H.S. Joo

Enhanced replication of porcine reproductive and respiratory syndrome (PRRS) virus in a homogeneous subpopulation of MA-104 cell line.

SummaryTwo different cell populations, high- (MARC-145) and low-permissive cell clones (L-1) to porcine reproductive and respiratory syndrome (PRRS) virus, were derived from MA-104 cell line (parent cell: P) by cell cloning. Maximum virus yields in MARC-145, P, and L-1 cell clones were 108.5, 103.5, and 102.5 tissue culture infective dose 50 (TCID50)/0.1 ml, respectively. The MARC-145 cell clone supported replication of all 11 different porcine reproductive and respiratory syndrome virus isolates that were tested. These results indicated that the MARC-145 cells will be useful for PRRS virus replication.

Evaluation of the ELISA and comparison to the complement fixation test and radial immunodiffusion enzyme assay for detection of antibodies against Mycoplasma hyopneumoniae in swine serum

An enzyme-linked immunosorbent assay (ELISA) was evaluated for detection of antibodies (Ab) against Mycoplasma hyopneumoniae and M. flocculare in sera from swine experimentally infected with these agents. In addition, the ELISA was compared with the complement fixation test (CFT), and radial immunodiffusion enzyme assay (RIDEA) for the demonstration of Ab against M. hyopneumoniae. Twenty two 6-week-old swine from a respiratory disease-free herd were divided into five groups. Two or three pigs from each of the four groups were inoculated, respectively, with M. hyopneumoniae or with M. flocculare while two pigs in each group were contact exposed to the inoculated penmates. A fifth group, consisting of three pigs, served as inoculated controls. Pigs inoculated with M. hyopneumoniae began coughing 13 days post inoculation (PI). Antibodies were first detected 2 weeks PI with the CFT, 3 weeks PI with the ELISA, and 5 weeks PI with the RIDEA. With the ELISA and RIDEA, Ab were still detectable one year PI at a very low level. With the CFT, Ab were not detectable in sera from any swine beyond 5 months PI. At necropsy 1 year PI, no lesions were detected in lungs of any of the animals nor were mycoplasmas detected. M. flocculare inoculated or contact-exposed pigs never evidenced clinical signs. Antibodies against M. flocculare were first detected 5 to 12 weeks PI with CFT, and 6 to 12 weeks PI with the ELISA. Peak optical density (OD) values obtained in the ELISA with M. flocculare Ab were as high as the values obtained with peak M. hyopneumoniae Ab titers. Levels of Ab against M. flocculare were at relatively higher OD at 1 year PI than Ab against M. hyopneumoniae. Sera with high levels of Ab against M. flocculare cross-reacted slightly with M. hyopneumoniae antigen in immunoblotting and ELISA.

Prevalence of swine influenza virus subtypes on swine farms in the United States

Summary Serologic and virologic prevalence of infection with different swine influenza virus (SIV) subtypes was investigated using swine sera, nasal swabs and lung samples that had been submitted for a diagnosis to the Minnesota Veterinary Diagnostic Laboratory. A total of 111,418 pig sera were tested for SIV antibody between 1998 and 2000, and 25,348 sera (22.8%) were found to be positive by the hemagglutination inhibition (HI) test. Of the positive samples, 16,807 (66.7%) and 8,541 (33.7%) had antibody to H1 and H3 subtypes, respectively. Between January 1998 and May of 2001, a total of 3,561 nasal swabs or lung samples were examined for the presence of SIV, and SIV was isolated from 1,124 samples (31.7%). Of these isolates, 869 (77.3%) and 255 (22.7%) were subtyped as H1 and H3, respectively, by the HI method. For further characterization, 120 SIV isolates each from 1998 to 2001 were randomly selected from a culture collection and their hemagglutinin (HA) and neuraminidase genes examined by reverse transcription-PCR and sequencing. Of the 480 isolates, 322 (67.1%), 22 (4.6%) and 129 (26.9%) were subtyped as H1N1, H1N2 and H3N2, respectively. The remaining 7 samples (1.5%) were found to contain both H1N1 and H3N2 viruses. The SIV H1N2 subtype was isolated from 1, 8, and 13 samples in 1999, 2000, and 2001, respectively. The 22 H1N2 isolates originated from 9 different states of the United States. Genetic screening of the HA genes of 12 selected H1N2 isolates showed that 8 of them had a close phylogenetic relationship with the Indiana isolate of H1N2 (A/Swine/Indiana/9K035/99), while 4 isolates were closely related to classical SIV H1N1.

Detection and subtyping of swine influenza H1N1, H1N2 and H3N2 viruses in clinical samples using two multiplex RT-PCR assays

A total of 360 type A swine influenza virus-positive samples including cell culture isolates, nasal swabs or lung tissues along with 30 virus-negative samples were tested for the detection and subtyping of H1N1, H1N2 or H3N2 by two multiplex reverse transcription (RT)-PCR assays. The positive samples had been collected between 1999 and 2001 from pigs with respiratory diseases, and type A influenza virus was isolated and subtyped by hemagglutination inhibition (HI) test at the Minnesota Veterinary Diagnostic Laboratory (MVDL). Two multiplex RT-PCR assays specific for H1 and H3, and N1 and N2 were developed. RT-PCR products with unique sizes characteristic of each subtype of influenza A virus were sequenced, and the sequences were demonstrated to be specific for H1N1, H1N2 or H3N2. Genomic RNAs or DNAs from 12 common swine pathogens other than type A influenza viruses were not amplified when the PCR assays were performed with these primer sets. Positive amplification reaction could be visualized with RNA extracted from up to 10(-5) dilution of each reference virus with original infectivity titer of 10(5) TCID(50)/ml. Of the 360 samples tested, swine influenza virus H1N1, H1N2 and H3N2 were identified in 200, 13 and 139 samples, respectively. The remaining eight samples were positive for both H1N1 and H3N2 viruses. The results of multiplex RT-PCR were 100% in agreement with those of virus isolation. These results demonstrate the usefulness of multiplex RT-PCR for detection and identification of influenza A virus subtypes. The results also indicate an increased occurrence of H1N2 in US swine population.

Pathogenicity of a skin isolate of porcine parvovirus in swine fetuses

The pathogenic properties of a skin isolate of porcine parvovirus (PPV), designated Kresse isolate, were compared with NADL-8 isolate, a prototype isolate of PPV, by in utero inoculation of mid-term and late-term gestation swine fetuses. Fetuses from pregnant sows of mid-gestation were inoculated with either NADL-8 or Kresse virus. Both isolates were highly pathogenic to mid-gestation fetuses. In contrast, dramatic differences in pathogenicity between these 2 isolates were observed in fetuses inoculated late in gestation. Such fetuses from each of 4 sows were inoculated with NADL-8 or Kresse virus isolate and sacrificed at 10, 18, 21, or 23 days postinoculation (PI). NADL-8-inoculated fetuses were grossly normal. The pathogenic effects of Kresse isolate were evident by gross pathology in fetuses collected at 18, 21, and 23 days PI, but not at 10 days PI. Hemagglutination (HA) and fluorescent antibody (FA) methods were used to identify virus in various tissues of late-gestation fetuses collected at 10 and 21 days PI. At 10 days PI, HA antigens were detected only in livers of NADL-8-inoculated fetuses, but in all tissues examined of Kresse-inoculated fetuses, including the brain. PPV specific fluorescence was demonstrated in tissues of fetuses inoculated with NADL-8 and Kresse virus. The major difference was that virus antigen was found in the brains of fetuses inoculated with Kresse virus, but not in NADL-8 infected fetuses. At 21 days PI, HA antigen was not detected in any of the tissues of fetuses inoculated with NADL-8 virus, with PPV specific fluorescence by FA being found only in the kidney. However, fetuses inoculated with Kresse virus displayed HA antigen in liver and PPV-specific fluorescence in all tissues tested including the brain. Both isolates induced similar antibody responses, 1:128 to 1:256 at 10 days and 1:512 to 1:1024 at 21 days PI. In addition, immunoglobulin G (IgG) deposits were demonstrated in kidneys and skin of fetuses inoculated with Kresse virus and IgM in brain, but not in tissues from fetuses inoculated with NADL-8 virus.

Strategies to control PRRS: A summary of field and research experiences

Abstract Various methods for the control of PRRS virus have been published. The technology of nursery depopulation (ND) appears to effectively control the spread of virus between members of endemically infected populations. ND consists of a strategic adjustment in pigflow based on the presence of specific serologic patterns as detected by the indirect fluorescent antibody test. This pattern indicates a low seroprevalence of antibodies detected in the breeding herd and recently weaned piglets (≤ 10%), in contrast to a high (> 50%) seroprevalence in 8 to 10 week old piglets. ND has been carried out on swine farms in the US and results indicate improvements in nursery piglet growth rate and mortality levels. Three examples are provided in the following text. Recently a modified live virus vaccine (RespPRRS, NOBL Laboratories/Boerhinger Ingleheim) has become commercially available. It is currently approved for use in piglets from 3 to 18 weeks of age; however, potential for the use in adult animals is currently under investigation.

Indirect fluorescent IgM antibody response of pigs infected with porcine reproductive and respiratory syndrome syndrome virus

IgG and IgM antibody responses were examined by an indirect fluorescent antibody method in pigs following inoculation with different porcine reproductive and respiratory syndrome virus (PRRSV) isolates or a vaccine virus. Viremia was also examined in the pigs. The IgG antibody was first detected between 9 and 14 days post inoculation (PI) and maintained high titers for at least 7 weeks PI. No change in IgG antibody titers was observed when the pigs were reinoculated with PRRSV 35 days PI. IgM antibody was detected between 5 and 28 days PI in the pigs. Reinoculation at 35 days PI caused a short term rise of IgM antibody. Virus was isolated from sera collected between 2 and 21 days PI. The IgM antibody was detected regularly in sera collected during viremia and up to 1-2 weeks after the viremic periods. These results suggest that pigs with detectable IgM antibody are probably pigs with recent infection and that routine testing of IgM antibody in purchased breeding pigs from seropositive farms may be useful in identification of pigs with recent infection.

Potentiating effect of adjuvants on humural immunity to porcine parvovirus vaccines in guinea pigs

Fourteen different adjuvants, given either in single or combined form with another compound were compared in guinea pigs for their ability to potentiate humoral immunity to porcine parvovirus (PPV) antigen after 2 vaccinations. Two injections were given, the second 3 weeks following the initial vaccination. Antibody concentrations to PPV in sera from injected animals were measured over a 5-week period by the hemagglutination inhibition test. At the conclusion of the experiment, guinea pigs injected with the following adjuvants and PPV antigen: CP-20 961 (Avridin), 50% aluminum hydroxide gel, ethylene maleic anhydride (EMA), oil and water emulsion (O/W) and dimethyl-dioctadecyl-ammonium bromide (DDA) immunologically responded with high geometric mean HI titers (380, 224 and 427, 602, 512, 1202 respectively), whereas guinea pigs receiving Emulsan, sodium dodecyl sulfate (SDS), L-121, combinations of Emulsan/aluminum hydroxide, SDS/aluminum hydroxide and B. pertussis/aluminum hydroxide responded with low mean titers (54, 64, 18, 27, 11, 64, 14, 20 respectively). Guinea pigs injected with antigen without adjuvant responded weakly with geometric mean titers of 3.3 and 16 for the 2 groups tested. Prior to booster injection, guinea pigs immunized with 13 of the preparations had low (less than 4) or undetectable antibody titers. Antibody titers from guinea pigs receiving DDA adjuvant continued to rise throughout the duration of the experiment and at the conclusion had the highest mean titers of the groups tested (1202). The 2 groups immunized with 50% aluminum hydroxide gel had high mean titers (224, 427), but in both instances there was a wide range of titers within a group evidenced by high standard deviations. In contrast, guinea pigs receiving either DDA, CP-20 961, O/W or EMA had antibody titers within a narrow range and small standard deviation. The significance of aluminum hydroxide gel concentration on immunogenicity is discussed.

Antibody responses of guinea-pigs, rabbits and pigs to inactivated porcine parvovirus vaccines

Antibody responses were compared in guinea-pigs, rabbits and pigs following vaccination with inactivated porcine parvovirus (PPV) vaccines. Mean PPV hemagglutination inhibition (HI) antibody titers of 52, 56 and 36 at 1 week after first vaccination and 896, 640 and 512 at 2 weeks after second vaccination were detected in guinea-pigs, rabbits and pigs, respectively. PPV vaccines prepared with greater concentrations of virus, as determined by hemagglutination (HA) units, and of aluminum hydroxide gel adjuvant, induced higher HI antibody titers in guinea-pigs. Optimal concentrations for inducing consistently high antibody titers consisted of vaccine virus with a HA titer of 256/0.1 ml and gel adjuvant at a final concentration of 50%. A second vaccination at 4 weeks compared to 2 or 3 weeks after first vaccination resulted in higher mean HI titers. These data provide preliminary information on the use of guinea-pigs or rabbits as laboratory animal models for testing the potency of PPV vaccines.

Replication of two porcine parvovirus isolates at non-permissive temperatures.

SummaryPrevious studies have shown that replication in vitro of the porcine parvovirus (PPV) isolate, KBSH, was restricted at 39°C but not at 37°C. In contrast, replication of the Kresse isolate was restricted at 37°C but not at 39°C. In this study, Kresse and KBSH isolates were passaged up to ten times in swine testicle (ST) cells at non-permissive temperatures, and at subsequent passage viral protein synthesis, viral DNA synthesis, and progeny virus were evaluated. KBSH became adapted for replication at 39°C upon serial passages, displaying an appreciable increase in viral progeny, viral polypeptides, and viral DNA concentration. This finding was also observed with Kresse virus isolate continuously passaged at 37°C. Neither isolate became adapted for replication at 32°C. In an attempt to examine the effect of in vitro passage at non-permissive temperatures on pathogenicity in swine, KBSH passaged 10 times either at 37°C or 39°C was inoculated into swine fetuses. Two of four fetuses inoculated with 39°C-passaged KBSH were dead and hemorrhagic or mummified. All four fetuses inoculated with 39°C-KBSH contained viral antigen and viral DNA. In contrast, fetuses inoculated with 37°C-passaged KBSH, or with cell culture fluid were normal in appearance. Viral antigen and viral DNA were not demonstrated in fetuses inoculated with 37°C-KBSH or cell culture fluids. These findings suggest the possibility that the ability to replicate at 39°C is associated with virulence in swine fetuses.