Oral Phenotype and Salivary Microbiome of Individuals With Papillon-Lefèvre Syndrome.

Papillon-Lefèvre syndrome (PLS) is an autosomal recessive rare disease, main characteristics of which include palmoplantar hyperkeratosis and premature edentulism due to advanced periodontitis (formerly aggressive periodontitis). This study aimed to characterize the oral phenotype, including salivary parameters, and the salivary microbiome of three PLS sisters, comparatively. Two sisters were toothless (PLSTL1 and PLSTL2), and one sister had most of the teeth in the oral cavity (PLST). Total DNA was extracted from the unstimulated saliva, and the amplicon sequencing of the 16S rRNA gene fragment was performed in an Ion PGM platform. The amplicon sequence variants (ASVs) were obtained using the DADA2 pipeline, and the taxonomy was assigned using the SILVA v.138. The main phenotypic characteristics of PLS were bone loss and premature loss of primary and permanent dentition. The PLST sister presented advanced periodontitis with gingival bleeding and suppuration, corresponding to the advanced periodontitis as a manifestation of systemic disease, stage IV, grade C. All three PLS sisters presented hyposalivation as a possible secondary outcome of the syndrome. Interestingly, PLST salivary microbiota was dominated by the uncultured bacteria Bacterioidales (F0058), Fusobacterium, Treponema, and Sulfophobococcus (Archaea domain). Streptococcus, Haemophilus, and Caldivirga (Archaea) dominated the microbiome of the PLSTL1 sister, while the PLSTL2 had higher abundances of Lactobacillus and Porphyromonas. This study was the first to show a high abundance of organisms belonging to the Archaea domain comprising a core microbiome in human saliva. In conclusion, a PLST individual does have a microbiota different from that of the periodontitis' aggressiveness previously recognized. Due to an ineffective cathepsin C, the impairment of neutrophils probably provided a favorable environment for the PLS microbiome. The interactions of Bacteroidales F0058, Caldivirga, and Sulfophobococcus with the microbial consortium of PLS deserves future investigation. Traditional periodontal therapy is not efficient in PLS patients. Unraveling the PLS microbiome is essential in searching for appropriate treatment and avoiding early tooth loss.

The subgingival microbiota of Papillon-Lefèvre syndrome.

BACKGROUND: There is little information about the microbiologic profiles of periodontal lesions in Papillon-Lefèvre syndrome (PLS) and the significance of bacteria in the pathogenesis of periodontitis in these patients. This comprehensive analysis of the subgingival microbiota in patients with PLS used 16S ribosomal RNA (rRNA) clonal analysis and the 16S rRNA-based Human Oral Microbe Identification Microarray (HOMIM). METHODS: Thirteen patients with PLS from seven unrelated families volunteered for this microbiologic study. Subgingival plaque was collected with sterile paper points from multiple sites with ≥5 mm probing depth, and whole genomic DNA was extracted. The 16S rRNA genes were amplified, cloned, and sequenced. The samples were then probed for ≈300 predominant oral bacterial species using the HOMIM. RESULTS: The most commonly detected phylotypes in the clonal analysis were Gemella morbillorum, Gemella haemolysans, Granulicatella adiacens, Lachnospiraceae OT 100 (EI074), Parvimonas micra, Selenomonas noxia, and Veillonella parvula. As a group, streptococci were commonly detected in these individuals. In the HOMIM analysis, a total of 170 bacterial species/phylotypes were detected, with a range of 40 to 80 species per patient with PLS. Of these, 12 bacterial species were detected in medium to high levels in ≥50% of the individuals. The high-frequency strains were clustered into eight groups: Aggregatibacter actinomycetemcomitans, Campylobacter spp., Capnocytophaga granulosa, G. morbillorum, P. micra, Porphyromonas endodontalis, Streptococcus spp., and Tannerella forsythia. CONCLUSIONS: The subgingival microbiota in PLS is diverse. Periodontal pathogens commonly associated with chronic and aggressive periodontitis and opportunistic pathogens may be associated with the development of severe periodontitis in patients with PLS.

Distribution of Bacteroides forsythus genotypes in a Japanese periodontitis population.

Bacteroides forsythus is an important pathogen in periodontal diseases and has been associated with advanced and refractory periodontitis. The difficulties associated with culturing this species have meant that the distribution and pathogenic mechanisms of B. forsythus remain unclear. In this study, the arbitrarily primed polymerase chain reaction (AP-PCR) method was used to investigate the genotype distribution of B. forsythus in a Japanese periodontitis population, as well as the relationship between AP-PCR genotypes and periodontal status. B. forsythus reference strain, ATCC 43037T and 137 clinical bacterial isolates from 64 subjects were separated into 11 distinct AP-PCR genotypes using a single randomly-sequenced primer, 5'-CCGGCGGCG-3' (A-05). The majority (80.9%) of B. forsythus strains examined belonged to AP-PCR genotypes I, II, III and IV (accounting for 39.7%, 20.6%, 10.3% and 10.3%, respectively). Types I and III primarily consisted of isolates from chronic periodontitis subjects (80.8% and 85.7%, respectively), while Types II and IV consisted mainly of isolates from aggressive periodontitis subjects (85.7% and 100%, respectively). Except for three subjects who harbored two different B. forsythus genotypes in the oral cavity, all subjects only infected with one genotype intraindividually. These results demonstrate that the AP-PCR method is useful for genotypic analysis of B. forsythus. This species showed a genetic diversity among the investigated population. A clonal nature of B. forsythus infection is suggested. Furthermore, different AP-PCR genotypes of B. forsythus appear to be associated with different types of periodontitis.

A microbiological study of Papillon-Lefévre syndrome in two patients.

AIM: To analyse the microflora of subgingival plaque from patients with Papillon-Lefévre syndrome (PLS), which is a very rare disease characterised by palmar-plantar hyperkeratosis with precocious periodontal destruction. METHODS: Bacterial isolates were identified using a combination of commercial identification kits, traditional laboratory tests, and gas liquid chromatography. Some isolates were also subjected to partial 16S rDNA sequencing. Plaque samples were also assayed for the presence of Porphyromonas gingivalis, Prevotella intermedia, and Actinobacillus actinomycetemcomitans in a quantitative enzyme linked immunosorbent assay (ELISA) using monoclonal antibodies. RESULTS: The culture results showed that most isolates were capnophilic and facultatively anaerobic species-mainly Capnocytophaga spp and Streptococcus spp. The latter included S. constellatus, S. oralis, and S. sanguis. Other facultative bacteria belonged to the genera gemella, kingella, leuconostoc, and stomatococcus. The aerobic bacteria isolated were species of neisseria and bacillus. Anaerobic species included Prevotella intermedia, P. melaninogenica, and P. nigrescens, as well as Peptostreptococcus spp. ELISA detected P gingivalis in one patient in all sites sampled, whereas A. actinomycetemcomitans was detected in only one site from the other patient. Prevotella intermedia was present in low numbers. CONCLUSIONS: Patients with PLS have a very complex subgingival flora including recognised periodontal pathogens. However, no particular periodontopathogen is invariably associated with PLS.

Papillon-Lefèvre syndrome: serum immunoglobulin G (IgG) subclass antibody response to periodontopathic bacteria. A case report.

BACKGROUND: Papillon-Lefevre syndrome (PLS) is a rare autosomal recessive disorder which is characterized by palmar-plantar hyperkeratosis and rapid periodontal destruction of both primary and permanent dentitions. In this case report, we present clinical features, and microbiological and immunological findings of 40 month-old Thai male PLS patient. METHODS: Microbiological examinations consisted of bacterial culture methods utilizing selective media, morphological identification, and biochemical tests. In addition, the specific serum IgG subclass antibody titers reactive with etiologic periodontal bacteria were determined by the dot-blot immunological analysis and enzyme linked immunosorbent assay (ELISA). RESULTS: The examinations revealed that the patient harbored 3 major suspected periodontopathic microorganisms, A. actinomycetemcomitans, P. gingivalis, and P. intermedia. The patient's serum IgG1, IgG2, and IgG3, but not IgG4, titers against A. actinomycetemcomitans were dramatically increased. The predominant IgG subclass was IgG1. In contrast, the IgG titers against other tested bacteria, P. gingivalis, P. intermedia, and F. nucleatum, appeared to be similar to those of a healthy control. CONCLUSIONS: A. actinomycetemcomitans seems to play a pivotal role in the bacteria-host interaction in PLS periodontal pathogenesis. Response of the specific serum IgG subclass antibody titers against the A. actinomycetemcomitans antigen has been demonstrated. This association warrants further investigation.

Combined systemic and local antimicrobial therapy of periodontal disease in Papillon-Lefèvre syndrome. A report of 4 cases.

4 patients, 2 pairs of siblings, suffering from Papillon-Lefèvre syndrome were treated for periodontal disease. Following extraction of hopeless teeth, the children received scaling and adjunctive systemic antibiotics (metronidazole and amoxicillin for 7 to 10 days). In addition, they performed supragingival pulsated jet irrigation with 0.06% chlorhexidine digluconate 1 x daily. In 2 siblings, A. actinomycetemcomitans was suppressed subgingivally below detectable levels, pocket probing depths were reduced to 4 mm or less, and plaque and bleeding indices were low. No further disease progression was seen over a 3-year-period. Another female patient also showed clinical improvement and suppression of subgingival A. actinomycetemcomitans and B. forsythus up to the 9-month-follow-up, while her sister showed further attachment loss over the course of 4 years. The present case reports indicated that in some patients suffering from Papillon-Lefèvre syndrome periodontal disease may be arrested by means of (i) oral hygiene instruction, (ii) extraction of severely diseased teeth, (iii) scaling, (iv) systemic antibiotics and (v) long-term antimicrobial irrigation.

Microbiological features of Papillon-Lefèvre syndrome periodontitis.

Papillon-Lefevre syndrome patients exhibit hyperkeratosis palmo-plantaris and severe periodontitis. The syndrome is an autosomal recessive trait, but the mechanism of periodontal destruction is not known. This report presents the clinical and microbiological features of an 11-year old girl with Papillon-Lefèvre syndrome. Clinical examination included conventional periodontal measurements and radiographic analysis. In samples from 3 deep periodontal lesions, the occurrence of major suspected periodontopathic bacteria was determined by selective and non-selective culture and polymerase chain reaction (PCR) identification, and the presence of cytomegalovirus and Epstein-Barr type 1 virus by a nested-PCR detection method. 10 of 22 available teeth demonstrated severe periodontal breakdown. Major cultivable bacteria included Actinobacillus actinomycetemcomitans (3.4% of total isolates), Prevotella nigrescens (16.4%), Fusobacterium nucleatum (14.3%) and Peptostreptococcus micros (10.6%). A. actinomycetemcomitans, P. nigrescens, Porphyromonas gingivalis and Eikenella corrodens were identified by PCR analysis. The patient's non-affected parents and older brother revealed several periodontal pathogens but not A. actinomycetemcomitans. The viral examination demonstrated cytomegalovirus and Epstein-Barr type 1 virus in the subgingival sample of the Papillon-Lefèvre syndrome patient. The father and brother yielded subgingival cytomegalovirus but not Epstein-Barr type 1 virus. We hypothesize that human herpesviruses in concert with A. actinomycetemcomitans play important rôles in the development of Papillon-Lefèvre syndrome periodontitis.

Subgingival microbial profile of Papillon-Lefèvre patients assessed by DNA-probes.

The prevalence of 18 selected bacterial species was assessed by means of "checkerboard" DNA-DNA hybridisation in a group of 12 Saudi-Arabian adolescents with Papillon-Lefèvre syndrome. A total of 36 tooth sites were investigated. The patients exhibited severe periodontal disease with deep pockets. All 12 patients harboured the putative bacterial pathogens P. intermedia, F. nucleatum, P. micros and S. intermedius while T. denticola, B. forsythus, P. nigrescens, E. corrodens, S. noxia and C. rectus were recovered from 11 patients. P. gingivalis was recovered from 9 patients and 18 sites while corresponding figures for A. actinomycetemcomitans were 8 and 19, respectively. A number of the investigated species (B. forsythus, T. denticola, P. intermedia, C rectus) reached high levels (> or =10(6) cells) in more than 1/2 of the patients. On the other hand, bacteria such as A. actinomycetemcomitans and P. gingivalis were infrequently encountered at high levels in these subgingival samples. In conclusion, the analysis failed to demonstrate a PLS-specific profile of the subgingival infection, since the bacterial composition of the sampled sites closely resembled that characterising deep pockets in adult periodontitis patients.

Palmar plantar keratosis and unusual periodontal findings. Observations from a family of 4 members.

The connection between palmar plantar hyperkeratosis and severe periodontal disease was first reported in 1924 by Papillon and Lefevre. The 2 major components of this syndrome (PLS) can also occur as distinct entities. The literature describes a number of cases which do not fit the classical disease descriptions. In this paper, we report on a family with an atypical PLS. The father had marked palmar plantar hyperkeratosis but a very late onset of destructive marginal periodontitis. The son also had palmar plantar hyperkeratosis, but despite the fact that he initially harboured Actinobacillus actinomycetemcomitans, Prevotella intermedia and Porphyromonas gingivalis, he did not develop periodontal disease over a seven-year observation period when improved oral hygiene and professional tooth cleaning were instituted.

Follow-up of two cases of Papillon-Lefèvre syndrome and presentation of two new cases.

Periodontitis resulting from Papillon-Lefèvre Syndrome has been known to cause early loss of primary dentition with subsequent involvement of the permanent dentition. In this study, two Papillon-Lefèvre Syndrome patients were followed for 3 years after initial treatment and improvement of their periodontal condition. In addition, two new cases of Papillon-Lefèvre Syndrome are presented. The follow-up treatment of the first two patients included monitoring the oral hygiene and performing ultrasonic scaling. Their present clinical appearance is very satisfactory. The periodontal condition of the third (new) patient was brought under control by extracting the involved deciduous teeth under antibiotic coverage, and by scaling and root planing the already erupted permanent teeth as well as by maintaining a high standard of oral hygiene. In the fourth case, all permanent teeth had erupted and the periodontium had already been severely destroyed. Actinobacillus actinomycetemcomitans was not detected by microbiologic examination after the periodontal conditions improved, except in the fourth case. Western blot analysis showed that the three first three patients had positive antibody response to the same antigens of Actinobacillus actinomycetemcomitans. Phagocytosis by polymorphonuclear neutrophil leukocytes) had not decreased, but the expression of surface receptors of polymorphonuclear neutrophil leukocytes was within the normal limits.

Genetic studies of syndromes with severe periodontitis and palmoplantar hyperkeratosis.

The Papillon-Lefèvre and Haim Munk syndromes are characterized by the presence of both palmoplantar hyperkeratosis (PPK) and severe early onset periodontitis. It is the early onset periodontal disease component that distinguishes these from other more common forms of PPK. It has been proposed that the periodontal disease component may be a casual association in individuals with PPK. Genetic syndromes with palmoplantar keratosis and severe ealry onset periodontitis may be due to specific bacterial infections in individuals with PPK. Recently, keratin gene mutations have been identified in several conditions typified by palmoplantar keratosis. The present study sought to test the hypothesis that a keratin gene defect similar to those previously identified in other PPK conditions is responsible for the Haim Munk and the Papillon. Lefèvre syndromes. We have performed genetic linkage studies to test for linkage between polymorphic DNA loci within 2 cytokeratin gene families and the disease phenotype in Haim Munk syndrome and Papillon-Lefèvre syndrome. Families with individuals segregating for the Haim Munk syndrome and the Papillon-Lefèvre syndrome were examined to determine disease status, and genotyped for microsatellite DNA markers closely linked to the acidic (type I) and the basic (type II) cytokeratin genes on chromosomes 12 and 17. Genotype data were evaluated for microsatellite allele homozygosity in affected individuals. Results of these preliminary genetic studies suggest that the gene defect in Haim Munk syndrome is not due to a gene defect in either the type I or the type II keratin gene clusters. These findings suggest that Haim Munk syndrome may be genetically distinct from other more common forms of PPK that have been linked to the cytokeratin gene families, and suggest that mutations in genes other than keratin genes are responsible. Additional family studies are needed to confirm these preliminary findings.

Microbiological and serological investigations of oral lesions in Papillon-Lefèvre syndrome.

Microbiological and serological (enzyme linked immunosorbent assay) investigations were carried out, including karyotyping, on two Asian children with Papillon-Lefèvre syndrome. In case 1, a girl aged four years, the most prevalent putative periodontopathogens were Eikenella corrodens, Fusobacterium nucleatum, Porphyromonas gingivalis, Prevotella intermedia (deciduous dentition) and Bacteroides gracilis, E corrodens and F nucleatum (permanent dentition). In case 2, a boy aged nine years, they were F nucleatum, P intermedia and P loeschii and E corrodens. Serum from case 2 showed a raised specific IgG antibody response to Actinomyces actino-mycetemcomitans serotype b. Thus, a wider range of species than hitherto reported may be associated with Papillon-Lefèvre syndrome, including A actino-mycetemcomitans and F nucleatum.

Occurrence of Actinobacillus actinomycetemcomitans and anti-leukotoxin antibodies in some members of an extended family affected by Papillon-Lefèvre syndrome.

Eighteen (18) members of an extended family in which numerous individuals have Papillon-Lefèvre syndrome (PLS) were examined. In all, 6 affected members and 12 non-affected members were included. All patients underwent a clinical examination which, in the dentate persons, included plaque index, bleeding on probing, probing depth, and periodontal attachment loss and a set of full mouth periapical x-rays. Subgingival bacterial samples were also collected from 2 teeth in the dentate patients for cultures and identification of Actinobacillus actinomycetemcomitans. Serum samples were collected from all participants and assayed for antileukotoxin antibodies. The results indicate that there is a high prevalence of leukotoxic strains of A. actinomycetemcomitans in persons suffering from PLS, as well as in unaffected family members. The ubiquitous presence of A. actinomycetemcomitans in the family units suggests a close association between A. actinomycetemcomitans and the periodontal disease associated with the syndrome; it also suggests that A. actinomycetemcomitans by itself is not sufficient for the expression of periodontal disease and that other factors, some of which must be genetic, are necessary for lesion development.

Clinical, bacteriological, and immunological examinations and the treatment process of two Papillon-Lefèvre syndrome patients.

Papillon-Lefèvre Syndrome (PLS) is a rare disease associated with the early onset of periodontal breakdown in deciduous and permanent dentition. The etiology of the destruction has not been completely clarified. Two female patients (ages 4 and 7 years) with severe destruction of the periodontal structures were examined. Except for palmar and plantar hyperkeratosis, dermatologic examination revealed no other medical disorders. On immunological analysis, measurement of serum antibody titers to 7 periodontopathic bacteria including Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans was performed by enzyme-linked immunosorbent assay (ELISA). Further immunoblot analysis of A. actinomycetemcomitans and microbial culture of samples collected from deep periodontal pockets and mouthrinse solution were performed. The serum of the two patients showed high IgG titer against A. actinomycetemcomitans. Immunoblot results of the two patients against sonicated extract of A. actinomycetemcomitans Y4 strain exhibited a similar pattern. The band pattern differed from that observed in other forms of early onset periodontitis patients or periodontally healthy subjects. Moreover, A. actinomycetemcomitans colonies were cultured in high percentages from the pocket samples. Antibiotic therapy was instituted in addition to conventional periodontal therapy. In the younger patient, all deciduous teeth were extracted as part of the treatment and A. actinomycetemcomitans was no longer detected. All four permanent first molars and 8 permanent incisors subsequently erupted with healthy periodontium. However, the older patient did not improve after periodontal and antibiotic (minocycline and erythromycin) treatments and A. actinomycetemcomitans was consistently detected. Ofloxacin medication finally eliminated A. actinomycetemcomitans from the periodontal pockets. This antibiotic was also associated with reduced gingival inflammation and probing depth.(ABSTRACT TRUNCATED AT 250 WORDS)

Late onset Papillon-Lefèvre syndrome? A chromosomic, neutrophil function and microbiological study.

We had the opportunity to study a family with one of the most destructive forms of periodontal disease known, the Papillon-Lefèvre syndrome. The parents had no consanguinity and were not affected, and were therefore to be considered carriers of the disease. 2 sisters, the eldest and youngest, showed periodontal breakdown and hyperkeratotic skin lesions, but their deciduous dentition was not affected. 2 brothers had skin lesions only and another brother and sister were healthy. Furthermore, 2 babies died at birth one after a 9-month pregnancy and the other after a 6-month pregnancy, and the mother also suffered 3 miscarriages. For 4 years, we studied the family: in the case of both sisters, mechanical periodontal treatment and antibiotics were unable to control the disease. In the chromosomic study of the 2 sisters affected, the GTG banding technique found no trace of anomalies in the cells analyzed, whose chromosomic formation was 46,XX. Before treatment, the chemotaxis of the PMN, the phagocytosis of opsonized Staphylococcus aureus, and production of superoxide radicals by PMN was significantly impaired in both sisters. Despite scaling and root planing, the periodontal lesions still progressed, but the PMN functions evaluated were now normal in both sisters. An orally asymptomatic but dermatologically affected brother showed no significant defect in the phagocytic activity and the production of superoxide radicals.

Papillon Lefevre syndrome: treatment of two cases with a clinical microbiological and histopathological investigation.

Papillon Lefevre syndrome is presented in the cases of two female patients of the ages of 7 and 9, who exhibited all typical symptoms of the disease. Microbiological and histopathological studies were done and treatment provided. Actinobacillus actinomycetemcomitans, which is suspected as a pathogenic factor in the disease was identified as well as some other gram negative microorganisms and an antibiogram was performed in which amoxycillin plus clavulanic acid was most effective. Histopathological investigation also confirmed the presence of gram negative bacteria. Granular cell infiltration was predominant in the surface epithelium. Prosthetic appliances were provided for the patients after mechanical and chemical plaque control. In addition to this, antibiotics (amoxycillin plus clavulanic acid) were prescribed every six months. No tooth loss was observed in both patients after more than two years follow-up period. At the moment only one patient is under review and because she is uncooperative, mild periodontal inflammation is still present around the teeth which erupted before the antibiotic regime, but not in the other teeth.

[Clinical, bacteriological, and immunological examination and treatment of two Papillon-Lefevre syndrome patients].

Papillon-Lefèvre Syndrome (PLS) is a rare disease accompanied by palmo-plantar hyperkeratosis and rapidly progressive periodontal breakdown of deciduous and permanent dentition. Two unrelated female PLS patients, four and seven years old, with severe periodontal destruction were examined. Antibody titers against seven strains by the enzyme-linked immunosorbent assay (ELISA), microbial cultures from deep periodontal pockets and mouth rinse samples and immunoblotting analysis were performed. Titers against Actinobacillus actinomycetemcomitans (A.a.) were found to be high by the ELISA test. Microbial cultures of A. a. were found in high percentage and immunoblotting results against sonicated extracts of an A. a. Y4 strain had similar patterns. All deciduous teeth were extracted from the younger patient, later permanent dentition erupted uneventfully and A. a. colonies could not be detected. However, the older patient did not exhibit improvement with conventional periodontal therapy and antibiotic (minocycline/erythromycin) treatment and A. a. colonies could be consistently cultured. After a subsequent ofloxacin medication, A. a. colony detection was suppressed. Furthermore, a reduction of gingival inflammation and pocket depth were observed. Therefore, A. a. was associated as an important pathogen in the etiology of periodontal disease in these PLS patients.

Periodontitis associated with Papillon-Lefèvre syndrome.

The predominant subgingival microflora, host immune response, and genetic history of a 14-year-old girl with Papillon-Lefèvre Syndrome (PLS) are reported. The patient had high counts of Actinobacillus actinomycetemcomitans and surface translocating bacteria. She had significantly raised levels of antibodies to five of the bacterial species studied with the levels to A. actinomycetemcomitans remaining high after antibiotic therapy. The polymorphonuclear leukocytes (PMN) also released significantly increased amounts of O2 compared to controls. The data presented support a role for A. actinomycetemcomitans and PMN dysfunction in the pathogenesis of PLS.

Outer membrane proteins of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus studied by SDS-PAGE and immunoblotting.

This investigation characterized and compared outer membrane proteins (OMP) of the closely related Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus by means of SDS-PAGE patterns and reactions on immunoblots with rabbit antiserum against A. actinomycetemcomitans FDC Y4. Reactions with serum from a patient with Papillon Lefévre syndrome (PLS), from whom periodontal wild strains of A. actinomycetemcomitans had been isolated, were also studied. OMP were purified with selective solubilization from lyophilized cells of 10 wild and 4 reference strains of A. actinomycetemcomitans and 4 reference strains of H. aphrophilus. OMP profiles from wild and reference strains of A. actinomycetemcomitans were similar while those from A. actinomycetemcomitans and H. aphrophilus differed. The most prominent difference was absence of a heat modifiable protein in H. aphrophilus strains. Immunoblotting revealed strong common antigens in most strains, including a heat modifiable protein with mol wt 34 kDa, as well as a 29 kDa and a 16.5 kDa protein. Treatment with pronase and sodium periodate confirmed the protein nature of the major OMP antigens.