Application of polymerase chain reaction for the correlation of Salmonella serovars recovered from greyhound feces with their diet.

The polymerase chain reaction was employed to correlate Salmonella serovars isolated from fecal material of greyhounds suffering from gastroenteritis with those isolated from the diet fed to the greyhounds prior to onset of diarrhea. Kennels around the Abilene, Kansas, area were contacted and supplied with materials needed to collect a portion of the diet each day. With the onset of diarrhea, the kennels were instructed to ship the fecal material and diet from the previous 10 days to the laboratory for testing. Forty-one fecal samples and corresponding diets were screened for Salmonella, Clostridium perfringens, Campylobacter jejuni, Staphylococcus aureus, Staphylococcus intermedius, and pathogenic (piliated) Escherichia coli by direct culture using standard procedures. The fecal material was also screened for coronavirus and parvovirus using electron microscopy. Thirty-five "normal" fecal samples were screened for all of the above mentioned microorganisms as a control. In addition, the fecal material was screened for E. coli verotoxins I and II and clostridial enterotoxins. A total of 61 Salmonella isolates were recovered from the 41 samples of feces and diet submitted for testing; 31 were recovered from the feces and 30 from the diet. Four Salmonella isolates were recovered from the normal fecal samples. Results obtained by PCR, plasmid profiles, antigenic analysis, and antibiogram profiles indicated that 16 of the 31 isolates recovered from the fecal material were the same strain as that recovered from the diet.

Serotype identification of Actinobacillus pleuropneumoniae by arbitrarily primed polymerase chain reaction.

Rapid and accurate determination of the Actinobacillus pleuropneumoniae serotype involved in a disease outbreak is important both in limiting the severity of an outbreak and for tracing the source of the infecting organism. This study describes the use of arbitrarily primed polymerase chain reaction (AP-PCR) as a rapid, precise, and genetically based procedure to identify A. pleuropneumoniae. AP-PCR amplification of bacterial genomic DNA results in specific DNA profiles, which can be used to differentiate currently recognized serotypes. This technique is especially useful for identifying previously nontypeable and serologically cross-reactive A. pleuropneumoniae field isolates. Consecutive passages of isolates on different media, freezing, and subsequent infection of pigs did not alter the AP-PCR genomic profile. We propose the use of M13 and T3-T7 oligodeoxynucleotide primers for diagnostic and epidemiological identification of A. pleuropneumoniae by AP-PCR techniques.

Identifying bovine respiratory syncytial virus by reverse transcription-polymerase chain reaction and oligonucleotide hybridizations.

An assay to identify tissue culture cells infected with bovine respiratory syncytial virus (BRSV) that utilizes reverse transcription (RT), the polymerase chain reaction (PCR), and a synthetic oligonucleotide hybridization probe has been developed. The RT-PCR assay uses a BRSV-specific negative-sense oligonucleotide primer to synthesize cDNA from a BRSV fusion protein mRNA template and another BRSV-specific oligonucleotide primer (positive sense) upstream from the negative-sense primer for PCR amplification. In the presence of mRNA templates of BRSV isolates originating from locations throughout the United States, the BRSV RT-PCR assay resulted in amplified products (381 bp) that were specific to BRSV, as demonstrated in hybridizations with a positive-sense oligonucleotide probe complementary to internal sequences and in sequence comparisons with the F protein of BRSV 391-2. In analysis of the BRSV RT-PCR assay with prototype strains of human RSV subgroups A and B, amplification of a similar 381-bp RT-PCR product was not evident, and no RT-PCR product hybridized with the internal probe. We conclude that the specific ability to amplify DNA sequences of BRSV F protein mRNA by RT-PCR and then to demonstrate the presence of the amplified product with a BRSV-specific oligonucleotide probe will greatly add to the speed, sensitivity, and specificity of BRSV diagnostics.

Pleuropneumonia caused by Actinobacillus pleuropneumoniae biotype 2 in growing and finishing pigs.

Actinobacillus pleuropneumoniae biotype 2 was isolated in pure culture or as the predominant isolate from the lungs of 9 growing and finishing pigs with pleuropneumonia. Gross and microscopic lesions resembled those caused by A. pleuropneumoniae biotype 1 serotypes (Nos. 1, 5, and 7) traditionally seen in the United States. The overall mortality rate for growing and finishing pigs on this 1,200-sow farrow-to-finish farm ranged from 0.37% to 0.84% per month from July 1990 to February 1991, and mortality due to respiratory disease ranged from 0.17% to 0.52% per month for the same period. This Actinobacillus species did not require V factor (no satellitism on blood agar with a Staphylococcus streak), was strongly beta-hemolytic, and demonstrated restriction fragment length polymorphisms in hybridization studies with A. suis, A. lignieresii, and A. equuli. Biochemically, the isolate most closely resembled A. pleuropneumoniae, and a DNA fragment considered specific for A. pleuropneumoniae biotypes 1 and 2 was demonstrated using polymerase chain reaction. Necrohemorrhagic pleuropneumonia similar to that caused by A. pleuropneumoniae biotype 1 was reproduced experimentally in 2 4-week-old pigs inoculated intratracheally with broth cultures of the A. pleuropneumoniae biotype 2. This study demonstrated the presence of A. pleuropneumoniae biotype 2 in the United States.

Human recombinant interleukin-2 augments porcine natural killer cell cytotoxicity in vivo.

Immunological parameters of porcine peripheral blood mononuclear cells after in vivo injections of human recombinant interleukin-2(125) (HrIL-2) were studied. Eighteen pigs (6 pigs/group) were injected with either 10(4) or 10(5) units/kg body weight of human rIL-2 or an equivalent volume of sterile physiological saline (control) on days 0 through 4. All pigs were immunized with an E coli J5 bacterin on day 0. Cytolytic activity to porcine fibroblasts (PK-15) was increased (P less than 0.02) in pigs treated with HrIL-2 when compared to control animals. Cytotoxicity to K-562 cells also showed a tendency (P less than 0.08) towards increased cytolytic activity in the HrIL-2-treated pigs. Lymphocyte blastogenesis, IL-2 production, and serum iron concentrations did not differ between treatment groups. Antibody concentrations to E coli J5 antigens increased significantly (P less than 0.05) in all groups after immunization, but there were no differences between treatment groups. These data suggest that in vivo injections of Hr-IL-2 increase natural cytotoxic cell activity in pigs without influencing other immune activities.

Isoprinosine and levamisole immunomodulation in artificially reared neonatal pigs.

An experiment was conducted to determine the capability of isoprinosine (ISO) and levamisole (LEV) to augment delayed-type hypersensitivity and lymphocyte blastogenic reactions in artificially reared pigs. Sow-reared pigs (n = 15) were kept with their dams; artificially reared pigs (n = 15) were removed from sows within 2 days after parturition and reared artificially for 21 days. Isoprinosine was administered orally (75 mg/kg/day) from days 0 to 10. Levamisole (2 mg) was injected subcutaneously on days 5 and 10. Control pigs were given distilled water orally from days 0 to 10 and injected subcutaneously with 0.15M NaCl on days 5 and 10. Lymphocyte proliferative responses to phytohemagglutinin, concanavalin A, and pokeweed mitogen were evaluated at week 2. The phytohemagglutinin skin-test responses were evaluated in all pigs at weeks 1 and 3 of the trial. Hematologic values, body weight, and mortality were evaluated each week. The skin-test responses and mitogen-induced lymphocyte proliferative responses were lower (P less than 0.05) in artificially reared controls when compared with responses in sow-reared pigs. However, ISO and LEV enhanced (P less than 0.05) the responses in the artificially reared pigs to values comparable with those of the sow-reared controls. Body weight was greater (P less than 0.01) in sow-reared pigs than in artificially reared pigs; drug treatment did not influence weight gain. These data indicated that immunopotentiation of the cellular immune responsiveness of artificially reared pigs may be possible with ISO or LEV.