Determination of antigenic specificity and relationship among Haemophilus pleuropneumoniae serotypes by an indirect hemagglutination test.

Serological properties of antigens extracted from strains of Haemophilus pleuropneumoniae belonging to seven different serotypes were investigated. Antisera were prepared in rabbits against Formalin-treated whole cell suspensions as well as autoclaved cell suspensions. Saline and heat extracts and their alcohol precipitate antigens of H. pleuropneumoniae were used in the indirect hemagglutination test. All the antigens used were easily adsorbed directly onto sheep erythrocytes. Saline extract antigen showed maximum type specificity. Heating of the whole cell suspension revealed the cross-reactive minor antigenic determinants. Thus, the heat extract preparations had both type-specific and species-specific antigens. It is suggested that the indirect hemagglutination test may be useful for both serotyping and serodiagnosis of H. pleuropneumoniae infections in pigs.     

Genome scale comparison of Mycobacterium avium subsp. paratuberculosis with Mycobacterium avium subsp. avium reveals potential diagnostic sequences

The genetic similarity between Mycobacterium avium subsp. paratuberculosis and other mycobacterial species has confounded the development of M. avium subsp. paratuberculosis-specific diagnostic reagents. Random shotgun sequencing of the M. avium subsp. paratuberculosis genome in our laboratories has shown >98% sequence identity with Mycobacterium avium subsp. avium in some regions. However, an in silico comparison of the largest annotated M. avium subsp. paratuberculosis contigs, totaling 2,658,271 bp, with the unfinished M. avium subsp. avium genome has revealed 27 predicted M. avium subsp. paratuberculosis coding sequences that do not align with M. avium subsp. avium sequences. BLASTP analysis of the 27 predicted coding sequences (genes) shows that 24 do not match sequences in public sequence databases, such as GenBank. These novel sequences were examined by PCR amplification with genomic DNA from eight mycobacterial species and ten independent isolates of M. avium subsp. paratuberculosis. From these analyses, 21 genes were found to be present in all M. avium subsp. paratuberculosis isolates and absent from all other mycobacterial species tested. One region of the M. avium subsp. paratuberculosis genome contains a cluster of eight genes, arranged in tandem, that is absent in other mycobacterial species. This region spans 4.4 kb and is separated from other predicted coding regions by 1,408 bp upstream and 1,092 bp downstream. The gene upstream of this eight-gene cluster has strong similarity to mycobacteriophage integrase sequences. The GC content of this 4.4-kb region is 66%, which is similar to the rest of the genome, indicating that this region was not horizontally acquired recently. Southern hybridization analysis confirmed that this gene cluster is present only in M. avium subsp. paratuberculosis. Collectively, these studies suggest that a genomics approach will help in identifying novel M. avium subsp. paratuberculosis genes as candidate diagnostic sequences.     

Multilocus short sequence repeat sequencing approach for differentiating among Mycobacterium avium subsp. paratuberculosis strains

We describe a multilocus short sequence repeat (MLSSR) sequencing approach for the genotyping of Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) strains. Preliminary analysis identified 185 mono-, di-, and trinucleotide repeat sequences dispersed throughout the M. paratuberculosis genome, of which 78 were perfect repeats. Comparative nucleotide sequencing of the 78 loci of six M. paratuberculosis isolates from different host species and geographic locations identified a subset of 11 polymorphic short sequence repeats (SSRs), with an average of 3.2 alleles per locus. Comparative sequencing of these 11 loci was used to genotype a collection of 33 M. paratuberculosis isolates representing different multiplex PCR for IS900 loci (MPIL) or amplified fragment length polymorphism (AFLP) types. The analysis differentiated the 33 M. paratuberculosis isolates into 20 distinct MLSSR types, consistent with geographic and epidemiologic correlates and with an index of discrimination of 0.96. MLSSR analysis was also clearly able to distinguish between sheep and cattle isolates of M. paratuberculosis and easily and reproducibly differentiated strains representing the predominant MPIL genotype (genotype A18) and AFLP genotypes (genotypes Z1 and Z2) of M. paratuberculosis described previously. Taken together, the results of our studies suggest that MLSSR sequencing enables facile and reproducible high-resolution subtyping of M. paratuberculosis isolates for molecular epidemiologic and population genetic analyses.     

DNA fingerprinting of Pasteurella multocida recovered from avian sources

Repetitive sequence-based PCR (rep-PCR) and amplified fragment length polymorphism (AFLP) were used to characterize a sample of 43 field isolates and 4 attenuated vaccine strains of Pasteurella multocida recovered from multiple avian species. Both rep-PCR and AFLP assays were rapid and reproducible, with high indices of discrimination. Concordance analyses of rep-PCR and AFLP with somatic serotyping indicate that, in general, somatic serotyping is a poor indicator of genetic relatedness among isolates of P. multocida. In addition, the data provide evidence of host specificity of P. multocida clones. Overall, the results of our study indicate that the rep-PCR and AFLP techniques enable rapid fingerprinting of P. multocida isolates from multiple avian species and enhance the investigation of fowl cholera outbreaks.     

Bactericidal activity of rabbit serum containing immunoconglutinin

When lymphocytes from hypersensitive animals are incubated with antigen, biologically active substances are formed which inhibit the migration of mesenchymal cells from normal animals.

These substances were tested by intradermal injection in guinea-pigs and rabbits. The supernatants from incubation of lymphocytes with a high dose of antigen caused immediate pallor which lasted several hours. Later there was a macroscopic inflammation maximal at 24 hours. The histology was characteristic of a delayed hypersensitivity reaction.

The injection of the supernatant from hypersensitive lymphocytes incubated with a small dose of antigen caused little or no pallor and was not followed by a delayed inflammatory reaction. Injection of this supernatant together with the antigen did not potentiate or alter the reaction, in contrast to in vitro experiments where the inhibition of the migration by this supernatant was potentiated by antigen.

Besides this factor a distinct factor occurs in extracts and supernatant fluids of lymphocytes cultivated without antigen and those from control animals, which increases vascular permeability. This substance is probably identical with the lymph node permeability factor (LNPF). The possible role of these biologically active substances in the mechanism of delayed type hypersensitivity is discussed.

     

Molecular epidemiology of Mycobacterium avium subsp. paratuberculosis isolates recovered from wild animal species

Mycobacterial isolates were obtained by radiometric culture from 33 different species of captive or free-ranging animals (n = 106) and environmental sources (n = 3) from six geographic zones within the United States. The identities of all 109 isolates were confirmed by using mycobactin J dependence and characterization of five well-defined molecular markers, including two integration loci of IS900 (loci L1 and L9), one Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis)-specific sequence (locus 251), and one M. avium subsp. avium-specific marker (IS1245), as well as hsp65 and IS1311 restriction endonuclease analyses. Seventy-six acid-fast isolates were identified as M. paratuberculosis, 15 were identified as belonging to the M. avium-M. intracellulare complex (but not M. paratuberculosis), and the remaining 18 were identified as mycobacteria outside the M. avium-M. intracellulare complex. Fingerprinting by multiplex PCR for IS900 integration loci clustered 67 of the 76 M. paratuberculosis strains into a single clade (designated clade A18) and had a Simpson's diversity index (D) of 0.53. In contrast, sequence-based characterization of a recently identified M. paratuberculosis short sequence repeat (SSR) region enabled the differentiation of the M. paratuberculosis isolates in clade A18 into seven distinct alleles (D = 0.75). The analysis revealed eight subtypes among the 33 species of animals, suggesting the interspecies transmission of specific strains. Taken together, the results of our analyses demonstrate that SSR analysis enables the genetic characterization of M. paratuberculosis isolates from different host species and provide evidence for the host specificity of some M. paratuberculosis strains as well as sharing of strains between wild and domesticated animal species.     

Sperm priming reveals specific autoreactive lymphocyte clones

Human spermatozoa from 18 donors were used to prime lymphocytes from 15 donors in a total of 60 different combinations. The sperm primed lymphocytes were tested in second culture against a total of 60 autologous and 180 allogeneic x-ray irradiated cells. We found that lymphocytes primed for 10–12 days in first culture with allogeneic spermatozoa showed highly significant responses in second culture to leukocytes autologous to the responder. The degree of autologous reactivity between specific subjects ranged from weak to significantly higher than the usual preferential primed lymphocyte typing reaction seen in a secondary mixed leukocyte culture reaction. These findings indicate that healthy human volunteers possess a set of self-reactive lymphocytes that can be detected by priming with allogeneic sperm in specific combinations.     

A Computer Reconstruction of the Entire Coronary Arterial Tree Based on Detailed Morphometric Data

A rigorous analysis of blood flow must be based on the branching pattern and vascular geometry of the full vascular circuit of interest. It is experimentally difficult to reconstruct the entire vascular circuit of any organ because of the enormity of the vessels. The objective of the present study was to develop a novel method for the reconstruction of the full coronary vascular tree from partial measurements. Our method includes the use of data on those parts of the tree that are measured to extrapolate the data on those parts that are missing. Specifically, a two-step approach was employed in the reconstruction of the entire coronary arterial tree down to the capillary level. Vessels > 40 μm were reconstructed from cast data while vessels < 40 μm were reconstructed from histological data. The cast data were reconstructed one-bifurcation at a time while histological data were reconstructed one-sub-tree at a time by “cutting” and “pasting” of data from measured to missing vessels. The reconstruction algorithm yielded a full arterial tree down to the first capillary bifurcation with 1.9, 2.04 and 1.15 million vessel segments for the right coronary artery (RCA), left anterior descending (LAD) and left circumflex (LCx) trees, respectively. The node-to-node connectivity along with the diameter and length of every vessel segment was determined. Once the full tree was reconstructed, we automated the assignment of order numbers, according to the diameter-defined Strahler system, to every vessel segment in the tree. Consequently, the diameters, lengths, number of vessels, segments-per-element ratio, connectivity and longitudinal matrices were determined for every order number. The present model establishes a morphological foundation for future analysis of blood flow in the coronary circulation.     

Koilocytosis preceding squamous cell carcinoma in situ of uterine cervix

Association of koilocytic changes with cervical squamous cell carcinoma is well documented. The studies of such concurrent association may miss those cases of papillomavirus infection where cytomorphologic expression of virus has disappeared by the time carcinoma appears. The authors studied cytologic material before the diagnosis was made of cervical squamous cell carcinoma in situ in 25 patients. Twenty-two (88%) of the 25 patients showed koilocytosis compared with only 6 out of 57 (10.5%) in the control group. The findings of this study support a possible predisposing role of papillomavirus in squamous cell carcinoma of the cervix.