P. C. Gauger

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.

Live Attenuated Influenza Vaccine Provides Superior Protection from Heterologous Infection in Pigs with Maternal Antibodies without Inducing Vaccine-Associated Enhanced Respiratory Disease

ABSTRACT 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

ABSTRACT 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.

Mortality in pigs given porcine circovirus type 2 subgroup 1 and 2 viruses derived from dna clones

PORCINE circovirus type 2 (PCV-2) is a ubiquitous swine virus. Following natural infection, the clinical presentation ranges from subclinical to one of several disease states, known collectively as porcine circovirus disease (PCVD). In late 2005, a disease spread rapidly through fattening barns in pig-dense areas of the USA, which was recognised mainly by the acute onset of high mortality. Investigations into cases found the following: a sudden increase in mortality that was at least two to three times the expected weekly rate; a high incidence of unthrifty, anorexic, dyspnoeic and listless pigs; and the onset of this condition was noticed four to six weeks after placement in the barn. Most of the affected animals, if not all, were non-responsive to a variety of treatments and eventually died. By the end of the fattening phase, the mortality rate ranged from 5 to 50 per cent in a given barn of pigs, with case fatality rates reaching 100 per cent when euthanasia and culling for salvage were included (Baker 2006, Henry and Tokach 2006). At postmortem examination, the most consistent gross lesions were enlarged lymph nodes, icterus, pneumonia, pulmonary oedema, and variable amounts of pleural and peritoneal effusions. The most consistent microscopic lesions were lymphoid depletion with granulomatous inflammation, non-suppurative interstitial pneumonia, and nephritis. PCV-2 was consistently recovered from affected pigs, and the high mortality syndrome was associated with PCVD. Genetic analysis of the isolates revealed that they belonged to PCV-2 group 1, a PCV-2 subgroup that had not previously been recognised in the USA (Cheung and others 2007). The PCV-2 subgroup 1 and subgroup 2 definition is based on the classification by Olvera and others (2007). This short communication describes inoculation of germ-free pigs with virus derived from infectious DNA clones constructed from subgroup 1 and 2 isolates recovered from affected pigs in a North Carolina herd (Cheung and others 2007). Twelve germ-free pigs, raised four to an isolator, were given an oronasal inoculation at 10 days of age with one of three inocula: isolator 1 pigs (group 1) received an inoculum derived from an infectious clone of PCV-2 subgroup 1 (1 ml volume of approximately 3·8 x 103 immunohistochemical units); isolator 2 pigs (group 2) received a similar challenge with an inoculum derived from an infectious clone of PCV-2 subgroup 2; and the pigs in isolator 3 (group 3) received a sham inoculum (PK-15 cell culture lysate free of PCV-2). The experimental design included observing the pigs for 35 days postinfection (dpi), at which time the pigs were euthanased and examined postmortem. The pigs in group 1 were normal in appearance and behaviour until 22 dpi, when, within 12 hours of each other, two pigs became listless and dyspnoeic. Both were euthanased and underwent a postmortem examination. Gross lesions consisted of extensive amounts of pleural and peritoneal effusions that were clear in one pig and an opaque yellow in the second pig. The lungs were diffusely affected with a dark-red congestion and severe interlobular pulmonary oedema. Generalised lymphadenopathy was seen, with the tracheobronchial lymph nodes being most affected. One of the two remaining pigs in the group was found dead 27 dpi and the fourth pig was dyspnoeic and euthanased the same day. All group 1 pigs had similar gross lesions. On 23 dpi all of the pigs in group 2 were normal in appearance and behaviour. Two of these pigs were euthanased and both were normal in appearance except for an increase in the size of lymph nodes associated with their lungs. At 35 dpi, one pig was normal in behaviour and the only lesions noted were enlarged lymph nodes. The other pig was normal until the morning of 35 dpi, when it was dyspnoeic and listless. Postmortem examination revealed gross lesions in this pig that were similar to those observed in the group 1 pigs. Two of the four pigs in group 3 were euthanased at 23 dpi and the other two at 35 dpi. All the pigs were normal in appearance and behaviour throughout the study, and no gross lesions were observed at postmortem examination. The most striking microscopic lesions in the affected pigs included severe hepatic necrosis and a depletion of germinal centres in lymph nodes and tonsils. Abundant staining for PCV-2 antigen was seen in these tissues by immunohistochemistry. This study is the first description of germ-free pigs being infected with PCV-2 subgroup 1 virus derived from a DNA clone. The lesions and clinical signs observed are comparable to experiments in which germ-free pigs infected with PCV-2 subgroup 2 virus received an immune-stimulation event – coinfection with porcine parvovirus or injection with adjuvant. The pigs manifested severe disease with distinctive lesions (Krakowka and others 2000, 2001). The conclusion of those studies was that a co-factor was needed to induce clinical disease, as pigs infected with only PCV-2 did not develop severe disease. Instead, they remained clinically normal but did have enlarged lymph nodes with lymphoid depletion, a microscopic lesion considered to be one of the hallmark lesions for PCVD. Sera and tissues from the pigs in the present study tested negative for porcine parvovirus, bovine viral diarrhoea virus, porcine reproductive and respiratory syndrome virus, and unknown cytopathic agents that could propagate in porcine cell lines. All 12 pigs remained free of detectable aerobic and anaerobic bacteria. No infectious agent that would account for an immunostimulatory co-factor was identified. PCV-2 was the only infectious agent recovered from the group 1 and group 2 pigs. Based on the nucleotide sequence, the virus recovered from each group was the same virus that was given to the group. The presence of severe disease without the detection of any co-factor in four of four, and one of four pigs given the PCV-2 subgroup 1 and 2 viruses, respectively, suggests one of three things: there was an unidentified exogenous factor that acted in synergy with the PCV-2 infection; there was an unknown endogenous factor that behaved in a similar way; or PCV-2 infection with virus derived from a DNA clone was capable of inducing severe disease without a co-factor. Additional studies will be required to understand the observed phenomenon in this gnotobiotic pig study.

Vaccine-associated enhanced respiratory disease is influenced by haemagglutinin and neuraminidase in whole inactivated influenza virus vaccines.

Multiple subtypes and many antigenic variants of influenza A virus (IAV) co-circulate in swine in the USA, complicating effective use of commercial vaccines to control disease and transmission. Whole inactivated virus (WIV) vaccines may provide partial protection against IAV with substantial antigenic drift, but have been shown to induce vaccine-associated enhanced respiratory disease (VAERD) when challenged with an antigenic variant of the same haemagglutinin (HA) subtype. This study investigated the role the immune response against HA, neuraminidase (NA) and nucleoprotein (NP) may play in VAERD by reverse engineering vaccine and challenge viruses on a common backbone and using them in a series of vaccination/challenge trials. Mismatched HA between vaccine and challenge virus was necessary to induce VAERD. However, vaccines containing a matched NA abrogated the VAERD phenomenon induced by the HA mismatch and this was correlated with NA-inhibiting (NI) antibodies. Divergence between the two circulating swine N2 lineages (92u2009% identity) resulted in a loss of NI cross-reactivity and also resulted in VAERD with the mismatched HA. The NP lineage selected for use in the WIV vaccine strains did not affect protection or pathology. Thus the combination of HA and NA in the vaccine virus strains played a substantial role in vaccine protection versus immunopathology, suggesting that vaccines that target the HA protein alone could be more prone to VAERD due to the absence of cross-protective NI antibodies.

Effect of pelleting on survival of porcine epidemic diarrhea virus-contaminated feed

Abstract Porcine epidemic diarrhea virus (PEDV) is a heat-sensitive virus that has devastated the U.S. swine industry. Because of its heat sensitivity, we hypothesized that a steam conditioner and pellet mill mimicking traditional commercial thermal processing may mitigate PEDV infectivity. Pelleting, a common feed processing method, includes the use of steam and shear forces, resulting in increased temperature of the processed feed. Two thermal processing experiments were designed to determine if different pellet mill conditioner retention times and temperatures would impact PEDV quantity and infectivity by analysis of quantitative reverse transcription PCR and bioassay. In Exp. 1, a 3 · 3 · 2 factorial design was used with 3 pelleting temperatures (68.3, 79.4, and 90.6°C), 3 conditioning times (45, 90, or 180 s), and 2 doses of viral inoculation (low, 1 · 102 tissue culture infectious dose50 (the concentration used to see cytopathic effect in 50% of the cells)/g, or high, 1 · 104 tissue culture infectious dose50/g). Noninoculated and PEDV-inoculated unprocessed mash were used as controls. The low-dose PEDV–infected mash had 6.8 ± 1.8 cycle threshold (Ct) greater (P < 0.05) PEDV than the high-dose mash. Regardless of time or temperature, pelleting reduced (P < 0.05) the quantity of detectable viral PEDV RNA compared with the PEDV-inoculated unprocessed mash. Fecal swabs from pigs inoculated with the PEDV-positive unprocessed mash, regardless of dose, were clinically PEDV positive from 2 to 7 d (end of the trial) after inoculation. However, if either PEDV dose of inoculated feed was pelleted at any of the 9 tested conditioning time · temperature combinations, no PEDV RNA was detected in fecal swabs or cecum content. Based on Exp. 1 results, a second experiment was developed to determine the impact of lower processing temperatures on PEDV quantity and infectivity. In Exp. 2, PEDV-inoculated feed was pelleted at 1 of 5 conditioning temperatures (37.8, 46.1, 54.4, 62.8, and 71.1°C) for 30 s. The 5 increasing processing temperatures led to feed with respective mean Ct values of 32.5, 34.6, 37.0, 36.5, and 36.7, respectively. All samples had detectable PEDV RNA. However, infectivity was detected by bioassay only in pigs from the 37.8 and 46.1°C conditioning temperatures. Experiment 2 results suggest conditioning and pelleting temperatures above 54.4°C could be effective in reducing the quantity and infectivity of PEDV in swine feed. However, additional research is needed to prevent subsequent recontamination after pelleting as it is a point-in-time mitigation step.

Determining the impact of commercial feed additives as potential porcine epidemic diarrhea virus mitigation strategies as determined by polymerase chain reaction analysis and bioassay1

Abstract 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.

Evaluation of the effects of flushing feed manufacturing equipment with chemically treated rice hulls on porcine epidemic diarrhea virus cross-contamination during feed manufacturing1

Abstract 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.

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

Abstract 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.