Clinical investigation into feed-related hypervitaminosis D in a captive flock of budgerigars (Melopsittacus undulatus): morbidity, mortalities, and pathologic lesions.

The Blank Park Zoo began suffering mortalities in the spring of 2012 within a flock of 229 captive budgerigars (Melopsittacus undulatus) housed in an interactive public-feeding aviary. Clinical signs in affected birds included weakness, posterior paresis, inability to fly, or acute death. Gross and microscopic lesions were not initially apparent in acutely affected deceased birds. Many birds had evidence of trauma, which is now hypothesized to have been related to the birds' weakness. Investigation into the cause(s) of morbidity and mortality were complicated by the opening of a new interactive enclosure. For this reason, environmental conditions and husbandry protocols were heavily scrutinized. Microscopic examination of dead budgies later in the course of the investigation revealed mineralization of soft tissues consistent with hypervitaminosis D. Pooled serum analysis of deceased birds identified elevated vitamin D3 levels. Vitamin D3 analysis was performed on the feed sticks offered by the public and the formulated maintenance diet fed to the flock. This analysis detected elevated levels of vitamin D3 that were 22.5-times the manufacturer's labeled content in the formulated diet. These findings contributed to a manufacturer recall of more than 100 formulated diets fed to a wide variety of domestic and captive wild animal species throughout the United States and internationally. This case report discusses the complexities of determining the etiology of a toxic event in a zoologic institution.

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.

Sequence-optimized and targeted double-stranded RNA as a therapeutic antiviral treatment against infectious myonecrosis virus in Litopenaeus vannamei.

Infectious myonecrosis virus (IMNV) is a significant and emerging pathogen that has a tremendous impact on the culture of the Pacific white shrimp Litopenaeus vannamei. IMNV first emerged in Brazil in 2002 and subsequently spread to Indonesia, causing large economic losses in both countries. No existing therapeutic treatments or effective interventions currently exist for IMNV. RNA interference (RNAi) is an effective technique for preventing viral disease in shrimp. Here, we describe the efficacy of a double-stranded RNA (dsRNA) applied as an antiviral therapeutic following virus challenge. The antiviral molecule is an optimized dsRNA construct that targets an IMNV sequence at the 5' end of the genome and that showed outstanding antiviral protection previously when administered prior to infection. At least 50% survival is observed with a low dose of dsRNA administered 48 h post-infection with a lethal dose of IMNV; this degree of protection was not observed when dsRNA was administered 72 h post-infection. Additionally, administration of the dsRNA antiviral resulted in a significant reduction of the viral load in the muscle of shrimp that died from disease or survived until termination of the present study, as assessed by quantitative RT-PCR. These data indicate that this optimized RNAi antiviral molecule holds promise for use as an antiviral therapeutic against IMNV.

dsRNA provides sequence-dependent protection against infectious myonecrosis virus in Litopenaeus vannamei.

Viral diseases are significant impediments to the sustainability of shrimp aquaculture. In addition to endemic disease, new viral diseases continue to emerge and cause significant impact on the shrimp industry. Disease caused by infectious myonecrosis virus (IMNV) has caused tremendous losses in farmed Pacific white shrimp (Litopenaeus vannamei) since it emerged in Brazil and translocated to Indonesia. There are no existing antiviral interventions, outside of pathogen exclusion, to mitigate disease in commercial shrimp operations. Here, we describe an iterative process of panning the genome of IMNV to discover RNA interference trigger sequences that initiate a robust and long-lasting protective response against IMNV in L. vannamei. Using this process, a single, low dose (0.02 µg) of an 81 or 153 bp fragment, with sequence corresponding to putative cleavage protein 1 in ORF1, protected 100 % of animals from disease and mortality caused by IMNV. Furthermore, animals that were treated with highly efficacious dsRNA survived an initial infection and were resistant to subsequent infections over 50 days later with a 100-fold greater dose of virus. This protection is probably sequence dependent, because targeting the coding regions for the polymerase or structural genes of IMNV conferred lesser or no protection. Interestingly, non-sequence specific dsRNA did not provide any degree of protection to animals as had been described for other shrimp viruses. Our data indicate that the targeted region for dsRNA is a crucial factor in maximizing the degree of protection and lowering the dose required to induce a protective effect against IMNV infection in shrimp.

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.

Complete genome sequence and pathogenicity of two swine parainfluenzavirus 3 isolates from pigs in the United States.

Two novel paramyxoviruses, 81-19252 (Texas81) and 92-7783 (ISU92), isolated from the brains of pigs in the United States in the 1980s and 1990s, were characterized. The complete genome of Texas81 virus was 15,456 nucleotides (nt) in length, that of ISU92 was 15,480 nt, and both genomes consisted of six nonoverlapping genes, predicted to encode nine proteins, with conserved and complementary 3' leader and 5' trailer regions and conserved gene starts, gene stops, and trinucleotide intergenic sequences similar to those in paramyxoviruses. The corresponding genes from these two viruses were similar in length, except for the F genes, of which the ISU92 form had an additional 24-nt U-rich 3' untranslated region. The P genes of swine viruses were predicted to produce V and D mRNAs by RNA editing (one to four G insertions in Texas81 and one to nine G insertions in ISU92) or C mRNA by alternative translation initiation. Sequence-specific features related to virus replication and host-specific amino acid signatures indicated that these viruses originated from bovine parainfluenzavirus 3 (bPIV3). Phylogenetic analysis of individual genes suggested that these viruses are novel members of the genus Respirovirus of the Paramyxovirinae subfamily and may be grouped into two subgenotypes of genotype A of bPIV3. Our comprehensive studies revealed that these swine PIV3 are variants of bPIV3 and were possibly transferred from cattle to pigs but failed to establish an active enzootic state. These two viruses were mildly pathogenic to conventionally reared pigs, and results from a limited enzyme-linked immunosorbent assay-based serosurvey of swine farms in Minnesota and Iowa in 2007 and 2008 were negative.

Viral reassortment and transmission after co-infection of pigs with classical H1N1 and triple-reassortant H3N2 swine influenza viruses.

Triple-reassortant swine influenza viruses circulating in North American pigs contain the internal genes derived from swine (matrix, non-structural and nucleoprotein), human [polymerase basic 1 (PB1)] and avian (polymerase acidic and PB2) influenza viruses forming a constellation of genes that is well conserved and is called the triple-reassortant internal gene (TRIG) cassette. In contrast, the external genes [haemagglutinin (HA) and neuraminidase (NA)] are less conserved, reflecting multiple reassortant events that have produced viruses with different combinations of HA and NA genes. This study hypothesized that maintenance of the TRIG cassette confers a selective advantage to the virus. To test this hypothesis, pigs were co-infected with the triple-reassortant H3N2 A/Swine/Texas/4199-2/98 (Tx/98) and the classical H1N1 A/Swine/Iowa/15/1930 viruses and co-housed with a group of sentinel animals. This direct contact group was subsequently moved into contact with a second group of naïve animals. Four different subtypes (H1N1, H1N2, H3N1 and H3N2) of influenza virus were identified in bronchoalveolar lavage fluid collected from the lungs of the experimentally infected pigs, with most of the viruses containing TRIG from the Tx/98 virus. Interestingly, only the intact H3N2 Tx/98 virus was transmitted from the infected pigs to the direct-contact animals and from them to the second contact group of pigs. These results demonstrated that multiple reassortments can occur within a host; however, only specific gene constellations are readily transmissible. It was concluded that certain HA and NA gene pairs, in conjunction with the TRIG cassette, may have a competitive advantage over other combinations for transmission and maintenance in swine.

Identification and characterization of a highly virulent triple reassortant H1N1 swine influenza virus in the United States.

A highly virulent H1N1 influenza A virus, A/Swine/Kansas/77778/2007 (KS07), which caused approximately 10% mortality in finishing pigs, was isolated from herds in the Midwestern United States. Molecular and phylogenic analysis revealed this swine isolate was a triple reassortant virus, similar to an H1N1 virus that infected humans and pigs at an Ohio county fair in August 2007. A pig challenge model was developed to evaluate the pathogenicity and transmission capacity of the KS07 virus. The results confirmed that the KS07 virus is highly virulent in pigs and easily transmitted to sentinel animals. The KS07 virus failed to cross-react with a panel of H1-specific swine sera. Interestingly, the KS07 virus shed for a prolonged period up to 7 days in infected pigs, indicating that this virus can spread efficiently between animals. The highly virulent H1N1 swine influenza virus is further evidence of reassortment among avian, human and swine influenza viruses and justifies the need for continued surveillance of influenza viruses in swine.

Identification of H2N3 influenza A viruses from swine in the United States.

Although viruses of each of the 16 influenza A HA subtypes are potential human pathogens, only viruses of the H1, H2, and H3 subtype are known to have been successfully established in humans. H2 influenza viruses have been absent from human circulation since 1968, and as such they pose a substantial human pandemic risk. In this report, we isolate and characterize genetically similar avian/swine virus reassortant H2N3 influenza A viruses isolated from diseased swine from two farms in the United States. These viruses contained leucine at position 226 of the H2 protein, which has been associated with increased binding affinity to the mammalian alpha2,6Gal-linked sialic acid virus receptor. Correspondingly, the H2N3 viruses were able to cause disease in experimentally infected swine and mice without prior adaptation. In addition, the swine H2N3 virus was infectious and highly transmissible in swine and ferrets. Taken together, these findings suggest that the H2N3 virus has undergone some adaptation to the mammalian host and that their spread should be very closely monitored.

Molecular characterization of glycoprotein genes and phylogenetic analysis of two swine paramyxoviruses isolated from United States.

Two swine paramyxoviruses (SPMV)-(81-19252 (Texas-81) and 92-7783 (ISU-92)-were isolated from encephalitic pigs in the United States in 1981 and 1992. Antigenic, morphologic, and biological characteristics of these two viruses were essentially similar to members of the family Paramyxoviridae. Antigenic analysis by indirect fluorescent antibody, immunoblot, and one-way cross-neutralization tests placed these viruses along with bovine parainfluenza 3 (BPIV3) viruses. Purified virions were 50-300 nm in size and morphologically indistinguishable from other paramyxoviruses. These two viruses hemagglutinated red blood cells and had neuraminidase activity. The gene junctions of fusion (F) and hemagglutinin (HN) glycoprotein genes of these viruses contained highly conserved transcription start and stop signal sequences and trinucleotide intergenic regions similar to other Paramyxoviridae. The F gene of ISU-92 was longer than Texas-81 due to insertion of a 24-nucleotide "U"-rich 3' untranslated region. Structure-based sequence alignment of glycoproteins of these two SPMVs indicated that they are essentially similar in structure and function to parainfluenzaviruses. The Texas-81 strain was closely related to BPIV3 Shipping Fever (SF) strain at nucleotide and amino acid level, while the ISU-92 strain was more closely related to BPIV3 910N strain. The envelope glycoproteins of ISU-92 had only approximately 92 and approximately 96% identity at nucleotide and amino acid levels with BPIV3-SF strain, respectively. The high sequence identities to BPIV3 indicated cross-species infection in pigs. Phylogenetic analyses based on both F protein and HN protein suggested the classification of these viruses into the subfamily Paramyxovirinae, genus Respirovirus, and genotype A of BPIV3.

Characterization of a newly emerged genetic cluster of H1N1 and H1N2 swine influenza virus in the United States.

H1 influenza A viruses that were distinct from the classical swine H1 lineage were identified in pigs in Canada in 2003­2004; antigenic and genetic characterization identified the hemagglutinin (HA) as human H1 lineage. The viruses identified in Canadian pigs were human lineage in entirety or double (human­swine) reassortants. Here, we report the whole genome sequence analysis of four human-like H1 viruses isolated from U.S. swine in 2005 and 2007. All four isolates were characterized as triple reassortants with an internal gene constellation similar to contemporary U.S. swine influenza virus (SIV), with HA and neuraminidase (NA) most similar to human influenza virus lineages. A 2007 human-like H1N1 was evaluated in a pathogenesis and transmission model and compared to a 2004 reassortant H1N1 SIV isolate with swine lineage HA and NA. The 2007 isolate induced disease typical of influenza virus and was transmitted to contact pigs; however, the kinetics and magnitude differed from the 2004 H1N1 SIV. This study indicates that the human-like H1 SIV can efficiently replicate and transmit in the swine host and now co-circulates with contemporary SIVs as a distinct genetic cluster of H1 SIV.

Naturally occurring influenza infection in a ferret (Mustela putorius furo) colony.

Tissue samples from 2 juvenile ferrets (Mustela putorius furo) from a colony that was undergoing an outbreak of respiratory disease were submitted to the Iowa State University Veterinary Diagnostic Laboratory. Microscopic examination of lung samples revealed bronchointerstitial pneumonia with necrotizing bronchiolitis. Influenza A virus was detected in sections of formalin-fixed lung by immunohistochemistry and reverse transcription polymerase chain reaction assay. A field investigation of the premises and analysis of additional samples led to the confirmation and characterization of an influenza virus with high homology to contemporary reassortant H1N1 swine influenza viruses. Although ferrets have been used extensively to research the virulence and transmissibility of avian, human, and swine influenza virus strains, no published information exists on naturally occurring outbreaks of swine influenza in ferrets.

Failure of protection and enhanced pneumonia with a US H1N2 swine influenza virus in pigs vaccinated with an inactivated classical swine H1N1 vaccine.

Two US swine influenza virus (SIV) isolates, A/Swine/Iowa/15/1930 H1N1 (IA30) and A/Swine/Minnesota/00194/2003 H1N2 (MN03), were evaluated in an in vivo vaccination and challenge model. Inactivated vaccines were prepared from each isolate and used to immunize conventional pigs, followed by challenge with homologous or heterologous virus. Both inactivated vaccines provided complete protection against homologous challenge. However, the IA30 vaccine failed to protect against the heterologous MN03 challenge. Three of the nine pigs in this group had substantially greater percentages of lung lesions, suggesting the vaccine potentiated the pneumonia. In contrast, priming with live IA30 virus provided protection from nasal shedding and virus replication in the lung in MN03 challenged pigs. These data indicate that divergent viruses that did not cross-react serologically did not provide complete cross-protection when used in inactivated vaccines against heterologous challenge and may have enhanced disease. In addition, live virus infection conferred protection against heterologous challenge.

The immune response and maternal antibody interference to a heterologous H1N1 swine influenza virus infection following vaccination.

This study investigated the efficacy of a bivalent swine influenza virus (SIV) vaccine in piglets challenged with a heterologous H1N1 SIV isolate. The ability of maternally derived antibodies (MDA) to provide protection against a heterologous challenge and the impact MDA have on vaccine efficacy were also evaluated. Forty-eight MDA(+) pigs and 48 MDA(-) pigs were assigned to 8 different groups. Vaccinated pigs received two doses of a bivalent SIV vaccine at 3 and 5 weeks of age. The infected pigs were challenged at 7 weeks of age with an H1N1 SIV strain heterologous to the H1N1 vaccine strain. Clinical signs, rectal temperature, macroscopic and microscopic lesions, virus excretion, serum and local antibody responses, and influenza-specific T-cell responses were measured. The bivalent SIV vaccine induced a high serum hemagglutination-inhibition (HI) antibody titer against the vaccine virus, but antibodies cross-reacted at a lower level to the challenge virus. This study determined that low serum HI antibodies to a challenge virus induced by vaccination with a heterologous virus provided protection demonstrated by clinical protection and reduced pneumonia and viral excretion. The vaccine was able to prime the local SIV-specific antibody response in the lower respiratory tract as well as inducing a systemic SIV-specific memory T-cell response. MDA alone were capable of suppressing fever subsequent to infection, but other parameters showed reduced protection against infection compared to vaccination. The presence of MDA at vaccination negatively impacted vaccine efficacy as fever and clinical signs were prolonged, and unexpectedly, SIV-induced pneumonia was increased compared to pigs vaccinated in the absence of MDA. MDA also suppressed the serum antibody response and the induction of SIV-specific memory T-cells following vaccination. The results of this study question the effectiveness of the current practice of generating increased MDA levels through sow vaccination in protecting piglets against disease.

Pine needle abortion biomarker detected in bovine fetal fluids.

Pine needle abortion is a naturally occurring condition in free-range cattle caused by the consumption of pine needles from select species of cypress, juniper, pine, and spruce trees. Confirmatory diagnosis of pine needle abortion has previously relied on a combined case history of pine needle consumption and detection of isocupressic acid in a sample from the dam. Stable metabolites of isocupressic acid include agathic acid, dihydroagathic acid, and tetrahydroagathic acid, which have been shown to be present in the serum of mature animals for a few days following consumption of pine needles. As maternal serum is infrequently submitted for diagnosis of cattle abortions, a diagnostic assay capable of confirming isocupressic acid exposure in other matrices would be desirable. To the authors' knowledge, no previous investigations have indicated whether these stable metabolites of isocupressic acid cross the placenta or are detectable in fetal tissues. Therefore, the presence of agathic acid, dihydroagathic acid, and tetrahydroagathic acid was evaluated using gas chromatography-mass spectroscopy on fetal thoracic fluid and stomach contents collected from 2 aborted bovine fetuses with a recent herd history of pine needle consumption by the dams and a subsequent abortion outbreak in the herd. Only tetrahydroagathic acid was detected in the fetal thoracic fluid and fetal stomach contents. The current study encourages diagnosticians to collect fetal thoracic fluids to permit the detection of tetrahydroagathic acid in cases of suspected pine needle abortion.

Comparison of a commercial enzyme-linked immunosorbent assay with hemagglutination inhibition assay for serodiagnosis of swine influenza virus (H1N1) infection.

A commercial indirect swine influenza virus (SIV) H1N1 enzyme-linked immunosorbent assay (ELISA) was compared with the hemagglutination inhibition (HI) assay by testing 72 samples from experimentally infected pigs and 780 field samples of undefined SIV status. The HI assay was performed using SIV isolates A/Swine/IA/73 for H1N1 and A/Swine/IA/8548-1/98 for H3N2. The ELISA used an SIV isolated in 1988. The results showed that HI and ELISA detected an antibody in 11 and 6, respectively, of 72 serum samples collected from pigs experimentally infected with a 1992 SIV isolate (A/Swine/IA/40776/92). The presence of antibodies in these experimental samples was confirmed by HI tests in which all 72 samples were positive against the homologous virus, a more recent H1N1 SIV isolate (A/Swine/NVSL/01) supplied by National Veterinary Services Laboratories, Ames, Iowa, and a 1999 H1N1 isolate currently used in a commercial vaccine. On testing 780 field samples, an overall agreement of 85.5% was generated between the HI and ELISA. This study demonstrated that the ELISA is a useful serodiagnostic screening test at herd level for detecting swine antibodies against SIV. However, a new SIV isolate representing current SIV strains circulating in the field is needed to replace the older isolates used in the HI and ELISA to increase the test accuracy for serodiagnosis of SIV.

Comparison of a commercial H1N1 enzyme-linked immunosorbent assay and hemagglutination inhibition test in detecting serum antibody against swine influenza viruses.

Recently a commercial enzyme-linked immunosorbent assay (ELISA) kit for detecting antibody against H1N1 swine influenza virus (SIV) has been made available to diagnosticians and veterinary practitioners. Because the hemagglutination inhibition (HI) test has been considered the standard test for SIV serology, diagnostic performance of the new ELISA was evaluated using positive (n = 60) and negative (n = 188) serum samples from young pigs with known status of SIV infection and compared with that of the HI test. Both ELISA and HI test identified all negative animals correctly. None of the serum samples (n = 64) from pigs inoculated with H3N2 SIV was positive by ELISA for SIV antibody. The H1N1 SIV antibody detectable by ELISA appears to develop more slowly in comparison with antibody detectable by HI test. Although antibody was detected by HI test in all inoculated animals (n = 20) by day 7 postinoculation (PI), antibody was detected by ELISA in 0%, 75%, and 100% of the inoculated animals on days 7, 14, and 28 PI, respectively. Discrepancy in test results between the 2 serologic tests appeared to be because of differences in antibody isotypes detected by each test. Enzyme-linked immunosorbent assay mainly detected IgG antibody, whereas the HI test detects IgM antibody very efficiently as well as IgG antibody. Collectively, the commercial ELISA is highly specific for antibody to H1N1 SIV but may not identify positive animals at the early stage of infection as effectively as the HI test, particularly when SIV is introduced to a naïve swine population.

Vitamin E and selenium concentrations in livers of pigs diagnosed with mulberry heart disease.

During a 2-year period from January 1, 1999, to December 31, 2000, 77 diagnoses of mulberry heart disease (MHD) were documented at the Iowa State University Veterinary Diagnostic Laboratory. Mean (+/- SD) liver vitamin E concentrations were lower (P < 0.05) in pigs with MHD (3.12 +/- 1.12 ppm, wet weight) than in pigs that died of causes other than MHD (4.80 +/- 3.2 ppm, wet weight). The majority of the pigs affected with MHD ranged in age from 3 to 7 weeks. Statistical influence of age was found on the concentration of vitamin E (P < 0.01) but not on concentration of selenium in liver in pigs with MHD. Concentrations of vitamin E below 2 ppm were considered deficient. Hepatic vitamin E concentrations below 2 ppm were measured in 25% of the pigs with gross and microscopic lesions of MHD. In contrast, liver selenium concentrations were adequate in all pigs.

A prolonged outbreak of polioencephalomyelitis due to infection with a group I porcine enterovirus.

A commercial swineherd in Indiana experienced high death loss of nursery pigs with neurologic disorders for a prolonged period. Polioencephalomyelitis was the consistent histopathological lesion in affected animals. A porcine enterovirus (PEV) classified to group I was isolated from spinal cords and brains collected from the affected animals. The isolate (ISUVDL 200103183) was determined to belong to either serogroup 5 or 6 at the National Veterinary Services Laboratories, Ames, IA. No other significant viral or bacterial agents were isolated from or detected in the animals. A prospective longitudinal serological monitoring of pigs in the index herd for the PEV isolate revealed that colostrum-derived neutralizing antibodies to the virus rapidly declined, and by the age of 21 days the majority of piglets had no or minimal neutralizing antibody against the virus. Seroconversion to the virus then coincided with increased mortality in the herd. Results of diagnostic and cohort observations supported a diagnosis of PEV infection as the cause of the prolonged outbreak of "polio." Investigation into factors that may be contributing to the prolonged problem is currently in progress.

Issues encountered in development of enzyme-linked immunosorbent assay for use in detecting Influenza A virus subtype H5N1 exposure in swine.

A potential mechanism by which highly pathogenic avian Influenza A virus subtype H5N1 could more readily infect human beings is through the infection of and adaptation in pigs. To detect the occurrence of such infection, monitoring of pig populations through serological screening would be highly desirable. In the current study, hemagglutination inhibition assays were able to detect antibodies against H5N1 developed in pigs, but because of antigenic variation between clades, the use of multiple virus strains were required. Whole recombinant virus and recombinant hemagglutinin antigen enzyme-linked immunosorbent assays (ELISAs) were generated that could detect antibody against multiple H5N1 strains, but which also detected antibody against endemic swine influenza viruses. A recombinant hemagglutinin antigen-based ELISA was as effective as the whole virus antigen ELISAs in detecting antibody against the H5N1 virus strains used and eliminated nearly all of the cross-reactivity with non-H5N1 virus antibody. The current study also highlighted the difficulty in establishing a decision (cutoff) value that would effectively counterbalance nonspecific reactivity against sensitivity. The results provide important information and considerations for the development of serological screening assays for highly pathogenic avian H5N1 viruses.