Alicia V. Trigo

Bio: Alicia V. Trigo is an academic researcher from Bayer. The author has contributed to research in topics: Nested polymerase chain reaction & Seroprevalence. The author has an hindex of 2, co-authored 2 publications receiving 162 citations.

Application of a nested polymerase chain reaction assay to detect Mycoplasma hyopneumoniae from nasal swabs.

Abstract: The porcine respiratory disease complex (PRDC) is an increasingly important cause of decreased swine productivity and is characterized by slow growth, decreased feed efficiency, anorexia, cough, and dyspnea. Mycoplasma hyopneumoniae is among the most prevalent and important infectious agents associated with PRDC. Understanding of mycoplasmal pneumonia has been hindered by inadequate diagnostic methods. Many of the currently available tests are relatively insensitive or nonspecific when used in a diagnostic laboratory setting or are too costly or difficult for routine diagnostic use. Several polymerase chain reaction (PCR) assays have been described, but they are not sensitive enough to detect the microorganisms in live pigs, from either nasal or tracheal swabs. A nested PCR using 2 species-specific sets of primers from the 16S ribosomal DNA gave positive results with as little as 80 microorganisms and did not cross-react with other mycoplasma species or with other microorganisms commonly found in the respiratory tract of pigs. This assay was better suited for detection of M. hyopneumoniae from nasal swabs than was conventional PCR. Nasal swab samples were taken at different time periods following experimental challenge of 10 susceptible pigs. Only 2 of the 55 swabs examined gave a positive result with conventional PCR, whereas 30 of the 55 swabs gave a positive result using the nested PCR. Twenty of 40 (50%) nasal swabs from pigs experiencing a respiratory disease outbreak where M. hyopneumoniae had been diagnosed also gave a positive result with the nested PCR. To confirm that the amplified product was specific, 4 nested PCR products were purified, sequences were determined and aligned, and they were confirmed to be from M. hyopneumoniae.

Seroprevalence of indirect fluorescent antibody to porcine reproductive and respiratory syndrome virus in selected swine herds

Abstract: Porcine reproductive and respiratory syndrome (PRRS) virus has recently been identified in the USA as a cause for reproductive failure in pregnant sows and respiratory disease in young pigs. 1-4,6 The syndrome has been commonly referred to as swine infertility and respiratory syndrome in this country but there was a general agreement to adopt the name PRRS at an international PRRS symposium (St. Paul, MN, May 1992). A virus designated as Lelystad virus has also been reported to cause a similar syndrome in several European countries. 1,5 Both viruses can cause significant economic problems for swine producers because of acute reproductive losses and piglet mortality. Chronic production losses involving increased mortality and reduced growth rate in pigs at postweaning age have also been reported on some infected farms. 1 In our recent study, an indirect fluorescent antibody (IFA) test was developed to detect and quantitate PRRS virus antibody in swine sera. 7 This IFA test for swine sera collected between 1981 and 1991 revealed evidence of PRRS virus infection in this country as early as April 1986. The purpose of the present study was to report the recent Seroprevalence status of IFA to PRRS virus by testing sera collected from selected swine herds during the first 6 months of 1992. Swine sera submitted January-June 1992 for serodiagnosis to the Oxford Veterinary Laboratory, Worthington, Minnesota, were used. Since October 1988, the laboratory has provided a swine antibody profile service to practicing veterinarians for different pathogens. The service has included serology for Actinonobacillus pleuropneumonia serotypes 1, 5, and 7, encephalomyocarditis virus, porcine parvovirus, Leptospira spp., and group D Streptococcus. The IFA test for PRRS virus was included starting in January 1992. Between January and June 1992, a total of 2,787 sera from 263 swine farms (an average of 10.6 serum samples per farm), were submitted from 13 different states. Veterinarians provided a brief farm history and requested serology for specific agents. Of the 263 farms, 225 requested PRRS virus serology. For the PRRS virus IFA test, aliquots of each sample were sent to the swine virology laboratory, University of Minnesota. ethanol was used to fix the cell monolayers in each well. Test sera, diluted 4-fold serially from 1:4 to 1:1,024, were added to test plates. The plates were incubated, washed, and incubated again with an optimal dilution of rabbit anti-swine IgG conjugated with fluorescein isothiocyanate. The plates were then washed and examined under a fluorescent microscope. Negative and positive reference sera and an uninfected SAM control for each serum were included in each test. The IFA titers were recorded as the highest serum dilutions with specific cytoplasmic fluorescence, and titers of ≤ 1:4 and ≥ 1: 16 were considered negative and positive, respectively. Of 2,787 sera from 263 swine herds tested, 979 sera (35.1%) had PRRS IFA titers of 21: 16 and 148 farms (56.3%) had 1 or more pigs that were PRRS virus IFA test positive (Table 1). Of 1,726 sera from 148 farms with PRRS virus seropositive pigs, 747 sera (43.3%) had IFA titers of ≤ 1:4 and 979 sera (56.7%) had the titers of ≥ 1:16. Of 979 seropositive samples, 222 (22.7%) 314 (32.1%), 283 (28.9%), and 160 (16.3%) samples had IFA titers of 1:16, 1:64, 1:256, and ≥ 1: 1,024, respectively. Of 225 farms requesting PRRS serology, 129 (57.3%) had seropositive pigs, whereas 19 of 38 farms (50.0%) that did not request PRRS virus serology were positive. Results of the farms with seropositive pigs were 1 1/16 (68.8%), 16/41 (39.0%) 35/65 (53.8%) 21/35 (60.0%), 37/61 (60.7%), and 28/45 (62.2%) farms for January, February, March, April, May, and June, respectively. No significant difference for seropositive farms was found between the months by chisquare analysis (P = 0.190). The positive farms were located in 11 different states.

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

Abstract: 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 α2,6Gal-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.

Development of a PCR test to diagnose Haemophilus parasuis infections.

Abstract: A polymerase chain reaction (PCR) test was developed in order to improve the accuracy and speed of diagnosis of Haemophilus parasuis, an economically important respiratory pathogen that affects swine. The gene sequence of the 16S small subunit ribosomal RNA of H. parasuis (GenBank M75065) was compared with 56 16S sequences of related bacteria, including those frequently isolated from pig tissues. Two species-specific primers were designed: HPS forward and HPS reverse. The predicted size of the amplified PCR product was 821 bp. The PCR test could detect a minimum of 102 bacteria and 0.69 pg of DNA. Thirty-one H. parasuis isolates, including 12 different serovars and 19 field isolates, were positive using the PCR test. No amplification was observed when the test was run using DNA from 15 other bacterial species commonly isolated from swine tissues. A weak band was observed when the PCR test was performed using Actinobacillus indolicus DNA as template. Clinical samples tested by PCR included tissues and swabs from 5 animals naturally infected with H. parasuis and 1 experimentally infected animal. The PCR was positive in 26 of 30 clinical samples. Four samples showed weak bands, and these results were not considered positive. Haemophilus parasuis was isolated from 18 of 30 of these samples. Tissues from specific pathogen-free (SPF) pigs and from unrelated species were negative for H. parasuis isolation and PCR. The developed PCR was successfully used in the diagnosis of H. parasuis infection, especially when compared with traditional microbiology techniques.

Update on Mycoplasma hyopneumoniae infections in pigs: Knowledge gaps for improved disease control.

Abstract: Mycoplasma hyopneumoniae (M. hyopneumoniae) is the primary pathogen of enzootic pneumonia, a chronic respiratory disease in pigs. Infections occur worldwide and cause major economic losses to the pig industry. The present paper reviews the current knowledge on M. hyopneumoniae infections, with emphasis on identification and analysis of knowledge gaps for optimizing control of the disease. Close contact between infected and susceptible pigs is the main route of M. hyopneumoniae transmission. Management and housing conditions predisposing for infection or disease are known, but further research is needed to better understand M. hyopneumoniae transmission patterns in modern pig production systems, and to assess the importance of the breeding population for downstream disease control. The organism is primarily found on the mucosal surface of the trachea, bronchi and bronchioles. Different adhesins and lipoproteins are involved in the adherence process. However, a clear picture of the virulence and pathogenicity of M. hyopneumoniae is still missing. The role of glycerol metabolism, myoinositol metabolism and the Mycoplasma Ig binding protein-Mycoplasma Ig protease system should be further investigated for their contribution to virulence. The destruction of the mucociliary apparatus, together with modulating the immune response, enhances the susceptibility of infected pigs to secondary pathogens. Clinical signs and severity of lesions depend on different factors, such as management, environmental conditions and likely also M. hyopneumoniae strain. The potential impact of strain variability on disease severity is not well defined. Diagnostics could be improved by developing tests that may detect virulent strains, by improving sampling in live animals and by designing ELISAs allowing discrimination between infected and vaccinated pigs. The currently available vaccines are often cost-efficient, but the ongoing research on developing new vaccines that confer protective immunity and reduce transmission should be continued, as well as optimization of protocols to eliminate M. hyopneumoniae from pig herds.