigs in pens with un-medicated feed had significantly worse clinical scores than those of the two antibiotic treatments. Over the entire study, the medicated pigs had significantly better average daily gain and feed efficiency, but did not differ from each other significantly. ® Registered trademark of ECO Animal Health Ltd.
Topic: Bacterial Disease Presenter: Leite, Fernando Abstract #: 7 Title: Vaccination Against Lawsonia intracellularis Leads to Decreased Salmonella enterica serovar Typhimurium Shedding in Co-infected Animals Authors: Fernando Leite1, Connie Gebhart1, Randall Singer1, Richard Isaacson1 1University of Minnesota, St. Paul, MN USA
Salmonella enterica serovar Typhimurium and Lawsonia intracellularis are two of the most prevalent intestinal pathogens of swine. L. intracellularis causes proliferative enteropathy, a disease which leads to decreased weight gain, diarrhea and production loss. Salmonella Typhimurium causes diarrhea but also results in subclinical persistent colonization of pigs and can lead to food borne illnesses. Salmonella enterica is responsible for over 1 million cases of food borne illness per year and is also the leading cause of death due to food borne illnesses (Scallan et al., 2011). It has been estimated that the economic losses due to salmonellosis in the US exceeds $3.5 billion per year (Brogden et al., 2005). Strategies aimed at reducing the burden of Salmonella enterica in all meats are crucial, including pork. L. intracellularis infection has been found as a risk factor for increased S. Typhimurium shedding in swine (Beloeil et al., 2004). In this study we investigated whether vaccination against L. intracellularis could lead to decreased S. Typhimurium shedding. To test this hypothesis, groups of nine animals were housed separately in three pens and assigned to five different treatments. Pigs were either challenged with S. Typhimurium, S. Typhimurium and L. intracellularis, S. Typhimurium and vaccinated against L. intracellularis, or S. Typhimurium L. intracellularis and vaccinated against L. intracellularis. A non-infected control group served as a negative control (n=6). The groups immunized against L. intracellularis were vaccinated with Enterisol® Ileitis (Boehringer Ingelheim Vetmedica) at three weeks of age. Animals were infected orally with a pure culture of 2 x 109 L. intracellularis organisms at 6 weeks of age and with 1x108 Salmonella Typhimurium at 7 weeks of age. Fecal shedding of S. Typhimurium was monitored using a most probable number method two days after infection and weekly thereafter until animals reached the age of 14 weeks. Fecal scores and rectal temperatures were also recorded. Two days post S. Typhimurium inoculation, the group challenged with Salmonella alone shed the most bacteria at 3 Log10 organisms per gram of feces while the group vaccinated and co-infected with L. intracellularis shed the least at 1.97 Log10 organisms. One week post infection is when the greatest differences among groups were observed and the vaccinated co-challenged group shed significantly less Salmonella (p>0.05) than the group co-infected without vaccination and the group challenged with Salmonella alone. These differences were of 1.63 and 2.12 Log10 organisms per gram of feces, respectively. Following the first week, the co-infected vaccinated group maintained the tendency of having the lowest shedding, however this was no longer statistically significant as shedding from all groups decreased as expected with infection. No significant changes among fecal scores and rectal temperatures were observed between the groups. These results indicate that vaccination against L. intracellularis may aid in the control of S. Typhimurium in animals co-infected with L. intracellularis.
References: Beloeil PA, Fravalo P, Fablet C, Jolly J.P., Eveno E., Hascoet Y., Chauvin C., Salvat G., Madec F. 2004. Risk factors for Salmonella enterica subsp. enterica shedding by market-age pigs in French farrow-to- finish herds. Prev Vet Med 63:103-120.
Brogden K.A., Guthmiller J.M., Taylor C.E. 2005. Human polymicrobial infections. Lancet 365:253- 255.
Scallan E., Hoekstra R.M., Angulo F.J., Tauxe R.V., Widdowson M.A., Roy S.L., Jones J.L., Griffin P.M. 2011. Foodborne illness acquired in the united states--major pathogens. Emerging Infectious Diseases 17:7-15
Topic: Bacterial Disease Presenter: Resende, Rita Abstract #: 8 Title: A Novel Diagnostic Platform for in situ Detection and Subtyping of Emerging and Endemic of Swine Pathogens Authors: TP Resende1; D Marthaler2; FA Vannucci2 1 Department of Veterinary and Biomedical Sciences 2 University of Minnesota Veterinary Diagnostic Laboratory College of Veterinary Medicine, University of Minnesota, St. Paul, MN, USA
Introduction In situ hybridization (ISH) is a nucleic acid- based method that allows the detection of a particular DNA or RNA sequence within the tissue sections. A novel ISH RNA-based chromogenic technique (RNAScope) describes single-molecule visualization through the use of hybridization-based signal amplification system. Using a probe design process similar to the PCR, this technique is able to specifically identify and differentiate virus subtypes within the lesions. Additionally, ISH can be valuable as a rapid diagnostic response to emerging pathogens, since it does not require the development of specific antibodies. The objective of this study was to describe the use ISH-RNA platform for detection of Senecavirus A (SVA), differential detection of Rotaviruses (groups A, B and C) and subtyping of Influenza A (H1 and H3) in infected tissues.
Materials and Methods Probes targeting specific sequences of the Senecavirus A (VP1 gene), Rotavirus (RV) RVA, RVB and RVC (VP6 gene), and Influenza A (H1 and H3 genes) were developed based on validated PCR primers currently used at the University of Minnesota Veterinary Diagnostic Laboratory (UMN-VDL). Formalin-fixed paraffin embedded tissues were selected based on PCR results. Duplex assays were developed with probes for Rotavirus group A and B, Rotavirus A and C, and Influenza H1 and H3. Hybridization signal was detected as green and red colorimetric staining followed by counterstaining with hematoxylin. PCR negative samples were used as negative controls, and potential cross-reactivity was performed using positive samples for PRRS, PCV2, PED and TGE.
Results Senecavirus A. Fifty-nine PCR positive samples were tested by ISH. Snout vesicles from six affected sows were analyzed and all of them were ISH positive. Necrotizing glossitis was observed in three piglets, and SVA was detected within these lesions. The virus was also detected in other tissue with no apparent lesions (small intestine, liver, heart, spleen, tonsil and lymph node) from sows and piglets. ISH was able to detect SVA with PCR up to 33. Rotaviruses. A total of 30 samples of small intestines were evaluated. Four samples were positive by PCR for the three Rotavirus groups with Ct ranged from 22 to 27 for RVA, 27 to 30 for RVB, and 23 to 28 for RVC. One sample was positive for RVB (Ct 30) and RVC (Ct 23). Two samples were positive for RVA (Ct 20 and 22). Twenty- three samples were positive for RVA and RVC with Ct ranged from 16 to 28 for RVA and from 21 to 31 for RVC. Influenza A. A total of 16 lung tissues were evaluated. Eight were positive for both subtype H1 and H3 by PCR with Ct values ranged from 15 to 33. Four were positive for H1 and four positives for H3.
Discussion and Conclusion The designed probes were successfully able to detect SVA, differentially detected rotaviruses (RVA, RVB and RVC), and subtyped Influenza A (H1 and H3). The lack of non-specific staining demonstrated the 100% specificity. ISH showed promising results regarding its applicability for a rapid diagnostic response to emerging pathogens, especially when there are no antibodies available. The establishment of this platform will be important to build preparedness for future challenges regarding rapid diagnostic responses. The information we have collected will help to optimize the sample collection for SVA investigation. Moreover, this new ISH platform will provide a better understanding of contribution of the viral subtypes in animals co-infected with Influenza A and Rotaviruses. Finally, this information is crucial for driving decisions in the field regarding the method of control to be applied.
Acknowledgements: CAPES, MNVDL
Topic: Bacterial Disease Presenter: Roos, Luiza R. Abstract #: 9 Title: Preliminary Results on Tonsillar Colonization of Dams and Their Offspring by M. hyorhinis and M. hyosynoviae prior to weaning Authors: Luiza R. Roos1, Maria Pieters1 1Veterinary Population Medicine Department, College of Veterinary Medicine, University of Minnesota, St. Paul, MN USA
Introduction Mycoplasma hyorhinis is recognized as a causative agent of polyserositis and arthritis in young pigs, and it is presumed to be transmitted from dams to piglets shortly after birth. Mycoplasma hyosynoviae causes arthritis in finishing pigs, and it has been suggested that the microorganism is not capable of being transmitted to pigs younger than 4-8 weeks of age. However, recent studies indicate that M. hyosynoviae tonsillar colonization of suckling pigs may occur prior to weaning. Although colonization prevalence prior to weaning by M. hyorhinis and M. hyosynoviae has been described using different samples and tests, such as culture of tonsil tissue and synovial fluids, serum tested by ELISA and complement fixation, and nasal swab samples tested by real time PCR, it has yet to be described using tonsillar swab samples. The aim of this study was to characterize and compare colonization of dams and their offspring by M. hyorhinis and M. hyosynoviae prior to weaning in tonsillar swabs tested by real time PCR. Materials and methods: Tonsillar swabs were collected from 29 dams of various parities and 120 piglets randomly selected from their litters at 1 and 3 weeks after farrowing, at a sow farm with history lameness in the production system. Tonsillar swabs were processed for DNA extraction and tested by real-time PCR to detect M. hyorhinis and M. hyosynoviae genetic material separately. Proportions of dams and piglets with M. hyorhinis and M. hyosynoviae genetic material detected on tonsillar swabs were compared within weeks of sampling using a two-sample test for equality of proportions, and association between M. hyorhinis and M. hyosynoviae in dams and piglets colonization status was evaluated by Pearson’s chi-squared.
Results One week after farrowing, M. hyorhinis was detected in 72% of dams and 8.3% of piglets’ samples. M. hyosynoviae was detected in 55% of dams and none of piglets’ samples. At week 3 after farrowing, M. hyorhinis was detected in 65% of dams and 50% of piglets’ samples; in contrast, M. hyosynoviae was detected in 48.3% of dams and 0.9% of piglets’ samples. No statistical difference on dams’ colonization status by M. hyorhinis or M. hyosynoviae, and no association were observed in either weeks of sampling. Piglet colonization by M. hyorhinis was significantly higher at week 3, and higher than by M. hyosynoviae at both weeks of sampling (p<0.05). No association between M. hyorhinis and M. hyosynoviae piglet colonization status was observed in either weeks of sampling.
Discussion Dams seemed to be consistently colonized with both M. hyorhinis and M. hyosynoviae. In contrast, piglets appeared to be significantly more colonized at week 3 prior to weaning and with M. hyorhinis only. The observed colonization status of piglets might reflect the timing of disease development reported in nursery stage for M. hyorhinis and finishing stage for M. hyosynoviae. Higher prevalence of M. hyorhinis on dams and piglets was observed in this study with the use of tonsillar swabs rather than other samples types previously reported. To our knowledge, this is the first investigation to evaluate M. hyorhinis and M. hyosynoviae dual colonization of dams and their offspring prior to weaning using tonsillar swabs.
Topic: Bacterial Disease Presenter: Rouillier, Jessica Abstract #: 10 Title: Retrospective Study Evaluating the Efficacy of Ingelvac MycoFLEX in 16 French Farms Authors: Rouillier J.1, Spindler C.1, Gauthier N.1, Jagu R.2, Messager I.2 1SELAS Vétérinaire de la Hunaudaye, 22640 Plestan, France, 2Boehringer Ingelheim France, 51100 Reims, FRANCE
Introduction Enzootic pneumonia caused by Mycoplasma hyopneumoniae is a widespread respiratory disease in pigs which contributes significantly to economic losses (1). Mycoplasma hyopneumoniae interacts with other respiratory pathogens such as e.g. PRRS and Influenza and is considered to play a central role in the Porcine Respiratory Disease Syndrome (2). Since 1995, several vaccines against M. hyopneumoniae have been marketed in France. More than 90% of French herds are vaccinated against M. hyopneumoniae (2). Ingelvac MycoFLEX was launched in 2009. It was the first Mycoplasma vaccine that can be mixed with a PCV2 vaccine, Ingelvac CircoFLEX. Although Ingelvac MycoFLEX is widely used against enzootic pneumonia worldwide, the French veterinarians remain reluctant to use it, mainly because of efficacy concerns. Therefore, the aim of this retrospective study was to evaluate the efficacy of Ingelvac MycoFLEX, when freshly mixed with Ingelvac CircoFlex, in herds that use this vaccine for many years.
Materials and methods This retrospective study was conducted together with the veterinarians from the SELAS de la Hunaudaye, a French animal health organization specialized in porcine management and medicine. The herds selected for the study were farms in which Ingelvac MycoFLEX is used for many years and for which regular lung lesions assessments are performed and economic data are available. For each included herd, the characteristics and the health status of the farm focused on several respiratory pathogens were described and economic data were collected. The slaughterhouse inspection data were investigated on 4073 lungs. The lung scoring system used to assess pneumonia-like gross lesions was the Madec and Kobisch system (total score = 28) (3).
Results In total, 16 Farrow to Finish herds with a usage of Ingelvac MycoFLEX for 4 years and 4 months on average, were included in the study. These farms were located in western France, an area with high pig density. The mean number of sows per herd was 254 (100 to 600 sows). The included herds were representative for the herds located in this area. About 50% of the herds were PRRS positive and 25 to 30% were SIV positive. The slaughter inspection data showed that the mean pneumonia-like gross lesions was 1.52, compared to 2.14 considering the whole data set of the SELAS de la Hunaudaye in 2014. The percentage of lungs without lesions was 68.18 %. The ADG8-115kg (g/day) was 707g compared to 704g when considering the whole panel of farms followed up by the SELAS de la Hunaudaye. The FCR8-115kg (g/kg) was 2.47 compared to 2.42 and the mortality rate was 5.83% compared to 6.12%.
Conclusion and Discussion Even if the herds included in this study were not randomly selected, they were representative of herds from western France in terms of type and management (4) as well as on respiratory health status (2). The pneumonia-like lesions scores in these 16 farms using Ingelvac MycoFLEX for many years were within the range expected in vaccinated herds (5) and were comparable to the lesions observed by the SELAS de la Hunaudaye on overall. The 16 included herds showed similar performances compared to the total panel of farms monitored by the SELAS de la Hunaudaye. This study shows that Ingelvac MycoFLEX is an effective alternative in the control of Mycoplasma hyopneumoniae infection.
References Pagot E. et al: Relationship between growth during the fattening period and lung lesions at slaughter in swine, Revue de Médecine Vétérinaire, 2007. Fablet C. et al: Infectious agents associated with respiratory diseases in 125 farrow-to-finish pig herds: A cross- sectional study, Veterinary Microbiology, 2012. Madec F., Kobisch M.: Bilan lésionnel des poumons de porcs charcutiers a` l’abattoir, Journées de la Recherche Porcine, 1982 Chambre d’Agriculture Bretagne, IFIP, UGPVB: Résultats Porcs Bretagne 2014, June 2015. 5. Leneveu P. et a: Lung lesions in pig at slaughter: a 2 year epidemiological study in France, IJARVM, 2005.
Topic: Bacterial Disease Presenter: Sato, Jose Paulo Abstract #: 11 Title: Low Pathogenicity of an Atypical Brachyspira hyodysenteriae Isolate Authors: JPH Sato; CER Pereira; AGS Daniel; NR Macedo; MP Gabardo; LVA Otoni; MR Andrade; RP Laub; JAB Zarate; RMC Guedes; Animal Pathology Laboratory, Department of Veterinary Clinic and Surgery, Veterinary School, Universidade Federal de Minas Gerais, BRAZIL
Introduction: Brachyspira hyodysenteriae is the etiologic agent of Swine Dysentery (SD), which is characterized by severe mucohemorrhagic diarrhea (1). Up to date, only a few B. hyodysenteriae isolates have being categorized as being of low virulence (2,3). In Brazil, this is the first report of an atypical strain, isolated from a healthy animal belonging to a SD-free herd. Therefore, the aim of this study was to evaluate the pathogenicity of this atypical strain compared to a known virulent B. hyodysenteriae strain in experimentally inoculated pigs.
Materials and Methods: Forty-eight 5-week-old crossbred pigs, free of Brachyspira-associated disease or other confunding enteropathogen, were randomly separated into 3 groups: control (CTRL, n=16), virulent (VIR, n=16) and atypical (ATYP, n=15). The VIR strain used in this study was isolated from a pig clinically affected with SD from a herd facing an outbreak of the disease, while the ATYP strain was isolated from a healthy pig from a SD-free-Brazilian herd. VIR and ATYP groups were inoculated by gavage with 50mL of culture broth containing 108 B. hyodysenteriae/mL on three consecutive days. CTRL pigs were sham inoculated with culture broth only. All animals were monitored daily for the presence of mucohemorrhagic diarrhea during 18 days. At necropsy, the intestinal tract was evaluated for the presence and distribution of catarrhal exsudate and hemorrhagic, necrotic and/or fibrinonecrotic lesions in the mucosa. Clinical signs and gross lesions were compared between groups by Kruskal-Wallis test. A p<0.05 was considered significant.
Preliminary Results: The proportion of pigs presenting clinical signs and lesions of SD was statistically higher in the VIR group compared to the ATYP and CTRL groups (Table 1). Histological evaluations are in process. Additionally, mucohemorrhagic diarrhea was first observed 7 days post-inoculation (DPI) in pigs from VIR group, while only one pig from ATYP group had diarrhea starting later at 15 DPI. Most of gross lesions in the intestinal tract were observed in the spiral colon. Pigs from the control group remained healthy throughout the study and had no lesions at the necropsy.
Table 1. Number of pigs per group presenting clinical signs and lesions of swine dysentery after Brachyspira hyodysenteriae inoculation
Clinical signs* Necropsy findings* Fecal blood First Excessive Mucosal Fibrinonecrotic Groups and/or mucus observation luminal mucus hemorrhage exudate a a a a Control 0/16 - 0/16 0/16 0/16 b b b b Virulent 8/16 7 DPI 9/16 9/16 8/16 a a a a Atypical 1/15 15 DPI 3/15 1/15 1/15 *Number of affected pigs/total number of pigs per group a,b Different letters indicate statistical differences between groups
Topic: Bacterial Disease Presenter: Senn, Michael Abstract #: 12 Title: Comparison of Duration of Efficacy of Ceftiofur Crystalline Free Acid Versus Enrofloxacin in an Actinobacillus pleuropneumoniae Challenge Model Authors: Bobby Cowles, DVM, MS, MBAa, Michael Senn, DVM, MSa, Deborah Amodie MSa, Adam Mueller DVMb, aZoetis Inc. Florham Park NJ USA, bSwine Services Unlimited INC, Rice MN USA
Introduction: Actinobacillus pleuropneumoniae (APP) is frequently used in bacterial respiratory disease models to investigate the efficacy of treatment regimens. This study was conducted to compare the duration of efficacy of EXCEDE for Swine and Baytril. The study was undertaken to determine whether the sustained-release formulation of EXCEDE provides a longer duration of clinical activity against the death loss and severe morbidity caused by APP.
Materials and Methods: Animals were assigned to treatments (7 treatment groups with 20 animals each) and pens according to randomized block design. One complete block of animals was housed in a single pen. On each of Days -7, -5, and -3, pigs in groups T2, T3, and T4 were administered Baytril, and pigs in T5, T6, and T7 were administered EXCEDE, whereas pigs in control group T1were administered saline on Day -3. All administrated treatments were done according to labels. On Day 0, all pigs in all study groups were challenged intratracheally with inoculum containing a strain of APP serotype 5 demonstrated to be susceptible to both Baytril and EXCEDE. Mortalities were documented throughout the study, and animals were necropsied upon death, euthanasia, and at the end of the study. On necropsy examination, individual lung lobe scores were assigned to the lung and a total percent lung score was determined. Data recording was performed by personnel masked to treatment. Statistical analyses were performed only for the primary variables, at the 5% level of significance. The entire study was conducted under veterinary supervision and was pre-approved by an Institutional Animal Care and Use Committee.
Results: Pigs in the EXCEDE treatment groups had significantly (P ≤ 0.05) less mortality when compared with pigs in the groups treated with saline and with pigs in all groups treated with Baytril. Pigs in groups treated with Baytril on Day -3 had significantly (P ≤ 0.05) less mortality when compared with pigs in the groups treated with saline and with pigs treated with Baytril on Day -5 and Day - 7. ( Day -3 – Saline 14/20, Baytril 7/20, EXCEDE 0/20) (Day -5 – Baytril 13/20, EXCEDE 0/20) (Day -7 – Baytril 14/20, EXCEDE 1/20). Animals treated with EXCEDE on all assessment days had significantly (P ≤ 0.05) lower total lung lesion scores when compared with animals in all of the other treatment groups.
Conclusions: The APP challenge model employed in this study demonstrated that the sustained-release formulation of EXCEDE for Swine provided a longer duration of clinical activity against the death loss and severe morbidity caused by APP than did Baytril 100.
Topic: Bacterial Disease Presenter: Smutzer, Megan Abstract #: 13 Title: Duration of Immunity to Mycoplasma hyopneumoniae Following Intramuscular Administration of an Experimental PCV MH Vaccine Authors: M. Smutzer BS1, G. Nitzel MS1, J. Johnson DVM1, Y. Diamondidis, DVM1, L. Taylor MS1, M. Bandrick DVM, PhD1 ; Zoetis Inc., Veterinary Medical Research and Development, Kalamazoo, MI, USA
Introduction: A research study was conducted to evaluate the duration of immunity to Mycoplasma hyopneumoniae (M. hyo) after vaccination with a single dose of an experimental PCV MH vaccine given at weaning (20-22 days of age). Pigs were challenged 23 weeks post- vaccination and lung lesions were measured as the primary variable.
Materials and Methods: On Day 0, 80 clinically healthy 20-22 days of age, susceptible pigs were enrolled. Pigs were blocked by litter and randomly assigned to a sentinel (NTX) group or one of two treatment groups. Treated pigs were administered intramuscularly 2 mL of one of the following experimental PCV MH vaccines: (T01) without M. hyo, and (T02) with M. hyo. Three of the NTX pigs were euthanized and necropsied during the vaccination phase (Day 84) to monitor for exposure to M. hyo. The remaining sentinel pigs were euthanized and necropsied prior to challenge (Day 154) to confirm absence of exposure to M. hyo. Twenty-three weeks after vaccination treated pigs were challenged intra- tracheally on two consecutive days with a live, pathogenic M. hyo lung homogenate. Pigs were euthanized and necropsied four weeks after challenge. At necropsy lungs were scored for lesions typical of M. hyo disease. Serum samples were collected from all pigs prior to vaccination, prior to challenge, and at necropsy to test for M. hyo specific antibodies by ELISA. Lung tissues of all pigs were aseptically swabbed at necropsy and cultured for common swine bacterial pathogens. A sample of lung tissue was collected from all pigs, fixed in formalin and tested for the presence of M. hyo antigen by IHC.
Results: Prior to challenge, pigs did not show clinical signs of a confounding disease. There was no indication of exposure to M. hyo during the pre-challenge phase of the study. All pigs were serologically negative to M. hyo on Day -1 (prior to vaccination). Prior to challenge, on Day 159, there were no NTX or T01 pigs M. hyo serologically positive. Lungs from the NTX pigs were negative for M. hyo by IHC at the time of necropsy (pre-challenge). The T02 group had significantly lower (P=0.0316) mean percent lung with lesions as compared to the T01 group at time of necropsy (Day 189) (Fig.1). The stratified mitigated fraction for T02 compared to T01 was 0.41 with 95% confidence interval of 0.053 – 0.786. Prior to challenge (Day 159) and at necropsy (Day 189), the T01 group had significantly lower (P=0.0028) ELISA values as compared to the T02 group. At final necropsy, 53.1% to 80% of the lung tissues collected from all remaining pigs were IHC-positive for M. hyo demonstrating M. hyo in lungs.
Conclusion: The experimental PCV MH vaccine was effective in mitigating lung lesions caused by M. hyo and was shown to significantly reduce the percent of the lung with lesions compared to the control, demonstrating a duration of immunity of at least 23 weeks against M. hyo.
Acknowledgement: The authors acknowledge the excellent contributions of the Zoetis ARS, SSG, and CO staff for study support, and Zoetis’s IACUC for review. In vivo procedures occurred according to state, national, or international regulations.
Topic: Bacterial Disease Presenter: Takeuti, Karine Abstract #: 14 Title: Dynamics of Mycoplasma Hyopneumoniae Infection of Replacement Gilts of Brazilian Multipler Herds Authors: K L Takeuti¹, C P Andrade¹, L L de Almeida², D C L Linhares3, D E S N Barcellos¹ ¹UFRGS, Brazil ²IPVDF, BRAZIL, 3Iowa State University, USA
Introduction: Young parity sows appear to have a greater risk to transmit Mycoplasma hyopneumoniae (Mhyo) to their offspring compared to higher parity sows¹. ELISA and PCR are useful tools to provide information about dynamics of Mhyo infection², especially the detection by real-time PCR obtained from laryngeal swabs³. The objective of this study was to understand the infection dynamics of Mhyo of replacement gilts introduced in Brazilian multiplier farms from their introduction until their first weaning.
Materials and Methods: Two multiplier pig herds (Farm A: 3700 sows; Farm B: 2200 sows) with respiratory symptoms were conveniently selected. Mhyo vaccination was made at 21 and 42 days of age and a third dose was given after the entry of the gilts (150 days of age) on the farms. Samples were taken on two different moments on farm A: Visit 1: 95 gilts were sampled right after their introduction on the multiplier farm; Visit 2: Forty sows were sampled six months after visit 1 (one day before weaning) and five piglets per litter. On farm B, three collections were performed: Visit 1: 103 gilts were sampled right after their introduction on the multiplier farm; Visit 2: 90 dams were swabbed four months after; Visit 3: 79 gilts were collected six months after the first visit (one day prior to weaning) and five piglets per litter were also sampled. Laryngeal swabs were obtained at all collections for DNA extraction (QIAGEN®) and real-time PCR testing for Mhyo shedding detection. Blood samples were collected for ELISA (IDEXX®) in Visit 1 for both farms. The shedding status was classified as: High shedding (Ct<32); Low shedding (Ct between 32 and 39.5), and No shedding when Ct was above 39.5.
Results: Mhyo seroprevalences right after the introduction of gilts on the multiplier herds were 98.9% on farm A and 94.2% on farm B. PCR results on farms A and B are shown in Figure 1 and Figure 2, respectively.
Discussion: Seroprevalences on both farms show that gilts have already been exposed to Mhyo before their introduction on multiplier herds. ELISA is an important tool as an indicator of previous contact with Myho, but it cannot distinguish vaccine induced antibodies from natural exposure. Moreover, it cannot indicate the infectious status of the animals. On the other hand, shedding status in the early phases of infection can be assess by real-time PCR, as well as the duration of Mhyo infection. Farm A and B showed a high prevalence of positive dams in Visit 1, but some of those gilts already had a low shedding status. No positive dams or piglets were detected in Farm A one day before weaning, indicating that all gilts stopped shedding between visits. A higher prevalence of negative gilts was detected in Visit 2 on farm B compared to Visit 1, and only one animal was positive in Visit 3. No evidence of vertical transmission was detected in dams and litters tested. Our results suggest that gilts arrived already infected on Brazilian multiplier farms and they were not capable to transmit Mhyo infection for their offspring during lactation. The knowledge of the infection profile of Mhyo in gilts is an important tool for better understanding of the infection chain on farms and to select the best control measures, such as vaccination and acclimation protocols.
Figure 1. Mhyo shedding status evaluated by real-time PCR in gilts and piglets from farm A.
Farm A
) 100 % (
e 80 v
iti 60 s o
p 40 20 0 Mhyo Mhyo Piglets Visit 1 Visit 2 0 High Shedding 49,5 0 Low Shedding 17,9 0 0 No shedding 32,6 100 100
Figure 2. Myho shedding status evaluated by real-time PCR in gilts and piglets from Farm B.
Farm B
100
)
% 80
(
e v 60
iti s
o 40 p 20
Mhyo
Visit 1 Visit 2 Visit 3 Piglets High Shedding 43,7 2,2 0 0 Low Shedding 13,6 12,2 1,3 0 No shedding 42,7 85,6 98,7 100
References: 1. Calsamiglia M & Pijoan C. 2000. Veterinary Record 530- 532. 2. Calsamiglia M et al. 1999. Swine Health Prod 263-268. 3.Pieters M & Rovira A. 2013. Allen D. Leman Swine Conference. 75-76.
Topic: Feed/Nutrition Presenter: Hendel, Erika Abstract #: 15 Title: Annual Occurrence of Mycotoxins in US Corn Darvested from 2012 to 2015 Authors: Erika Hendel;1 Raj Murugesan1, S. Maria Mendoza1, Timothy Jenkins2 1BIOMIN America, Inc., San Antonio, TX, USA; 1BIOMIN Holding GmbH, Getzersdorf, AUSTRIA
Mycotoxins are harmful secondary metabolites produced by fungal species capable of infesting various commercial crops. These fungal species are roughly divided into two groups: those predominantly producing mycotoxins on the field (e.g. Fusarium spp.) and those predominantly occurring in storage (e.g. Aspergillus and Penicillium spp.). Mycotoxin contamination of feed materials is a global concern, as exposure to mycotoxins significantly impacts animal health and productivity. BIOMIN has been conducting an annual global survey of commodity crops and completed feeds since 2004, including an annual corn survey in the United States since 2012. A total of 994 samples were analyzed from the 2012 to 2015 corn harvest, including corn and corn silage. Samples were analyzed utilizing HPLC and LC-MS/MS methodologies. The six major mycotoxin groups analyzed comprised of aflatoxins, type B trichothecenes such as deoxynivalenol (vomitoxin), ochratoxin-A, fumonisins, type A trichothecenes such as T-2 toxin, and zearalenone derivatives. A high proportion of samples contained at least one detected mycotoxin type (84% ± 5; mean % positive over all years ± SE), with multi- mycotoxin contamination of many samples (≥2 mycotoxins detected in 42% ± 6 of samples annually; mean ± SE). The most prevalent mycotoxins found within samples from 2012 and 2013 included fumonisins (72% in 2012; 54% in 2013) and type B trichothecenes (33% in 2012; 27% in 2013). In the 2014 and 2015 crop, type B trichothecenes became most prevalent (62% in 2014; 75% in 2015), followed by fumonisins (56% in 2014; 53% in 2015). The occurrence of aflatoxins in the tested samples had a decreasing trend over the years (2012-15; 30%, 14%, 19%, 1%), while zearalenone remained steady (overall mean of 17% ± 3; mean ± SE). The percent of positive samples of fumonisins over 1000 ppb (2012-15: 20%, 35%, 49%, 40%) and percent of positive samples of type B trichothecenes over 1000 ppb (2012-15: 32%, 14%, 35%, 16%) varied annually, emphasizing the importance of comprehensive monitoring programs for mycotoxin risk assessment.
Topic: Feed/Nutrition Presenter: Lyoo, Kwang-Soo Abstract #: 16 Title: Effect of Vaccination with a Modified Live Porcine Reproductive and Respiratory Syndrome Virus Vaccine on Growth Performance in Fattening Pigs under Field Conditions Authors: Kwang-Soo Lyoo1), Jong-Young Choi2), Hye-Kwon Kim2), Tae-Wook Hahn2) 1)Korea Zoonosis Research Institute, Chonbuk National University, 2)College of Veterinary Medicine, Kangwon National University, SOUTH KOREA
Introduction Porcine reproductive and respiratory syndrome virus (PRRSV) control or elimination by vaccination is economically meaningful as slow growth rates and decreased feed efficiency due to persistent PRRSV infection represent addition production costs in growing/finishing pigs. It is generally speculated that PRRSV vaccination may be a potential strategy to reduce not only the virus shedding to environment but also the severity of respiratory signs of growing pigs. The aim of this study was to examine serologic responses and growth performance parameters in growing/finishing pigs with or without a commercial PRRSV MLV vaccination under conventional conditions.
Materials and Methods Four barns were designated for the four study groups (A, B, C, and D) in the growing-to- finishing site. Subsequently, all pigs (between 11 and 12 weeks old) of barn “A” were vaccinated with a commercial PRRSV MLV (Ingelvac PRRS MLV; Boehringer Ingelheim Vetmedica Inc,), and all pigs of the age of barns B, C, or D, as control groups, were given sterile saline.
An ELISA kit (HerdChek PRRS X3, IDEXX) was used to determine the PRRSV serological response. The presence or absence of PRRSV antibodies was calculated by sample to positive (S/P) ratio (≥ 0.4). To calculate average daily weight gain (ADG), the body weights of the 20 ear-tagged pigs of each group at moving and the pigs at marketing (between 26 and 27 weeks old) were recorded. The mean ADG (kg/day) was statistically analyzed using one way ANOVA test using the SPSS 10.0.
Results Overall, most pigs suffered clinical respiratory symptoms ranging from mild to severe in all facilities. The pigs of barn A were evaluated for relatively mild disease symptoms than the pigs in the other barns by the swine practitioner.
Discussion The present study reports the growth performance by the PRRSV MLV vaccine effect in growing/finishing pigs under field conditions. In particular, the growing pigs were obtained from a PRRSV-free population, and the vaccinated pigs and infected pigs without vaccination at 2nd site showed different body weight gain at marketing. We frequently experienced that most farm managers tended to avoid PRRSV vaccination as it was not convenient to estimate and compare the economic advantage of the vaccination to farms. The results suggest that PRRSV vaccination in growing pigs can be a potential strategy to enhance growth performance.
Topic: Feed/Nutrition Presenter: Manner, Klaus Abstract #: 17 Title: Apparent Ileal Digestibility & Growth Performance in Piglets as Influenced by Supplementation with Isoquinoline Alkaloids or Naringin Authors: Klaus Männer1, Valeria Artuso2, Tobias Steiner2, Armin Müller2, Jürgen Zentek1 1Free University of Berlin, Königin- Luise-Straße 49, 14195 Berlin, Germany 2Phytobiotics Futterzusatzstoffe GmbH, Wallufer Straße 10a, 65343 Eltville, GERMANY
Introduction: Appropriate nutrient digestibility is key to optimize animal health and growth performance, particularly in post- weaning pigs. The use of anti-inflammatory and anti- oxidative compounds has demonstrated to improve growth performance and to enhance the integrity of the intestinal mucosa. Particularly, isoquinoline alkaloids (IQ) derived from the Papaveraceae plant Macleaya cordata and naringin (NRG), a bitter-tasting flavanone glycoside from citrus fruits have shown anti-inflammatory and anti-oxidative properties, respectively. The objective of the present study was to determine the effects of IQ or NRG on apparent ileal digestibility and growth performance of post-weaning piglets.
Materials and Methods: Fifty-six post-weaning piglets (Danbred, Denmark; barrows and gilts; average initial BW: 6.39 ± 0.4 kg; weaning: 25 ± 2 days of age) were selected for this 6- week feeding study. Piglets were blocked by BW, litter, and gender and randomly assigned to 4 treatments with 7 replicate pens per treatment group and 2 piglets per pen. Treatments were 1) Control (basal diet; n = 14), 2) IQ-1 (basal diet + 60 mg IQ/kg; n = 14), 3) IQ-2 (basal diet + 120 mg IQ/kg; n = 14), 4) NRG (basal diet + 50 mg NRG/kg; n = 14). IQ consisted in a commercial product (Sangrovit® Extra, Phytobiotics Futterzusatzstoffe GmbH, Eltville). Piglets had ad libitum access to mash feed and drinking water. Piglets were fed a starter (25-38 days of age) and grower diet (39-66 days of age). Fecal scores and growth performance were determined weekly. Apparent ileal digestibility coefficient (AID) was determined on twenty- eight piglets (n = 7 per treatment group) at 66 days of age by using TiO2 (5 g/kg) as an indigestible marker.
Results Apparent Ileal Digestibility (AID): AID of protein, phosphorus and total amino acids was improved in IQ-2 group (80.1 ± 1.47%, 59.9 ± 4.36% and 81.3 ± 2.1%, respectively) as compared to Control group (75.2 ± 5.51%, 52.3 ± 4.68%, 76.4 ± 4.18%, respectively; p < 0.05), whereas AID of phosphorus, alanine, aspartic acid and leucine was improved in the NRG group (59.6 ± 4.29%, 75.8 ± 2.85%, 79.0 ± 2.05%, 83.4 ± 1.12%, respectively) as compared to Control group (52.3 ± 4.68%, 67.7 ± 8.29, 73.5 ±5.08, 79.7 ± 4.28%, respectively; p < 0.05).
Growth Performance: Fecal scores did not differ between treatments (p > 0.05), indicating no major health issues in the piglets. IQ-1, IQ-2 and NRG improved weight gain in the first two weeks of the experiment (p < 0.05), whereas FCR was improved in the entire experimental period (Control: 1.47, IQ-1: 1.43, IQ-2: 1.40, NRG: 1.42; p < 0.001). In addition, pigs receiving NRG showed a highest overall weight gain as compared to Control group (p = 0.023).
Conclusion: Supplementing post-weaning pigs with IQ and NRG may increase the capacity for nutrient absorption by improving ileal digestibility of nutrients. Increased digestibility was reflected in improved feed efficiency. Therefore, IQ and NRG may represent good strategies to support gut integrity and growth performance in piglets.
Topic: Feed/Nutrition Presenter: Nielsen, Bea Abstract #: 18 Title: Improvement in Piglet Performance and Gut Health with new Bacillus subtilis Probiotic Authors: Nielsen Bea, PhD, Team Manager, Innovation Dept., Cernat R.C., PhD. Senior Research Scientist Chr. Hansen A/S, Bøge Alle 10-12, 2970 Hørsholm, DENMARK
The need for cost-effective, high efficacy feed additives, such as probiotics, is increasing these days as focus on the use of antibiotic growth promoters has intensified globally. Probiotics consisting of spore forming Bacillus spp. have previously shown to positively affect performance and health in pigs. 260 new spore-formers isolated from fermented food, feces from healthy pigs, different culture collections and soils were screened in vitro. The two best candidates were selected for further in vivo trials. One of these candidates, identified as Bacillus subtilis subsp. subtilis by 16S rDNA, gyrB, and rpoB gene sequencing, is presented herein.
Minimal inhibitory concentration (MIC) was used for antimicrobial susceptibility testing of the strains and the MIC values were found to be acceptable according to EFSA’s breakpoint values. Sporulation and antimicrobial activity against Clostridium perfringens Type A and Type C, Salmonella typhimurium, Staphylococcus aureus and porcine pathogenic E. coli strains belonging to serotypes O147:K89:F4, O149:K91:F4 and O101:K-:F5 were also analyzed along with growth studies in different medium, bile concentrations and pH values. In vitro adhesion to porcine jejunal IPEC-J2 cell line, human Caco-2 and mucus-secreting HT-29 MTX cell lines was also examined. The probiotic protective effect on small intestinal epithelium was further investigated with in vitro challenge models where IPEC-J2 cell line and the porcine pathogenic E. coli strains were used. The in vivo trial consisted of 219 newly weaned piglets randomly distributed into control or Bacillus subtilis (DSM25841) treatment group balanced for gender and live weight. The groups were fed standard diets based on corn, soybean and barley, no bacillus spp or with bacillus, respectively.
In vitro attachment of Bacillus subtilis vegetative cells to human mucus-secreting HT-29 MTX cells was 12.7 ± 0.9, followed closely by the porcine jejunal IPEC-J2 cells (12.2 ± 1.93) whereas the attachment to Caco-2 cells was only 10.7 ± 1.5. Only few in vitro adhesion studies on other probiotic Bacillus spp. have been conducted so far and our findings support these studies and highlight the ability of our selected spore former to attach in vitro as well as the differences in characteristics and functions displayed by the three intestinal cell lines.
The Bacillus subtilis candidate also showed in vitro probiotic and pathogen specific protective effect on the porcine IPEC-J2 intestinal cells challenged with porcine pathogenic E. coli. Particularly, the in vitro adherence of E.coli O101:K-:F5 significantly decreased (P < 0.05) from 14.1 ± 1.1 to 0.4 ± 0.8 in presence of Bacillus subtilis.
Compared to control group in vivo results were numeric or significant in terms of daily gain in piglets offered feed supplemented with Bacillus spp. DSM 25841 strain (235 g/day vs. 218 g/day) together with feed conversion (1.15 kg/kg vs. 1.21 kg/kg; P<0.05) and improved faecal scoring (P < 0.01).
Topic: Feed/Nutrition Presenter: Palomo Yagüe, Antonio Abstract #: 19 Title: Narasin Toxicosis in Lactation Sows Author: A Palomo Yagüe, SETNA NUTRICIÓN S.A.U.- InVivo NSA . PI Santa Ana c/El Clavo, 1 Madrid, SPAIN
Introduction An outbreak of antagonism poisoning with narasin and tiamulin in swine is described. A total of 156 lactating sows sudden death over a period of two weeks after being drink tiamulin and eat feed ration a accidentally contaminated with narasin. The farm have an swine dysentery acute case on sows and decided to treatment with pleutomutilin on water at 60 mg/l or 8 mg/kg of life weight.
Materials and Methods A farm with 3.000 sows open cycle in the north of Spain had a swine dysentery outbreak and Vet decide a tiamulin oral treatment during one week. On second day, start died lactation sows on acute form previous neurological clinical signs (posterior paresis, skeletal muscle weakness, depression, ataxia, progressing to lateral recumbency), anorexia, lethargy and dyspnea (1,2). Body temperature stay in normal ranges. We stopped the water treatment and take serum, faeces and feed for analytical study of micotoxins and antibiotics antagonist. The feed samples with liquid chromatography revealed levels of 85 to 110 ppm of narasin . At same moment we studied the feeding production process and determined the sow feed contamination with poultry premixes than included narasin at 100 ppm. Feed samples were negative to micotoxins , microbiological contaminations and others carboxylic ionophores ( monensin , salinomycin and lasalocid ). Serum samples were PRRSv negative and high level of creatinine kinase ( 12.146 to 17.875 U per L ) and aspartate transferase ( 439 to 612 U per L ).
Table I – Sows dead by day
Discussion and Conclusions It appears that ionophore narasin was accidentally included in the sow lactation feed at a feed mill where poultry feed is also processed . The toxicity of ionophores antibiotics with pleuromutilin antagonism . is believed to be due to interference with the Na:K transport pump across cellular membranes, in particular of skeletal muscle fibres . This leads to increased intracellular calcium concentrations with consequent calcification and swelling of mitochondria, swelling of the sarcoplasmic or endoplasmic reticulum, cell membrane damage and eventual cell death.
Results At necropsy of 20 dead sows, we had lesions on heart , kidney , urinary vesicle , skeletal muscles and digestive system. We report skeletal muscle degeneration and necrosis.
References
1. CARPENTER ( 2005 ) . Tiamulin and narasin toxicosis in nursery pigs. J. Swine Health Prod. 13 , 333-336 2. KAVANAGH (1990). Salinomicin toxicity in pigs . Vet. Record 127:507 3. PLUMLEE (1995) . Acute salinomicyn toxicosis of pigs . J. Vet. Diagn. Invest. 7 ,419-422 4. STURUS, M ( 2016 ). Narasin toxicosis in finishing pigs. Journal of Swine Health and Production – Volume 24, Number 4 205- 211
Topic: Feed/Nutrition Presenter: Sandberg, Fredrik Abstract #: 20 Title: Improved Livability with the Use of Wean Fuel™ for Nursery Pigs Fed Either a Simple High Soybean Meal Diet or a Complex High Lysine Diet Authors: F. Sandberg1, S. England1, C. Phillips2, B. Mitteness2, S. Anderson2, and M. Bible1 1Furst-McNess Company, Freeport, IL; 2Camas, Inc., Le Center, MN USA
Introduction: Wean Fuel™ is a combination of polyclonal egg antibodies and phytonutrients. With changing rules of the use of antibiotics extensive research has been conducted into the development and use of polyclonal egg antibodies, and a growing body of evidence exists showing the practical use of this technology (1, 2, 3) to support livability, and protect gut structure for life time performance such as feed efficiency. Pigs fed an antibiotic free vegetarian diet containing Wean Fuel™ had a significantly improved livability (93.5%) compared with controls (88.7%) and numerically greater than positive control (91.8%) which contained both animal plasma and antibiotics (3). Previous work has shown that protection of gut structure in terms of improved crypt and villi during a controlled challenge study can lead to improved overall feed efficiency (2). As part of a continuous program two separate trials are reported where the responses to Wean Fuel is characterized in pigs raised in a commercial research facility (2400 head wean to finish barns) under normal health challenge conditions, on growth performance and health status of nursery pigs.
Materials & Methods: In two separate experiments, the pigs were blocked by weight and barn environment, and stratified by sex and sow farm. The pigs were placed on dietary treatments immediately upon arrival. Experiment 1 (Exp. 1) used 409 weaned commercial pigs (19 days of age, 13.4 lb; 7 reps; 27-31 pigs/pen). Control (CON1) was a simple diet containing a relatively high soybean meal inclusion for a first stage starter ration (30%), and a treatment diet where 30 lbs per ton of Wean Fuel™ was added to control (WF1). Experiment 2 (Exp. 2) utilized 425 weaned commercial pigs (19 days of age; 13.1 lb; 7-8 reps; 25-34 pigs/pen) with 2 dietary treatments. The treatments were a control (CON2) formulated with 15% soybean meal and to contain 0.1% higher SID Lys than its estimated requirements and the same diet with 30 lbs per ton of Wean Fuel™ added (WF2). Each experiment utilized a 3-phase meal form feeding program, where the Wean Fuel™ was fed in phases 1 and 2 for approximately the first 21 days, and phase 3 was common within each experiement. A FANCOM feeding system was used to deliver feed. Pens of pigs were weighed and feed disappearances were recorded on d 0, 21, and 49. This data was used to calculated ADG, ADFI, and F:G. In order to simulate commercial practice, pigs were treated if required, and those that did not respond to injectable antibiotic were tagged, removed from their pen, and placed in sick pens. At the end of each experiment, the true number of dead (% mortality) and viable pigs (% morbidity) were calculated. Data were analyzed as a randomized complete block design using the GLM procedure in Minitab with Fisher’s t-test to determine differences between dietary treatments.
Results and Discussion: For d 0-21 Exp. 1 results, there were no differences (P≥0.768) observed in ADG, ADFI, or F:G. No differences were observed for d 0-21 mortality per pen or treats per pen. There were no differences (P≥0.735) observed for d 0-21 ADG, ADFI, or F:G for Exp. 2. Also, no differences (P≥0.148) were observed for morbidity, mortality, or treats for d 0-21.
Table 1. Exp. 1 D 0-49 Results and D 21-49 F:G
CON1 WF1 SE P-value ADG, lb 1.12 1.11 0.066 0.643 ADFI, lb 1.66 1.64 0.122 0.550 F:G 1.47 1.48 0.028 0.500 % Morbidity/pen 1.77 5.11 3.490 0.158 % Mortality/pen 3.81a 0.92b 2.475 0.099 Treats/pen 42.3 40.1 9.35 0.705 d 21-49 F:G 1.57 1.59 0.204 0.257
Table 2. Exp. 2 D 0-49 Results and D 21-49 F:G
CON2 WF2 SE P-value ADG, lb 1.16 1.16 0.088 0.957 ADFI, lb 1.67 1.65 0.091 0.545 F:G 1.44 1.42 0.041 0.279 % Morbidity/pen 1.77 1.88 2.487 0.934 % Mortality/pen 3.65 1.63 3.183 0.281 Treats/pen 38.6 37.6 10.22 0.861 d 21-49 F:G 1.56 1.52 0.055 0.163
In Exp. 1 overall mortality was reduced from 3.81% to 0.92% (P<0.1; Table 1) and in Exp. 2 mortality was numerically reduced from 3.65% to 1.63% (P=0.281; Table 2). In Exp 2. the use of Wean Fuel lead to a numerical improvement of 2.6% on feed efficiency from day 21-49 (P=0.163), but this was not the case where 30% soybean meal was used. This may suggest that another type of damage occurred to the gut from soybean meal that could not be protected by the use of Wean Fuel. Further research will focus on the interactions between feeding ingredients and the use of Wean Fuel in supporting livability and improving life time feed efficiency.
Conclusions: Wean Fuel may be a significant tool for reducing mortality in swine production, and further testing to support life time feed efficiency is warranted.
References: 1. Diraviyam et al. (2014) PLoS ONE 9(5): e97716. 2. Owusu-Asiedu et al. (2003) J Anim Sci: 81:1790-1798. 3. Sandberg et al. (2015) J Anim Sci: 93(2):140.
Acknowledgements: We thank Mr. Dale Rieck for his extremely hard work to ensure quality of research, and for care of the animals.
Topic: Feed/Nutrition Presenter: Sparks, Chris Abstract #: 21 Title: Effects of Hostazym X on Pigs during Late Nursery and Finishing on Growth Performance and Death Loss Authors: C. Sparks, R. Cabrera and D. Nolan; Huvepharma US Peachtree City, Georgia USA
Abstract Xylanases have been used for with improved digestibility in poultry and swine with sometimes mixed results. In a retrospective analysis of data it was noticed that each trial has shown at least a numerical reduction of death loss. This study was set up to measure the effects of Hostazym X on death loss and weight sold. A total 960 head of 11 lbs. weanling pigs (PIC 1050 X DNA 600) were used for this experiment. Pigs were separated by gender and were randomly allotted to pens of 26 pigs per pen. Treatments started at approximately 25 lbs. of weight. Pens were blocked by sex and location and treatment was randomly assigned to one of two treatments. A total of 36 pens were utilized for 18 replications. The 2 dietary treatments included control diet (C) and diet supplemented with 0.01% Hostazym X (HX). Hostazym treatment diets were formulating with equal Lys:kcal as the control. DDGS and choice white grease were kept the same in each treatment. Rations were typical industry rations with approximately 20% DDGS in each phase. Choice white grease was added at 2%. Hostazym X was given a matrix value of 180,000 kcal ME per pound. Because fat was held constant, the HX diet had the lysine level adjusted to maintain an equal ratio of lysine to energy as the control. Pigs fed HX had a slightly improved ADG (1.74 vs 1.73 lbs.) and FG (2.48 vs 2.50) compared to the control from the start of treatment to first removal of pigs for harvest. Pigs fed HX ended up being 1.66 lbs. heavier than control pigs. Pigs fed HX had a greater survival rate than control fed pigs 92.93% vs 90.33%. With a heavier weight and less death loss (7.07 vs 9.67 %), HX fed pigs had a greater amount of pig weight sold per pen than the control (6464.3 vs 6257.1 lbs. /pen). These results are indicative of the 13 other fields trials reviewed in the retrospective analysis; in that they all have demonstrated a reduced death loss and more pigs marketed as top value pigs. While the exact mechanism is still unknown, it is clear that Hostazym X will improve your profitability by giving you more pigs, and more weight to sell with less feed cost.
Topic: Feed/Nutrition - #22 Title: In vitro Cytotoxicity Test and Antiviral Activity of Tumeric Extract Against PRRS Virus Authors: Pemika Anantikulchai*, Pandhira Emprom*, Kidsadagon Pringproa**, Panuwat Yamsakul* *Dapartment of Food Animal Clinic, Faculty of Veterinary Medicine, Chiang Mai University, Thailand **Department of Veterinary Bioscience and Veterinary Public Health, Faculty of Veterinary Medicine, Chiang Mai University, Thailand
Introduction Porcine Reproductive and Respiratory Syndrome or PRRS is an important outbreak of swine and can cause an immense damage in worldwide swine industries. At present, the defensive and control of PRRS mostly depend on farm biosecurity, gilt management and vaccination program which give an uncertain result. Nowaday, herb extraction in Thailand is very popular for against pathogens in swine. Tumeric has the active ingredient called Curcumin which is the polyphenolic compound and is reported to have the antiviral activity, antioxidant activity and many more. The objective of this study was to evaluate the cytotoxicity and antiviral activity of turmeric extract against PRRS virus in vitro.
Materials and Methods Curcumin extraction used method from solid dispersion technique. The cytotoxicity test, we cultured marc-145 cells in 96 wells plates for 2 plate and 5000 cells per well. Then we add the turmeric extract which is diluted with media in two-fold serial dilutions, by starting at 100 ug/ml. Then incubated in 5% CO2 AT 37c for 24 hr. The first plate will be tested for MTT assay and the second plate will be stained with crystal violet. We will used the highest concentration of the extract that doesn’t cause the cytopathic effect. The next step is to do the antiviral activity test, we cultured marc-145 cells in 96 wells plates and 5000 cells per well. Then incubated in 5% CO2 AT 37c for 24 hr. and infected the 10 multiplicity of infection of PRRSv for 1 hr at 37c. After that we pipete the virus out of the wells and replace with the diluted turmeric extract at the concentration follow the cytotoxicity test and two more lower dilutions. After that, incubate them in in 5% CO2 AT 37c and collect the supernatant at 24, 48 and 72 hpi follow with the immunoperoxidase monolayer assay to find virus titer. The data were analyzed with one-way ANOVA with a Tukey-Kramer HSD and for the antiviral activity test we use two-way ANOVA and compare the differences by using Tukey-kramer HSD.
Results Cytotoxicity test found that at the concentration of 0.00005 – 1.56 µg/mL has no cytotoxicity effect (Fig.1). Then the antiviral activity test, we found that turmeric extract can inhibit PRRS virus replication at 1.56 µl/mL in vitro study (Fig.2).
Fig.1 The result of cytotoxicity test for turmeric extract
Fig.2 The results of antiviral activity test for turmeric extract
Conclusion This result is interesting in use herb extraction against PRRS infection and should be study with in vivo later.
Topic: Other Presenter: Ajuwon, Kolapo Abstract #: 23 Title: Analysis of the Efficacy of Alquernat Immuplus as a Natural Immune Booster vs. Levamisole (Well-Known Chemical Immune Booster at a Specific Dose) Author: Ajuwon, K.M. and Yan, H., Purdue University; Biovet, S.A., SPAIN
Introduction Farm animals often face chronic immune challenge in the form of disease in the production environment of typical farms. The ability of animals to mount an appropriate immune response determines whether they get a disease or not. The status of the immune system in conferring protection against infection is affected by genetics and on-farm management system. Therefore, boosting the animals’ immune response is a strategy to enhance their disease resistance.
Objective The objective of the study was to analyze the efficacy of Alquernat Immuplus as a natural immune booster by evaluating the immune response of pigs after parvovirus vaccination, vs Levamisole (chemical immune booster at a specific dose).
Materials and Methods Double blind method Animals: nursery pigs at six weeks old (10 replicates, 4 pigs per replicate, total of 40 pigs per treatment). Treatments: 1. Control‐no vaccination, no immune booster 2. Vaccinated with inactivated parvovirus vaccine 3. Vaccinated with inactivated parvovirus vaccine + Alquernat Immuplus at 1kg per ton of feed. 4. Vaccinated with inactivated parvovirus vaccine + Levamisole HCl in feed (4g per ton of feed). Measurement parameters: 1. Parvovirus titer in serum at day 70 2. Record mortality 3. Feed intake and body weight at day 42, 56 and 70 for determination of growth performance and feed conversion.
Results Productive parameters were better in Alquernat Immuplus group, with a Feed Conversion Rate 9 points lower than Levamisole group. Titer counts show that animals in the Alquernat Immuplus group have a higher maintenance in percentage in the number of titers from day 42 to day 70: 72.72% of the animals keep the 1:80, compared to a 61.11% in Levamisole group and 58.82% in control group. The Alquernat Immuplus group had fewer number of animals with a reduction in the number of titers up to <1:20. This number increased from 18 animals at day 42 to 29 animals at day 70 (161.1% increase) in the Alquernat Immuplus group, compared to 225% increase in the Levamisole group and 233% in the control group.
Conclusion There was improved growth and immune response performance to Alquernat Immuplus. This suggests that the product can serve an immune booster as well as enhancing productive parameters. It does not leave residues in the animal and and does not require a withdrawal period. In a previous study done at Fujian University in China, it was also proved that Alquernat Immuplus increased the amount of immunoglobulins (IgM, IgG, IgA), as well as complement C3 and complement C4 in serum. In this case the product was tested vs Astragalus, which is a recognized botanical immune booster.
Topic: Other Presenter: Bernau, Maren Abstract #: 24 Title: Magnetic Resonance Imaging as a Tool to Detect Local Tissue Reactions After Vaccination in Piglets Author: Bernau, Maren; Kraus, A.S.; Schwanitz, S.; Scholz; Livestock Center Oberschleissheim, Ludwig-Maximilians-University Munich, GERMANY
Introduction Local (tissue) reactions are regularly described, when working with adjuvanted vaccines. Imaging methods like magnetic resonance imaging (MRI) can be used to evaluate the whole extent of a local reaction repetitively in vivo. This method was used to evaluate the local extent of two combined porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae (MH) vaccines in piglets.
Materials and Methods The study was performed in a commercial pig farm. A total of 16 piglets was used. They were randomly assigned to 2 treatment groups (n=8 per group): A = Ingelvac CircoFLEX®/Ingelvac MycoFLEX® mixed prior to administration (aqueous carbomer adjuvant); B = Porcilis® PCV MH (oil based adjuvant). Both vaccines had an injection volume of 2 ml and were used according to the manufacturer’s instructions. The piglets were vaccinated at day 25 of age using a sterile single-use needle (21 G x 5/8”). MRI was used to detect the local tissue reaction at day 1, 8, 15, 22, 29, 36 & 43 after vaccination. In this study, an open low-field MRI system (Siemens Magnetom Open) was used, performing T1- (TR 841ms; TW 17ms) and T2- (TR 5690ms; TW 102ms) weighted coronary Spin-Echo sequences, in order to illustrate a wide spectrum of pathologic conditions. A regional image analysis (Able 3D Doctor) was performed to evaluate the volume of the regions with increased signal intensity at the vaccination side (VS) and at the control side (CS; see Fig. 1). Finally, the volume difference (vol_diff) between both sides was calculated.
Results Significant differences were found for vol_diff depending on group and days after vaccination. Group B showed significant vol_diff several days after vaccination. The maximum extent was found at day 22 (T1cn: 3.74 ± 0.88, evaluating 5 images; p<0.01). The detected vol_diff´s in group B were larger than the vol_diff´s in group A.
Conclusions and Discussion Using MRI, differences between local tissue reaction sizes and distributions can be detected at the living animal, repetitively and three-dimensionally over a long period of time. In the present study, significant differences were detected between both groups, regarding the variable vol_diff (A < B). As both vaccines contain the same injection volume and similar antigens, the detected differences are likely to be related to the different adjuvants. Further tests including only the adjuvant would be required to confirm this hypothesis. In terms of safety assessment, MRI seems to be an appropriate method to evaluate local tissue reaction sizes at the living animal, repetitively and in three-dimensional extent.
Acknowledgements: This study was funded by Boehringer Ingelheim. Topic: Other Presenter: Bernau, Maren Abstract #: 25 Title: Growth Performance in Piglets After Vaccination Authors: Bernau, Maren; Kraus, A.S.; Schwanitz, S.; Scholz; Livestock Center Oberschleissheim, Ludwig-Maximilians-University Munich, GERMANY
Introduction Porcine circovirus type 2 (PCV2) and Mycoplasma hyopneumoniae (MH) are two pathogens which have huge economic impact on the global pork industry. Two vaccine combinations are currently commercially available in Europe which include both antigens. The aim of the present study was to evaluate the impact of these two vaccine combinations on weight gain after vaccination.
Materials and Methods The trial was performed in a randomized, blinded, negative controlled study design on a commercial pig farm. A total of 201 crossbred pigs (Piétrain x German Landrace) were used. At the age of 21 ± 1 days, animals were weighed (weight I), checked for general health and equally distributed among three treatment groups (A = Ingelvac CircoFLEX®/Ingelvac MycoFLEX® mixed prior to vaccination; B = Porcilis® PCV M Hyo; and C = sodium solution) taking into consideration initial body weight and litter assignment. The products were administered according to the manufacturer’s instruction using a sterile single-use needle (21 G x 5/8“; 0.8 x 16 mm). Pigs were weaned at 32 ± 1 days of age, weighed (weight II) and moved into a nursery barn where treatment groups were kept commingled in pens of 15 to 22 animals. All pens were enriched with balls, metal chains, and straw racks. During the study pigs were fed with a conventional feed (11-13 MJ/kg of ME age-related). At 74 ± 1 days of age all pigs were weight again (weight III) (see Fig. 1).
A GLM analysis using the SAS software package was performed to evaluate differences using the variables weight and average daily gain (ADG). ADG was calculated for the time period between vaccination/injection and weaning (period I), between weaning and end of nursery (period II), and between vaccination/injection and end of nursery (period III).
Results Table 1 shows the results for the ADG calculation. Significant differences were found in period II and period I. Group B showed significant lower ADG than the other groups. No significant difference was found between group A and C.
Table 1 – Results of the GLM analysis, representing the ADG of each group and time period including standard deviation. Period I = vaccination - weaning; period II = weaning – end of nursery; period III = vaccination/injection – end of nursery.
Gain - LSM ± standard error of estimation [g] period I period II period III A 174.02 ± 6.97 493.46 ± 22.10a 431.77 ± 18.44a B 168.89 ± 6.84 458.38 ± 21.93b 402.26 ± 18.30b C 180.44 ± 7.03 495.98 ± 22.09a 434.90 ± 18.43a
Conclusions and Discussion Significant differences were found regarding ADG between the two vaccines (A and B). As all animals were kept under the same husbandry conditions and as the challenge with PCV2 and MH only occurred after the nursery period on this farm, the differences in weight gain can be attributed to the reactivity of the vaccines. The findings of this study are in line with a study that was carried out in parallel which measured the local reactivity of the two vaccine combinations over a six week period using magnetic resonance imaging (1).
Funding This study was funded by Boehringer Ingelheim.
References: (1) Bernau et al.: submitted to Leman conference 2016
Topic: Other Presenter: Boyer, Perle Abstract #: 26 Title: Porcine Reproductive and Respiratory Syndrome Genetic Resistance: An Online Course at Destination of Swine Experts and Professionals Author: Perle Boyer, Montserrat Torremorell, University of Minnesota, College of Veterinary Medicine, St. Paul, MN USA
Abstract Porcine reproductive and respiratory syndrome (PRRS) is deeply affecting the US and global swine industries. PRRS is commonly managed on farms through both immunization protocols to decrease the severity of clinical signs, and biosecurity measures to prevent the introduction of new strains. However, there are limitations to those control actions. The high genetic variation among PRRS viral strains can allow some strains to evade the defense mechanisms created by the immune system even after vaccination. Moreover, it has been shown that location of the farm and the number of other swine operations around it are the most important factors that determine risk of new infections. Selection of animals genetically resistant to F-18 and K-88 E.coli has been successful on swine farms. Efforts to genetically select animals that show resistance against PRRSV infections are undergoing and show promising results. However, genetics is a fast-evolving field and it can become challenging for a swine professional to stay current on all the advances made on a daily basis. Furthermore genetic selection programs are complex and require a comprehensive understanding of the methods and strategies employed by breeding stock companies to deliver genetic improvement. For this reason, the University of Minnesota in association with Iowa State University is developing an online course centered on educating animal health professionals on genetic selection programs, from concepts to genetic improvement of health with a focus on the PRRS virus. The class is designed in four (4) self-paced modules available to the learner from the first day. Module 1 will introduce the main notions of infectious biology while module 2 will define what genetics and genetic selection are. The third module will focus on the implementation of genetic selection to disease. Lastly, the latest research on PRRSv resistance will be explained in module 4. Class material designed collaboratively by a team of experts in genetics and education, will be in the form of audio files, essential reading, and interviews of reference people in the field. By the time a learner finishes the online class, he/she will be able to discuss the various concepts and strategies involved in genetic selection and will be able to apply those concepts to disease resistance and health improvement. Lastly, the learner will also be able to evaluate the impact that genetic selection on disease may have on disease epidemiology and pathogen evolution. More specifically, the learner will have a thorough understanding on the application of those concepts to PRRSv. The class launching is planned for January 2017.
Topic: Other Presenter: Canning, Paisley Abstract #: 27 Title: Concentration of Tylvalosin in the Synovial Fluid and Plasma of Pigs When Administered Orally in the Drinking Water for Five Consecutive Days at Label Dose Authors: P. Canning,1 DVM; K. Skoland,1 BS; J. Bates,1 DVM; R. Kaptur,2 DVM; D. Rosener,2 DVM; S. Rajewski,3 BS, PhD; J Coetzee,3 BVSc, Cert CHP, PhD, DACVCP; A. Ramirez, 1 DVM, MPH, PhD, DACVPM; L. Karriker,1 DVM, MS, DACVPM; 1Swine Medicine Education Center, Iowa State University, Ames, USA; 2Pharmgate Animal Health, Ames, USA; 3Pharmacology Analytical Support Team (PhAST), Iowa State University, Ames, USA
Introduction Infectious arthritis caused by Mycoplasma hyosynoviae and M. hyorhinis is a welfare and economic concern for swine operations (Neto et al., 2012). Field observations suggest that Aivlosin® Water Soluble Granules (62.5% Tylvalosin) are effective against M. hyosynoviae. Aivlosin® WSG is not indicated for hyosynoviae and there is limited information available about its efficacy for this use. Canning et al 2016 reported that tylvalosin (TVN) and its metabolite, 3-acetyltylosin (3AT), can be found in the joints of healthy grower pigs after oral gavage at label dose, however, gavage studies are not representative of administration under field conditions. This study reports the concentration of TVN and 3AT in the joints and plasma of healthy grower pigs during oral administration of Aivlosin® WSG through the drinking water for five consecutive days at label dose (50 ppm).
Materials and Methods Sixty 45kg mixbreed commercial pigs were housed individually at an Iowa State University research site. All pigs were clinically healthy at selection and did not show signs of lameness. Pigs were not exposed to other antibiotics for at least 28 days before enrollment. Pigs were provided water ad libitum. Water disappearance was measured daily. Aivlosin WSG® (62.5% Tylvalosin) was mixed daily into the drinking water at 50ppm and administered daily for 5 days (120 hours). At 10 timepoints during the medication administration, six pigs were euthanized and plasma and joint fluid were collected from each pig. The timepoints were as follows: 0 (07:00 Day 1), 48, 60, 72, 84, 96, 102, 108, 114 and 120 hours on medication. Medicated water, plasma and synovial fluid were analyzed for concentrations of TVN and 3AT using high-pressure liquid chromatography with mass spectrometry detection by the Pharmacology Analytical Support Team at Iowa State University.
Results and Discussion Mean TVN concentration in the medicated water was 60.6 ppm and mean individual water consumption was 4.5 liters per day. No TVN or 3AT were detected in the synovial fluid and plasma at time 0. Peak mean ±SD plasma concentrations of TVN were identified at hour 60 (6.42 ng/ml ±2.68), 84 (5.91 ng/ml ±4.05) and 102 (6.94 ng/ml ±4.38) and followed a diurnal pattern with highest and lowest concentrations detected in the evenings and mornings, respectively, for hours 48 to 108. In synovial fluid, mean TVN concentrations remained similar at hours 48, 60, 72 and 84, ranging from 2.87 ng/mL ±3.0 to 3.96 ng/mL ±5.30. The lowest mean TVN synovial fluid concentration was seen at hour 96 (0.55 ng/ml ±0.94) while the peak mean TVN concentration was at hour 102 (4.61 ng/ml ±4.03) and then concentrations decreased steadily until hour 120 (1.83 ng/mL ± 0.90). For hours 72, 96 and 108, there was at least one pig per group with 0ng/mL of TVN detected in the synovial fluid. Mean 3AT concentrations were similar to TVN for plasma and synovial fluid. Rosener et al. (2013) report that 90% of M. hyosynoviae isolates have in vitro MICs for tylvalosin of less than 60ng/mL. Tylvalosin MIC data for M. hyorhinis isolates from Thailand show the MIC90 to be 190ng/mL (Thongkamkoon et al., 2005). Concentrations detected in this study are less than reported MICs, however, MICs can be poor predictors of clinical efficacy for macrolides.
References 1. Neto JC et al.: 2012, JSHAP 20:82. 2. Canning et al 2016 JVPT doi: 10.1111/jvp.12309. 3. Rosener et al.: 2013, AASV p. 213. 4. Thongkamkoon et al.: 2005, APVS p. 244. ® Registered trademark of ECO Animal Health Ltd.
Topic: Other Presenter: Iglesias, Irene Abstract #: 28 Title: Control Measures for Foot-and-Mouth Disease in Swine Farms in the USA Estimated Using Simulation Modeling Authors: Irene Iglesias1*, Kimberly VanderWaal1, Fernando Sampedro1, Amy Kinsley1, Tim J.Goldsmith1, Andres Perez1; 1 Department of Veterinary Population Medicine, University of Minnesota, St. Paul, MN USA.
Traditional strategies for controlling foot-and-mouth disease (FMD) epidemics are based on the implementation of surveillance activities and movement restrictions in affected geographical zones. Because the USA has not suffered an FMD epidemic for almost 100 years, it is quite uncertain how the implementation of alternative surveillance strategies would affect the course of an FMD incursion in the country. The purpose of the study was to estimate the impact of alternative control measures on the duration and size of hypothetical FMD epidemics affecting swine farms in the USA.
Epidemics were simulated in three States (Minnesota, Oklahoma and North Carolina) with distinct demographic and ecological conditions and including 10,468 swine farms. A stochastic spatial simulation model (InterSpread Plus, v. 6.1.2.25) was used to simulate six scenarios reflecting variations of control measures (radii of control zones, periods between veterinary visits, and efficacy of movement restrictions). Each scenario was initiated in 18 different premises and running 100 iterations for each one of them. In the baseline scenario, control strategies were based on current USDA guidelines. The model was parameterized using data collected from the peer-reviewed literature and through elicitation of expert opinion. Data on size and type of production and data on animal movement were collected from USDA databases and private companies respectively. The predicted mean number of infected farms (PNIF) was compared for each scenario to the baseline scenario using the Wilcoxon test.
For the baseline scenario, the PNIF was 183 (IC 95% 177.67-188.33). Increasing the control zones (20%; 50%) reduced the PNIF by 50% and by 76%, respectively. Increasing the periods between veterinary visits (20%; 50%) increased significantly the PNIF by 13% and by 43% respectively. Decreasing the efficacy of movement restriction ( 20%) increased the PNIF farms significantly by 62%.
Results demonstrate, in quantitative terms, the importance of effectiveness of surveillance and control strategies in controlling FMD epidemics. Results suggest that radii of control zones are extremely important in determining the size of epidemics predicted. These results will help to inform the selection of control schemes in the face of a hypothetical FMD epidemic in the US.
Topic: Other Presenter: Resende, Rita Abstract #: 29 Title: Co-localization of Lawsonia intracellularis and Proliferative Markers in Affected Pigs Demonstrated by Dual-Color RNA in situ Hybridization Authors: TP Resende1; FA Vannucci2; CJ Gebhart1,2 1 Department of Veterinary and Biomedical Sciences 2 University of Minnesota Veterinary Diagnostic Laboratory College of Veterinary Medicine, University of Minnesota, St. Paul, MN, USA
Introduction Proliferative enteropathy (PE), caused by Lawsonia intracellularis is an important disease in swine herds worldwide. PE is characterized by proliferation of enterocytes, resulting in decreased nutrient absorption and, thus, poor performance. The understanding of PE pathogenesesis, especially the mechanism of enterocyte proliferation, may broaden our options for diagnosis, prevention and control of PE. Increased mitosis rate and expression of Rho GTPases in PE affected pigs have previously been reported, but they were not evaluated at the infection site. The objective of this study was to use a novel dual-color RNA in situ hybridization (ISH) to evaluate the co- localization and distribution of L. intracellularis (detected through the expression of a housekeeping gene, aspA) and two proliferative markers involved in the progression of cell division (Ki-67 and RhoA) in porcine enterocytes using intestinal samples from experimentally infected pigs, euthanized at 21 days post infection.
Materials and Methods Probes targeting specific mRNA sequences for Ki-67, RhoA and aspA were developed. Formalin-fixed paraffin embedded ileum sections from five experimentally infected pigs were selected based on the percentage of infected crypts, as determined by immunohistochemistry (IHC) scores (0 – 4). Duplex RNA in situ hybridization assays were used to co-localize the Ki-67 with L. intracellularis and similarly RhoA with L. intracellularis aspA expression. Hybridization signals were detected as green (Ki-67 and RhoA) and red (aspA) colorimetric staining followed by counterstaining with hematoxylin.
Results The ileum sample with IHC score equal to 0 did not show any staining for aspA, and therefore was used as a normal intestinal morphologic reference. As previously described for mammals, Ki-67 positive signal was mostly observed in closer association with nuclei of immature enterocytes in the base of normally proliferating crypts. RhoA signal, on the other hand, was visualized as diffuse cytoplasmic staining along the entire crypt length, including goblet cells. All four L. intracellularis IHC positive samples were also positive for aspA. In these L. intracellularis positive samples, Ki-67 staining was more diffusely distributed along the entire crypt, in contrast to the distribution in negative samples. In addition, clusters of Ki-67 positive cell staining were observed in association with L.intracellularis infected cells, reinforcing the association between L. intracellularis infection and enterocyte proliferation. The relationship, however, between RhoA and L. intracellularis was not clear due to the fact that RhoA had diffuse staining through the cytoplasm of all cells of the crypts.
Discussion and Conclusion Although enterocyte proliferation has been recognized as a hallmark of L. intracellularis infection, the in situ evaluation and co- localization of the bacterium with proliferative markers has not been reported. IHC for Ki-67 is broadly employed in numerous studies. However, this new ISH platform offers the opportunity to co-localize two different mRNA sequences within the tissue, highlighting the increased mitoses rate in L. intracellularis affected samples. RhoA GTPase is a signaling G protein that can be involved in proliferation. Increased RhoA gene expression in pig cells was previously detected by PCR during in vivo L. intracellularis infection after 21 days. In our findings, there was not a clear difference between RhoA positive signals in negative and infected samples. A more detailed study, using ISH in association with image software for quantification of positive signal is required to detect any difference. Nevertheless, these results demonstrate co-localization of L. intracellularis and Ki-67 in proliferative lesions and highlight the use of ISH to understand PE pathogenesis.
Acknowledgements: CAPES, MNVDL
Topic: Production Presenter: Furutani, Aina Abstract #: 30 Title: Impact of Porcine Epidemic Diarrhea (PED) on Reproductive Performance in F1 Crossbred Sows Authors: Aina Furutani1, Tadahiro Kawabata2, Yosuke Sasaki1 1University of Miyazaki, 2Kagoshima Prefectural Economics Federation of Agricultural Cooperatives, JAPAN
Introduction and Objectives Porcine epidemic diarrhea (PED) is an emerging disease of pigs in a number of countries of North Americas, Europe and Eastern Asia. In October 2013, the first PED outbreak was reported in Japan after an interval of seven years. The clinical signs of PED virus infection include anorexia and watery diarrhea in the gilts and sows and a high mortality rate in piglets. However, reproductive performance in individual sows exposed to PED virus have not been reported in Japan. Therefore, the objectives of the present study were to compare reproductive performance of individual sows exposed to PED virus at different periods of production stage.
Materials and Methods The present study was carried out for PED positive three farrow-to-wean commercial farms in Kagoshima Prefecture, southern part of Japan. In these farms, PED outbreak was observed in farrowing barn in 31st December 2013 in Farm A, 3rd January 2014 in Farm B, and 4th January 2014 in Farm C. These farms were identified as PED case farm that confirmed by RT-PRC and immunohistochemistry, and herd immunization (feedback) was carried out for all gilts and sows except for those in farrowing barn. Production records were obtained from sows existed in the day of PED outbreak in each farm. Sows were categorized into six groups based on the period exposed to PED virus: being exposed during 0-30 days of pregnancy (G1), 31-60 days (G2), 61-90 days (G3), 91 days or later (G4), being exposed during lactating (L), and being exposed during postweaning (W). In addition, control group was constructed by obtaining farrowing records between March 2012 and March 2013 (non-infected group). Data used in the present study contained 1056 sows in Farm A, 1137 sows in Farm B, and 1035 sows in Farm C. A mixed effect linear model was used for statistical analysis.
Results Compared to non-infected sows, no reductions on total pigs born and pigs born alive were found in G1, G2, G3, and G4 sows. However, G4 and L sows had lower number of pigs weaned and higher preweaning mortality than non-infected sows (P < 0.05; Table 1). G4 sows had longer weaning-to-first- mating interval than non-infected sows in Farm A and C (P < 0.05), but no difference of weaning-to- first-mating interval between sow groups was found in Farm B. In addition, there was no difference of farrowing rate between G4, L, and non-infected sows. Regarding subsequent productivity, there was no difference of pigs born alive at subsequent parity between sow groups.
Conclusions The present study showed that PED outbreak decreased productivity. However, impact of PED on reproductive performance in individual sows was different among periods of production stage exposed to PED virus. Additionally, our result indicates that there was no negative effect of herd immunization on subsequent performance.
Sow groups Farm A Farm B Farm C
Number of pigs weaned, pigs Non-infected sows 10.3±0.1a 10.1±0.1a 10.5±0.1b G1 sows 10.0±0.2a 10.6±0.2a 10.9±0.2ab b G2 sows 9.3±0.2b 10.2±0.2a 11.0±0.1a c G3 sows 9.0±0.3c 10.2±0.1a 10.6±0.2ab G4 sows 1.6±0.3e 3.2±0.5c 2.6±0.3d L sows 6.0±0.6d 4.3±0.6b 5.6±0.6c Preweaning mortality, % Non-infected sows 6.4±0.3d 6.8±0.3d 9.5±0.4e G1 sows 12.4±1.7c 10.3±1.2c 11.2±1.3d G2 sows 14.2±1.6c 12.7±1.2c 9.3±0.8f G3 sows 16.3±1.9c 10.9±1.1c 12.6±1.3c G4 sows 83.1±3.2a 74.8±3.6a 75.2±3.1a L sows 40.4±5.4b 63.4±5.1b 49.0±5.2b
Table 1. Comparison of reproductive performance by sow groups. a-e Values without the same superscripts within column differed significantly (P < 0.05). Topic: Production Presenter: Harsh, Bailey Abstract #: 31 Title: The Effects of Improvest® on Carcass Cutability and Belly Quality: A Meta-Analysis Authors: B.N. Harsh*, B. Cowles†, R.C. Johnson‡, D.S. Pollmann#, A.L. Schroeder†, A.C. Dilger, D.D. Boler*; * Department of Animal Sciences, University of Illinois, Urbana-Champaign, IL 61801 † Zoetis, Inc., Kalamazoo, MI, 49007; ‡ Choice Genetics USA, Des Moines, IA, 50266; # DSP Consulting LLC, Alpine, UT, 84004 USA
Immunological castration (with Improvest®, gonadotropin releasing factor analog-diphtheria toxoid conjugate) provides an effective alternative to physical castration (PC) for the reduction of boar odor. Although meta-analyses have been conducted to evaluate the effects of immunological castration (IC) on live performance, these reviews have not evaluated the effects on carcass cutability and belly quality. Therefore, the objectives of this meta-analysis were to assess the value of IC barrows compared with PC barrows and evaluate the effect of hot carcass weight (HCW) on IC barrow cutability and value.
Pigs in all studies were administered Improvest according to manufacturer’s recommendations and only studies following U.S. cutting standards were included. Lean cutting yield (LCY) was defined as: LCY = [whole ham + trimmed loin + Boston butt + picnic + spareribs)/chilled side wt] x 100. Carcass cutting yield (CCY) was determined using the following equation: CCY = [(LCY components + natural fall belly)/chilled side wt] x 100. To evaluate the effects of HCW of IC barrows on carcass cutting yields, IC barrows were grouped by HCW as light (< 90.9 kg), average (90.9 - 97.7 kg), or heavy (> 97.7 kg). The effect of HCW in PC barrows was not estimated. Belly thickness, length, width, and iodine value were used as indicators of belly quality. Commercially processed bacon slicing yield was determined using the equation: No. 1 slice yield = (sliced wt/cooked wt of belly) x 100. A five year average of USDA Agriculture Marketing Service prices was used to calculate total value difference between carcasses of equal weights from IC and PC barrows using cutting yield estimates from this meta-analysis. Data were analyzed using the MIXED procedure of SAS with fixed effects of Improvest treatment or HCW group. Study was included as a random effect.
Lean cutting yield of IC barrows was 1.39% units greater (P < 0.0001) than PC barrows (70.89 vs. 69.50%). Similarly, CCY of IC barrows was 1.24% units greater (P < 0.001) compared with PC barrows (86.80vs. 85.56%). Therefore, based on CCY, value of IC barrows was increased by $2.44 compared with PC barrows. As HCW of IC barrows increased, both CCY and LCY declined linearly (L; P < 0.01), with lighter IC barrows having a 1.46% unit advantage in CCY compared with heavy IC barrows (P < 0.01). Natural fall bellies of PC barrows comprised a greater (P < 0.05) percentage of side weight than those from IC barrows (15.80 vs 15.50%). Bellies from IC barrows were thinner (P < 0.0001) than PC barrows, but thicker (P < 0.03) than gilts (3.55 vs. 3.83 & 3.23 cm, respectively). Slicing yield of bacon from IC barrows was 3.42% less (P < 0.0001) compared with PC barrows (84.24 vs. 87.66%). However, belly yield and slicing yield differences were minimized when IC barrows were marketed at a heavier weight.
Improvest increased LCY, CCY and overall carcass value compared with PC barrows. However, this cutability advantage decreased as IC barrows were slaughtered at heavier weights. Although bellies from IC barrows were thinner (at lighter weights) and had reduced bacon slicability compared with bellies of PC barrows, bellies from IC barrows were thicker than gilts and exhibited increased bacon slicing yield when slaugååhtered at heavier weights.
Topic: Production Presenter: Lowe, Erin Abstract #: 32 Title: Evaluating Disease Behavior and Health Interventions Using Pattern Classification of In-Process Performance in Animal Cohorts for Flows and Systems Authors: Erin Lowe, D Polson; Boehringer Ingelheim Vetmedica, Inc., MO USA
Introduction Producers typically evaluate records to determine if farm, flow and system performance are meeting expectations and to detect problems. For both reproductive sites and growing sites, these evaluations typically involve assessing time-series reports as well as cohort-based reports (e.g., breeding and growing animal groups). Time-series reports can be useful, but are inadequate for enabling timely and targeted responses to detected problems. Cohort-based reports are better suited for timely detection of in- process problems, but lack objective rigor, appropriate orientation or both. A model was developed to categorize cohort performance patterns and detect in-phase shifts of tracked measures (e.g., mortality, morbidity, clinical events, feed/water consumption).
Materials and Methods To construct the algorithm for pattern characterization, cohort start date is treated as time zero, and interval segments are user- defined. For each interval segment, a time series chart is generated and statistical process control (SPC) calculations are applied to each set of data, resulting in a time-series SPC chart for each interval. All control limits are standardized (0,1) and standardized values are then calculated for all individual data points. Using a rule-based algorithm, all standardized data points are classified as Low (L), Middle (M) and High (H). A three-interval pattern classification matrix was developed for all combinations of LMH, resulting in a total of 27 patterns. These 27 patterns were then ordinally scaled from least (LLL) to most (HHH) serious, and were further consolidated into eight distinct categories. A pilot project was conducted utilizing daily mortality data obtained from two producers. Cohorts were classified by type of mortality pattern and category within specific production phases.
Results The model, in conjunction with diagnostic testing, can be used to better inform disease diagnosis as well as enable improved design of interventions. Further, it can be utilized to assess the performance and financial impact of interventions following implementation by enabling the quantification of changes in, for example, mortality patterns post vs pre-intervention.
Discussion To evaluate and leverage the methodology, the cohort model will be further developed to include dynamically captured data for leading, early clinical, lagging clinical and ending cohort indicators with systematic animal and environmental sample/diagnostic testing results; the objective being to improve operational decision-making and intervention design. Topic: Production Presenter: Newcom, Doug Abstract #: 33 Title: Impact of Piglet Birthweight on Post-weaning Performance and System Profitability Author: D. W. Newcom, National Swine Registry, West Lafayette, IN USA
With the increase in litter size through genetic selection, piglet size at birth has become smaller and generally more variable. It is well documented smaller pigs at birth have a higher probability of mortality prior to weaning and reduced probability of becoming a full-value market pig. Reduced growth rate or more days on feed are a drag on the profitability of the system even if there are “more pigs out the door.” The goal of this analysis is to show “more pigs out the door” of a sow unit may not be the most profitable in farrow-to-finish or integrated systems.
Data from the National Swine Registry’s (NSR) Swine Testing and Genetic Evaluation System (STAGES™) were utilized to determine the impact of piglet birth weight on post-weaning performance and system profitability. Records included birth-, weaning-, and off-test weight from purebred Yorkshire, Landrace, and Duroc piglets; data from piglets not performance tested was not included. A total of 22,109 pigs were included. Data was broken down by breed and weight at birth: < 1.0 kg, 1.0 – 1.5 kg, 1.5 – 2.0 kg, and > 2.0 kg. Data was combined to evaluate the impact on commercial performance (½ Yorkshire ½ Landrace females bred to Duroc boars). Summary statistics are shown in Table 1 . Table 1. Summary Statistics Class Wt Range Avg BW, lb Avg WW, lb TNB NW Wean % 1 <1.0 kg 1.93 12.90 15.88 12.32 78% 2 1.0 – 1.5 kg 2.91 13.91 15.13 11.79 78% 3 1.5 – 2.0 kg 3.79 15.39 13.93 11.49 82% 4 >2.0 kg 4.77 17.46 14.42 10.97 88%
Class Wean Age, d Mkt/Litter % Mkt OT Age, d OT_WT, lb Days to 250 lb 1 26.3 11.34 71% 179.87 244.1 184.5 2 23.2 11.32 75% 174.14 250.2 175.3 3 22.2 11.26 81% 170.85 256.5 168.6 4 21.5 10.86 87% 167.77 263.7 162.4 Results indicate larger pigs at birth are from smaller litters, but wean a higher percentage of pigs that are heavier at weaning, grow faster to market weight, and market a greater percentage of pigs compared to lighter pigs at birth.
An economic analysis, extrapolated to 2,500 sows is shown in Table 2.
Table 2. Economic analysis of birthweight impact on farrow-to-finish revenue Class Pigs $/Lactation NPD/litter Total mkt wt Days on feed Feed cost Δ Revenue 1 ------2 -71 $9.59 $4.38 $190,436.17 $148,603.59 $(161,489.87) $254,345.36 3 -420 $12.86 $5.87 $387,705.85 $256,808.62 $(328,774.56) $418,730.72 4 -2587 $15.02 $6.86 $600,492.54 $350,900.68 $(509,217.67) $562,509.38
As you can see, larger pigs at birth does lead to fewer pigs marketed annually, but the reduced cost of feeding sows less days during lactation, fewer NPD, more total pounds of pork marketed, and fewer days on feed show an larger economic return. Smaller pigs at birth will eat less feed, but will be marketed lighter (or stay longer in a finisher), adding to finisher costs at a detriment to the pig flow of the system as a whole.
Topic: Production Presenter: Philips, Reid Abstract #: 34 Title: Virulence of Currently Circulating PRRSV Isolates under Experimental Conditions Authors: A Patterson, G Haiwick, J Victoria, J Hermann, R Philips Boehringer Ingelheim Vetmedica, Inc., MO, USA
Introduction In 2014 in North Carolina, anecdotal reports of a virulent PRRSV isolate (RFLP cut pattern 1-7-4) were reported by practicing veterinarians. Reports indicated that the isolate was more transmissible, had higher levels of viremia for longer time periods and was more virulent in comparison to other circulating PRRSV strains. The objective of this study was to evaluate PRRS 1-7-4 isolates under experimental challenge conditions.
Materials and Methods Sixty, PRRSV negative conventional, three week old pigs were randomized into four treatment groups (n=15/group). On D0, animals in Group 1 and 2 were inoculated intranasally with 2ml of one of two viral harvests derived from clinical samples collected in 2014 (RFLP pattern 1-7-4; lineage 1); animals in Group 3 were challenged with a positive control isolate (RFLP pattern 1-18-2; lineage 1); animals in Group 4 were inoculated with 1X PBS. Serum was collected on D0, 1, 3, 5, 7, 14, 28 and 35. On D14, 10 animals from each group were necropsied; remaining animals were necropsied on D35. At the time of necropsy, lungs were scored for macroscopic lesions and bronchoalveolar lavage (BAL) was collected. Serum samples and BAL were tested by RT-PCR for the presence of PRRSV RNA and by ELISA for the presence of anti-PRRSV antibodies. BAL was cultured for the presence of bacteria.
Results Least square mean lung lesion scores (with 95% confidence range) at D14 by group are listed in Table 1. Group mean cycle quantification values (Cq) on serum samples by day and group are presented in Figure 1. Viremia occurred in 13/15 animals in Group 2 and 3 by D1; all animals became viremic by D3. In Group 1, 13/15 animals were viremic by D3; all animals were viremic by D7. Viremia was detectable in all remaining challenged animals at D35.
All challenged animals had detectable PRRSV antibodies by D14; antibodies persisted through D35. Animals challenged with placebo material remained seronegative throughout the trial. Additional results were compiled but not reported.
Table 1. Least square mean (LSM) lung scores
Group % Range 1 6.9a 0.00 - 24.3 2 14.2a,b 1.1 - 39.3 3 17.0b 3.1 - 48.1 4 0.0c 0.0 - 0.5 Letters indicate significant differences (p<0.05) among groups
Figure 1. Viremia (Cq) by day and group
Discussion Two 1-7-4 PRRSV isolates recovered in 2014 from farms experiencing severe clinical signs associated with PRRSV were compared to a virulent 1-18-2 PRRSV isolate recovered in 2008. Numerical differences in lung lesions and onset of viremia were observed between the two 2014 isolates. No significant differences were detected between the 2014 isolate (Group 2) and the 2008 isolate (Group 3) within the same lineage.
Topic: Production Presenter: Pierdon, Meghann Abstract #: 35 Title: Lesion Scores and Productivity throughout Gestation for Sows in a Large Dynamic Group Fed Via Electronic Sow Feeder Authors: Meghann Pierdon1, VMD, Raymond Boston1, PhD, Thomas Parsons1, VMD, PhD 1University of Pennsylvania School of Veterinary Medicine
Introduction The objective of this study was to better understand the welfare and productivity of sows gestating in pens, in particular, those housed in dynamic groups and fed via electronic sow feeding (ESF) stations. Both the degree of familiarity and the method of introduction of new sows into the pen were evaluated using skin lesions as a measure of aggressive interactions. As more sows move out of stalls and into groups it will be important to have simple reliable animal based metrics to measure the impact on welfare in order to guide management strategies.
Materials and Methods The experiment used a 2 x 2 factorial arrangement with the degree of familiarity among the new sows as the first factor and the method by which new sows are introduced into the established, dynamic group as the second factor. The cohort of new sows was either familiar with each other because of pre-mixing (PMIX) in a pen during the week post weaning or not pre-mixed and unfamiliar with each other (UMIX) and left in stalls for the week post weaning. The cohort of incoming sows was introduced to the large dynamic pen either as a batched unit, in which all new sows were introduced into the dynamic group in the ESF pen together (BAT), or individually, in which the new sows were introduced into the dynamic group singly (IND).
Lesions were assessed on Days 0 (weaning), 7 (day before mixing), 8 (day of mixing), 11, 15, 20, 34, 48, 62, 76, 90 and 113 (loading into farrowing). Animals were scored for body lesions using a standardized scale measuring quality and quantity of three body regions anterior (cranial to the caudal aspect of the shoulder), side (between the caudal shoulder and cranial hip), and, posterior (from the cranial hip caudally). The quality of each lesion was scored from 1-3 based on the size and depth of the lesion. The score recorded for each region was the score from the highest scoring, most severe, lesion. The quantity of the lesions was counted and given a standardized score from 1-5 for each region. Farrowing information was collected on all sows.
All statistical analysis was done with STATA v13.1. Quantity of lesion scores was analyzed using mixed effect poisson regression due to the count nature of the data. Quality of lesion scores was analyzed using a mixed effect ordinal regression model. Treatment, parity, region and day were included as fixed effects in the models and sow id was included as the random effect. The significant interactions, treatment by day and parity by day were also included in the models.
Farrowing rate was treated as a binary outcome and analyzed using a mixed effect logistic regression model with parity and treatment as fixed effects and sow id as random effect. Post-hoc linear comparisons were adjusted for significance using the Bonferoni method.
Results Lesion quantity: There was no treatment effect but a significant treatment by day interaction (P<.001) where premixed animals had higher lesions than unmixed animals prior to introduction to the large pen. There were significantly more lesions in the anterior region than the side or posterior region (P<.001). There was a significant treatment by parity interaction (p<.001) where younger parity animals peaked higher early in gestation and older animals peaked lower and had fewer lesions later in gestation. Lesion quality: Quality showed the same significant interactions and trends as quantity except the quality in the side region was not different from the anterior region. Farrowing success: There was no effect of treatment or parity on the whether or not sows farrowed (P>.05). The farrowing rate was 88% for the 211 animals in the trial.
Discussion and Conclusions Lesions are indicative of sows facing aggressive interactions with other sows, and those with more severe lesions likely have lower welfare. In this pen we see that the environment is not the same for everyone and that younger animals may have more lesions and maintain them for longer than older animals. This indicates that adaptation to the pen is not a rapid process for these younger animals but rather one that takes place over several parities and supports increased parity segregation in group housing. The impact of a measure like lesion scores must be weighed against the increased freedom of movement provided by pens and more attention paid to the management of younger animals in pen gestation. Notably despite the increased lesions after introduction to the pen, productivity was not negatively impacted. Often we use measures of productivity such as farrowing rate as an assessment of animal welfare. This study implies that animals may have similar productivity but have a different level of welfare.
Acknowledgments: This work was funded by the National Pork Board.
Topic: Production Presenter: Valdes-Donoso, Pablo Abstract #: 36 Title: Assessment of Porcine Reproductive and Respiratory Syndrome (PRRS) Impact in US Sow Farms Authors: Valdes-Donoso, P.12, Alvarez, J.1, Morrison, R.1, Jarvis, L.S. 2, and Perez. A1. 1Department of Veterinary Population Medicine, College of Veterinary Medicine, University of Minnesota, St. Paul, MN, USA; 2Department of Agricultural and Resource Economics, University of California Davis, Davis, CA, USA
Background Porcine reproductive respiratory syndrome (PRRS) is an endemic swine disease causing large industry impact. The complex epidemiology of the disease, coupled with a lack of complete information and the diverse clinical outcomes observed in infected farms, have hampered efforts to quantify PRRS’ impact on production over time.
Objective To measure the impact of PRRS outbreaks in routinely vaccinated sow farms.
Methods Longitudinal production data, regularly collected on 16 sow farms from a single system that had been affected with PRRS, were used to build a fixed-effects model to evaluate the post-outbreak production of weaned pigs. Seven indicators of farm performance (litter size, number of stillbirths, number of pre-wean piglets and sows dead, number of sows with abortions, number of sows with repetition of services, and sows farrowing) were also assessed. Pre-outbreak data were used to establish a baseline that was used to estimate the decrease in weaned pigs produced, and losses were translated into a revenue decrease assuming the average market price of $45.40 / weaned pig.
Results Production declined one week before the outbreak was reported, and the decline was greatest between 5 and 6 weeks after. Recovery was not monotonic, and a new decay was observed between the 11th and 18th week post-outbreak. By the end of the study (35 weeks post- outbreak) a trend towards recovery was observed in all performance indicators, although baseline levels were never reached. Abortions increased significantly the week before the outbreak was reported, and stillbirths remained significantly higher than baseline even in the 35th week after the outbreak. Through the 36 weeks following an outbreak, PRRS caused a decrease of 1.9 weaned pigs per sow, leading to decrease revenue in a standard farm of this system of approximately $350,000 through the same period.
Conclusions PRRS caused a significant decrease in weaned pig production and the negative effect lingered. Around 25% of total losses occurred within the first six weeks of the outbreak, 50% of losses by the 12th week post-outbreak. We were only able to measure production losses and not the full economic impact of PRRS. Still, our estimates indicate a larger loss than previously estimated, i.e., a decrease of 1.90 weaned pigs over 36 weeks compared with the 1.44 per sow year reported elsewhere (Holtkamp et al. 2013), even though farms here were performing routine PRRS vaccination.
Relevance Metrics of PRRS-associated losses here demonstrate, in quantitative terms, the impact of the disease, and would be useful for informing cost-benefit analyses and bio-economic models of interventions. The methods presented here may also be applied to measure impacts in other type of farms, such as farms without vaccination protocols, for comparative purposes.
Acknowledgement: We thank the system, which participates in the SHMP, for sharing the information used for these analyses.
Topic: Reproduction Presenter: Almeida, Fernando Abstract #: 37 Title: Impacts of Birth Weight on Ovarian Follicle Development in 150-day Old Gilts Author: Almeida, Fernando, Federal University of Minas, Gerais; Alvarenga-Dias, Ana Luisa, Federal University of Uberlandia; Moreira, Leticia, Federal University of Minas Gerais Aparecida; Fiuiza, Aparecida, Federal University of Minas Gerais; Chiarini- Garcia, Helio, Federal University of Minas Gerais, BRAZIL
Background In the last decades, selection for improved prolificacy has resulted in higher litter sizes, and has thereby increased the proportion of low birth weight piglets. It is well documented that LW piglets have lower growth performance, muscle accretion and poor carcass quality. However, little is known about the relations of birth weight with subsequent reproductive performance in gilts. This study investigated the effects of birth weight on reproductive tract and ovarian follicle development in 150-d old gilts. Twenty eight female pigs of different birth weight ranges (high-HW: 1.8-2.2 kg; low- LW: 0.8-1.2 kg) from higher parity commercial sows were reared until 150 days of age, and their body weights were recorded at weaning, end of nursery and end of the grower- finisher phase. The animals were killed and their reproductive tracts collected for biometrical and histomorphometrical analysis.
Results LW gilts showed significantly lower body weights and growth rates during all phases of production compared to their HW counterparts (P < 0.01). Most biometrical measurements of the reproductive tract were similar between the experimental groups, except vaginal length, which was shorter in LW gilts (P < 0.05). LW females also showed a lower number of medium size (3-5 mm; P < 0.01) ovarian follicles, pre-antral follicles (P < 0.07) and a greater number of atretic follicles per ovarian cortex area (P < 0.05).
Conclusions Besides the effects on postnatal growth performance birth weight affects vaginal length and the follicular dynamics process, which may impair the reproductive performance of replacement gilts.
Topic: Reproduction Presenter: Arend, Lidia Abstract #: 38 Title: Administration of Melatonin during the Follicular and Early Luteal Phase to Mimic Short Days and Minimize Seasonal Infertility for Prepubertal Gilts Housed under Differing Hours of Light and Heat Authors: Arend, Lidia; Knox, Robert; Duangkamol, Phoophitphong; University of Illinois Urbana-Champaign; USA
It is well-documented that fertility in pigs is reduced in late summer and early fall compared to winter and spring. This seasonal infertility is characterized by delayed puberty, pregnancy failure, reduced litter size, and delayed return to estrus after weaning. These problems are associated with abnormalities in follicular development and the luteal phase in early gestation. It is thought to be mediated, at least in part, through seasonal changes in the duration of nighttime secretion of melatonin. Coinciding with the periods of reduced fertility, nocturnal melatonin is minimal in late summer and early fall. In addition, numerous studies have associated heat stress with reproductive failures in the same seasons. To determine whether seasonal infertility during heat stress might be mediated through changes in photoperiod, this study was designed to test if exogenous melatonin could mimic short days and minimize seasonal infertility problems in the follicular and early luteal phases. The experiment was performed in a single replicate with terminal line PIC gilts that were selected at 165 d of age and randomly assigned by age and weight (116.5 kg) to treatment. On the first day of the experiment, (Day 1), gilts (n=36) were allocated in a 2 x 3 factorial treatment design to receive melatonin (MEL) or Placebo (PLA) and housing in an environmental room for a total of 13 d. Housing occurred in one of three rooms that provided: 1) 8 h of light (240 lux) and heat (32°C, 8H); 2) 16 h light and heat (16H); or 3) 24 h light and heat (24H). Gilts were fed 1.8 kg in the AM and then MEL (5 mg, Sigma) or PLA treatment orally as a top dress on the PM feed (0.9 kg). On Day 6, gilts received an i.m. injection of PG600 and fenceline boar exposure once daily until Day 13. Trans-rectal ultrasound was performed every other day from Day 6 to 10 to assess follicles. Gilts in estrus were inseminated twice on each day standing. On Day 14, all gilts were fed a control gestation diet once daily in the AM (2.7 kg), and all rooms were adjusted to similar lighting (16L:8D) and temperature (22°C). On Day 47 animals were slaughtered and reproductive tracts were collected to assess d 33 pregnancy, litter size, fetal and placental measures. Continuous and categorical data were analyzed using the GLM and GENMOD procedures of SAS for the main effects of treatment and room. Rooms set for 8H, 16H and 24H, had average 24 h temperatures of 26.5, 28.0 and 30.0°C, 49, 35 and 46% humidity, and 74, 75, and 79 Temperature Humidity Index (THI), respectively. Luminosity averaged 73, 157 and 266 lux and ammonia levels averaged 19, 8 and 11 ppm. There was no effect of treatment or room (P>0.10) on estrus (91.6±11.8%), interval from PG600 to estrus (4.1±0.2d), gilts with large follicles (91.6±15.9%), or large follicles/gilt (14.3±1.5). However, there was a tendency (P=0.08) for MEL (87.8%) to improve pregnancy rate compared to PLA (63.3%) but no effect of room. Total fetuses (15.3±3.1), healthy fetuses (14.7±2.9), abnormal fetuses (0.7±0.5) and placental efficiency (0.09±0.01, fetus wt./placenta wt.) were not affected by treatment or room (P > 0.10). In this study, gilts were exposed to differing hours of heat stress throughout the day during the follicular and luteal phases, but responses were near optimal for large follicle development, estrus expression, and litter size. This suggests that under heat stress, a PG600 induced follicular phase may negate melatonin effects on endogenous hormone release. However, the trend for improved pregnancy, suggests luteal, embryo or uterine response to melatonin supplementation during the first week of gestation. To better understand how melatonin might minimize effects of seasonal infertility, further studies in the follicle and luteal periods are needed.
Topic: Reproduction Presenter: Lopes, Marcos Abstract #: 39 Title: Using Crossbred Field Reproduction Data to Select Pure Line Animals Authors: Marcos S. Lopes1, John Eggert2, John W.M. Bastiaansen3, Egbert F. Knol1 (1)Topigs Norsvin Research Center, Beuningen, the Netherlands, (2)Topigs Norsvin, Burnsville, USA, (3) Wageningen University, Wageningen, NETHERLANDS
Abstract Text: In pig breeding, selection takes place in purebred lines. Further, genetic evaluations have been performed mainly using information collected on purebreds, in high- health environments, although the final product of the pig industry is a crossbred animal. This strategy may not be optimal when the objective is to improve crossbred performance. Therefore, different strategies need to be considered to accelerate genetic progress at crossbred level. As shown in previous studies, marker effects estimated in one breed do not accurately predict performance in another breed. This might be due to breed-specific effects caused by differences in linkage disequilibrium between the marker and the QTL, as well as differences in allele frequencies and in genetic background of the different breeds. Breed- specific effects are observed when the same allele of a given marker has a different effect depending on its breed origin, resulting in different allele substitution effects across breeds. When this is the case, single-breed breeding values may not be efficient to predict crossbred performance. Our aim was to estimate the contribution of alleles from each parental breed to the genetic variance of traits measured in crossbred offspring, and to compare the prediction accuracies of breeding values from a traditional genomic selection model (GS), trained on purebred or crossbred data, with accuracies of breeding values from a model that accounts for breed-specific effects (BS), trained on crossbred data. Traits evaluated were litter size (LS) and gestation length (GL) of pigs. The genetic correlation between purebred and crossbred performance for both LS and GL was 0.90. For both traits, the additive genetic variance was higher for alleles inherited from the Large White (LW) compared to alleles inherited from the Landrace (LR) breed (0.74 and 0.56 for LS, and 0.42 and 0.40 for GL, respectively). Highest accuracies of predicting crossbred performance were obtained when training on crossbred data. For LS, prediction accuracies were the same for GS and BS breeding values (0.23), while for GL, prediction accuracy for BS was slightly greater than accuracy of GS breeding values (0.53 and 0.52, respectively).Evidence of breed-specific effects for LS and GL was demonstrated. In future studies, traits with lower genetic correlation between purebred and crossbred performance (e.g. mothering ability and feed intake) should be evaluated to confirm the potential advantages use of BS models in genomic predictions.
Keywords: Pigs, Crossbred performance, breed-specific effects
Topic: Reproduction Presenter: Palomo Yagüe, Antonio Abstract #: 40 Title: VEGF-receptor System Immunoreaction in Subepithelial Endometrium Area in Healthy and Arresting Iberian Pig Attachment Sites Authors: A Palomo Yagüe , MA Sánchez, RA García-Fernández, B Sánchez, P García-Palencia, C Naranjo, JM Flores, Dept. Animal Medicine and Surgery, Faculty Veterinary Sciences, Complutense University Madrid, SPAIN
Introduction The Iberian pig is an autochthonous Mediterranean breed characterized by a lower prolificacy. Recently, it has been reported that 23 % of the conceptuses die during the first 30 days of gestation (1).VEGF (Vascular Endothelial Growth Factor)- receptor system seems to play an important role in the vascular events during the peri-implantation period in pigs (2,3). The objective of this study was to analyze the expression of VEGF-receptor system in the vessels of subepithelial area of endometrium of Iberian pigs at healthy and arresting embryo attachment sites at gestation day (gd)22 and gd32.
Materials and Methods The reproductive tracts of Iberian sows from Guijuelo (Salamanca, Spain) at gd22 (n=20) and gd32 (n=20) were collected after slaughtering. Embryos were grouped as healthy (H) or arresting (A) based on standard criteria (4,5). Endometrial samples were obtained from H and A attachment sites. Expression of VEGF, VEGFR1 and VEGFR2 were analyzed by immunohistochemistry using anti-VEGF antibody (Santa Cruz Biotechnology), anti- VEGFR1 antibody (Abcam®) and anti-VEGF R2 antibody (Bioss Inc.,). Quantification was performed in 10 fields, counted at 400X magnification. Results obtained were analyzed using SPSS Statistics 19 Software for Windows (SPSS-Ibérica Inc., Spain).
Discussion and Conclusions VEGF, VEGFR1 and VEGFR2 were detected in endometrium samples at gd22 and gd32 in lean breeds (3,4,6). Our results confirm that VEGF system is expressed in a similar way in Iberian pigs. Immnunoreaction from VEGF and VEGFR1 in the subepithelial capillary vascular network increased in healthy conceptus-attachment sites as pregnancy advanced. This may show a significant role for VEGF system in embryo viability.
Results Results regarding VEGF-receptor system vascular density are compiled in Figure 1. The density of VEGF positive capillary vessels in the subepithelial area of endometrium increased in healthy conceptus-attachment sites and decreased in arresting fetal sites as pregnancy advanced. VEGFR1 expression was higher in the subepithelial capillary endometrium from healthy sites and increased from gd22 to gd32. The density of VEGFR2-positive capillary vessels decreased slightly in healthy attachment sites from gd22 to gd32, whereas in arresting sites an obvious decrease in VEFGR2 immunostaining was observed on gd32 as compared with gd22.
Figure 1. VEGF-receptor system positive vessels/mm2 in subepithelial capillary area of endometrium associated with healthy (H) and arresting (A) conceptuses at gd22 and gd32 (mean ± s.e.m).
Acknowledgments Supported by a grant of the Ministry of Economy and Competitiveness of Spain: AGL2010-17021
References 1. González-Añover et al. 2011. Theriogenology 75(1): 34. 2. Przala et al. 2006. Biol Reprod 6(Suppl 1): 59. 3. Kaczmarek et al. 2008. Mol Reprod Dev 75(2): 362. 4. Tayade et al. 2006. J Immunol 176: 148. 5. Wessels et al. 2007. Am J Reprod Immunol 58: 470. 6. Winther et al. 1999.Placenta 20:35.
Topic: Reproduction Presenter: Ribeiro, Abstract #: 41 Title: The Influence of Air Contact on the Quality of Extended Boar Semen Authors: CV Ribeiro1, JL Moroni1, MB Menegat1, APG Mellagi1, ML Bernardi2, I Wentz1 and FP Bortolozzo1 1Setor de Suínos, Favet-Universidade Federal do Rio Grande do Sul, Porto Alegre-RS, Brazil; 2Departamento de Zootecnia, FAgro- UFRGS, Porto Alegre-RS, BRAZIL
Introduction It has been suggested that motility parameters and pH of liquid stored boar semen can be affected when preserved in contact with high amount of ambient air1. However, the presence of air within insemination doses is not routinely prevented in Artificial Insemination Centers. The aim of the present study was to evaluate the influence of different amounts of air on porcine semen quality during the storage period.
Materials and Methods Four ejaculates from 5 boars were used in this study. Each ejaculate (n=20) was diluted (30x106/mL) in a BTS-extender and allocated into three groups, on a split sample basis, differing on the amount of air entrapped within the polystyrene tubes (100 mL). In group AIR-0 the tubes were completely filled with extended semen (0% air); in group AIR-25, 75% of the tube volume was filled with extended semen and 25% with air; in group AIR-50, 50% of the tube volume was filled with extended semen and 50% with air. Insemination doses were stored at 17.3 ± 0.5 ºC for 120 h. Using a CASA system (AndroVision®, Minitüb GmbH), the motility parameters were assessed at 24, 72 and 120 h of storage. The evaluation of acrosomal integrity was performed by examining formalin- fixed samples with phase-contrast microscopy (1000x) at 72 and 120 h of storage. The pH was measured at 24, 48, 72 and 120 h of storage with a digital pH-meter (QUIMIS® Q400AS). At 120 h, samples were incubated in water-bath at 38 °C for the thermoresistance test and sperm motility was assessed after 30 min of incubation. Data were analyzed as repeated measures using the GLIMMIX procedure of SAS® Software. Boar and week of collection were included as random effect in the model. Groups were compared using the Tukey-Kramer test at a significance level of 5%.
Results The pH was influenced by the amount of air entrapped within the tubes (Figure 1; P<0.01). At 120 h of storage, total motility, progressive and rapid motility were higher (P<0.05) in AIR-0 (75.3%; 65.1%; 42.0%, respectively) when compared to AIR-50 (49.3%; 38.1%; 21.4%, respectively). Group AIR-25 showed intermediate values (63.3%; 50.7%; 30.5%, respectively) and did not differ from the others (P>0.05). There was no difference in acrosome integrity and slow, circular and local motility among groups (P>0.05). After the thermoresistance test, total motility, progressive motility and rapid motility were higher in group AIR- 0 compared to AIR-25 and AIR-50 (Table 1).
Figure 1. The pH mean of insemination doses containing different amounts of air during storage for 5 days. Groups with a different letter were statistically different (P<0.01).
Table 1. Motility parameters after the thermoresistance test at 38 °C for 30 min according to the volume of air within the tubes. Motility, % AIR-0 AIR-25 AIR-50 SEM
Total 62.47a 51.50b 42.07b 9.03
Progressive 49.98a 38.09b 29.62b 8.29
Rapid 28.22a 18.45b 13.82b 4.69 SEM: standard error of mean; a,b in the row (P<0.05)
Conclusions The presence of air within tubes affects the quality of extended semen during storage, and the amount of entrapped air is relevant. Although the effect on sperm motility was only evident on the last day of storage (120 h), the differences in pH between groups were clear from 24 h onwards.
References 1. Vyt P: 2007, Reprod Dom Anim 42 (2), p. 218-220. Topic: Viral Disease Presenter: Arruda, Andreia Abstract #: 42 Title: Investigation of Land Coverage and Elevation as Risk Factors for PRRS Breaks Author: Arruda, Andreia, Morrison, Robert, Vilalta, Carles, Perez, Andres; University of Minnesota USA
Introduction Porcine reproductive and respiratory syndrome (PRRS) is the most impactful disease in the North American swine industry, and even though there has been substantial PRRS research over the past years, incidence does not appear to be decreasing (Betlach et al., 2016). The Swine Health Monitoring Project (SHMP) is a national volunteer project that aims to monitor and aid in control of PRRS and other swine diseases. It currently represents approximately 42% of the sow population of the United States. The objective of this study was to use the SHMP database to investigate the association between environmental factors (e.g. land elevation and land coverage) and the incidence of PRRS breaks.
Materials and Methods All swine sites participating in the SHMP from 2009 to 2016 and that shared weekly PRRS status were enrolled in this study. Number of PRRS breaks, years of participation and site location were collected from the SHMP database. Environmental features considered for analysis included land coverage (cultivated areas, shrubs and trees), land altitude (in meters above sea level) and slope (in degrees compared to surrounding areas). Other risk factors of importance included region, production system, farm size and swine density. The environment-related variables and pig density were captured in raster format from different sources (slope and altitude: SRTM [CGIAR-CSI 2006), GTOPO30 [USGS 2002]; land coverage: Global Land Cover 2000 Project [JRC, European Commission]) and extracted to points (farm locations) using ArcMap 10.2.2. A multilevel mixed-effects Poisson regression was fit using STATA/ IC 14.1. Clustering of swine sites within production system was accounted for using a random effect. Statistically significant associations were declared at P<0.05.
Results A total of 706 swine sites were enrolled in the study. The mean number of outbreaks per year was 1.38 (SD: 1.65), and the median was 1 (IQR: 2). The maximum number of outbreaks per year was 9, and approximately 40% of the farms did not report any break. The final multivariable model included swine density and herd size as being positively associated with incidence of outbreaks (P < 0.01), which has been previously reported for PRRS (Lambert et al., 2012; Velasova et al., 2012). Even though altitude (meters above sea level) was not significant in the final model, farms located in terrains with a slope of 9% or higher were “protected” compared to farms located in terrains with slopes of 1% or lower (P<0.01). Finally, being located in an area of shrubs/ herbaceous cover and trees had a “protective” effect on PRRS incidence compared to being located in a cultivated/ managed areas (P<0.05).
Conclusions and Discussion In conclusion, highly inclined terrains were associated with fewer PRRS breaks, as was the presence of shrubs and trees when compared to cultivated/ managed areas. Prospective studies are needed to substantiate these findings in the future.
Acknowledgements SHMP participants for sharing information and the Swine Health Information Center for funding.
References Betlach et al. 2016. National Hog Farmer, available in: http://nationalhogfarmer.com/ health/swine- health-monitoring-project- serving-swine-disease-surveillance-system Lambert et al. 2012. Prev Vet Med, 104:84. Velasova et al. 2012. BMC Vet Res, 8:814.
Topic: Viral Disease Presenter: Bandrick, Meggan Abstract #: 43 Title: Efficacy of Porcine Epidemic Diarrhe a Vaccine When Administered to Sows and Evaluated by Piglet Challenge at One Day of Age Authors: Bandrick, Meggan, D. Fredrickson, L. Taylor, J. Johnson, S. Zager, J. Marx, V. Rapp-Gabrielson; Zoetis Inc., Veterinary Medical Research and Development, Kalamazoo, MI, USA
Introduction Pigs are susceptible to PEDv infection and disease immediately following birth. Severity of clinical signs decreases with age. An initial outbreak on a naïve farm results in 80% to 100% mortality in newborn piglets. Therefore, to reduce pre-weaning mortality due to PEDv, it is key to protect piglets immediately following birth. The objective of this study was to evaluate the efficacy of an experimental Porcine Epidemic Diarrhea Vaccine when administered to pregnant sows, with a PEDv challenge in newborn piglets.
Materials and Methods A total of 21 sows/litters were enrolled and randomly allotted to one of two treatment groups. Treatment groups included T01, control (adjuvant only) and T02, experimental Porcine Epidemic Diarrhea Vaccine; n=10 sows/treatment. (One T01 sow was found dead on Study Day 4, and was replaced on Day 7; 20 litters were challenged.) Sows were housed 2/room with each treatment represented per room. Treatment administration (2 mL, IM) occurred at 5 (Day 0) and 2 (Day 21) weeks pre-farrowing. The sow/litter was the experimental unit. T01=94 piglets, T02=78 piglets. Blood and fecal swabs were collected from sows on various days for PEDv FFN antibody and PEDv RT-qPCR determination, respectively. All farrowings were monitored as reasonably possible; piglets were individually identified, and litters were standardized to up to 10 piglets/ litter. Litters were challenged with virulent PEDv (PEDV_IA_2013) when all piglets per litter were 1-12 hr of age. Pigs were observed for 7 days post- challenge (dpc). On 7 dpc (or at the time of removal if prior to 7 dpc), pigs were euthanized, necropsied, gastroenteritis lesions were scored, and a fecal swab was collected for PEDv RT-qPCR. Piglet mortality was attributed to PEDv unless there was specific evidence to suggest the mortality was not due to PEDv; piglet mortality was not attributed to PEDv if the animal completed the study.
Results Sows were negative for PEDv FFN antibodies pre-treatment; T01 remained at baseline for the study entirety. Vaccination induced an increase in least squares mean (LSM) PEDv FFN antibodies in T02 sows (p=0.0001). The back-transformed (bt) LSM percent litter mortality attributable to PEDv was significantly greater in T01 litters (100%) compared to T02 litters (40.5%), p=0.0029.
Figure 1. Litte r Mortality Due to PEDv
Of the 10 T01 litters, no litters/piglets completed the study; the last pig was removed on 5 dpc. Of the 10 T02 litters, litters had piglets that completed the study. The mitigated fraction of mortality in T02 compared to T01 was 0.70, 95% confidence interval: 0.400 – 1.000. Further, btLSM PEDv in fecal swabs at necropsy was significantly less in T02 litters (27,124) than T01 (811,136), p=0.026.
Conclusions and Discussion Vaccination resulted in significantly less mortality and fecal shedding in litters of vaccinated sows compared to control sows. Therefore, under the conditions of this study, the experimental Porcine Epidemic Diarrhea Vaccine is efficacious.
Acknowledgement The authors acknowledge the excellent contributions of the Zoetis ARS, SSG, and CO staff for study support, and Zoetis’s IACUC for review. In vivo procedures occurred according to state, national, or international regulations.
Topic: Viral Disease Presenter: Baumgardner, Eric Abstract #: 44 Title: Use of Phylogenetic Analysis of Concatenated Structural Sequences as a Means of Differentiating PRRSV Strains Authors: Baumgardner, Eric; Lawrence, P.K., Newport Laboratories, Inc., Worthing, MN USA
Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is one of the most economically significant swine pathogens in the United States (1). The virus is broadly categorized into Type I and Type II virus genotypes. However, since its emergence, the virus has evolved a significant amount of genetic diversity. Nucleotide identity within the open reading frame encoding the major surface glycoprotein, GP5 can be less than eighty percent within genotype II and less than seventy percent within genotype I (2). High antigenic variation among the circulating field strains creates a major problem in selecting vaccine strains that could be included in autogenous vaccines. Several methods have been used to categorize PRRSV including RFLP typing, GP5 sequencing, MJ typing, and, most recently, complete genome sequencing (2). These methods are highly variable and differ significantly in their ability to predict whether the strains are antigenically distinct and could afford broad neutralization.
At Newport Laboratories we recently compared RFLP typing and MJ typing to different methods of phylogenetic analysis coupled with whole genome analysis for selecting vaccine strains. We sequenced the complete genome of several recent field isolates including many that can be associated with the recent 1-7-4 PRRSV outbreak. The genome of each virus was used to determine the in silico RFLP pattern, MJ type, and GP5 sequence of each strain. Additionally, the sequence of the open reading frames encoding structural proteins GP2, GP3, GP4, GP5, and M were translated and the amino acid sequences concatenated in the order they are encoded in the PRRSV genome. Phylogenetic trees were constructed using the complete genome (not shown here), the amino acid sequence of GP5 alone, and concatenated structural protein sequences (Figure 1). Figure 1: Phylogenetic analysis of Concatenated structural PRRSV proteins (A) and GP5 (B). 14- 00549-1 strain circled.
As illustrated in Figure 1, strains cluster differently when additional sequence information is included in the analysis. MJ type does not appear to correlate well with phylogenetic classification. Both RFLP typing and MJ typing focus on very specific portions of the GP5 open reading frame. This can result in misclassification of viral strains as illustrated by the 14-00549-1 strain in Figure 1. This strain could be expected to belong to the 1-7-4 strain group based on its RFLP pattern and the presence of a D4 MJ type in this cluster. However, it is very dissimilar to strains associated with the 1- 7-4 outbreak and does not possess deletions in nsp2 like most of the recent 1-7-4 strains. Potentially important differences in structural proteins within the 1-7-4 cluster were also apparent during our analysis. We compared the amino acid sequence of structural proteins within the 1-7-4 group and found that GP5 sequences shared 97% identity while identity within GP3 was as low as 90.6%. Phylogenetic analysis based on whole genomes can bias strain selection because variations in non- structural regions can mask critical variation in structural genes. Newport Laboratories' analysis uses sequence information from structural proteins that play a critical role in active immunity and excludes genomic regions that may not be relevant.
References 1. Holtkamp et al.: 2013, J Amer Vet Med Assoc, 21:72. 2. Murtaugh et al.: 2010, Virus Res. 154:18.
Topic: Viral Disease Presenter: Canning, Paisley Abstract #: 45 Title: Impact of Bacillus subtilis C-3102 (Calsporin®) on Enteric Histopathology in Nursery Pigs after Challenge with Porcine Epidemic Diarrhea Virus (PEDv) Authors: P. Canning1, C. Ruston1, D. Madson2, J Bates1, K. Skoland1, J. Davenport1, C. Wang3, Q. Chen2,J. Zhang2, L. Karriker1; 1Swine Medicine Education Center, Iowa State University College of Veterinary Medicine, 2412 Lloyd Veterinary Medical Center, Ames, IA 50011 2Veterinary Diagnostic Laboratory, Iowa State University 3Veterinary Diagnostic and Production Animal Medicine, Iowa State University College of Veterinary Medicine, 2412 Lloyd Veterinary Medical Center, Ames, IA 50011
This study examined the effects of feeding Bacillus subtilis C-3102 (Calsporin® Calpis Co. Ltd., Japan) at on intestinal health in weaned pigs after challenge with Porcine Epidemic Diarrhea virus (PEDv).
Materials and Methods A two by three factorial design composed of three diets containing 0 CFU/g or 500 000 CFU/g or 1 000 000 CFU/g of Calsporin® and PEDv or sham challenge was conducted. Pigs were 14 days old at the start of the study and PEDv naïve. Ten pigs were randomly allocated to each treatment group and were housed in groups of five. Pigs were fed the treatment diets for 23 days. On day 19, pigs were challenged with PEDv positive or PEDv negative cell culture by oral gavage and necropsied at four days post inoculation (dpi). Five small intestine segments were collected per pig using a standardized technique. Histology slides were prepared for measurement of villi to crypt height ratios (VCR), and PEDv immunohistochemistry (IHC) and atrophic enteritis (AE) scoring for each section. For IHC, PEDv antigens were detected and semi-quantitatively scored based on the percentage of enterocytes showing a positive signal. Atrophic enteritis scores were based on the presence and severity of enteritis. All scoring was completed by a single blinded veterinary pathologist. Average daily gain (ADG) was compared across treatment groups as well. Responses were analyzed using linear mixed models. Comparisons among groups were assessed using F-tests followed by Tukey's t-tests for multiple comparisons. Differences were considered significant at the level of P < 0.05. All analysis was performed on SAS® 9.4.
Results There were significant differences in IHC, AE and VCR between PEDv positive and PEDv negative pigs fed 0 CFU/g Calsporin®. Within PEDv positive groups, there were significant reductions in IHC and AE between pigs fed 0 CFU/g Calsporin® and treatment groups that did receive Calsporin®. A reduction in IHC and AE scores corresponds to a reduction in PEDV antigen signal and in severity of enteritis. VCR was greater in PEDv positive pigs fed Calsporin® compared to pigs on the 0 CFU/g diet. There were no significant differences in average daily gain between groups.
Conclusions There appears to be an association between administration of Calsporin® and improved IHC scores, histology and villi to crypt ratios in nursery pigs challenged with PEDv compared to cohorts that did not receive Calsporin®. The impact of these parameters on growth during the entire finishing period is unknown and should be assessed with an additional study of longer duration and larger sample size.
Topic: Viral Disease Presenter: Dee, Scott Abstract #: 46 Title: Modeling the Transboundary Risk of Feed Ingredients Contaminated with Porcine Epidemic Diarrhea Virus Authors: Dee, Scott; Neill, Casey; Spronk, Gordon; Pipestone Applied Research, Singrey, Aaron; Clement, Travis; Christopher- Hennings, Jane; Nelson, Eric; Animal Disease Research and Diagnostic Laboratory, South Dakota State University, Chochrane, Roger; Jones, Cassandra; Department of Grain Science, Kansas State University, Patterson, Gilbert; Center for Animal Health and Food Safety, University of Minnesota, USA
Background This study describes a model developed to evaluate the transboundary risk of PEDV-contaminated swine feed ingredients and the effect of two mitigation strategies during a simulated transport event from China to the US.
Methods Ingredients imported to the USA from China, including organic & conventional soybeans and meal, lysine hydrochloride, D-L methionine, tryptophan, Vitamins A, D & E, choline, carriers (rice hulls, corn cobs) and feed grade tetracycline, were inoculated with PEDV. Control ingredients, and treatments (ingredients plus a liquid antimicrobial (SalCURB, Kemin Industries (LA) or a 2% custom medium chain fatty acid blend (MCFA)) were tested. The model ran for 37 days, simulating transport of cargo from Beijing, China to Des Moines, IA, US from December 23, 2012 to January 28, 2013. To mimic conditions on land and sea, historical temperature and percent relative humidity (% RH) data were programmed into an environmental chamber which stored all containers. To evaluate PEDV viability over time, ingredients were organized into 1 of 4 batches of samples, each batch representing a specific segment of transport. Batch 1 (segment 1) simulated transport of contaminated ingredients from manufacturing plants in Beijing (day 1 post- contamination (PC)). Batch 2 (segments 1 and 2) simulated manufacturing and delivery to Shanghai, including time in Anquing terminal awaiting shipment (days 1-8 PC). Batch 3 (segments 1, 2 and 3) represented time in China, the crossing of the Pacific and entry to the US at the San Francisco, CA terminal (day 1-27 PC). Batch 4 (segments 1-4) represented the previous events, including transport to Des Moines, IA (days 1-37 PC). Samples were tested by PCR, VI and swine bioassay to measure whether viable PEDV could be detected across ingredients and throughout time of the simulated journey.
Results Across control (non-treated) ingredients, viable PEDV was detected in soybean meal (organic and conventional), Vitamin D, lysine hydrochloride and choline chloride. All other samples were VI and swine bioassay negative. In contrast, viable PEDV was not detected by any method in all samples treated with LA or MCFA.
Conclusions These results demonstrate the ability of PEDV to survive in a subset of feed ingredients using a model simulating shipment from China to the US. This is proof of concept suggesting that contaminated feed ingredients could serve as transboundary risk factors for PEDV, along with the identification of effective mitigation options. Future studies will investigate the survivability of surrogate viruses representing foreign animal disease pathogens under similar conditions.
Topic: Viral Disease Presenter: Diel, Diego G. Abstract #: 47 Title: Infection Dynamics and Pathogenesis of Senecavirus A Swine Authors: Diel, Diego G.; Joshi, Lok R.; Fernandes, Maureen V.H.; Clement, Gravis; Lawson, Steven; Nelson, Eric A.; Animal Disease Research and Diagnostic Laboratory, Department of Veterinary and Biomedical Sciences, South Dakota State University, Resende, Tatine P.; Vanucci, Fabio A.; Veterinary Diagnostic laboratory, University of Minnesota, St. Paul, MN USA
Senecavirus A (SVA), a picornavirus of the genus Senecavirus, has been recently associated with vesicular disease and neonatal diarrhea and mortality in swine the US and in Brazil. Many aspects of SVA infection biology and pathogenesis remain unknown. In this study, the infection dynamics and pathogenesis of SVA were investigated in pigs. Twelve finishing pigs (~100 lb) were randomly allocated into two experimental groups as follows: Group 1: Mock-control group (n = 4) and Group 2: SVA-inoculated group (n = 8). Animals were inoculated intranasally and orally with a contemporary SVA strain and monitored daily for characteristic clinical signs and lesions associated with SVA infection. Viremia was assessed in serum and virus shedding was monitored in oral and nasal secretions and feces. Samples collected on days 0, 3, 5, 7, 10, 14, 21, 28 and 35 post-inoculation (pi) were tested by SVA real-time reverse transcriptase PCR (rRT- PCR) and virus isolation. Clinical signs were first observed on day 4 pi, and were characterized by lameness and lethargy. Vesicular lesions were observed on the snout, coronary bands, dewclaws and sole of the hooves of inoculated animals. Lameness and vesicular lesions were observed between days 4 and 14 pi. Viremia was detected between days 3 and 10 pi, whereas virus shedding was detected between days 1 and 28 pi in oral and nasal secretions and feces. Notably, rRT-PCR and in situ hybridization performed on tissues collected on day 38 pi revealed the presence of viral RNA on the tonsil of all SVA infected animals. Serological responses were monitored by virus neutralization and indirect immunofluorescence assays. Animals developed an early and robust neutralizing antibody (NA) response to SVA, with NAs being first detected on day 5 pi and peaking on day 10 pi. High levels of NAs were still detected on day 38 pi. SVA specific IgG antibodies were first detected by IFA on day 10 pi, peaked on day 14 pi and presented a slight decline on days 35 and 38 pi. These results demonstrate the pathogenicity of a contemporary SVA strain and provide significant insights on the infection dynamics, pathogenesis and shedding patterns of SVA in swine.
Topic: Viral Disease Presenter: Diel, Diego G. Abstract #: 48 Title: Evidence of Senecavirus A Replication in Tonsillar Lymphoid Cells Authors: TP Resende1; LR Joshi2; M Sturos1; EA Nelson2; FA Vannucci1 DG. Diel2 1 University of Minnesota Veterinary Diagnostic Laboratory College of Veterinary Medicine, University of Minnesota, St. Paul, MN, USA 2 Animal Disease Research and Diagnostic Laboratory, Department of Veterinary and Biomedical Sciences, South Dakota State University, Brookings, 57007, SD, USA
Introduction Senecavirus A (SVA) has been recently associated with vesicular disease and neonatal mortality in swine in Brazil and USA 1. Vesicular lesions associated with SVA infections are indistinguishable from important foreign animal vesicular diseases, including foot-and-mouth disease virus (FMDV). Moreover, SVA was histologically demonstrated within vesicular lesions using in situ hybridization (ISH-RNA) 2. This technique is able to detect transcriptionally active virus encoding SVA-VP1 mRNA. The objective of this exploratory study was to evaluate the presence and distribution of SVA in the tonsils and skin biopsies of pigs experimentally- infected with SVA.
Materials and Methods A total of 15 formalin-fixed paraffin embedded tissues (12 tonsils and 3 skins) were submitted to the University of Minnesota Veterinary Diagnostic Laboratory (UMN-VDL). Tissues were collected during the necropsy on day 38 after experimental inoculation conducted by ADRDL-SDSU. Additionally, three skin biopsies were obtained from vesicular lesions of the 12 experimentally- infected animals. Probe targeting specific genomic regions of the SVA (VP1 gene) were developed as previously described 2. Hybridization signals were detected as red colorimetric staining followed by counterstaining with hematoxylin. In addition, immunohistochemistry (IHC) targeting immune cell markers for macrophages (Iba-1), T cells (CD3) and B cells (CD79a) were performed in order to histologically map the cell population present within the SVA infected area. Four tonsils were used as negative controls.
Results SVA was demonstrated in the three skin biopsies of sows showing vesicular disease. Although the presence of SVA was predominately observed in acantholytic keratinocytes forming intraepidermal vesicles in the stratum spinosum, one sample revealed significant amount of virus in the basal cell layer. SVA was also observed in the superficial dermis, and it coincides with the presence of Iba-1 positive cells, suggesting the presence of SVA in the cytoplasm of macrophages. A total of 11 tonsil tissues were positive for SVA. The virus was predominately found in the basal layer of the crypt epithelium and in the subepithelial area. Aggregation of Iba-1 positive cells was present in the subepithelial regions of the tonsils crypts where SVA were detected. Interestingly, six tonsils revealed presence of SVA in the tonsillar lymphoid follicles. This histological region showed abundant presence of CD3 positive cells indicating expression of VP1 mRNA by SVA within T cells.
Discussion and Conclusion The pathogenesis of SVA infections remains unknown. During FMDV infection in pigs, tonsils have been inculpated as the primary site for virus replication and the major source of shedding 3. Our findings showed the presence of SVA RNA in tonsillar epithelium and lymphoid cells of infected pigs 38 days after exposure.
References 1. Joshi LR et al, 2016. J. Clin. Microbiol, 54(6):1536-45. 2. Resende T et al, 2016. 24th IPVS, Dublin, Ireland, p. 109. 3. Stenfeldt C et al, 2014. PLoS One, 9(9):e106859. 4. Stenfeldt C et al. Transbound Emerg Dis, 2016. 63(2):152-64.
Topic: Viral Disease Presenter: Dvorak, Cheryl Abstract #: 49 Title: Development of an Indirect Enzyme-linked Immunosorbent Assay for the Identification of Antibodies to Senecavirus A in Swine Authors: Cheryl M.T. Dvorak1*, Zeynep Akkutay-Yoldar 2, Suzanne R. Stone1, Steven J. Tousignant3, Fabio Vannucci4, and Michael P. Murtaugh;1 1Department of Veterinary and Biomedical Sciences, University of Minnesota, 1971 Commonwealth Ave, St. Paul, MN 55108, USA; 2Ankara University, Faculty of Veterinary Medicine, Department of Virology, Diskapi 06110, Ankara, Turkey; 3Swine Vet Center P.A., 1608 S. Minnesota Ave, St. Peter, MN 56082, USA; 4Veterinary Population Medicine, University of Minnesota, 1365 Gortner Ave, St. Paul, MN 55108, USA
Background Seneca Valley virus (SVV), a member of the family Picornaviridae, genus Senecavirus, species Senecavirus A (SVA), is a recently identified single- stranded RNA virus closely related to members of the Cardioviruses genus. SVV was originally identified as a cell culture contaminant and was not associated with disease until 2007 when it was first observed in pigs with Idiopathic Vesicular Disease (IVD). Vesicular disease is sporadically observed in swine, is not debilitating, but is significant due to its resemblance to foreign animals diseases, such as foot and mouth disease, whose presence would be economically devastating to the United States. IVD disrupts swine production until foreign animal diseases can be ruled out. Identification and characterization of SVA as a cause of IVD will help to quickly rule out infection by foreign animal diseases.
Results We have developed and characterized an indirect ELISA assay to identify serum antibodies to SVA. Viral protein 1, 2 and 3 (VP1, VP2, VP3) were expressed, isolated, and purified from E. coli and used to coat plates for an indirect ELISA. Sera from pigs with and without IVD symptoms as well as a time course following animals from an infected farm, were analyzed using this indirect ELISA to determine the antibody responses to VP1, VP2, and VP3. Antibody responses to VP2 were higher than VP1 and VP3 and showed high affinity binding on an avidity ELISA. The quantitative ELISA results were compared with an IFA assay, also in development, showing similar results.
Conclusions This assay can now be used to help differentially diagnose IVD due to SVA and helping to quickly rule out the presence of economically devastating foreign animal diseases.
Topic: Production Presenter: Homann, Taylor Abstract #: 50 Title: Characterizing Piglet Loss from PRRS Outbreak Authors: Taylor Homann1, Carles Vilalta1, DVM, PhD, Bob Morrison1, DVM, MBA, PhD 1 University of Minnesota
Introduction PRRS is a disease that causes high economic impact, particularly in sow farms with the main implication being a decrease in pigs weaned. This decrease can come from one of three sources: a decrease in litters farrowed (i.e. decreased conception rates, increased sow death, and increased resorptions / abortions), a decrease in live born per litter (i.e. decreased ovulation, increased resorption, increased stillborn and mummified fetuses) and an increase in pre wean mortality. This study had the objective of quantifying the percent that can be attributed to each source.
Methods 24 farms were selected as a convenience sample of herds enrolled in Swine Health Monitoring Project (SHMP). Farms had clinical signs and reported to SHMP as a new break. Farms had to have recovered to baseline between 2013 and 2016 according to the method reported by Linhares et al (2015)1. Use data from cohort farrowing report or equivalent to calculate average sows farrowed, live born per litter, and average of pre-wean mortality for 21 weeks prior to break. Determine decrease in wean pigs due to loss in litters, in live born and in pre wean mortality per week during the outbreak. Sum loss and determine percent attributed to each. Note: if one factor outperformed the average prior to the break, it was characterized as not contributing to overall loss.
Results Average attribution of total loss across all farms was: 38% from reduced litters farrowed, 24% from decreased live born and 38% from increased pre wean mortality. While the percent of each type of loss is approximately equally distributed, individual farms do not exhibit this distribution. (Figure 1).
References Linhares DCL. Comparison of time to PRRSv-stability and productive losses between two exposure programs to control PRRSv in sow herds. PVM. 2014; 116:111-119
Figure 1. Percentage of attribution of pig weaning loss in all the participant farms.
Topic: Viral Disease Presenter: Jarvis, Matthew Abstract #: 51 Title: Evolution and Protein Changes in Porcine Epidemic Diarrhea Virus in the United States Authors: Matthew Jarvis1 , Ham Lam1 , Martha Nelson2 , Michael Murtaugh3 , Douglas Marthaler1 1Department of Veterinary Population Medicine, University of Minnesota, 2Division of International Epidemiology and Population Studies, Fogarty International Center, National Institutes of Health, Bethesda, 3Department of Veterinary and Biomedical Sciences
Due to the introduction of porcine epidemic diarrhea virus (PEDV) into North and South America, PEDV has caused severe economic losses for global swine producers. Historically, shorter viral genes, including the envelope, membrane, nucleocapsid, or the S1 portion of the spike gene were sequenced to understand the evolution of PEDV. However, two‐thirds of the virus is composed of a single large open reading frame (ORF1), which encodes for 16 non‐ structural proteins (NSPs). In this study, we sequenced the whole genome of 93 PEDV strains from the US to understand the origin and phylogenetic relationships compared to global PEDV strains.
Between January and December 2014, PEDV positive samples were selected based on viral concentration and geographical distribution within the US. In total, 93 whole genome sequences were generated, which were sequenced by the University of Minnesota, Iowa State University, and the Ohio State University. The newly generated sequences were combined with the 126 PEDV whole genome sequences from GenBank, which exclude vaccine strains. Nucleotide and amino acid entropy analyses were performed. A concatenated genome was constructed for each sequence, and recombination and Bayesian analysis was performed. In addition, the putative pAPN receptor‐binding residues were analysized and modeled to compare differences between American and global PEDV strains.
Entropy analysis of the PEDV nucleotide and amino acid sequences revealed that the NSP2 and NSP3 region of the genome, as well as the S1 domain, were more variable compared to the rest of the genome. Recombination was found in NSP2, NSP3, NSP14 through NSP16, the S1 domain, and the nucleocapsid gene. The regions were removed prior to Bayesian analysis, which estimated a time to most recent common ancestor (tMRCA) of September 2010 ‐ August 2012, and July 2009 – July 2011 for the major and minor clades of PEDV sequences, respectively. An evolutionary rate of 6.2 x 10‐4 substitutions/site/year was determined for the complete PEDV genome, which is not significantly different than the S1 region’s substitution rate, 1.5 x 10‐3 substitutions/site/year. Modeling of the pAPN receptor binding domain (RBD) revealed that Asian PEDV strains contained more amino acid differences in key binding areas than US PEDV strains. However, vaccine strain DR13 was found to be more similar to US sequences than to Asian strains in the pAPN RBD. Substitutions occurred at 8 and 13 different positions within the pAPN RBD for US and Asian sequences, respectively.
Higher entropy levels in the NSP2 and NSP3 regions, as well as the S1 domain, suggest a higher evolutionary selection pressure on those regions, While the sequence data from Asia and Europe is limited, our analysis still points to an Asian origin for the American PEDV introduction and subsequent epidemic. Similarities in the pAPN regions between the DR13 vaccine strain and US sequences could suggest some efficacy of vaccine strains to protect against US PEDV. However, a higher evolutionary rate in the S1 domain makes developing a long‐term vaccine problematic, regardless of geographical origin. Continued research into the dynamics of key regions within the PEDV genome, as well as the role of recombination in evolution, will reveal better treatment options and help to protect the industry from future outbreaks.
Topic: Viral Disease Presenter: Kingsley, Keith Abstract #: 52 Title: Classification and Description of 1-3-4 PRRS Virus Outbreaks Using MJPRRS Technology Authors: K. Kinsley1, DVM, P. Novak2, L. Hanna2, and BK Kim2, PhD 1 Phibro Animal Health Corporation, Teaneck, NJ 07666 2 MJ Biologics, Mankato, MN 56001
Tracking PRRS viruses by their RFLP has been a common practice for many years. At times it has helped to catalogue virus identification, but has failed to explain clinical changes that arise from small changes to the viral DNA. MJ PRRS Grouping Technology is a tool that is a step closer to correlating clinical changes on farm to changes in the ORF 5 sequences (DNA, RNA, and ultimately Amino Acid).
With the increasing number of cases identified with the 1-3-4 RFLP we began a process of summarizing these sequences not only by RFLP, but additionally through the lens of MJ Biologics patented Grouping Technology. Interestingly, as MJPRRS virus grouping is applied to this RFLP classification, several patterns have been identified.
1. For the last 6 – 7 years, MJPRRS Grouping Technology has classified 82 – 85% of all PRRS ORF5 sequences as a D-group. This would cross nearly all RFLP families during that same timeframe. MJ Grouping of the 1-3-4 RFLP family indicated that viruses first identified as a 1-3-4 have appeared as an S-group 72% of the time. This is a noticeable change from the historical annual trends of S-groups being identified 12 – 15% of the time. 2. D- and S- groups (and their associated sub-groupings) are viewed by MJPRRS Grouping Technology as independent physical presentations of the PRRS virus to a population. With prior low identification of S-grouped viruses, it is reasonable to assume that population exposure to these viruses is likely to have been low and that prior immunity to these physical presentations is not apparent. 3. Stepping beyond just the D- and S- grouping down to the sub-group level (D-1 → D-8, including D-4b, and S-1 → S-8) presentations, a further pattern of virus identification reveals itself through the MJPRRS Grouping Technology. Within the heavy S-group identification of the 1-3-4 RFLP, S-2 and S-8 are identified as the most common presentations. A further comparison of clinical signs has associated the S-2 presentation with consistent high morbidity and mortality across different ages and stages of production regardless of geographic location.
While RFLP comparisons will place these viruses close, it is easy to see why reports of neighboring farms with widely varied clinical signs, despite the same RFLP, are common. MJPRRS Grouping Technology identifies virus presentations independent of RFLP and additively helps to catalogue and compare virus challenges between farms. Further consistency can be gained by utilizing these two tools (RFLP and MJPRRS Grouping Technology) together to better understand and respond to developing PRRS virus epidemics.
Topic: Viral Disease Presenter: Knutson, Todd Abstract #: 53 Title: NGS Informatics Pipelines for Unknown Pathogen Detection, Assembly, and Genome Evaluation Authors: Todd Knutson, Matthew Jarvis, and Douglas Marthaler University of Minnesota, Department of Veterinary Population Medicine, Veterinary Diagnostic Laboratory. St. Paul, MN
Porcine viral outbreaks in the United States cause substantial animal, economic, and agricultural loss to the swine industry. Rapid detection of common viral pathogens by qPCR is effective, yet these methods often fail to identify highly mutable viral subtypes and cannot identify unknown viruses causing clinical disease. Nextgeneration sequencing (NGS) of porcine samples followed by bioinformatics analysis has allowed us to survey the entire virome of an animal and provide rapid diagnostic information, including full genome assembly and comparative genomics with previously described viral strains, which was accomplished using custom software pipeline for analysis of the raw NGS data.
Pathogen detection begins with RNA/DNA isolation from various animal sources, followed by library preparation and sequencing via the Illumina MiSeq generating >16 million paired end (PE) reads per lane of 250 bp length. We assess the quality of sequencing and library preparation using FastQC and remove adapters and low quality sequences using Trimmomatic. High quality PE reads are classified using an ultrafast kmer search strategy employed by Kraken software and a database of known species. We created a custom database containing fulllength RefSeq genomes from all bacteria, viruses, archaea, fungi, protozoa, and plasmids. We also included all viral genomes in GenBank and update our database with new viral sequences identified at our institution. Kraken software can classify over 4 million reads per minute, which is 900 times faster than Megablast and generates an output that can be easily interpreted and displayed with interactive pie charts via Krona software. Individual read classification allows for isolation of speciesspecific reads that greatly facilitates de novo genome assembly. We employ three virusspecific de novo short read assemblers: IVA, PRICE, and A5, combining their results for most accurate sequences. Fulllength viral assemblies are verified by remapping short reads to the assembly. Finally, newly assembled genomes are examined in the context of previously annotated viral subtypes. We have a curated database of viral sequences (from GenBank, ViPR, and inhouse sources) that are used to annotate and evaluate new sequences for single nucleotide variants, indels, and phylogenetic analysis. We also determine amino acid conservation statistics, ORF detection, annotation, and perform recombination analysis.
We used our informatics pipeline to examine a set of 12 porcine fecal samples from the University of Texas, A&M that all displayed enteric disease from an unidentified pathogen. These samples contained an average of 2,100 (0.47%) viral reads, 409,000 (61%) bacterial reads, and 50,000 (9.1%) porcine reads. Among the viral reads, the dominant species was porcine enterovirus G and one sample contained enough data (5,847 reads) to generate a fulllength genome assembly with 116x coverage, which was manually verified. Comparative genomics revealed that it contained a single ORF, was highly divergent among subtypes in GenBank (closest BLASTn result had 80% identity and 91% coverage), and included a 647 nt insertion that mapped to a region of a torovirus replicase protein. The other samples also contained this enterovirus Gtorovirus hybrid. The development of our bioinformatics pipeline allowed for not only rapid pathogen identification of the likely causative agent in a porcine herd, but automated de novo genome assembly and annotation revealed a novel recombinant viral species circulating in the United States.
Title: Viral Disease Presenter: Lawrence, Paul Abstract #: 54 Title: Use of an Autogenous Vaccine to Improve Protection against a 1-7-4 PRRSV Strain Authors: PK Lawrence1, EA Bumgardner1 1Newport Laboratories, Inc, Biological Development and Research Department, Worthington, MN, USA
Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) is among the most important pathogens affecting swine industry worldwide (1). Since its emergence the virus has evolved, resulting in a significant amount of diversity (2). The antigenic variability of strains circulating in the field serves as a barrier to the development of effective vaccines. Currently available commercial vaccines offer good protection against disease caused by homologous strains. However, they often exhibit reduced efficacy against heterologous field strains (3). Inactivated autogenous vaccines can be manufactured with strains homologous to those emerging in the field. However, their efficacy has been debated (3).
In a recent study, we evaluated the use of two different modified live vaccines when delivered in combination with a PCV2 vaccine and a bivalent autogenous vaccine. Four-week old weaned pigs were divided into five treatment groups as follows- A) Commercial MLV vaccine; B) Commercial MLV vaccine rehydrated in Circovac® (Merial Inc.), concurrently with an autogenous PRRSV vaccine delivered at a separate site; C) Experimental MLV Vaccine; D) Experimental MLV mixed with Circovac®, concurrently with an Autogenous PRRSV vaccine delivered at a separate site; E) Mock-vaccinated controls; F) Non- treated/non- challenged controls. Six weeks after their initial vaccination animals were challenged with a virulent 1-7-4 RFLP PRRSV strain homologous to one of the strains included in the autogenous vaccine. Two weeks after the challenge was administered the serum was drawn from all animals and pigs were humanely euthanized. The lung tissue was excised and sent for examination by PRRSV specific immunohistochemistry (IHC) at Iowa State University. Serum was sent for analysis by fluorescent focus neutralization (FFN) assay at South Dakota State University. The results of this experiment are briefly outlined in Figures 1 and 2. FFN results were analyzed using a Student’s t-test to determine whether significant differences were present between the groups. Analysis of the FFN data revealed a significant increase in neutralizing antibody titer against the challenge virus in both groups that received autogenous PRRSV vaccine. The results of the IHC analysis demonstrated that fewer animals were IHC positive in groups that also received the autogenous vaccine in comparison to the corresponding groups that received only MLV.
In conclusion, the autogenous vaccine used in this study was able to enhance the protective capacity of both MLV vaccines against a virulent heterologous field strain.
References 1. Holtkamp et al.: 2013, J Amer Vet Med Assoc, 21:72. 2. Shi et al.: 2010, Virus Res, 154:7. 3. Murtaugh et al.: 2010, Vaccine, 29:8192. ®Circovac is a registered trademark of Merial, Inc.
Topic: Viral Disease Presenter: Lenz, M Corinne Abstract #: 55 Title: Efficacy of an Experimental Swine Influenza Vaccine, Killed Virus, in Pigs Challenged With a Cluster IV-A H3N2 Swine Influenza A Virus Authors: MC Lenz, VJ Rapp-Gabrielson. A Heinz, LP Taylor, J Johnson, T Hildebrand, K Segur, A Kaliyati, Y Diamondidis; Veterinary Medicine Research & Development, Zoetis, Kalamazoo, MI USA
Introduction Characterization of Cluster IV H3N2 influenza A viruses (IAV) from US swine has demonstrated the emergence of six phylogenic clades, IV-A through IV-F.1-3 The average pairwise nucleotide distance between clades is 5.4% to 7.6% and the most prevalent clade is IV-A, which represented 76.68% of the H3N2 viruses in the NVSL-USDA-APHIS- VS Influenza Surveillance database from January 2013 to March 2014.3 The objective of this study was to evaluate the efficacy of an experimental Swine Influenza Vaccine, Killed Virus, H1N1, H1N2 & H3N2, in pigs challenged with a heterologous H3N2 Cluster IV-A field virus.4
Materials and Methods Forty-five weaned, 3-week-old swine IAV-negative pigs were randomly assigned to treatment using a generalized block design. Pigs were vaccinated twice, on Days 0 and 14, with either an Amphigen® placebo (T01, n=21), the experimental swine influenza vaccine (T02, n=21), or held as non- vaccinated, non-challenged controls (NTX, n=3). NTX pigs were necropsied on Day 27 and not included in the analysis. The T01 and T02 pigs were challenged on Day 28 with A/swine/Indiana/ A01271853/2012 (H3N2). This virus, received from NVSL, Ames Iowa, is a Cluster IV-A field isolate that contains the pH1N1 M gene. The HAs of the vaccine and challenge virus are 97.3% similar. Pigs were necropsied 5 days post-challenge. The primary variable analyzed was lung lesions at necropsy. Virus isolation from nasal swabs and bronchial aveolar lavage fluids (BALF), clinical observations, rectal temperatures, and hemagglutination inhibition (HI) antibody titers were analyzed as secondary variables. The level of significance was set at α=0.05. This study was conducted in accordance with guidelines of the Zoetis Kalamazoo IACUC.
Results Vaccinated pigs had a significantly lower percentage of lung with lesions at necropsy, significantly higher HI antibody titers to the challenge virus, a significantly lower percentage of pigs ever shedding virus, and a significantly lower percentage of pigs positive for virus isolation from BALF at necropsy (Table 1). The LS Mean log10 TCID50/mL virus titers from nasal swabs of vaccinates were significantly lower compared to placebo on all five days post-challenge and by area under the curve analysis (P ≤0.0001; data not shown). In addition, the LS Mean rectal temperatures on post-challenge Days 29, 31 and 33 were significantly lower for vaccinates compared to controls (P≤0.0002; data not shown).
Group % Lung HI % Pigs Ever Postive Lesions Titer** * Nasal BALF Sheddin g Placebo 16.15% 5.0 100% 100% Vaccine 4.67% 95.1 15% 0% P value <0.0001 <0.0001 0.0001 0.0001
Table 1. Results of Cluster IV-A H3N2 swine IAV challenge of vaccinated and placebo pigs *Back transformed LS Mean **Back transformed geometric mean HI titer to challenge virus A/swine/Indiana/A01271853/2012 (H3N2) on Day 27 Conclusions Under the conditions of this study, vaccination of pigs with the experimental Swine Influenza Vaccine helped to protect against challenge with a Cluster IV-A virus representing the most prevalent H3N2 clade currently circulating in US swine. References 1. Kitikoon P, et al. 2013. J. Gen. Virol. 94, 1236–1241, doi.org/10.1099/vir.0.51839-0. 1. Anderson TK, et al. 2013. Influenza Other Respir. Viruses 7 (Suppl. 4),42–51, doi.org/10.1111/irv.12193. 2. Anderson TK, et al. 2015. Virus Res. 201:24-31, doi.org/10.1016/j.virusres.2015.02.009 3. Data on file, Study Report No. B820R-US-14-436, Zoetis Inc.
Topic: Viral Disease Presenter: Lenz, M Corinne Abstract #: 56 Title: Antigenic and Genetic Diversity of Cluster IV H3N2 Influenza A Viruses of Swine Authors: VJ Rapp-Gabrielson1, MC Lenz1, MR Culhane;2 1Veterinary Medicine Research & Development, Zoetis, Kalamazoo, MI and 2University of Minnesota College of Veterinary Medicine, St. Paul, MN USA
Introduction Characterization of Cluster IV H3N2 influenza A viruses (IAV) from US swine has demonstrated the emergence of six phylogenic clades, IV-A through IV-F.1,2,3 In addition, hemagglutination inhibition (HI) antibody cross-reactivity and antigenic cartography has been used to quantify antigenic differences among contemporary H3N2 viruses and to identify the genetic basis for these differences.4,5 In a previous report we also evaluated genetic and antigenic diversity of swine H3N2 viruses.6 In this study we further characterized swine H3N2 viruses representing major genetic and antigenic clusters, in the context of amino acid substitutions at antigenic Sites A and B that are associated with major cluster transitions for swine H3N2 viruses.4,7
Materials and Methods The viruses evaluated were from Zoetis’ collection of US field isolates or viruses obtained from NVSL- USDA-APHIS-VS as part of the national voluntary swine influenza surveillance program. Subtyping and sequencing were performed at the University of Minnesota Veterinary Diagnostic Laboratory (VDL) or, for the viruses obtained from NVSL, the HA gene sequence data were obtained from the NIAID Influenza Research Database through the web site http://www.fludb.org.8 The sequences were aligned using Clustal W. Phylogenetic analyses were conducted using MEGA version 6.9 To evaluate HI cross-reactivity, polyclonal antisera were produced by inoculating weaned IAV- negative pigs with inactivated, adjuvanted whole- virus monovalent vaccines.10 The animal phase of these studies was conducted under the guidelines of the Zoetis Kalamazoo IACUC.
Results & Conclusions HI cross-reactivity testing (Table 1) and amino acid substitutions at key cluster-transition sites A and B of the HA1 protein (Table 2) of viruses representing Clades IV, IV-A, IV-B, IV-C and IV-F highlighted the diversity between and among clades These data are consistent with previous reports showing considerable antigenic diversity among and within the H3N2 Cluster IV clades that may be associated with amino acid substitutions at the key antigenic sites of the HA1 protein of H3N2 viruses.
Table 1. HI Cross-reactivity of H3N2 swine IAV
Swine IAV H3N2 Antisera (Clade/Virus) Antigen to: IV IV-A IV-B IV-C IV-F MO/069 NC/394 NC/548 IN/853 MN/872 MN/731 IL/355 TX/555 MO/069 2560 80 <10 80 40 20 <10 40 NC/394 160 320 <10 80 40 10 <10 <10 NC/548 40 20 1280 20 320 <10 <10 40 IN/853 320 160 40 160 160 80 <10 20 MN/872 80 40 320 80 1280 20 <10 80 MN/731 320 160 <10 80 80 160 <10 10 IL/355 10 10 <10 <10 10 <10 640 <10 TX/555 640 80 640 40 320 20 <10 320
Table 2. Amino acid substitutions at key antigenic sites A & B of the HA1 protein of H3N2 swine IAV
Amino Acid Substitution at Sites A & B Clade Virus 145 155 156 158 159 189 IV MO/069 N H N D Y R NC/394 N Y N N Y K IV-A NC/548 K Y N N D K IN/853 N Y N N Y K MN/872 K Y H N Y K IV-B MN/731 N Y N N Y K IV-C IL/355 N Y H G H E IV-F TX/555 N/K H N N Y R
References 1. Kitikoon P, et al. 2013. J. Gen. Virol. 94, 1236–1241, doi.org/10.1099/vir.0.51839-0 1. Anderson TK, et al. 2013. Influenza Other Respir. Viruses 7 (Suppl. 4),42–51, doi.org/10.1111/irv.12193 2. Anderson TK, et al. 2015. Virus Res. 201:24-31, doi.org/10.1016/j.virusres.2015.02.009 3. Kitikoon P, et al. 2013. Influenza Other Respir. Viruses 7 (Suppl.4), 32-41, doi.org/10.1111/irv.12189 5. Lewis NS, et al. 2014. J. Virol. 88:4752-4763, doi:JVI.03805-13 6. Rapp-Gabrielson VJ, et al. 2014. Presentation at VAAVV Conference, Iowa State University, Ames, IA, June 18-20. 7. Koel BF, et al. 2013. Scinece 342:976-979, doi.10.1126/science.1244730 1. Squires RB, et al. 2012. Influenza Other Respir. Viruses 6.6: 404–416. PMC. Web. 11 May 2016 2. Tamura K, et al. 2013. Molecular Biology and Evolution 30:2725-2729. 3. Data on file, Study Report Nos. B820R-US-12-099, B821R-US-13-155, B821R-US-13-153 and B820W-US- 13-236, Zoetis Inc.
Topic: Viral Disease Presenter: Miller, Timothy Abstract #: 57 Title: Two-Year Stability of Bovine Serum Albumin Formulated with VaxLiant™ Adjuvants in CF-1 Mice Authors: Tim Miller, Laura Trygstad, Melissa Inman, Mary Ann Pfannenstiel, William Swafford
A new class of adjuvants capable of being customized to the antigen is being developed by VaxLiant, LLC. Stability studies were conducted to assess physical parameters and immunological responses to antigens formulated with these adjuvants when stored at refrigerated or ambient temperatures. One of the more comprehensive methods for assessing the stability of an adjuvanted vaccine is to measure its ability to elicit a serological response in an animal model over time. Therefore, this study was conducted to assess the ability of the ENABL 1™ and ENABL™ 6 adjuvants combined with 50ug/dose bovine serum albumin (BSA) to elicit a serological response in female CF-1 mice at five time points over two years.
Female CF-1 mice were inoculated with the BSA + adjuvant (stability) vaccines on the day of preparation and again following 6, 12, 18 or 24 months of storage at refrigerated or ambient temperatures. A new group of mice was inoculated at each time point. Mice were observed daily for health and blood was collected approximately two weeks after inoculation. The level of anti-BSA response was measured by ELISA and the geometric mean (GMT) for each group was calculated using the average ELISA titer.
As expected, the immune response to BSA was measurably greater when the BSA was formulated with adjuvant. Mice inoculated with vaccines containing the ENABL™ adjuvants had 100% serconversion to BSA at every time point, whereas an average of 70% seroconversion was obtained in the mice inoculated with BSA prepared in phosphate buffered saline (PBS). The GMT values obtained for all vaccines in which the BSA and adjuvants were stored combined (stability vaccines) did not diminish over the 24 month study period at either storage temperature. The GMT values also did not diminish over 24 months for mice inoculated with vaccines prepared fresh on Day 0 of each time point (fresh vaccines) using adjuvant stock solutions stored at each temperature.
The overall conclusion of this study is that both VaxLiant™ adjuvant formulations assessed for stability appear to be capable of storage at either ambient or refrigerated temperatures up to 24 months without loss in the ability to elicit an immune response in vivo. These adjuvants were stored combined with an antigen or stored separately without loss in activity.
Topic: Viral Disease Presenter: Neira, Victor Abstract #: 58 Title: Cross Protection Evaluation and Antigenic Diversity of Influenza A Viruses Prevalent in Intensive Swine Farms in Chile Authors: Rodrigo Tapia1, Victoria García1, Miguel Vivar1, Sergio Bucarey1, Rafael Medina2, Víctor Neira1*. 1Facultad de Ciencias Veterinarias y Pecuarias, Universidad de Chile, Santiago, Chile. 2Facultad de Medicina, Pontificia Universidad Católica de Chile, Santiago, Chile
Introduction Influenza A virus (IAV) is widely distributed in Chilean intensive swine farms, where pandemic H1N1 (pH1N1), human-like H1N2, and human-like H3N2 have been identified using full genome sequencing. Genetic distances suggest at least 5 different clusters of IAV, 3 of them are the most prevalent. The objective of this study was a) to determine cross protection between Chilean IAVs, and b) to determine antigenic diversity of IAV from surveillance samples of sera or IAV isolates.
Material and Methods IAV vaccines were prepared using 3 Chilean reference viruses: pH1N1, Chilean A H1N2, and Chilean B H1N2, representing the most prevalent viruses in the country. To select these viruses, phylogenetic analysis was used to identify the virus with HA sequence more similar to the consensus sequence for each cluster. Groups of four guinea pigs were vaccinated with each vaccine and another group was mock vaccinated. Animals were negative against IAV antibodies at day 0. Intramuscular vaccination was performed at day 1 and booster at day 14. At day 28, animals were euthanized by C02 and the sera were obtained. Hemagglutination inhibition (HI) assay was performed with the homologous and viruses from the same and other clusters. After, 50 new IAV isolates obtained in 2015 were evaluated by HI to determinate antigenic variability using the reference sera previously obtained. Also, 607 serum samples from 13 farms obtained in 2015-2016 were evaluated by HI using reference viruses in order to determine circulating viruses. In most of the farms included, IAV was previously confirmed only by ELISA.
Results and Discussion All sera reacted with viruses from the same cluster, with HI titers over 1/160. All challenges with viruses from other clusters were negative. Then, different clusters have not cross protection between them (pH1N1, Chilean A H1N2 and Chilean B H1N2). All IAV isolates were subtyped, 26 out 50 IAV isolates were subtyped as a Pandemic H1 (52%), 22 out total (44%) was subtyped as Chilean H1 cluster A and only 2 isolates were subtyped as Chilean H1 cluster B. One thousand, one hundred and forty-nine HI assays were performed out 607 serum samples in order to determine circulating viruses in 13 farms. From a total samples, 139 react against pH1N1, representing 11 farms. Thirty-five samples react against Chilean A H1N2, representing 4 farms. One farm reacts against H3N2 and another against Chilean B H1N2. All farms were positive at least one virus and four of them had antibodies against 2 or more viruses. HI results demonstrate predominance of pH1N1 and secondly Chilean A H1N2 cluster in swine farms in 2015-2016. HI assay is still very useful and complementary of sequencing. This is study confirms the circulation of swine IAV variants in Chile and demonstrates fail in cross protection between Chilean clusters, which would be important in vaccine development.
Funded by the grant Anillo de Investigación en Ciencia y Tecnología - PIA ACT1408 from CONICYT Chile, HHSN272201400008C NIH-NIAID, FONDEF ID14I10201.
Topic: Viral Disease Presenter: Niederwerder, Megan C Abstract #: 59 Title: Microbiome Associations in Pigs with the Best and Worst Clinical Outcomes Following Co-infection with Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and Porcine Circovirus Type 2 (PCV2) Authors: Megan C. Niederwerdera,b, Crystal J. Jaingc, James B. Thissenc, Ada Giselle Cino-Ozunaa,b, Kevin S. McLoughlind, and Raymond R. R. Rowlanda aDepartment of Diagnostic Medicine/Pathobiology, bKansas State Veterinary Diagnostic Laboratory, College of Veterinary Medicine, Kansas State University, 1800 Denison Avenue, Manhattan, Kansas 66506, USA. cPhysical & Life Sciences Directorate, dComputations Directorate, Lawrence Livermore National Laboratory, 7000 East Avenue, Livermore, California 94550, USA.
Abstract On a world-wide basis, co-infections involving porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) are common and contribute to a range of polymicrobial disease syndromes in swine. Both viruses compromise host defenses, resulting in increased susceptibility to infections by primary and secondary pathogens that can affect growth performance as well as increase morbidity and mortality. An experimental population of 95 nursery pigs was co-infected with PRRSV and PCV2. At 70 days post-infection (dpi), 20 representative pigs were selected as having the best or worst clinical outcome based on average daily gain (ADG) as well as the presence and severity of clinical disease. Worst clinical outcome pigs had prolonged and greater levels of both PRRSV and PCV2 viremia as measured by qPCR. Serum, lung and fecal samples collected at 70 dpi were analyzed using a comprehensive DNA microarray technology, the Lawrence Livermore Microbial Detection Array, which can detect over 8,000 microbes. Bacterial species, such as Bacillus cereus, were detected at a significantly higher rate in the serum of worst performing pigs. At the level of the fecal microbiome, the overall microbial diversity was significantly lower in the worst clinical outcome group. Members of the Proteobacteria phylum, specifically Escherichia coli, were detected at a significantly higher rate in the feces of best performing pigs. The results reinforce the importance of pathogen load in determining clinical outcome and suggest an important role of microbial diversity as a contributing factor in disease. With growing pressure to eliminate antibiotic use and innovate new management tools for infectious diseases in food animals, this work provides foundational knowledge about the microbiome characteristics that may contribute to performance enhancement in the presence of common viral pathogens.
Topic: Viral Disease Presenter: Philips, Reid Abstract #: 60 Title: Evaluation of PRRSV Challenge Dose in Vaccinated Pigs Authors: G Haiwick, A Neubauer, J Herman, M Roof, B Fergan, R Philips Boehringer Ingelheim Vetmedica, Inc. MO
Introduction The infectious dose of PRRSV has been shown to be very low, therefore highly infectious. The objective of this study was to evaluate the effect of PRRSV challenge dose in vaccinated pigs.
Materials and Methods The study was performed in ninety, three-week-old pigs from a PRRS naïve and PCR negative source. Groups 1-5 (n=10) were vaccinated (Day 0) with Ingelvac PRRS® MLV (2ml IM). Forty pigs served as matched non-vaccinated challenge controls (NVC- Groups 1-4; n=10 per group). Groups 1-4 were challenged on Day 28 intranasally with 2.0 ml of virulent PRRSV SDSU-73 at 4log, 3log, 2log or 1log10TCID50/ml, respectively. Group 5 was not challenged. Temperature (Day 28-42), viremia and ADWG (Day 28- 70) were evaluated and statistically analyzed.
Results At all challenge doses, Ingelvac PRRS® MLV vaccinated pigs demonstrated a significant decrease in days pyrexic compared to NVC groups (P<0.05). At PRRSV challenge doses of ≤2logs, the average temperatures and days pyrexic of the vaccinated challenged pigs were similar to the non-challenge control (Table 1).
As compared to the NVC, there was a significant increase in ADWG (P<0.05) in the 3, 2 and 1log groups, and at P<0.07 in the 4log group. ADWG in vaccinated groups challenged with ≤2logs of PRRSV were numerically similar to the ADWG of control. There was a measurable negative impact on ADWG in the NVC groups with no difference across all challenge doses (Table 2).
Vaccinated pigs demonstrated fewer percent PCR positive pigs than NVC pigs at all challenge doses. As challenge dose decreased the percentage of viremic pigs in vaccinated groups decreased, with viremia in vaccinated pigs challenged with ≤2logs similar to the non- challenge control (Figure 1). At all challenge doses, the NVC pigs show similar post-challenge viremia profile.
Discussion In this study, at all challenge doses, Ingelvac PRRS® MLV vaccinated pigs demonstrated a reduction in post-challenge viremia, temperature and increased ADWG as compared to NVC pigs. Based on challenge dose (≤2logs), the consequences in vaccinated pigs were similar to non-challenged pigs. The post-challenge viremia and ADWG of NVC pigs were similar across all challenge doses, indicating a measurable negative impact. Implementation of vaccine for PRRS control can mitigate the consequences of PRRSV infection subsequently improving health and performance.
Topic: Viral Disease Presenter: Philips, Reid Abstract #: 61 Title: Efficacy of Ingelvac PRRS® MLV Against a Heterologous PRRSV 1-7-4 RFLP Challenge Authors: A. Patterson, B. Fergen, J. Hermann, G. Haiwick, R. Philips Boehringer Ingelheim Vetmedica, Inc., Ames, USA
The use of Ingelvac PRRS® vaccines can significantly reduce lung lesions following challenge with heterologous isolates (86-94% ORF5 nucleotide similarity) in the three-week-old pig respiratory challenge model.1 However, the efficacy of Ingelvac PRRS® MLV vaccine against current virulent PRRSV isolates , such as RFLP 1-7-4, has not been reported to date. This experiment was designed to evaluate the efficacy of two commercially available PRRSV vaccines in a three-week- old pig respiratory challenge model, using a heterologous RFLP 1-7-4 field isolate from 2014.
Materials and Methods At approximately three weeks of age (Day 0 of the study), 154 PRRSV naïve piglets pigs were randomized into groups, and intramuscularly vaccinated with 2 ml of either a placebo (challenge controls n=64), Ingelvac PRRS® MLV (n=45) or Fostera® PRRS (n=45). Pigs were housed in rooms by group during the vaccination period. At day 28 of the study (D28), all pigs were comingled and challenged with 2.0 mL intramuscularly and 2.0 mL intranasally (1 mL per nostril) with 104.6 TCID50/mL of PRRSV RFLP 1-7-4. Serum samples, weights, and temperatures were collected periodically from D0 through termination of the study on D42. On D42 (14 days post-challenge), all pigs were necropsied and lungs were scored for the presence of macroscopic lesions and BALF samples were collected. Serum samples were tested by RT-PCR for the presence of viremia and by ELISA for the presence of anti-PRRSV antibody. A subset of samples were assayed by bead- based multiplex assay for multiple cytokines including IFN-α. Data were analyzed using Generalized and Linear Mixed Models. Pairwise comparisons between groups were conducted as appropriate using a level of confidence of 0.05 to indicate statistical significance.
Results Table 1 summarizes lung lesions (percentage) for each group. Table 2 summaries average daily weight gain (ADWG) for the post- challenge period by group. Figure 1 displays the percentage of animals with detectable amounts of INF-α at D29 and D35. Additional data analysis is in progress at the time of abstract preparation.
Table 1. Day 42 Percent Lung Lesions (Median)
Group Treatment Lung Lesions 1 Ingelvac PRRS® 8.4a MLV 2 Fostera® PRRS 12.9a 3 Placebo 25.4b
a significantly different from the placebo at P≤0.05
Group Treatment ADWG in lbs. 1 Ingelvac PRRS® 0.61a MLV 2 Fostera® PRRS 0.49a 3 Placebo 0.24b Table 2. Post-challenge ADWG
a significantly different from the placebo at P≤0.05
Figure 1. IFN-alpha % positive by group and study day
A,B values are significantly different at P≤0.05
Conclusion The pigs vaccinated with Ingelvac PRRS® MLV had significantly reduced lung lesions, and increased ADWG, in comparison to placebo vaccinated pigs, following challenge with a recent PRRSV RFLP 1-7-4 isolate. In addition, vaccination with Ingelvac PRRS® MLV resulted in a significantly lower percentage of animals with an IFN-α response as compared to placebos at D29 and D35.
References 1. Patterson, A., et al. 2013. Proc Leman Swine Conf.
Topic: Viral Disease Presenter: Sasaki, Yosuke Abstract #: 62 Title: Epidemiological Information of Porcine Epidemic Diarrhea During the First Epidemic Year (2013 to 2014) in the Southern Part of Kyushu Island, Japan Authors: Y. Sasaki, J. Alvarez, A. Perez, S. Sekiguchi and M. Sueyoshi University of Miyazaki, Miyazaki 889-2192, Japan
Introduction and Objectives Porcine epidemic diarrhea virus (PEDv) was first reported in Japan in the 1990s, and a PED live vaccine was approved in 1996 (Sueyoshi et al., 1995). Since then, only sporadic and relatively unimportant outbreaks were recorded. However, in 2013 and following the first PEDv case reported in Japan in seven years, detected in Okinawa prefecture in October, the virus rapidly spread across the country with 817 PEDv cases confirmed across 38 prefectures as of August 31, 2014. Miyazaki and Kagoshima prefectures are located on the southern island of Japan and are the major swine-producing area in Japan. In these prefectures, during the first year of the epidemic, PED was detected from December 2013 to July 2014. The objective of the study here was to investigate the epidemiological information of PED during the first year of the epidemic (2013 to 2014) in the southern part of Kyushu Island, Japan.
Material and Methods The present study was conducted in the southern part of Kyushu Island, Miyazaki and Kagoshima prefectures, Japan. Information on the location and characteristics of all swine farms in these prefectures was obtained from the prefecture database (including farm size and farm operation type). The database included 1269 pork producers. Between- farm distance and Kernel density was calculated using ArcGIS V10.2 (ESRI, Redlands, California, USA). Data on PED incidence in the region between December 2013 and July 2014 was obtained from Miyazaki and Kagoshima Livestock Hygiene Service Center, and was available. PED was first suspected based on the presence of clinical signs, and all cases suspected were confirmed by laboratory testing. Statistical analyses were performed in SAS ver 9.4 (SAS Institute Inc., Cary, NC, USA). Presence of spatio-temporal clustering was assessed using the Bernouilli model of the spatial scan statistic.
Results and Discussion During the first epidemic year (2013 to 2014), 250 out of 1269 (19.7%) farms broke with PED. The first case was detected in middle December, and a sharp increase was observed in early February and middle March. The outbreak was mainly seen in the southeast part of the Kagoshima prefecture and the southwest part of the Miyazaki prefecture (Figure 1). Spatio-temporal clustering was found in both of those areas. Average between-farm distance was 1.056±0.06 km, and no difference between PED infected and non-infected farms was found. Number of neighbor farms within 1 km was higher in PED infected farms than non-infected farms (4.8 vs. 2.5 farms; P<0.05). In conclusion, PED spread in the studied region was related to farm density, but not between-farm distance. Further epidemiological studies are needed to identify risk factors associated with a PED outbreak.
References Sueyoshi et al., 1995. J. Comp. Pathol. 113, 59–67.
Topic: Viral Disease Presenter: Tapia, Rodrigo Abstract #: 63 Title: Phylogenetic and Antigenic Characteristics of the Hemagglutinin Gene of Swine-origin Influenza A Virus in Chile Author: Rodrigo Tapia1, Victoria García1, Sergio Bucarey1, Gonzalo Barriga2, Rafael Medina2, Víctor Neira1*. 1Facultad de Ciencias Veterinarias y Pecuarias, Universidad de Chile, Santiago, CHILE 2Facultad de Medicina, Pontificia Universidad Católica de Chile, Santiago, CHILE
Introduction Influenza A virus (IAV) is frequently detected in Chilean intensive swine farms, where the pandemic H1N1 (pH1N1), human-like H1N2, and human-like H3N2 genotypes have been identified. Commercial vaccines containing North American IAV strains are currently used in Chile; however, there is limited information regarding genetic and antigenic difference between North American and Chilean strains. The objective of this study was to analyze and compare genetic and antigenic characteristics of HA1 globular domain of Chilean and North American IAVs.
Material and Methods 48 sequences of the HA1 region of H1 and H3 IAVs, from 18 Chilean intensive swine farms, were used in this study. Phylogenetic trees were constructed using maximum likelihood method based on the general time reversible model with a variation rate among sites given by gamma distribution with invariant sites. Nucleotide and deduced amino acid sequences from antigenic sites (Sa, Sb, Ca1, Ca2 and Cb for H1; A, B, C, D and E for H3) were also analyzed by distance matrices and their amino acid signature patterns. Predictions of N-linked glycosylation sites on HA1 protein were made according to the Asn-X-Ser/Thr sequon. Reference sequences available in GenBank, corresponding to North American H1 (α, β, γ, and δ) and H3 (I and IV) clusters used in commercial vaccines, were also included in the analysis.
Results δ-cluster was the closest North American cluster to Chilean human-like H1 virus, however, they were over 9% distant at the sequence level. For HA1, the nucleotide and amino acid identities were 86.8–89.9% and 86.1–91%, respectively. In the case of the antigenic sites, the identities were 79.7–89.4% and 68.3–82.9%, respectively. The greatest difference in antigenic residues was noted in Sb and Ca1 sites. Furthermore, most Chilean human-like H1 have an additional glycosylation site at residue 291, with a total of 6 sites, as compared to δ-cluster. On the other hand, cluster I was the closest North American cluster to Chilean human-like H3 virus, with nucleotide and amino acid identities between 87.4–90.7% and 87.3–90% for HA1; and 83.1–90.2% and 73.8–83.6% for the antigenic sites, respectively. The greatest variation occurs in residues located in B and C antigenic sites. Similarly, the major difference between North American and Chilean pH1 strains occurs in the amino acid sequence of the antigenic sites, with an identity of 68.3-100%. Some Chilean pH1 strains have an additional glycosylation site at residue 123, near of Sa antigenic site, compared to North American pH1 strains.
Conclusion Chilean human-like H1 and H3 IAVs are not related to any North American cluster. The identities in antigenic sites were lower than in the rest of the HA1 region, and the identities of the amino acid sequences were lower than the nucleotide sequences, indicating the dominance of non-synonymous change in the antigenic sites, also seen in the amino acid signature pattern. These mutations may interfere with the immune response mediated by neutralizing antibodies, interference that can be enhanced by the presence of additional glycosylations near antigenic sites. This is consistent with preliminary results of hemagglutination inhibition assays, where viruses from the most prevalent Chilean cluster (human-like H1) did not show cross-immunity with North American strains (β, γ and δ), present in commercial vaccines (data not shown). Results from this study can be used for further cross-protection studies and to update the vaccines used in Chile.
PhD program funded by CONICYT-PCHA/Doctorado Nacional/2014-21140719. Project funded by the grant Anillo de Investigación en Ciencia y Tecnología - PIA ACT1408 from CONICYT Chile, HHSN272201400008C NIH-NIAID, FONDEF ID14I10201.
Topic: Viral Disease Presenter: van Geelen, Albert Abstract #: 64 Title: Comparative Pathogenesis and Characterization of Contemporary 1-7-4 PRRSV Isolates in Weanling Age Piglets Authors: A.G.M. van Geelen, Kay Faaberg, Phani Das, Nestor Montiel, Laura Miller, Vikas Kulshreshtha, Alexa Buckley, and Kelly Lager, VPRU, ARS-USDA, Ames, IA USA
Porcine respiratory and reproductive syndrome (PRRS) continues to be an economically important disease affecting commercial pig production in the United States and worldwide. Its considerable sequence and antigenic variation, coupled with the limited protection offered by current vaccine options impedes our abilities to control PRRS virus (PRRSV). Recently, PRRSV isolates of 1-7-4 lineage have been associated with clinical cases of severe maternal reproductive failure. Next-generation sequencing was used to analyze 22 contemporary PRRSV isolates that were all previously determined to have a restriction fragment length polymorphism (RFLP) 1-7-4 pattern. Four isolates were selected for a comparative pathogenesis study in weanling pigs that included a PRRSV isolate of known moderate pathogenicity in weanling age piglets. Eighty-five 4-week-old piglets were inoculated with 5 x 10E4 TCID50 intranasally. Although the isolates were 87.4% to 95.1% identical in genomic sequence for ORFs2-7, there was considerable difference in pathogenicity among the isolates. Pathogenicity was determined by analyzing average daily gain (ADG), pyrexia measured by intramuscular temperature, viral titers in serum and bronchiolar-alveolar lavage and gross lung lesions. We identified two isolates, not previously described that caused more severe disease than our reference pathogenic isolate. Both of these 1-7-4 isolates caused 14% mortality within 2 weeks. The group of pigs infected with NADC-34 isolate had the highest average body temperatures of 41C on 5, 8 and 9 days post infection (dpi)., a decrease in ADG of 98.4%, and peak serum titers of 10E2.9 at 4 dpi. Isolate ISU/2014/3 with peak viral titers at 7 dpi of 10E3.7 caused a 63.2% decrease of ADG, reached peak temperatures of 40.5 to 40.7 between 5-8dpi. On the other hand, isolate ISU/2014/2 caused no pyrexia, had a 4.6% decrease in ADG but still had peak viral titers of 10E2.6 at 7dpi. This study shows that there is considerable difference in disease outcome caused by PRRSV isolates grouped under the 1-7-4 lineage and that other parameters of pathogenicity remain to be identified for future evaluation of disease.
Topic: Viral Disease Presenter: Wetzell, Thomas Abstract #: 65 Title: Comparison of Full Dose Versus Partial Dose of a Modified Live PRRS Vaccine Authors: T Wetzell, G Anderson, R Philips; Boehringer Ingelheim Vetmedica, Inc., MO, USA
Introduction High mortalities are sometimes observed in pigs exposed to PRRSv, even when vaccination is administered. Vaccination protocols at only half the label dose of modified live PRRS vaccine are used infrequently in the field to reduce production costs. Half dosing PRRS vaccine may lead to more variation in the protective immune response to field virus PRRS infections when faced with highly pathogenic strains and/or early exposure compared to a full dose of modified live PRRS vaccine. The objective of the project was to determine if variation in grower finisher mortality could be reduced in a flow experiencing high mortalities by utilizing a full dose protocol of a modified live PRRS vaccine compared to a half dose protocol.
Materials and Methods Two flows experiencing higher than expected mortality in grower finisher pigs located in a hog dense portion of the upper Midwest were identified. Prior to June, all pigs received a half dose of a modified live PRRS vaccine 1-2 weeks post-weaning. Close out group data was collected from March through November for these barns. Starting in June all pigs in the same flows started receiving a full dose of a modified live PRRS vaccine 1-2 weeks post-weaning. Barn close out data on production performance was obtained. A total of 327 barns given the one half dose of a modified live PRRS vaccine and 136 barns given the full dose of a modified live PRRS vaccine were descriptively analyzed and statistical process control (SPC) charting conducted. Oral fluid (OF) samples were obtained from 100 barns vaccinated with a full dose at weaning; 7-8 weeks post-weaning, in the nursery, and 12-14 weeks post weaning in the finisher. Genomic sequencing of ORF5 was done on PCR positive samples.
Results OF results from 100 sites tested revealed 28% of the positive sites to be infected with field virus. Mortality rate of on 327 groups that received one half dose of a modified live PRRS vaccine compared to a mortality rate of 136 groups that received a full dose of a modified live PRRS vaccine are shown in Table 1.
Table 1. Before / After Average Closeout Results Treatment n % Mortality
Half Dose 327 5.69
Full Dose 136 4.12
Discussion Groups receiving the recommended full dose of a modified live PRRS vaccine had lower mortality and less variation than prior groups receiving a half dose of a modified live PRRS vaccine. If side by side comparisons are difficult to do, before and after Statistical Process Control (SPC) charting can provide a valuable tool in vaccine decisions when process changes are considered.
Topic: Viral Disease Presenter: Wetzell, Thomas Abstract #: 67 Title: Performance Comparison of Partial Dosing to Full Dosing of Ingelvac PRRS® MLV Authors: T Wetzell, R Philips, S. Playter; Boehringer Ingelheim Vetmedica, Inc., MO, USA
Introduction Increased mortalities have recently been observed in growing pig populations that have been infected with highly virulent strains of PRRSv, including isolates of RFLP 1-7-4. In addition, off-label use of modified live PRRS vaccines at partial dosing to reduce production input costs occurs. The objective of this project was to determine if differences in performance and mortality could be detected in nursery and finisher pigs when moving from a partial to full dose modified live PRRS vaccination protocol.
Materials and Methods Sixteen hundred weaned pigs received weekly from one sow farm into a wean-to-finish system in the Upper Midwest USA had experienced higher than expected mortalities and erratic performance for a prolonged period of time. Health issues that had been identified in the pigs included a high incidence of Porcine Respiratory Disease Complex (PRDC) including, PRRS, Influenza type A Virus (IAV) and Mycoplasma hyopneumoniae. All pigs included in the analysis were received as PRRS negative pigs from the sow farm. Pigs had received a partial dose of a modified live PRRS vaccine after arrival. A protocol change was implemented and pigs started receiving a full dose of Ingelvac PRRS® MLV after arrival. Close out information from October 2013 through December 2014 was obtained for 56 nursery groups and 51 finishing groups that had received partial dose of vaccine. Close outs were also obtained for 24 nursery groups and 37 finishing groups from November 2014 through December 2016 that received a full dose of Ingelvac PRRS® MLV. The close out data was compared by statistical process control charts (SPC). Oral fluid and serum was collected from 15 finishing sites and tested for PRRSV by PCR.
Results Eleven of fifteen finishing sites (73%) were positive for PRRS field type virus (based on sequencing). Mortality rates were reduced by 2% for nursery and finishing sites receiving the full dose protocol. Cull rates were also 0.7% lower for full dose finishing sites (Table 1). The full dosing protocol showed a SPC detection signal with ten consecutive points below the mean for finishing mortality by group representing a sustained process change (data not shown).
Table 1. Performance measurements in nursery pigs receiving partial vs full dose modified live PRRS vaccine
Groups Mortality Cull FCR
Partial* 56 4.82% 0 1.61
Full** 24 3.88% 0 1.58
* Number of groups Oct 2013 – Dec 2014 ** Number of groups Nov 2014 – Dec 2016 Table 2. Performance measurements in finisher pigs receiving partial vs full dose modified live PRRS vaccine
Groups Mortality Cull FCR
Partial* 51 5.87% 1.85% 2.87
Full** 37 4.91% 1.16% 2.79
* Number of groups Oct 2013 – Dec 2014 ** Number of groups Nov 2014 – Dec 2016
Discussion Compared to a full dose, partial dosing of a modified live PRRS vaccine may lead to more variation in the immune response and subsequent inconsistent performance when challenged with highly pathogenic PRRS strains. Following labeled dosing recommendations can lead to a significant return on investment when compared to partial dosing of a modified live PRRS vaccine. Topic: Viral Disease Presenter: White, Lauren Abstract #: 68 Title: Stochastic Mathematical Modeling of Management Interventions to Control Influenza A Virus in Swine Breeding Herds Authors: LA White1, M Torremorell2 & ME Craft2, 1 Department of Ecology, Evolution & Behavior, 2Department of Veterinary Population Medicine, University of Minnesota, St. Paul, U.S.A.
Introduction Influenza A virus (IAV) is a global, endemic infection that causes significant morbidity and productivity losses in swine and poses a substantial threat to public health. Recent modeling and empirical work on IAV suggests that vaccination (especially heterologous) can have limited efficacy, and that piglets play an important role as an endemic reservoir. The objective of this study is to test additional intervention strategies aimed at reducing the incidence of IAV in piglets overall and ideally, preventing piglets from becoming exposed in the first place. These interventions include biosecurity measures, vaccination, and management options that swine producers could employ individually or in combination to control IAV in their herds.
Methods We have developed a stochastic Susceptible-Exposed-Infectious-Recovered-Vaccinated (SEIRV) model of IAV dynamics in a swine breeding herd. The construction of this metapopulation model reflects the spatial organization of a standard breeding herd and accounts for the different classes of pigs therein including gilts, sows, and piglets in various production and immune stages. Notably, this model allows for loss of immunity for vaccinated and recovered animals, and for vaccinated animals to have distinct latency and infectious periods as suggested by the literature. The interventions tested include: (1) mass and pre-farrow vaccination strategies with different vaccine efficacy (homologous vs. heterologous), (2) gilt isolation (no indirect transmission to or from the gilt development unit), (3) gilt vaccination upon arrival to the farm, (4) early weaning, and (5) varied timing of gilt introductions to the breeding herd. Additionally, we conducted a global sensitivity analysis using Latin Hypercube Sampling (LHS) and partial rank correlation coefficient (PRCC) analysis to assess the relative importance of each parameter in the model.
Results None of the individual interventions tested here consistently eliminated infection from a medium sized herd. In contrast to previous modeling studies, mass homologous vaccination did not eliminate transmission completely in sows and gilts. In concert, however, mass vaccination, early weaning of piglets (removal 0-7 days after birth), gilt isolation, gilt vaccination, and longer periods between introductions of gilts (6 months) were the most effective at reducing prevalence: endemic prevalence overall was reduced by 51% relative to the null case, endemic prevalence in piglets was reduced by 74%, and IAV was eliminated completely from the herd in 23% of all cases. Based on a global sensitivity analysis, the incubation period, infectious period, duration of immunity, and transmission rate for piglets with maternal immunity consistently had the highest correlation with three separate measures of IAV prevalence, and therefore are parameters that warrant increased attention for obtaining empirical estimates.
Discussion Our findings support other modeling and empirical studies that suggest that piglets play a key role in maintaining IAV in breeding herds. We recommend a combination of biosecurity measures in combination with targeted homologous vaccination or vaccines that provide wider cross-protective immunity to prevent incursions of virus to the farm and subsequent establishment of an infected piglet reservoir.