Ann C. Vorwald

Diagnosis of Porcine Reproductive and Respiratory Syndrome

The most suitable tissue samples and test procedures for the etiologic diagnosis of porcine reproductive and respiratory syndrome (PRRS) were found to depend on several variables including the age of the pig from which tissues were collected, the stage of infection (acute or persistent), the available complement of diagnostic reagents, and the urgency in obtaining results. When the diagnosis involved acute infection of congenitally or neonatally infected pigs, and susceptible cell culture(s) was available for virus isolation, then both serum and alveolar macrophages (AM) were reliable samples. Alveolar macrophages flushed from infected lungs provided a temporal advantage, however, in that in addition to their use for virus isolation, i.e., from a lysate of AM, they could be cultured in vitro and examined for the presence of viral antigens by immunofluorescence microscopy (FA) as early as 1 hour after they were added to the culture vessel. The examination of AM in this manner also circumvented the need for additional cell cultures to test for infectious virus. Testing presuckhng sera by indirect FA for antibodies to PRRS virus also was of diagnostic value and, like FA with AM, could be completed soon after sample collection. For older pigs, AM were more reliable than serum, lungs, or any of 27 other tissues evaluated as diagnostic samples and were often the only samples in which infectious virus and viral antigens were detected when pigs were euthanized more than 3 weeks postexposure. A simple procedure for on-farm collection of AM as well as methods for testing AM for viral antigens and neonatal (presuckling) sera for homologous antibody in a modestly equipped laboratory, such as one that might be maintained by a veterinary practitioner, are described and discussed.

Genetic variation and phylogenetic relationships of 22 porcine reproductive and respiratory syndrome virus (PRRSV) field strains based on sequence analysis of open reading frame 5

SummaryPorcine reproductive and respiratory syndrome virus (PRRSV) strains from 13 states in the United States, Guatemala and Canada were used to compare the envelope glycoprotein gene (ORF 5) nucleotide and deduced amino acid sequences. The gene was the same size, 603 nt, for all the 22 field strains. These strains had 89–94% amino acid identity compared to reference strain VR 2332. A putative signal sequence and cleavage site between residues 31 and 32 was identified and three potential glycosylation sites were present on all but two strains. Hydrophobicity/hydrophilicity and surface probability analyses reveal a primary structure for the envelope glycoprotein (E protein) with six potential surface regions that could be antigenic sites. Similar E protein structural features are conserved for the prototype European PRRSV – Lelystad virus.

Strain specificity of the immune response of pigs following vaccination with various strains of porcine reproductive and respiratory syndrome virus.

The primary objective of the study was to determine strain specificity of the immune response of pigs following vaccination with selected strains of porcine reproductive and respiratory syndrome virus (PRRSV). The experimental design included five groups (I through V, six pigs per group) free of antibody for PRRSV at the beginning of the experiment (day 0). On day 0, groups III, IV, and V were vaccinated with attenuated versions of PRRSV strains 8, 9, and 14, respectively. On day 21, the immunity of group II (non-vaccinated/challenged controls) and groups III, IV, and V was challenged by exposing each pig to a composite of the virulent versions of these same three strains. On day 35, all pigs, including non-vaccinated/non-challenged pigs of group I, were necropsied. Lungs and selected lymph nodes were examined for lesions. Serum samples obtained at weekly intervals throughout the study and lung lavage fluids obtained at necropsy were tested for the presence of PRRSV and its strain identity. Before challenge the strain of PRRSV identified in the sera of vaccinated pigs was always that with which the particular pig had been vaccinated (i.e. homologous strain), whereas, with one exception, only heterologous strains were identified after challenge. This apparent strain exclusion as a result of vaccination was statistically significant (P = 0.004). The tendency for heterologous strains to predominate after challenge suggests that a pigs immune response to PRRSV has some degree of strain specificity. Whether this finding has any clinical relevance in regard to immunoprophylaxis remains to be determined.

Comparative safety and efficacy of attenuated single-strain and multi-strain vaccines for porcine reproductive and respiratory syndrome☆

The objective of this study was to compare the efficacy and safety of single-strain and multi-strain vaccines for the prevention of the respiratory facet of porcine reproductive and respiratory syndrome. The study comprised six groups of pigs (A through F, eight pigs per group). At the beginning of the study (Day 0) Groups C and D were vaccinated with a single-strain vaccine, and Groups E and F were vaccinated with a multi-strain vaccine. The multi-strain vaccine contained five attenuated strains of PRRSV including the strain used as the single-strain vaccine. On Day 28 Groups B (nonvaccinated/challenged control), D, and F were challenged with a highly virulent field strain of PRRSV that was unrelated to any of the strains used for vaccination. Group A was kept as a nonvaccinated/nonchallenged control. On Day 42 all pigs were necropsied. Their lungs and lymph nodes were examined for virus-induced changes. Serum samples obtained at weekly intervals during the study and lung lavage fluids obtained at necropsy were tested for the presence and titer of PRRSV. Serum samples were also tested for antibody. The presence and severity of clinical signs and lesions were the primary means by which vaccine efficacy and safety were evaluated. Both vaccines provided a high level of protective immunity to challenge. However, appreciable lymph node enlargement in pigs vaccinated with multi-strain vaccine, with or without subsequent challenge, raised a question as to its safety. Collectively these results indicate that both single-strain and multi-strain attenuated PRRSV vaccines can be effective immunogens, but additional studies in regard to safety are needed before multi-strain vaccines can be recommended for routine field use.

The effect of porcine parvovirus and porcine reproductive and respiratory syndrome virus on porcine reproductive performance

From a worldwide perspective, porcine parvovirus (PPV) and porcine reproductive and respiratory syndrome virus (PRRSV) are the most common viral causes of porcine reproductive failure. A typical epidemic of PPV-induced reproductive failure is presented as an increased number of mummified fetuses and sometimes, entire litters are mummified. If infection with PPV is very early in gestation, the number of liveborn pigs may be further reduced as a result of embryonic death and resorption. During the acute stage of infection gilts and sows have few, if any, clinical signs, and it is unlikely that PPV is ever the direct cause of abortion. In contrast, a typical epidemic of PRRSV-induced reproductive failure is presented as a broader spectrum of clinical features including abortions, late-term dead fetuses, stillborn pigs, and weakborn pigs. In the later stages of an epidemic, there may also be an increase in the number of mummified fetuses, but their prevalence is likely to be far less than during an epidemic of PPV-induced reproductive failure. During the acute stage of infection with PRRSV, gilts and sows may have few, if any, clinical signs, or they may be severely affected and even die. This difference largely reflects the relative virulence of the strain of PRRSV causing the epidemic. A timely and reliable laboratory diagnosis of either disease can be made when appropriate tests are performed with appropriate samples. Vaccines are available for prevention of both diseases.

Genomic sequence and virulence comparison of four Type 2 porcine reproductive and respiratory syndrome virus strains

Porcine reproductive and respiratory syndrome virus (PRRSV) is a ubiquitous and costly virus that exhibits substantial sequence and virulence disparity among diverse isolates. In this study, we compared the whole genomic sequence and virulence of 4 Type 2 PRRSV isolates. Among the 4 isolates, SDSU73, MN184, and NADC30 were all clearly more virulent than NADC31, and among the 3 more virulent isolates, there were subtle differences based on viral replication, lung lesions, lymphadenopathy, febrile response, decreased weight gains, and cytokine responses in the lung. Lesions consistent with bacterial bronchopneumonia were present to varying degrees in pigs infected with PRRSV, and bacteria typically associated with the porcine respiratory disease complex were isolated from the lung of these pigs. Genomic sequence evaluation indicates that SDSU73 is most similar to the nucleotide sequence of JA142, the parental strain of Ingelvac(®) PRRS ATP, while the nucleotide sequences of NADC30 and NADC31 are more similar to strain MN184. Both the NADC30 and NADC31 isolates of PRRSV, isolated in 2008, maintain the nonstructural protein 2 (nsp2) deletion seen in MN184 that was isolated in 2001, but NADC31 has two additional 15 and 36 nucleotide deletions, and these strains are 8-14% different on a nucleotide basis from the MN184 strain. These results indicate that newer U.S. Type 2 strains still exhibit variability in sequence and pathogenicity and although PRRSV strains appear to be reducing the size of the nsp2 over time, this does not necessarily mean that the strain is more virulent.

Lymphoid Hyperplasia Resulting in Immune Dysregulation Is Caused by Porcine Reproductive and Respiratory Syndrome Virus Infection in Neonatal Pigs

Amid growing evidence that numerous viral infections can produce immunopathology, including nonspecific polyclonal lymphocyte activation, the need to test the direct impact of an infecting virus on the immune system of the host is crucial. This can best be tested in the isolator piglet model in which maternal and other extrinsic influences can be excluded. Therefore, neonatal isolator piglets were colonized with a benign Escherichia coli, or kept germfree, and then inoculated with wild-type porcine reproductive and respiratory syndrome virus (PRRSV) or sham medium. Two weeks after inoculation, serum IgM, IgG, and IgA levels were 30- to 50-, 20- to 80-, and 10- to 20-fold higher, respectively, in animals receiving virus vs sham controls, although <1% was virus specific. PRRSV-infected piglets also had bronchial tree-associated lymph nodes and submandibular lymph nodes that were 5–10 times larger than colonized, sham-inoculated animals. Size-exclusion fast performance liquid chromatography revealed that PRRSV-infected sera contained high-molecular-mass fractions that contained IgG, suggesting the presence of immune complexes. Lesions, inflammatory cell infiltration, glomerular deposits of IgG, IgM, and IgA, and Abs of all three isotypes to basement membrane and vascular endothelium were observed in the kidneys of PRRSV-infected piglets. Furthermore, autoantibodies specific for Golgi Ags and dsDNA could be detected 3–4 wk after viral inoculation. These data demonstrate that PRRSV induces B cell hyperplasia in isolator piglets that leads to immunologic injury and suggests that the isolator piglet model could serve as a useful model to determine the mechanisms of virus-induced immunopathology in this species.

Diagnosis of porcine reproductive and respiratory syndrome using infected alveolar macrophages collected from live pigs

A highly sensitive method of detecting infection of live pigs with porcine reproductive and respiratory syndrome virus (PRRSV) was developed by testing alveolar macrophages collected by pulmonary lavage. Five pigs were exposed by oronasal inoculation or by contact to PRRSV when they were 10 (1 pig) or 14 weeks (4 pigs) of age. Diagnostic samples (alveolar macrophages and sera) were collected from each pig just before exposure to PRRSV. During the next 9 weeks sera were collected at weekly intervals and alveolar macrophages were collected at weeks 2 and 4-9. Both sera and alveolar macrophages were suitable for detecting early infection, but alveolar macrophages were clearly the better sample after longer intervals. Virus was last isolated from serum at week 4 (from 1 of 5 pigs), whereas it was isolated from the alveolar macrophages of 4 of the 5 pigs at week 4 and from at least 2 pigs at each of the weekly intervals thereafter (i.e. weeks 5, 6, 7, 8, and 9 postexposure). The most sensitive method of testing alveolar macrophages for PRRSV was cocultivation with MARC-145 cells. None of the pigs had any clinical signs after exposure to PRRSV or as a result of pulmonary lavage and there was no evidence that repeated pulmonary lavage caused anything other than a mild, transient (mild hyperemia) tissue reaction.

The role of biotechnologically engineered vaccines and diagnostics in pseudorabies (Aujeszky's disease) eradication strategies

Modern-day biotechnology has an almost unlimited number of possibilities for reducing the impact of hereditary and infectious diseases. To date one of its most visible and rewarding applications for veterinary medicine has been in the genetic engineering of vaccines and diagnostics to assist in the eventual eradication of pseudorabies (PR, Aujeszkys disease). In the following review we summarize some of the most pertinent issues relative to PR eradication and point out the present and potential role of biotechnology in achieving our goal.

Alveolar Macrophages as a Diagnostic Sample for Detecting Natural Infection of Pigs with Porcine Reproductive and Respiratory Syndrome Virus

In a previous series of studies focused on the diagnosis of porcine reproductive and respiratory syndrome (PRRS), we tested sera, alveolar macrophages, and various tissues for their suitability as diagnostic samples when collected from acutely and persistently infected pigs. 3,5 The results of these studies indicated that the causative virus (PRRS virus [PRRSV]) could usually be isolated from any of several kinds of samples (serum, tonsils, lungs, and alveolar macrophages) during acute infection but from only alveolar macrophages during later stages of infection. However, during a recent experiment that was initially unrelated to our investigations of PRRS, we found that alveolar macrophages may sometimes be the diagnostic sample of choice even during acute infection. Details of this fortuitous observation follow. A group of 20 8-week-old pigs was delivered from a commercial swine herd to our laboratory for use in an experiment on pseudorabies (PR). On the following day (day 0) we euthanized and necropsied 2 of the 20 pigs as noninfected controls for the PR experiment (not described here) and found small areas of consolidation in the lungs of both. Although we suspected that these lesions were the result of a bacterial pneumonia, we tested alveolar macrophages of both of the pigs for PRRSV. We also tested the sera collected on day 0 from these and the remaining 18 pigs for both PRRSV and homologous antibody. The detection of PRRSV and antibody in some of these initial samples led us to test additional samples collected at later times (Table 1). The methods used to identify PRRSV and homologous antibody are outlined briefly in the following paragraphs. Additional details can be found in previous reports. 5,2