Publications – Swine in Minnesota

Pioneering Structural Study of Porcine Coronavirus

Today, we are highlighting the research of a completely different team at the University of Minnesota. The Minnesota Supercomputing Institute provides advanced research computing infrastructure and expertise to advance and accelerate research and foster innovation and discoveries.

MSI PIs Wei Zhang (research associate professor, Diagnostic and Biological Sciences) and Fang Li (associate professor, Veterinary and Biomedical Sciences) have published a new paper that describes some of their continuing research into the structure of coronaviruses. These are a large group of viruses that includes such deadly diseases as SARS and MERS. Coronaviruses have four forms, known as α-, β-, γ-, and δ-coronavirus, which affect different hosts. For example, β-coronaviruses affect only mammals, while the δ form affects both birds and mammals.

The coronavirus structure includes a feature called a “spike protein,” which allows the virus to attach to the host’s cells. The spike proteins of α- and β-coronavirus have been well studied. The spike protein of the δ-coronavirus, however, is described for the first time in this paper. The researchers used cryo-electron microscopy, a fast-developing technology in which protein molecules are studied under ultra-cold temperatures with an electron microscope. This technology was used to determine the structure of the spike protein of porcine δ-coronavirus (PdCoV), a lethal virus infecting pigs, elucidating how PdCoV infects pigs cells and evades the host immune system. This is the first atomic-resolution cryo-electron microscopic study from the state of Minnesota, and is a milestone in the structural biology field at the University of Minnesota.

Zhang Li spike protein porcine deltacoronavirus

Image Description: Overall structure of PdCoV S-e in the prefusion conformation. (A) Schematic drawing of PdCoV S-e (spike ectodomain). S1, receptor-binding subunit. S2, membrane fusion subunit. GCN4-His₆, GCN4 trimerization tag followed by His₆ tag. S1-NTD, N-terminal domain of S1. S1-CTD, C-terminal domain of S1. CH-N and CH-C, central helices N and C. FP, fusion peptide. HR-N and HR-C, heptad repeats N and C. Residues in shaded regions (N terminus, GCN4 tag, and His₆ tag) were not traced in the structure. (B) Cryo-EM maps of PdCoV S-e with atomic model fitted in. The maps have a contour of 6.6 σ. (C) Cryo-EM structure of prefusion PdCoV S-e. Each of the monomeric subunits is colored differently. (D) Structure of a monomeric subunit in the prefusion conformation. The structural elements are colored in the same way as those in panel A. Image and description, J Shang et al., J Virol. 92:e01556-17 (2018). © American Society for Microbiology.

The paper was published in late 2017 on the website of the Journal of Virology: J Shang, Y Zhang, Y Yang, Q Geng,W Tai, L Du, Y Zhou, W Zhang, F Li. 2018. Cryo-Electronic Microscopy Structure of Porcine Deltacoronavirus Spike Protein in the Prefusion State. Journal of Virology 92 (4): e01556-17. doi: 10.1128/JVI.01556-17.

This report comes from the MSI research highlights.

Time-series analysis for porcine reproductive and respiratory syndrome in the United States

Today, we are sharing an open-access publication from Dr. Andreia Arruda, Dr. Ana Alba and members of the MSHMP team in the journal PlosOne.

This study was conducted using data collected from the Morrison Swine Health Monitoring Project. The main objective of this study was to use time-series analysis to investigate whether yearly patterns commonly described for PRRS were in fact conserved across different U.S. states.

Methods

The 268 breeding herds enrolled in this project were the ones that participated in the MSHMP from July 2009 to October 2016. PPRS status of each farm was reported weekly following the AASV guidelines. The five states examined included Minnesota (MN), Iowa (IA), North Carolina (NC), Nebraska (NE), and Illinois (IL).

Results

81 MN farms, 72 IA, 45 NC, 30 NE, 40 from IL, were enrolled in the study with a mean number of animals per site of 2,666; 3,543; 2,342; 4,041; and 4,018 respectively.

journal.pone.0195282.g002 — Graphs showing the prevalence (black line) and upper and lower 95% confidence intervals (grey dotted lines) of PRRS virus positive farms for the five different U.S. states participating in this study: A: Minnesota; B: Iowa; C: Nebraska, D: North Carolina and E: Illinois

The main finding of this study was that PRRS seasonality varies according to geographical region, and the commonly referred “PRRS season” is not necessarily the only time of increase in disease incidence.

Another interesting finding from this study was the presence of an alternating trend for all examined states within of the U.S., except for the state of Iowa, the largest pork producing states in the country (approximately 31.4% of the total US hog and pig inventory), which had an increasing linear trend over the examined years.

In conclusion, PRRS seasonal patterns are not homogeneous across the U.S., with some important pork producing states having biannual PRRS peaks instead of the previously reported winter peak. Findings from this study highlight the importance of coordinating alternative control strategies in different regions considering the prevailing epidemiological patterns, and the need to reinforce strict biosecurity practices beyond the typically described “PRRS season”.

You can also listen to Dr. Arruda present some of these research findings at the 2017 Leman conference.

Abstract

Industry-driven voluntary disease control programs for swine diseases emerged in North America in the early 2000’s, and, since then, those programs have been used for monitoring diseases of economic importance to swine producers. One example of such initiatives is Dr. Morrison’s Swine Health Monitoring Project, a nation-wide monitoring program for swine diseases including the porcine reproductive and respiratory syndrome (PRRS). PRRS has been extensively reported as a seasonal disease in the U.S., with predictable peaks that start in fall and are extended through the winter season. However, formal time series analysis stratified by geographic region has never been conducted for this important disease across the U.S. The main objective of this study was to use approximately seven years of PRRS incidence data in breeding swine herds to conduct time-series analysis in order to describe the temporal patterns of PRRS outbreaks at the farm level for five major swine-producing states across the U.S. including the states of Minnesota, Iowa, North Carolina, Nebraska and Illinois. Data was aggregated retrospectively at the week level for the number of herds containing animals actively shedding PRRS virus. Basic descriptive statistics were conducted followed by autoregressive integrated moving average (ARIMA) modelling, conducted separately for each of the above-mentioned states. Results showed that there was a difference in the nature of PRRS seasonality among states. Of note, when comparing states, the typical seasonal pattern previously described for PRRS could only be detected for farms located in the states of Minnesota, North Carolina and Nebraska. For the other two states, seasonal peaks every six months were detected within a year. In conclusion, we showed that epidemic patterns are not homogeneous across the U.S, with major peaks of disease occurring through the year. These findings highlight the importance of coordinating alternative control strategies in different regions considering the prevailing epidemiological patterns.

Detection of influenza A virus in aerosols of vaccinated and non-vaccinated pigs in a warm environment

Today we are sharing a publication from the Torremorell lab regarding the impact of vaccination (both homologous and heterologous) on the detection of swine influenza virus in aerosols. The full publication is available in open access online on the PlosOne website.

Influenza A virus can be transmitted by direct and indirect contact and aerosols. Indeed, the virus has been detected and isolated from aerosols generated from pigs with and without immunity. Since then, there has been increased evidence of the role of aerosols in influenza transmission among swine.

Vaccination is used in swine populations as a strategy to mitigate clinical effects and the economic impact of influenza infections. It has also been proven to reduce shedding in pigs. Additionally, a study on the transmission of influenza in ferrets showed that high temperature may decrease the risk of airborne transmission. Therefore, we wondered if combining vaccination and high temperature would affect the detection of influenza virus in the air.

The objective of this study was to assess the effect of vaccination on the generation of influenza A virus bioaerosols under warm conditions in pigs with varying degrees of cross-protective immunity.

Material and Methods

36 pigs of three weeks of age, seronegative for influenza were separated into four groups:

vaccinated with an influenza strain identical to the one used for the challenge (homologous)
vaccinated with a commercial vaccine containing multiple strains of influenza, all different from the challenge strain (heterologous, multivalent)
vaccinated with a commercial vaccine containing one influenza strain different from the challenge strain (heterologous, monovalent)
unvaccinated, which received an injection of saline instead

Pigs were challenged intranasally and intratracheally with a strain of H1N1 influenza virus, two weeks after the last vaccination.
Serum collected the day prior to the vaccination and at the end of the study 14 days post inoculation were tested via hemagglutination inhibition (HI) and ELISA.. Nasal swabs and oral fluids were collected and tested via PCR. Air samples were collected three times a day and tested via PCR and virus isolation. Temperature and humidity were recorded every five minutes.

Results

Hemagglutination inhibition and ELISA

Prior to infection, pigs in group 1 (Vaccinated, homologous) had significantly higher HI titers compared to the other three groups. In the group 3 (vaccinated, heterologous monvalent) 4 pigs had HI titers against the challenge strain, while pigs in groups 2 and 4 were negative against the challenge strain. All groups were HI positive against the challenge strain at necropsy, however HI titers were statistically different between group 4 and groups 1 and 3.

Proportion of pigs infected

The proportion of pigs infected was significantly higher in group 4 than in the vaccinated ones. Also, the percentage of infected pigs in group 1 was significantly lower than in group 2, but there was no difference with group 3.

Torremorell vacc pigs aerosol influenza proportion negative pigs — Proportion of negative pigs over time

Nasal swabs and oral fluids

Pigs in group 4 had higher amounts of nasal virus shedding most of the sampling days compared to vaccinated groups. Additionally, group 2 had higher levels of IAV compared with groups 1 and 4. Oral fluid results were in agreement with nasal swab.

Torremorell vacc pigs aerosol influenza nasal shedding — Nasal shedding over time

Air samples

All air samples in the vaccinated groups tested negative by RRT-PCR. Air samples collected at days 1, 2 and 3 from NON-VAC pigs tested positive by RRT-PCR but negative by virus isolation

Abstract

The 2009 influenza pandemic, the variant H3N2v viruses in agricultural fairs and the zoonotic poultry H5N9 infections in China have highlighted the constant threat that influenza A viruses (IAV) present to people and animals. In this study we evaluated the effect of IAV vaccination on aerosol shedding in pigs housed in warm environmental conditions. Thirty-six, three-week old weaned pigs were obtained from an IAV negative herd and were randomly allocated to one of 4 groups: 1) a homologous vaccine group, 2) a heterologous multivalent vaccine group, 3) a heterologous monovalent group and, 4) a non-vaccinated group. After vaccination pigs were challenged with the triple reassortant A/Sw/IA/00239/04 H1N1 virus. Environmental temperature and relative humidity were recorded throughout the study. Nasal swabs, oral fluids and air samples were collected daily. All samples were tested by RRT-PCR and virus isolation was attempted on positive samples. Average temperature and relative humidity throughout the study were 27°C (80°F) and 53%, respectively. A significantly higher proportion of infected pigs was detected in the non-vaccinated than in the vaccinated group. Lower levels of nasal virus shedding were found in vaccinated groups compared to non-vaccinated group and IAV was not detected in air samples of any of the vaccinated groups. In contrast, positive air samples were detected in the non-vaccinated group at 1, 2 and 3 days post infection although the overall levels were considered low most likely due to the elevated environmental temperature. In conclusion, both the decrease in shedding and the increase in environmental temperature may have contributed to the inability to detect airborne IAV in vaccinated pigs.

Vaccination against Lawsonia intracellularis decreases shedding of Salmonella enterica serovar Typhimurium in co-infected pigs changes the host gut microbiome

This is our Friday rubric: every week a new Science Page from the Bob Morrison’s Swine Health Monitoring Project. The previous editions of the science page are available on our website.

This week, we are sharing the summary of a publication by Dr. Fernando Leite who recently received his PhD from the University of Minnesota. The full scientific article regarding the effect of the vaccination against Lawsonia intracellularis on the shedding of Salmonella typhimurium and the host microbiome is available on open access in Nature.

Materials and Methods

A total of five treatment groups were used:

challenged with S. Typhimurium alone,
challenged with both S. Typhimurium and L. intracellularis,
challenged with S. Typhimurium and vaccinated against L. intracellularis,
challenged with both S. Typhimurium and L. intracellularis and vaccinated against L. intracellularis
a non-infected control.

Results

The greatest difference in shedding level between groups was found at 7 days post-infection. At this time point, the co-challenged animals from the vaccinated group shed statistically less S. Typhimurium per gram of feces than the animals from the non-vaccinated, co-challenged group. The co-challenged vaccinated group also shed significantly less S. Typhimurium than the singly infected S. Typhimurium group.
L. intracellularis vaccination did not have a significant impact on S. Typhimurium shedding when animals were singly infected with S. Typhimurium.

Leite Ileitis vaccination salmonelle co infection

At 7 days post-infection, different treatment groups had significant differences in their microbiome community structure. The co-infected vaccinated group clustered apart from all other treatment groups.

Conclusion

These results indicate that vaccination against L. intracellularis impacts the microbiome and reduces shedding of S. Typhimurium in co-infected animals.

Production Losses From an Endemic Animal Disease: PRRS in Selected Midwest US Sow Farms

In this publication in Frontiers in Veterinary Science, Drs. Valdes-Donoso from UC Davis and Andres Perez from the Center of Animal Health and Food Safety (CAHFS) at the University of Minnesota, measured the impact of Porcine Reproductive and Respiratory Syndrome (PRRS) on the production of weaned pigs.

To do so, they monitored 16 different sow farms, all parts of a single production system in the Midwest for 48 weeks and recorded a total of 8 indicators:

number of weaned pigs
number of stillbirths per litter
number of live births per litter
number of pre-weaned dead
number of sows farrowing
number of sows repeating service
number of sows aborting
number of sows dead

For each farm and each indicator, the 12 weeks before the outbreak served as a baseline for the farm performances and the data was recorded until 35 weeks post outbreaks. All of the outbreaks occurred during the second half of 2014. The inventory of the farms varied between 2,714 and 6,009 breeding females.

The following figure represented the weekly average for the 8 recorded parameters from 12 weeks pre-outbreak to 35-weeks post-outbreak.

Perez PRRS sow farm losses Midwest

Based on these results, it was estimated that a PRRS outbreak caused a 7.4% decrease in weaned pigs per sow year, i.e., 1.92 fewer weaned pigs per breeding unit. In an average sized farm of this firm, the slight reduction in farrowing yielded a decline of 249 fewer farrows per year. The chances that a sow repeats service increased by 37%, while aborted fetuses increased by 26% in a year with a PRRS outbreak.

The primary estimate (using 12 weeks as pre-outbreak period) is that PRRS reduced weaned pig production per farm by 7.4% on an annual basis, leading to a decrease in output value per sow year of $86.6, or $367,521 per farm year for an average sized farm. If instead we assume the outbreak began in t −1 (i.e., using 11 weeks as pre-outbreak period), the estimated reduction in weaned pig production was 7.6%, or $88.8 less per sow year and an average revenue loss of $376,773 among the farms studied.

Results showed that weaned pig production declined in week t − 1, although statistically insignificant, as did several performance indicators. The data suggest that the average PRRS outbreak in this set of farms began at least one week before it was announced.”

The rise in abortions was the strongest signal of PRRSV activity in our data. Increased surveillance, particularly to rising abortions, may allow farms to identify PRRS more quickly.

The length of PRRS outbreaks, as well as their effects over time, is highly variable. The results of this study demonstrate that PRRS has a negative effect on weaned pig production for a longer time than previously estimated. Indeed, the estimated means of weaned pig production remained below the baseline throughout the 35 weeks that we are able to observe following the outbreak.

For more details, read the open-access publication on the Frontiers in Veterinary Science website.

Abstract:

Porcine reproductive and respiratory syndrome (PRRS) is an endemic disease causing important economic losses to the US swine industry. The complex epidemiology of the disease, along with the diverse clinical outputs observed in different types of infected farms, have hampered efforts to quantify PRRS’ impact on production over time. We measured the impact of PRRS on the production of weaned pigs using a log-linear fixed effects model to evaluate longitudinal data collected from 16 sow farms belonging to a specific firm. We measured seven additional indicators of farm performance to gain insight into disease dynamics. We used pre-outbreak longitudinal data to establish a baseline that was then used to estimate the decrease in production. A significant rise of abortions in the week before the outbreak was reported was the strongest signal of PRRSV activity. In addition, production declined slightly one week before the outbreak and then fell markedly until weeks 5 and 6 post-outbreak. Recovery was not monotonic, cycling gently around a rising trend. At the end of the study period (35 weeks post-outbreak), neither the production of weaned pigs nor any of the performance indicators had fully recovered to baseline levels. This result suggests PRSS outbreaks may last longer than has been found in most other studies. We assessed PRRS’ effect on farm efficiency as measured by changes in sow production of weaned pigs per year. We translated production losses into revenue losses assuming an average market price of $45.2/weaned pig. We estimate that the average PRSS outbreak reduced production by approximately 7.4%, relative to annual output in the absence of an outbreak. PRRS reduced production by 1.92 weaned pigs per sow when adjusted to an annual basis. This decrease is substantially larger than the 1.44 decrease of weaned pigs per sow/year reported elsewhere.

Evaluation of a Partially De-oiled Microalgae Product in Nursery Pig Diets

The publication we are sharing today is a collaboration between the Department of Animal Science, the Department of Food Science and nutrition and the West Central Outreach and Research Center at the University of Minnesota. It is published in open access in the journal Translational Animal Science.

Objectives

The aim of this study was to evaluate the potential use of microalgae extract (MAE) as a feed ingredient in nursery pig diets.

Methods

300 weaned pigs were selected, blocked by initial body weight and allotted to 60 pens, with five pigs per pen. Ratio of gilts and barrows was balanced evenly. Pens within blocks were assigned randomly to one of five dietary treatments.

Dietary treatments included:

corn and soybean meal (CON),
CON with 1% MAE,
CON with 5% MAE,
CON with 10% MAE
CON with 20% MAE.

Diets were formulated to meet the nutrient requirements of nursery pigs and fed using a 3-phase program, where each phase consisted of a 2-wk period.

Average Daily Gain (ADG), Average Daily Feed Intake (ADFI) and Gain to Feed (G:F) were measured weekly.

After 42 days, 30 pigs were harvested and intestinal samples were collected to measure mucosal length and goblet cell quantifications.

Results

Final body weight of pigs among pens consuming MAE was greatest when consuming 1, 5, or 10% MAE compared with those fed the control diet, but feeding 20% MAE was not different from the control diet. The greater final body weight appeared to be the result of greater ADG from days 1 and 7, due to a higher ADFI.

There was no effect of feeding MAE on G:F during most weigh periods except during days 15 to 21 when G:F increased in pigs fed MAE.

Feeding diets with MAE did not result in changes in intestinal architecture measured by the height of the intestinal mucosal or presence of mucus-producing cells in the jejunum. In contrast, the ileum of pigs fed the 5% MAE diet tended to have reduced mucosal height compared with that of pigs fed 20% MAE diet. Goblet cell area of the ileum was not affected by dietary treatments.

Link to the full article

Abstract

Although microalgae can be used as a source of energy and macronutrients in pig diets, there is limited information on the use of partially de-oiled microalgae co-products in swine feeding programs. The objectives of this study were to evaluate the effects of a partially de-oiled microalgae extract (MAE) in nursery pig diets on growth performance and health status. A total of 300 pigs (initial BW = 6.3 ± 2.1 kg) were used in a 42-d experiment. Treatments included a standard corn-soybean meal control diet, and diets containing 1, 5, 10, or 20% MAE replacing primarily corn. The ME content of MAE was calculated from the chemical composition, and diets were formulated to meet or exceed nutrient requirements for nursery pigs. Pigs were stratified by weaning BW into 12 blocks in a randomized complete block design, with sex distributed evenly among blocks. Pens of pigs (5 pigs/pen) were assigned randomly within block to one of 5 dietary treatments. Pig BW and feed disappearance were recorded weekly. On d 42, thirty pigs were harvested and sections of the jejunum and ileum were collected for gut morphology analysis, and a liver sample was collected for metabolomic analysis using liquid chromatography-mass spectroscopy. Data were analyzed by ANOVA with diet as treatment effect, and contrasts were used to test linear or quadratic effects of dietary MAE inclusion level. Overall, pigs fed 1 and 5% MAE had the greatest (quadratic P < 0.05) ADG, resulting from greater (quadratic P < 0.05) ADFI. There was a tendency for a greater number of pigs requiring injectable treatments (P = 0.16) and a greater mortality (P = 0.14) in pigs fed the control diet than pigs in any of the diets with the MAE. Final BW increased (P < 0.05) for pigs fed 1 and 5% MAE diets. The improvements in ADG were not explained by differences in mucosa height or goblet cell count among dietary treatments. Pigs fed diets containing 1 or 5% MAE had relatively less concentration (P < 0.05) of ammonia in the liver and had changes in metabolites associated with the urea cycle. In conclusion, feeding MAE resulted in increased growth responses and may have beneficial health effects when fed to nursery pigs.

Understanding Tail-Biting in Pigs through Social Network Analysis

Today, we are sharing a publication on pig welfare by our colleagues in the Outreach and Extension center at the University of Minnesota, Drs. Li, Zhang, Johnston and Martin. More specifically, the researchers focused their study on the effect of social network on tail-biting in pigs. The full-text of the article is available in open-access on the website of the journal Animals.

We know that pigs are social animals and that they naturally form social structures to maintain a cohesive group. However, we have little understanding of how those group dynamics affect deleterious behavior like tail-biting. To answer the question of the association between social structure and incidence of tail-biting in pigs, the researchers created 18 groups of 8 pigs.

6 groups were Littermates: all the 8 pigs were born from and nursed by the same sow.
6 groups were Half-group of littermates: 4 pigs were born from the same sow whereas the 4 others came from the litter of another sow.
6 groups were Non-littermates: all 8 pigs were born from a different sow.

Each group was housed in a nursery pen after weaning where the pigs stayed for the duration of the study until they reached 10 weeks of age. Researchers analyzed growth performances, tail injuries, and behavior.

Growth performances did not differ among groups in this study. However, littermates showed a higher incidence of tail-biting with 15% of the pigs showing chewing or puncture wounds with visible blood but no infection.

Behavior was analyzed by videotaping the pigs 2 weeks after they were placed into their pens, 1 week later when each group was moved together to a new pen and 1 week after the move. The video recordings were viewed by a trained researcher to determine association interactions among pigs. Pigs were considered associated with each other if they were lying together frequently and with more than 50% or more of their bodies in contact with each other. For each pig (white circle in the figure above), researchers measured the direct association between each individual pig and its penmates (1) as well as the peripheral association among the penmates (2).

At the individual pig level, littermates had lower direct association than non-littermates and half-group of littermates, suggesting that littermates might be less socially connected directly among themselves. However, the indirect association among penmates did not vary.

Another interesting observation, although statistically insignificant, is that littermates appeared to spend less time in the lying posture than other groups.

Overall, littermates had a lower strength of social connections, more absent ties, and fewer weak ties, compared to non-littermates and half-group of littermates. Less social connection with pen-mates might predispose pigs in littermate pens to development of tail-biting. Regardless of litter origin, most pigs appeared connected by weak social ties and few pigs formed strong social ties with their pen mates.

Abstract

The objective of this study was to investigate the association between social structure and incidence of tail-biting in pigs. Pigs (n = 144, initial weight = 7.2 ± 1.57 kg, 4 weeks of age) were grouped based on their litter origin: littermates, non-littermates, and half-group of littermates. Six pens (8 pigs/pen) of each litter origin were studied for 6 weeks. Incidence of tail injury and growth performance were monitored. Behavior of pigs was video recorded for 6 h at 6 and 8 weeks of age. Video recordings were scanned at 10 min intervals to register pigs that were lying together (1) or not (0) in binary matrices. Half weight association index was used for social network construction. Social network analysis was performed using the UCINET software. Littermates had lower network density (0.119 vs. 0.174; p < 0.05), more absent social ties (20 vs. 12; p < 0.05), and fewer weak social ties (6 vs. 14, p < 0.05) than non-littermates, indicating that littermates might be less socially connected. Fifteen percent of littermates were identified as victimized pigs by tail-biting, and no victimized pigs were observed in other treatment groups. These results suggest that littermates might be less socially connected among themselves which may predispose them to development of tail-biting.