Detection and disease diagnosis trends (2017-2022) for Streptococcus suis, Glaesserella parasuis, Mycoplasma hyorhinis, Actinobacillus suis and Mycoplasma hyosynoviae at Iowa State University Veterinary Diagnostic Laboratory.

BACKGROUND: Accurate measurement of disease associated with endemic bacterial agents in pig populations is challenging due to their commensal ecology, the lack of disease-specific antemortem diagnostic tests, and the polymicrobial nature of swine diagnostic cases. The main objective of this retrospective study was to estimate temporal patterns of agent detection and disease diagnosis for five endemic bacteria that can cause systemic disease in porcine tissue specimens submitted to the Iowa State University Veterinary Diagnostic Laboratory (ISU VDL) from 2017 to 2022. The study also explored the diagnostic value of specific tissue specimens for disease diagnosis, estimated the frequency of polymicrobial diagnosis, and evaluated the association between phase of pig production and disease diagnosis. RESULTS: S. suis and G. parasuis bronchopneumonia increased on average 6 and 4.3%, while S. suis endocarditis increased by 23% per year, respectively. M. hyorhinis and A. suis associated serositis increased yearly by 4.2 and 12.8%, respectively. A significant upward trend in M. hyorhinis arthritis cases was also observed. In contrast, M. hyosynoviae arthritis cases decreased by 33% average/year. Investigation into the diagnostic value of tissues showed that lungs were the most frequently submitted sample, However, the use of lung for systemic disease diagnosis requires caution due to the commensal nature of these agents in the respiratory system, compared to systemic sites that diagnosticians typically target. This study also explored associations between phase of production and specific diseases caused by each agent, showcasing the role of S. suis arthritis in suckling pigs, meningitis in early nursery and endocarditis in growing pigs, and the role of G. parasuis, A. suis, M. hyorhinis and M. hyosynoviae disease mainly in post-weaning phases. Finally, this study highlighted the high frequency of co-detection and -disease diagnosis with other infectious etiologies, such as PRRSV and IAV, demonstrating that to minimize the health impact of these endemic bacterial agents it is imperative to establish effective viral control programs. CONCLUSIONS: Results from this retrospective study demonstrated significant increases in disease diagnosis for S. suis, G. parasuis, M. hyorhinis, and A. suis, and a significant decrease in detection and disease diagnosis of M. hyosynoviae. High frequencies of interactions between these endemic agents and with viral pathogens was also demonstrated. Consequently, improved control programs are needed to mitigate the adverse effect of these endemic bacterial agents on swine health and wellbeing. This includes improving diagnostic procedures, developing more effective vaccine products, fine-tuning antimicrobial approaches, and managing viral co-infections.

Complete Coding Genome Sequence of a Novel Porcine Reproductive and Respiratory Syndrome Virus 2 Restriction Fragment Length Polymorphism 1-4-4 Lineage 1C Variant Identified in Iowa, USA.

A porcine reproductive and respiratory syndrome virus 2 strain was identified in lung samples from nursery piglets associated with a 17.15% mortality rate on a swine farm in Iowa. Open reading frame 5 (ORF5) sequencing indicated that this strain is a restriction fragment length polymorphism (RFLP) 1-4-4 lineage 1C variant strain, and its complete coding genome sequence was determined.

Determining the impact of commercial feed additives as potential porcine epidemic diarrhea virus mitigation strategies as determined by polymerase chain reaction analysis and bioassay.

Mitigation of porcine epidemic diarrhea virus (PEDV) was assessed using two feed additives (0.5% inclusion of a benzoic acid [BA] product and 0.02% inclusion of an essential oil [EO] product; DSM Nutritional Products Inc., Parsippany, NJ), and combination of both products (0.5% BA and 0.02% EO) in spray-dried porcine plasma (SDPP) and a swine gestation diet (FEED) as determined by real-time quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and bioassay. Viral RNA quantification was performed at 7 sampling days post-laboratory inoculation (d 0, 1, 3, 7, 14, 21, and 42) and infectivity was assessed via bioassay with 10-d-old pigs. There was a tendency for treatment × feed matrix × day interaction (P = 0.094), in which the cycle threshold (Ct) value increased over time in FEED when treated with both feed additives, whereas there was no increase over time observed in SDPP treated with both feed additives. There was a feed matrix × day interaction (P < 0.001) in which Ct increased over time in FEED, whereas very little increase over time was observed in SDPP. A tendency for a treatment × feed matrix effect (P = 0.085) was observed where FEED treated with the combination of EO and BA had a greater (P < 0.05) PEDV Ct value than other FEED treatments, and all SDPP treatments had the lower PEDV Ct values compared to FEED treatments (P < 0.05). Overall, the combination of both feed additives was most effective at reducing the quantity of genetic material as detected by qRT-PCR (P < 0.001) compared to either additive alone or no feed additive. Virus shedding was observed in the d 7 postinoculation SDPP treatment that was treated with both feed additives, as well as d 0 untreated FEED and d 0 FEED treated with both feed additives. No other treatment bioassay room had detectible RNA shed and detected in fecal swabs or cecal contents. In summary, the combination of EO and BA enhanced the degradation of PEDV RNA in feed but had little impact on RNA degradation in SDPP. Both untreated feed and feed treated with the combination of EO and BA resulted in infection at d 0 post-laboratory inoculation; however, neither set of samples was infective at d 1 postinoculation. In addition, SDPP harbored greater levels of quantifiable RNA for a longer duration of time compared to FEED, and these viral particles remained viable for a longer duration of time indicating differences in viral stability exist between different feed matrices.

Feed batch sequencing to decrease the risk of porcine epidemic diarrhea virus (PEDV) cross-contamination during feed manufacturing.

Feed has been identified as a vector of transmission for porcine epidemic diarrhea virus (PEDV). The objective of this study was to determine if feed batch sequencing methods could minimize PEDV cross-contamination. Porcine epidemic diarrhea virus-free swine feed was manufactured to represent the negative control. A 50 kg feed batch was mixed in a pilot scale feed mill for 5 min, sampled, then discharged for 10 min into a bucket elevator and sampled again upon exit. Next, a pathogenic PEDV isolate was used to inoculate 49.5 kg of PEDV-free feed to form the positive control. The positive control was mixed, conveyed and sampled similar to the negative control. Subsequently, 4 sequence batches (sequence 1 to 4) were formed by adding a 50 kg batch of PEDV-negative feed to the mixer after the prior batch was mixed and conveyed; all sequences were mixed, conveyed, and sampled similar to the negative and positive control batches. None of the equipment was cleaned between batches within a replicate. This entire process was replicated 3 times with cleaning the feed mill between replicates. Feed was then analyzed for PEDV RNA by real-time reverse transcriptase semiquantitative polymerase chain reaction (rRT-PCR) as measured by cycle threshold (Ct) and for infectivity by bioassay. Sequence 1 feed had higher (P ˂ 0.05) rRT-PCR Ct values than the positive batch and sequence 2 feed had higher (P ˂ 0.05) Ct values than sequence 1, regardless of sampled location. Feed sampled from the mixer from sequence 2, 3, and 4 was rRT-PCR negative whereas feed sampled from the bucket elevator was rRT-PCR negative from sequence 3 and 4. Bioassay was conducted using 66 mixed sex 10-d-old pigs confirmed negative for PEDV allocated to 22 different rooms. Pigs were initially 10-d old. Control pigs remained PEDV negative for the study. All pigs from the mixer positive batch (9/9) and bucket elevator positive batch (3/3) were rRT-PCR positive on fecal swabs by the end of the study. One replicate of pigs from mixer sequence 1 was rRT-PCR positive (3/3) by 7 dpi. One replicate of mixer pigs from sequence 2 was rRT-PCR positive (3/3) by 7 dpi although no detectable PEDV RNA was found in the feed. The results demonstrate sequenced batches had reduced quantities of PEDV RNA although sequenced feed without detectible PEDV RNA by rRT-PCR can be infectious. Therefore, a sequencing protocol can reduce but not eliminate the risk of producing infectious PEDV carryover from the first sequenced batch of feed.

Evaluation of the effects of flushing feed manufacturing equipment with chemically treated rice hulls on porcine epidemic diarrhea virus cross-contamination during feed manufacturing.

Various strategies have been proposed to mitigate potential risk of porcine epidemic diarrhea virus (PEDV) transmission via feed and feed ingredients. Wet disinfection has been found to be the most effective decontamination of feed mill surfaces; however, this is not practical on a commercial feed production scale. Another potential mitigation strategy would be using chemically treated rice hulls flushed through the feed manufacturing equipment. Therefore, the objective of this study was to determine the effects of medium-chain fatty acids (MCFA) or formaldehyde-treated rice hull flush batches as potential chemical mitigation strategies for PEDV during feed manufacturing. Feed without evidence of PEDV RNA contamination was inoculated with PEDV. Based on polymerase chain reaction analysis, this feed had a cycle threshold (Ct) = 30.2 and was confirmed infective in bioassay. After manufacturing the PEDV-positive feed, untreated rice hulls, formaldehyde-treated rice hulls, 2% MCFA- (a 1:1:1 blend of hexanoic, octanoic, and decanoic acid) treated rice hulls, or 10% MCFA-treated rice hulls were flushed through laboratory scale mixers. For the untreated rice hulls, 3 of 6 samples had detectable PEDV RNA, whereas 1 of 6 formaldehyde-treated rice hull flush samples and 2 of 6 of the 2% MCFA rice hull flush samples had detectable PEDV RNA. However, PEDV RNA was not detected in any of the 10% MCFA rice hull flush samples. Then, rice hulls treated with 10% MCFA were mixed and discharged through a production scale mixer and bucket elevator following PEDV-positive feed. No rice hull flush or feed samples from the mixer following chemically treated rice hull flush had detectible PEDV RNA. However, one 10% MCFA rice hull sample collected from the bucket elevator discharge spout had detectible PEDV RNA. Dust collected following mixing of PEDV contaminated feed had detectable PEDV RNA (Ct = 29.4) and was infectious. However, dust collected immediately after the 10% MCFA rice hull flush batch had a reduced quantity of PEDV RNA (Ct = 33.7) and did not cause infection. Overall, the use of rice hull flushes effectively reduced the quantity of detectible RNA present after mixing a batch of PEDV-positive feed. Chemical treatment of rice hulls with formaldehyde or 10% MCFA provided additional reduction in detectible RNA. Finally, dust collected after manufacturing PEDV-inoculated feed has the potential to serve as a vector for PEDV transmission.

Emergence of Porcine epidemic diarrhea virus in the United States: clinical signs, lesions, and viral genomic sequences.

During the 10 days commencing April 29, 2013, the Iowa State University Veterinary Diagnostic Laboratory received the first 4 of many submissions from swine farms experiencing explosive epidemics of diarrhea and vomiting affecting all ages, with 90-95% mortality in suckling pigs. Histology revealed severe atrophy of villi in all segments of the small intestines with occasional villus-epithelial syncytial cells, but testing for rotaviruses and Transmissible gastroenteritis virus (Alphacoronavirus 1) were negative. Negative-staining electron microscopy of feces revealed coronavirus-like particles and a pan-coronavirus polymerase chain reaction (PCR) designed to amplify a conserved region of the polymerase gene for all members in the family Coronaviridae produced expected 251-bp amplicons. Subsequent sequencing and analysis revealed 99.6-100% identity among the PCR amplicons from the 4 farms and 97-99% identity to the corresponding portion of the polymerase gene of Porcine epidemic diarrhea virus (PEDV) strains, with the highest identity (99%) to strains from China in 2012. Findings were corroborated at National Veterinary Services Laboratories using 2 nested S-gene and 1 nested N-gene PCR tests where the sequenced amplicons also had the highest identity with 2012 China strains. Whole genome sequence for the virus from 2 farms in 2 different states using next-generation sequencing technique was compared to PEDV sequences available in GenBank. The 2013 U.S. PEDV had 96.6-99.5% identity with all known PEDV strains and the highest identity (>99.0%) to some of the 2011-2012 Chinese strains. The nearly simultaneous outbreaks of disease, and high degree of homology (99.6-100%) between the PEDV strains from the 4 unrelated farms, suggests a common source of virus.

Live attenuated influenza vaccine provides superior protection from heterologous infection in pigs with maternal antibodies without inducing vaccine-associated enhanced respiratory disease.

Control of swine influenza A virus (IAV) in the United States is hindered because inactivated vaccines do not provide robust cross-protection against the multiple antigenic variants cocirculating in the field. Vaccine efficacy can be limited further for vaccines administered to young pigs that possess maternally derived immunity. We previously demonstrated that a recombinant A/sw/Texas/4199-2/1998 (TX98) (H3N2) virus expressing a truncated NS1 protein is attenuated in swine and has potential for use as an intranasal live attenuated influenza virus (LAIV) vaccine. In the present study, we compared 1 dose of intranasal LAIV with 2 intramuscular doses of TX98 whole inactivated virus (WIV) with adjuvant in weanling pigs with and without TX98-specific maternally derived antibodies (MDA). Pigs were subsequently challenged with wild-type homologous TX98 H3N2 virus or with an antigenic variant, A/sw/Colorado/23619/1999 (CO99) (H3N2). In the absence of MDA, both vaccines protected against homologous TX98 and heterologous CO99 shedding, although the LAIV elicited lower hemagglutination inhibition (HI) antibody titers in serum. The efficacy of both vaccines was reduced by the presence of MDA; however, WIV vaccination of MDA-positive pigs led to dramatically enhanced pneumonia following heterologous challenge, a phenomenon known as vaccine-associated enhanced respiratory disease (VAERD). A single dose of LAIV administered to MDA-positive pigs still provided partial protection from CO99 and may be a safer vaccine for young pigs under field conditions, where dams are routinely vaccinated and diverse IAV strains are in circulation. These results have implications not only for pigs but also for other influenza virus host species.

Modifications in the polymerase genes of a swine-like triple-reassortant influenza virus to generate live attenuated vaccines against 2009 pandemic H1N1 viruses.

On 11 June 2009, the World Health Organization (WHO) declared that the outbreaks caused by novel swine-origin influenza A (H1N1) virus had reached pandemic proportions. The pandemic H1N1 (H1N1pdm) virus is the predominant influenza virus strain in the human population. It has also crossed the species barriers and infected turkeys and swine in several countries. Thus, the development of a vaccine that is effective in multiple animal species is urgently needed. We have previously demonstrated that the introduction of temperature-sensitive mutations into the PB2 and PB1 genes of an avian H9N2 virus, combined with the insertion of a hemagglutinin (HA) tag in PB1, resulted in an attenuated (att) vaccine backbone for both chickens and mice. Because the new pandemic strain is a triple-reassortant (TR) virus, we chose to introduce the double attenuating modifications into a swine-like TR virus isolate, A/turkey/OH/313053/04 (H3N2) (ty/04), with the goal of producing live attenuated influenza vaccines (LAIV). This genetically modified backbone had impaired polymerase activity and restricted virus growth at elevated temperatures. In vivo characterization of two H1N1 vaccine candidates generated using the ty/04 att backbone demonstrated that this vaccine is highly attenuated in mice, as indicated by the absence of signs of disease, limited replication, and minimum histopathological alterations in the respiratory tract. A single immunization with the ty/04 att-based vaccines conferred complete protection against a lethal H1N1pdm virus infection in mice. More importantly, vaccination of pigs with a ty/04 att-H1N1 vaccine candidate resulted in sterilizing immunity upon an aggressive intratracheal challenge with the 2009 H1N1 pandemic virus. Our studies highlight the safety of the ty/04 att vaccine platform and its potential as a master donor strain for the generation of live attenuated vaccines for humans and livestock.

Efficacy of inactivated swine influenza virus vaccines against the 2009 A/H1N1 influenza virus in pigs.

The gene constellation of the 2009 pandemic A/H1N1 virus is a unique combination from swine influenza A viruses (SIV) of North American and Eurasian lineages, but prior to April 2009 had never before been identified in swine or other species. Although its hemagglutinin gene is related to North American H1 SIV, it is unknown if vaccines currently used in U.S. swine would cross-protect against infection with the pandemic A/H1N1. The objective of this study was to evaluate the efficacy of inactivated vaccines prepared with North American swine influenza viruses as well as an experimental homologous A/H1N1 vaccine to prevent infection and disease from 2009 pandemic A/H1N1. All vaccines tested provided partial protection ranging from reduction of pneumonia lesions to significant reduction in virus replication in the lung and nose. The multivalent vaccines demonstrated partial protection; however, none was able to prevent all nasal shedding or clinical disease. An experimental homologous 2009 A/H1N1 monovalent vaccine provided optimal protection with no virus detected from nose or lung at any time point in addition to amelioration of clinical disease. Based on cross-protection demonstrated with the vaccines evaluated in this study, the U.S. swine herd likely has significant immunity to the 2009 A/H1N1 from prior vaccination or natural exposure. However, consideration should be given for development of monovalent homologous vaccines to best protect the swine population thus limiting shedding and the potential transmission of 2009 A/H1N1 from pigs to people.