Chronic Oral Inoculation of Porphyromonas gingivalis and Treponema denticola Induce Different Brain Pathologies in a Mouse Model of Alzheimer Disease.

Periodontitis is a chronic inflammatory disease driven by dysbiosis in subgingival microbial communities leading to increased abundance of a limited number of pathobionts, including Porphyromonas gingivalis and Treponema denticola. Oral health, particularly periodontitis, is a modifiable risk factor for Alzheimer disease (AD) pathogenesis, with components of both these bacteria identified in postmortem brains of persons with AD. Repeated oral inoculation of mice with P. gingivalis results in brain infiltration of bacterial products, increased inflammation, and induction of AD-like biomarkers. P. gingivalis displays synergistic virulence with T. denticola during periodontitis. The aim of the current study was to determine the ability of P. gingivalis and T. denticola, grown in physiologically relevant conditions, individually and in combination, to induce AD-like pathology following chronic oral inoculation of female mice over 12 weeks. P. gingivalis alone significantly increased all 7 brain pathologies examined: neuronal damage, activation of astrocytes and microglia, expression of inflammatory cytokines interleukin 1ß (IL-1ß) and interleukin 6 and production of amyloid-ß plaques and hyperphosphorylated tau, in the hippocampus, cortex and midbrain, compared to control mice. T. denticola alone significantly increased neuronal damage, activation of astrocytes and microglia, and expression of IL-1ß, in the hippocampus, cortex and midbrain, compared to control mice. Coinoculation of P. gingivalis with T. denticola significantly increased activation of astrocytes and microglia in the hippocampus, cortex and midbrain, and increased production of hyperphosphorylated tau and IL-1ß in the hippocampus only. The host brain response elicited by oral coinoculation was less than that elicited by each bacterium, suggesting coinoculation was less pathogenic.

Pathology and bacteria related to digital dermatitis in dairy cattle in all year round grazing system in Brazil.

Digital dermatitis (DD) is one of the main causes of lameness in dairy cattle worldwide, and it is frequently reported in high-yielding, free stall dairy herds from regions with a temperate climate. However, DD is also observed with high prevalence in grazing cattle with a low milk yield in tropical regions. To clarify whether these differences have an impact on the etiology of the disease, we studied DD lesions from all year round grazing cattle of mixed breed in Brazil using high-throughput 16S rRNA gene sequencing and fluorescent in situ hybridization. The study included samples from 66 skin lesions and 5 healthy skins collected from five farms. Both techniques showed Treponema spp. to be the most abundant bacteria, present in all but one of the samples with minimal epidermal alterations. We identified eleven different Treponema strains belonging to the six major phylotypes of Treponema which have all previously been identified in DD lesions. Furthermore, we identify Dichelobacter nodosus in DD lesions by gene sequencing and also by fluorescent in situ hybridization in almost half of biopsy specimens in areas with mild epithelial damage and together with Treponema. The present data support the hypothesis that Treponema constitutes the main pathogen responsible for DD, independent of the environment and region where cows are kept, and it further suggests D. nodosus as another potentially important pathogen.

Bovine digital dermatitis: Current concepts from laboratory to farm.

Bovine digital dermatitis (DD) is a severe infectious disease causing lameness in dairy cattle worldwide and is an important ruminant welfare problem that has considerable economic issues. Bovine DD is endemic in many regions worldwide and it is important to understand this major disease so that effective control strategies can be identified. There is substantial evidence that specific treponeme phylotypes play an important causative role in bovine DD. This review considers current research, including DD Treponema spp. investigations, associated DD pathobiology, and current and potential treatment and control options. Epidemiological data, alongside new microbiological data, help delineate important transmission routes and reservoirs of infection that allow effective interventions to be identified. Better on-farm housing hygiene, pasture access, routine footbathing and claw trimming with disinfected equipment need to be implemented to significantly reduce the incidence of DD. There is a paucity of peer reviewed research into both commonly used and novel treatments. In vitro antimicrobial susceptibility studies of DD treponemes and effective treatment of human treponematoses clearly indicate that antibiotics frequently selected for DD treatments are not the most efficacious. Whilst there are understandable concerns over milk withdrawal times in dairy cattle, more needs to be done to identify, license and implement more appropriate antibiotic treatments, since continued overuse of less efficacious antibiotics, applied incorrectly, will lead to increased disease recurrence and transmission. More research is needed into methods of preventing DD that circumvent the use of antibiotics, including vaccination and transmission blocking studies, to reduce or hopefully eradicate DD in the future.

Histopathological Characterization of the Lesions of Contagious Ovine Digital Dermatitis and Immunolabelling of Treponema-like Organisms.

Contagious ovine digital dermatitis (CODD) is a cause of severe lameness in sheep and the three Treponema phylogroups Treponema medium/Treponema vincentii-like, Treponema phagedenis-like and Treponema pedis have been associated with clinical disease. The aims of this study were: (1) to describe the histopathological changes associated with each previously established grade of clinical lesion, and (2) to investigate immunohistochemically the association of the Treponema-like organisms with the observed histopathological changes. Early lesions were characterized by lymphoplasmacytic infiltration of the distal digital skin, with suppurative coronitis and intracorneal pustules. In more advanced stages of the disease there was complete separation of the dorsal wall of the hoof with a necrotizing and fibrinosuppurative exudate and dermatitis. The later lesions were mostly resolved, but with milder suppurative changes remaining within the cornified layer and periosteal reaction of the dorsal aspect of the distal phalanx. Large numbers of Treponema-like organisms were identified within early grade lesions (as well as later, more advanced grade lesions) and were specifically associated with the observed histopathological changes. The results of this study provide some evidence in support of the hypothesis that the three CODD-associated Treponema phylogroups are involved in the aetiopathogenesis of this disease.

Mechanisms of IL-8 suppression by Treponema denticola in gingival epithelial cells.

The purpose of this study was to investigate the mechanism(s) of interleukin (IL)-8 suppression by Treponema denticola, one of the major periodontal pathogens, in gingival epithelial cells. Immortalized human gingival epithelial HOK-16B cells were infected with wild-type (WT), dentilisin-deficient (K1) or flagellin-deficient (flgE) T. denticola in the presence or absence of 2% human serum for 24 h. The levels of IL-8 expression were measured with real-time reverse transcription PCR and ELISA. In the absence of human serum, the WT and flgE, but not K1, substantially reduced not only the levels of IL-8 protein but also of IL-8 mRNA. Such downregulation of IL-8 mRNA was independent of bacterial invasion. Degradation of cytokine mixture by the WT, K1 and flgE revealed dentilisin-dependent preferential degradation of tumor necrosis factor (TNF)-α, an IL-8-inducing cytokine. WT and flgE significantly decreased the levels of TNFα secreted by HOK-16B cells, suggesting modulation of IL-8 through dentilisin-mediated degradation of TNFα. The addition of human serum to the culture potentiated the suppressive effect of T. denticola, resulting in substantial reductions of IL-8 and TNFα levels, even by K1. The serum-dependent effects of T. denticola were attributed to its ability to suppress the accumulation of intracellular reactive-oxygen species (ROS), a group of ubiquitous signaling molecules. Pretreatment with an antioxidant suppressed TNFα-induced IL-8 expression, confirming the role of ROS in TNFα signaling. Collectively, T. denticola targeted a key inflammatory cytokine and its signaling molecule to modulate the host innate immune response, which provides a new insight into modulation of host immunity by a periodontal pathogen.

Efficacy of salicylic acid in the treatment of digital dermatitis in dairy cattle.

Digital dermatitis (DD) is one of the most important causes of lameness in dairy cattle worldwide. The objective of this study was to evaluate the efficacy of salicylic acid in the treatment of the disease. A total of 201 DD lesions from 173 cows from four commercial dairy herds were evaluated at day 0 during routine hoof trimming and were allocated into two groups, namely, a control group given chlortetracycline spray, and a treatment group given 10 g of salicylic acid powder applied topically within a bandage. Pain, lesion size and clinical appearance (scored M0 to M4) were evaluated on days 3, 14 and 34 post-treatment. A change to M0 was defined as healing, while changes of M2 or M4 to M1 or M3 were classified as clinical improvements. Healing rates did not differ significantly between treatment groups at days 3 and 14. By day 34 the healing rate was fivefold better (P=0.01) for the treatment vs. the control group, with healing rates of 13.6% and 3.1%, respectively. By day 3, the rate of improvement was 2.5-fold better (P=0.02) for the controls. By day 34 the overall positive effect (i.e. healing and improvement) was 1.75-fold better (P=0.05) for the treatment group. Lesions from the control group were 2.2 times more likely (P=0.09) to have a pain score equal to 2 by day 14. The proportion of lesions getting smaller by days 14 and 34 was 2.5 times higher (P<0.08) for the treatment vs. the control group. The findings suggest salicylic acid should be considered as an alternative to chlortetracycline for the treatment of DD as it appears more efficacious and would assist in reducing antibiotic use.

Growth curves and morphology of three Treponema subtypes isolated from digital dermatitis in cattle.

Digital dermatitis (DD) is an infectious claw disease of cattle that causes painful lesions, principally along the coronary band of the claws. In the US alone, the estimated economic impact of DD is estimated to be $190 million. The etiology of DD remains unclear and there is no reliable laboratory test, so DD is most often diagnosed clinically. Spirochetal bacteria of the genera Treponema have been implicated in DD infections following their isolation using culture techniques, serological detection of bovine antibodies against treponemes, and amplification of treponemal 16s DNA sequences by PCR. During in vitro growth of spirochetes and treponemes isolated from DD, morphological changes have been observed indicating the presence of a spiral form and an encysted form. It is not known why encysted forms appear or what role they have in the progression of DD. The current study established growth curves for three subtypes of treponemes, Treponema denticola-like, Treponema phagedenis-like, and Treponema medium-like, while photographically monitoring changes in morphology. In addition to observing spiral and encysted forms, two intermediate forms were also observed. These appeared as either spiral forms with spherical bodies or as enveloped clusters of granules. The observation of encysted forms adds further support to the theory that treponemes causing recurrent infections deep in bovine skin have mechanisms to facilitate persistence and the chronic character of DD.

An experimental infection model to induce digital dermatitis infection in cattle.

Bovine digital dermatitis (DD), also known as papillomatous digital dermatitis (foot warts), has been recognized as a major cause of lameness in cattle, with important economic and welfare consequences. The evaluation of therapeutic and preventive interventions aiming to control DD infections in dairy cattle is often challenged by the complex multifactorial etiology of the disease. An experimental infection model to induce acute DD lesions in a controlled environment is proposed. The goal was to provide a standard way of reproducing DD infections independent of external factors that could confound the natural course of the disease, such as management practices or infection pressure, resulting in transmission of DD between animals. A group of 4 yearling Holstein heifers free of any clinical evidence of hoof disease was recruited from a commercial dairy farm and housed in an experimental facility in 1 pen with slatted flooring. The hind feet were wrapped to mimic conditions of prolonged moisture (maceration) and reduced access to air (closure) and inoculated at the heel and dewclaw areas with a homogenate of a naturally occurring DD lesion skin biopsy or a culture broth of Treponema spp. After a period of 12 to 25 d, 4 of 6 and 1 of 4 dewclaw areas inoculated with biopsied DD lesion or a Treponema spp. culture, respectively, had gross lesions compatible with DD. Histopathology confirmed the gross diagnosis in the sites inoculated with tissue homogenate. In the site inoculated with Treponema spp. culture broth, histopathology revealed an incipient DD lesion. Treponema spp. were detected by PCR in both naturally occurring DD homogenate and Treponema spp. culture broth inoculation sites. An experimental infection model to induce acute DD in cattle was developed, which may be used to evaluate interventions to control DD and study the pathogenesis of this infectious hoof disease in a controlled manner.

Host and environmental reservoirs of infection for bovine digital dermatitis treponemes.

Bovine digital dermatitis (BDD) is a global infectious disease causing lameness of cattle and is responsible for substantial animal welfare issues and economic losses. The causative agents are considered to be spirochetal bacteria belonging to the genus Treponema, which have consistently been identified in BDD lesions worldwide. One potential means of controlling infection is the disruption of transmission; however, the infection reservoirs and transmission routes of BDD treponemes have yet to be elucidated. To address these issues, we surveyed for evidence of BDD treponeme presence in the dairy farm environment, in bovine tissues and in bovine gastrointestinal (GI) tract contents. A total of 368 samples were tested using PCR assays specific for each of three currently recognised, isolated phylotypes of BDD treponemes. All environmental samples, together with insects and GI tract content samples were negative for BDD treponeme DNA from the three phylotypes. However, we identified BDD treponemes in two non-pedal bovine regions: the oral cavity (14.3% of cattle tested) and the rectum (14.8% of cattle tested). Whilst only single phylotypes were detected in the oral cavity, two of the rectal tissues yielded DNA from more than one phylotype, with one sample yielding all three BDD treponeme phylotypes. Whilst it might be considered that direct skin to skin contact may be a major transmission route of BDD treponemes, further studies are required to characterise and determine the potential contribution of oral and rectal carriage to BDD transmission.

Treponema pallidum distribution patterns in mucocutaneous lesions of primary and secondary syphilis: an immunohistochemical and ultrastructural study.

To study the different patterns of Treponema pallidum distribution in primary and secondary syphilis, 34 biopsy specimens of 8 patients with primary and 26 with secondary syphilis were assessed. Histopathological features, silver stain, and immunohistochemical T pallidum polyclonal antibody expression were investigated. The number and distribution of spirochetes were evaluated, and ultrastructural studies were performed. Spirochetes were identified with Warthin-Starry stain in 17 specimens (4/8 primary and 13/26 secondary syphilis), whereas immunohistochemical analysis disclosed spirochetes in 29 (8/8 primary and 21/26 secondary syphilis). In secondary syphilis, an epitheliotropic pattern characterized by abundant spirochetes in the lower mucosa/epidermis in an intercellular distribution was observed. In contrast, primary syphilis exhibited a mixed epitheliotropic and vasculotropic pattern further manifested by treponemes surrounding the vascular walls. These differences were statistically significant. Ultrastructural examination confirmed these results. Immunohistochemistry shows greater sensitivity when compared with Warthin-Starry staining. The immunohistochemical pattern of T pallidum distribution may permit the diagnostic differentiation of primary from secondary syphilis.

Nodal inflammatory pseudotumor caused by luetic infection.

Inflammatory pseudotumor of lymph nodes (IPT-LN) represents an unusual cause of lymphadenitis of unknown etiology. Upon the observation of a case of IPT-LN associated with Treponema pallidum (Tp) infection, we analyzed a series of 9 IPT-LN and 9 extranodal IPT (spleen, 4 cases; lung, orbit, gut, skin, and liver) for the presence of Tp, using a polyclonal antibody anti-Tp. At the time of biopsy, none of the patients was suspected for luetic infection, nor specific serologic tests were available. IPT-LN areas extensively involved the nodal parenchyma in 4 cases, whereas they were focal in the remaining 5 cases. Capsular thickening and inflammation (6/9), venulitis (3/9), small granulomas (3/9), and follicular hyperplasia (7/9) were observed in the associated lymphoid parenchyma. Tp were detected in 4/9 cases of LN-IPT and in none of the extranodal IPT. Tp were extremely abundant within the IPT areas and in the perivascular tissues in the surrounding parenchyma, whereas they were scattered within the capsule. In Tp+ cases, marked follicular hyperplasia was the single distinctively associated feature. Double immunostains revealed that Tp were predominantly contained in the cytoplasm of CD11c+ CD163+ macrophages, some of which co-expressed HLA-DR. In addition, scattered S100+ interdigitating dendritic cells also showed intracytoplasmic Tp. This study shows that a significant number of IPT-LN is associated with Tp infection. A spirochetal etiology can be suspected in cases of IPT-LN, independently from the extension of the lesions, especially when pronounced follicular hyperplasia is found. Infection by Tp of macrophages and dendritic cells are in keeping with in vitro data and indicate that immune mediated mechanisms may be involved in the pathogenesis of the lesions.

Association of Treponema spp. with canine periodontitis.

To evaluate the association of oral Treponema (T.) spp. with severity of canine periodontitis, subgingival plaque samples of dogs of various breeds undergoing surgery were investigated. A wide range of oral Treponema spp. was analysed by a molecular and culture-independent approach applying DNA-DNA dot blot hybridization analysis and fluorescence in situ hybridization using Treponema specific oligonucleotide probes specific for phylogenetic groups I-VII of oral treponemes as well as probes specific for T. socranskii and T. denticola. To assess the periodontal status of affected dogs clinical parameters were measured and the periodontal status was classified from grade 0 (physiological periodont) to 3 (severe periodontitis). The periodontal status correlated significantly with an increasing concentration of volatile sulfur compounds (VSC, r=0.854) determined with a Halimeter, indicating a positive correlation between the presence of VSC-producing bacteria and periodontitis. In this study Treponema spp. of phylogenetic groups III, V-VII were not detected in any sample, whereas T. denticola-like treponemes were found only in 2 of 51 animals. However, treponemes belonging to phylogenetic groups I, II and IV of oral treponemes or T. socranskii were found in up to 64.84% of the dogs. The detection rate of Treponema spp. was significantly associated with an increased periodontal status. Treponemes present in periodontal lesions were also visualized by fluorescence in situ hybridization of gingival biopsies showing Treponema spp. not only in the microbial biofilm but also within the gingival tissue. The data presented here indicate that oral Treponema spp. are associated with canine periodontitis. Similar to human periodontitis, treponemes of groups I, II and IV and T. socranskii were found more frequently the higher the degree of periodontitis was.

Lesion formation and antibody response induced by papillomatous digital dermatitis-associated spirochetes in a murine abscess model.

Papillomatous digital dermatitis (PDD), also known as hairy heel wart, is a growing cause of lameness of cows in the U.S. dairy industry. Farms with PDD-afflicted cows experience economic loss due to treatment costs, decreased milk production, lower reproductive efficiency, and premature culling. While the exact cause of PDD is unknown, lesion development is associated with the presence of anaerobic spirochetes. This study was undertaken to investigate the virulence and antigenic relatedness of four previously isolated Treponema phagedenis-like spirochetes (1A, 3A, 4A, and 5B) by using a mouse abscess model with subcutaneous inoculation of 10(9), 10(10), and 10(11) spirochetes. Each of the PDD isolates induced abscess formation, with strain 3A causing cutaneous ulceration. Lesion development and antibody responses were dose dependent and differed significantly from those seen with the nonpathogenic human T. phagedenis strain. Strains 3A, 4A, and 5B showed two-way cross-reactivity with each other and a one-way cross-reaction with T. phagedenis. Strain 5B showed one-way cross-reactivity with 1A. None of the isolates showed cross-reactivity with T. denticola. In addition, distinct differences in immunoglobulin G subclass elicitation occurred between the PDD strains and T. phagedenis. From these data, we conclude that spirochetes isolated from PDD lesions have differential virulence and antigenic traits in vivo. Continuing investigation of these properties is important for the elucidation of virulence mechanisms and antigenic targets for vaccine development.

Environmental modulation of oral treponeme virulence in a murine model.

This investigation examined the effects of environmental alteration on the virulence of the oral treponemes Treponema denticola and Treponema pectinovorum. The environmental effects were assessed by using a model of localized inflammatory abscesses in mice. In vitro growth of T. denticola and T. pectinovorum as a function of modification of the cysteine concentration significantly enhanced abscess formation and size. In contrast, growth of T. denticola or T. pectinovorum under iron-limiting conditions (e.g., dipyridyl chelation) had no effect on abscess induction in comparison to that when the strains were grown under normal iron conditions. In vivo modulation of the microenvironment at the focus of infection with Cytodex beads demonstrated that increasing the local inflammation had no effect on lesion induction or size. In vivo studies involved the determination of the effects of increased systemic iron availability (e.g., iron dextran or phenylhydrazine) on the induction, kinetics, and size of lesions. T. denticola induced significantly larger lesions in mice with iron pretreatment and demonstrated systemic manifestations of the infectious challenge and an accompanying spreading lesion with phenylhydrazine pretreatment (e.g., increases in circulating free hemoglobin). In contrast, T. pectinovorum virulence was minimally affected by this in vivo treatment to increase iron availability. T. denticola virulence, as evaluated by lesion size, was increased additively by in vivo iron availability, and cysteine modified growth of the microorganism. Additionally, galactosamine sensitized mice to a lethal outcome following infection with both T. denticola and T. pectinovorum, suggesting an endotoxin-like activity in these treponemes. These findings demonstrated the ability to modify the virulence capacity of T. denticola and T. pectinovorum by environmental conditions which can be evaluated by using in vivo murine models.

[Bejel: an unusual cause of stomatitis in the child]. / Le bejel: une cause inhabituelle de stomatite de l'enfant.

INTRODUCTION: Bejel (endemic syphilis) is usually encountered in children living in intertropical areas, although imported cases have been exceptionally reported in Europe. OBSERVATION: We report on a young girl aged 5 diagnosed in France, who had painless stomatitis and enlarged cervical nodes. Diagnosis of bejel was confirmed by serology, and spirochetes were shown inside mucous patches by biopsy specimen silver staining. Favorable clinical and serological outcome occurred following benzathin-penicillin therapy. Contamination is likely to have occurred in Mali where she had been living for several years, but secondary stage manifestations only appeared in France, where she was living for several months. DISCUSSION: Although rare in France, bejel should not be overlooked in children originating from countries where endemic syphilis has a high prevalence.

Possible paleopathological evidence of treponematosis from a megalithic site at Agripalle, India.

An Iron Age (megalithic) skull recovered from a cist burial complex at Agripalle, Andhra Pradesh, India, exhibits extensive erosion of the calvarium, areas of sclerotic diploe, irregular osteitic and periosteitic lesions, and deep ulcerations with a granulomatous appearance of nodular foci due to bone remodeling. These lesions are found over the entire surface, but are less severe in the temporal region and in the occipital region below lambda. There is extensive ulceration and destruction of the orbital roof and the nasopalatine region. A thick bony mass representing a healed lesion is present on the nasal margin. Comparison with the pathologic skeletal series of Ortner and Putschar ([1981] Smithson. Contrib. Anthropol. 28:180-218), Steinbock ([1976] Paleopathology: Diagnosis and Interpretation, pp.86-169), and Calvin ([1964] Bones and Disease: Evidence of Disease and Abnormality in Early Man) indicates that these findings warrant a diagnosis of an advanced stage of treponematosis. The material from Agripalle, together with similar specimens recovered from the sites of Bhimbetka (Iron Age) and Inamgaon (Chalcolithic), furnish additional evidence supporting the hypothesis of the prehistoric antiquity of treponemal disease in both the New and Old Worlds.

Burned-out endemic syphilis (Bejel): facial deformities and defects in Saudi Arabia.

Endemic syphilis (bejel) is an endemic, contagious, non-sexually transmitted treponematosis of primitive communities which, if not treated, can cause deformities and defects of the face in its late stage. Several burned-out cases from Saudi Arabia are shown, and the techniques of plastic surgical repair are presented by word and illustrations. We describe in detail a total rhinoplasty (with panfacial reconstruction) in which a scalping flap was used as outer cover and superimposed on a classical island forehead flap based on a subcutaneous pedicle containing both frontal vessels for inner lining.

Comparison of virulence of clones of two Trichomonas vaginalis strains by the subcutaneous mouse assay.

No statistical differences in virulence were found among five clones isolated from each of two Trichomonas vaginalis strains JH31A and Balt 42. The former strain, isolated from a patient showing no cervical epithelial abnormalities, caused relatively small subcutaneous lesions in mice [mean volume for the noncloned strain, 75.45 +/- 4.43 mm3 (n = 70); mean of means for cloned populations, 77.28 +/- 3.14 mm3 (n = 230)]. The latter, Balt 42, isolated from a woman with an in situ carcinoma of the cervix uteri, produced large subcutaneous abscesses in mice [mean volume for the noncloned strain, 202.28 +/- 12.53 mm3 (n = 55); mean of means for cloned populations, 200.48 +/- 13.72 mm3 (n = 264)]. The apparent homogeneity of T. vaginalis strains with regard to virulence reinforces the dependability of the subcutaneous mouse assay.

Electron microscopy of lymph nodes of hamsters experimentally infected with Treponema pertenue.

The morphology of lymph node tissue from normal hamsters and from hamsters experimentally infected with Treponema pertenue Gauthier was compared by means of light and electron microscopy. The capsules of the lymph nodes from infected hamsters showed an increased thickness in comparison with those of the non-infected animals. The infected lymph nodes differed from normal lymph nodes by small accumulations of neutrophilic leucocytes in the cortical areas. In addition, the amount of intercellular collagenous matrix present between large elongated cells was greatly increased in lymph nodes from infected animals. Electron microscopy of thin sections of infected lymph nodes showed intercellularly located treponemes in the leucocyte infiltration areas. These regions also showed the increased amounts of the collagenous matrix. Treponemes were occasionally found intracellularly in macrophages. These treponemes did not show their typically helical shape, but were present as spherical forms or cysts.