Introduction: Porcine pleuropneumonia inflicts important economic losses on most commercial herds. Detection of subclinical or chronic infection in animals still remains a challenge, as isolation and identification of A. pleuropneumoniae serotypes is difficult and quantification of the bacteria on agar plates is often almost impossible. The aim of the study was to develop and evaluate a serotype-specific quantitative TaqMan probe-based PCR for detection of serotype 2 in pig lungs, tonsils, and nasal swabs.Material and Methods: The primers were designed from the capsular polysaccharide biosynthesis genes of A. pleuropneumoniae serotype 2. PCR specificity and sensitivity were evaluated using reference strains and several other bacterial species commonly isolated from pigs.Results: The real-time qPCR for detection of A. pleuropneumoniae serotype 2 was highly specific and gave no false positives with other serotypes or different bacterial species of pig origin. The detection limit for pure culture was 1.2 × 10⁴ CFU/mL, for lung tissue and nasal swabs it was 1.2 × 10⁵ CFU/mL, and for tonsils - 1.2 × 10⁵ CFU/mL.Conclusion: The method can be used to serotype A. pleuropneumoniae isolates obtained during cultivation and to detect and identify A. pleuropneumoniae serotype 2 directly in nasal swabs and tonsil scrapings obtained from live pigs or lung tissue and tonsils collected post-mortem.

Pseudorabies virus (PRV) nucleic acids in the trigeminal ganglia and tonsils of swine latently infected with the virus were analyzed. By use of DNA-polymerase chain reaction (PCR), 14 of 14 trigeminal ganglia and 12 of 14 tonsils were positive for PRV genomes. By use of RNA-PCR, RNA containing the large latency transcript splice junction were detected in 4 of 4 trigeminal ganglia and 4 of 5 tonsils. In general, results of both PCR procedures indicated that the amounts of PRV DNA and RNA per microgram of cellular nucleic acids were higher in trigeminal ganglia than in tonsils. Identification of peripheral tissues that harbor latent PRV is an important asset for PRV research. The presence of large latency transcript in tonsil tissues, in the absence of virus replication, is a critical characteristic, which indicates that the tonsil is a site of PRV latency. For diagnostic purposes, animals need not be euthanatized to obtain their nervous tissue to determine latency; instead, tonsil biopsy specimens could be obtained from live animals for analysis. For pathogenesis studies, multiple specimens obtained sequentially from the same animal would be available for examination for the duration of the experiment.

The Rice strain of pseudorabies virus (PRV) was intranasally instilled in pigs that were seronegative to PRV. Cells were scraped or brushed from tonsillar surfaces biweekly until pigs were euthanatized at either 10 or 16 weeks after infection. The DNA extracted from tonsillar cells or parenchyma were subjected to polymerase chain reaction analysis, using either a single set of oligonucleotide primers or nested primers from the PRV gII glycoprotein gene. Pigs became seropositive to PRV by 3 weeks after infection. The virus was isolated from the trigeminal ganglia and tonsils of pigs that were euthanatized or died 1 to 2 weeks after infection, but not from pigs that were euthanatized 10 or 16 weeks after infection. The PRV gene products were consistently detected in trigeminal ganglia and tonsils of all pigs at 1, 10, and 16 weeks after infection, and sporadically in the nasal mucosa, lymph nodes, and lungs of pigs that were euthanatized or died during the first 2 weeks after infection. Cells collected biweekly from tonsillar surfaces were mostly nucleated, squamous epithelial cells with fewer lymphocytes and neutrophils. Polymerase chain reaction analysis of DNA extracted from these cells revealed PRV DNA in a large proportion of the samples when sufficient cells were collected to provide 1 microgram of extracted DNA for use in the reaction mixtures. A second group of pigs had PRV strain 4892 intranasally instilled. The virus was isolated from tonsillar swab specimens until 3 weeks after infection. Tonsillar brushing specimens were collected biweekly until 14 weeks after infection. Some brushing specimens contained all nucleated, squamous epithelial cells, whereas other specimens contained a mixture of epithelial cells and up to 15% neutrophils, lymphocytes, and small mononuclear cells. Results of polymerase chain reaction analysis of DNA extracted from tonsillar cells collected 5, 11, and 14 weeks after infection were consistently positive for PRV gene products. Intact cells collected from tonsillar surfaces were placed in polymerase chain reaction mixtures with nested oligonucleotide primers from the PRV gII glycoprotein gene and were subjected to multiple amplification cycles. Afterward, the specificity of the amplified PRV gene products was determined by hybridization procedures, using a virus-specific oligonucleotide probe. Most nucleated, squamous epithelial cells stained positive for PRV DNA, suggesting that these cells were the primary source of PRV gene products in tonsillar brushing specimens.

Vaccination of cattle with a tissue culture-derived Pasteurella haemolytica serotype 1 vaccine elicited a serotype-specific inhibition of nasal and tonsillar colonization by the homologous serotype under field conditions. Calves (n = 101) originated from a single farm, where half the calves were vaccinated. The calves were delivered to an order-buyer barn 105 days later, and given a second dose of vaccine. At the order-buyer barn, calves were mixed with 27 calves, some of which had clinical signs consistent with respiratory tract disease. Also 12 of the original calves were infected with P haemolytica serotype 1 by tonsillar instillation. After 6 days at the order-buyer barn, calves were shipped 1,600 km by truck to a feedyard, and arrived the next day. Tonsillar wash and nasal secretion aspiration specimens were collected for culture of P haemolytica on days 1, 8, and 29 at the feedyard. Inhibition of colonization was evidenced by lower frequency of isolations from the vaccinates than from the nonvaccinates after transport to the feedyard. Selectively lowering the frequency of colonization by P haemolytica serotype 1 could reduce losses attributable to pneumonic pasteurellosis.

Pseudorabies virus (PRV) latency was investigated, using polymerase chain reaction (PCR). A PCR protocol was developed that specifically amplified a 217-base pair sequence within the gene encoding the essential glycoprotein gp50 of PRV. Using this PCR procedure, the gp50 sequence was amplified from tissues of pigs infected with various doses of PRV (Becker strain). At postinoculation day 64, viral isolation was performed on nasal swab specimens and homogenates of tonsils and trigeminal nerve ganglia obtained from 11 PRV-convalescent, seropositive pigs. Results were negative in all cases. By use of PCR, 11 of 11 pigs had PRV-positive trigeminal nerve ganglia and brain stem, 10 of 11 pigs had PRV-positive tonsils, and 9 of 11 pigs had PRV-positive olfactory bulbs.

OBJECTIVE To evaluate the effects of dietary nonphytate phosphorus (NPP) content on ileal lymphocyte subpopulations and cytokine expression in the cecal tonsils and spleen of hens that were or were not inoculated with Salmonella Typhimurium. ANIMALS 64 Salmonella-free hens. PROCEDURES Hens were fed a diet with 0.22% (control; n = 32) or 0.42% (high-P; 32) NPP for 6 weeks and then orally inoculated with S Typhimurium (5 × 107 CFUs) or PBSS. Tissues were obtained from 8 S Typhimurium–inoculated and 8 PBSS-inoculated hens from each group at 2 and 7 days postinoculation (DPI). Percentages of ileal CD4+ and CD8+ lymphocytes were determined by flow cytometry. Cytokine mRNA expression was determined by quantitative real-time PCR assays. RESULTS For S Typhimurium–inoculated hens, plasma parathyroid hormone concentration was significantly increased and 1,25-dihydroxyvitamin D3 concentration was decreased in hens fed the high-P diet, compared with values in hens fed the control diet. Salmonella Typhimurium inoculation caused an increase in the percentage of ileal CD8+ lymphocytes and the expression of interleukin (IL)-1β, IL-6, IL-8, interferon-γ, IL-12, and IL-18 in the cecal tonsils and spleen and a decrease in the expression of IL-4 and IL-10 in the cecal tonsils. Hens fed the high-P diet had significantly increased splenic expression of interferon-γ at 2 DPI and IL-1β, IL-6, IL-12, and IL-18 at 7 DPI, compared with hens fed the control diet. CONCLUSIONS AND CLINICAL RELEVANCE Results suggested there was a T-helper 1 cytokine reaction in the cecal tonsils and spleen of S Typhimurium–inoculated hens, and dietary NPP content altered calcium regulation hormone concentrations and affected splenic cytokine expression.

Objective—To determine immunomodulatory effects of synbiotics administered in ovo on immune-related gene expression in adult chickens. Animals—30 Green-legged Partridgelike chickens. Procedures—On incubation day 12, eggs were injected with 3 synbiotics (Lactococcus lactis subsp lactis IBB SL1 with raffinose family oligosaccharides [RFOs; S1], Lactococcus lactis subsp cremoris IBB SC1 with RFOs [S2], and Lactobacillus acidophilus and Streptococcus faecium with lactose [S3]). Control eggs were injected with RFOs prebiotic or saline (0.9% NaCl) solution. Gene expression of 6 cytokines (interleukin [IL]-4, IL-6, IL-12p40, IL-18, interferon [IFN]-β, and IFN-γ) and 1 chemokine (IL-8) was analyzed in the cecal tonsils and spleen of 6-week-old chickens by means of reverse transcription quantitative PCR assays. Results—Gene expression for IL-4, IL-6, IFN-β, and IL-18 was significantly upregulated in the spleen of chickens in groups S2 and S3. In contrast, IL-12 expression was downregulated in group S2 and IFN-γ expression was downregulated in group S3. Expression of IL-8 did not change in chickens treated with synbiotics in ovo. Gene expression of all cytokines, except for IL-18, was downregulated in cecal tonsils. Conclusions and Clinical Relevance—In ovo administration of synbiotics activated the immune system in adult chickens. The intestinal immune system (cecal tonsils) had downregulation of expression for the cytokines evaluated, which indicated an increase in oral tolerance, whereas in the peripheral part of the immune system (spleen), expression of IL-4 and IL-6 was upregulated. Evaluation of immune-related gene expression patterns may be useful when monitoring the effectiveness of synbiotic selection with respect to immunobiotic properties.

This study investigated and compared the antimicrobial resistance patterns and ribotypes of Staphylococcus aureus isolated from pig tonsils and cow’s milk in China. A total of 90 isolates of S. aureus was included: 42 strains were isolated from tonsils of pigs and 48 from half-udder milk. The broth microdilution method and the double-disc diffusion test (D test) were used for antimicrobial susceptibility testing. The mecA gene for methicillin-resistant S. aureus (MRSA) and the ermA, ermB, ermC, and msrA genes for erythromycin-resistant strains were detected by polymerase chain reaction (PCR). The isolates were ribotyped with the Riboprinter system. The highest frequency of resistance was observed with clindamycin (91.1%), followed by penicillin (90.0%), and erythromycin (85.6%). All strains were susceptible to vancomycin and trimethoprim-sulfamethoxazole. The D test showed that 54.5% (42/77) of erythromycin-resistant isolates had the constitutive resistance phenotype and 45.5% (35/77) had the inducible resistance phenotype to clindamycin. A higher proportion of resistance to cephalosporins, macrolides, fluoroquinolones, and pleuromutilins was observed in pig isolates than in milk isolates (P < 0.05). The mecA gene was detected in all MRSA isolates; 89.6% of erythromycin-resistant strains harbored the ermC gene and 16.9% harbored the ermB gene. A total of 35 different ribogroups was found among the isolates investigated; 83.3% of pig strains belonged to 1 cluster with a similarity coefficient of 0.84. In contrast, 3 main clusters were observed among 68.8% of milk strains, which indicates a high degree of host specificity.