Digital dermatitis in Swedish dairy herds assessed by ELISA targeting Treponema phagedenis in bulk tank milk.

BACKGROUND: Digital dermatitis (DD) is a contagious hoof infection affecting cattle worldwide. The disease causes lameness and a reduction in animal welfare, which ultimately leads to major decreases in milk production in dairy cattle. The disease is most likely of polymicrobial origin with Treponema phagedenis and other Treponema spp. playing a key role; however, the etiology is not fully understood. Diagnosis of the disease is based on visual assessment of the feet by trained hoof-trimmers and veterinarians, as a more reliable diagnostic method is lacking. The aim of this study was to evaluate the use of an enzyme-linked immunosorbent assay (ELISA) on bulk tank milk samples testing for the presence of T. phagedenis antibodies as a proxy to assess herd prevalence of DD in Swedish dairy cattle herds. RESULTS: Bulk tank milk samples were collected in 2013 from 612 dairy herds spread across Sweden. A nationwide DD apparent prevalence of 11.9% (8.1-14.4% CI95%) was found, with the highest proportion of test-positive herds in the South Swedish regions (31.3%; 19.9-42.4% CI95%). CONCLUSIONS: This study reveals an underestimation of DD prevalence based on test results compared to hoof trimming data, highlighting the critical need for a reliable and accurate diagnostic method. Such a method is essential for disease monitoring and the development of effective control strategies. The novelty of ELISA-based diagnostic methods for DD, coupled with the disease's polymicrobial origin, suggests an avenue for improvement. Developing an expanded ELISA, incorporating antigens from various bacterial species implicated in the disease, could enhance diagnostic accuracy. The significance of this study is underscored by the extensive analysis of a substantial sample size (612). Notably, this investigation stands as the largest assessment to date, evaluating the application of ELISA on bulk tank milk for DD diagnosis at the herd level.

HIV with initial ocular Kaposi sarcoma / HIV com sarcoma de Kaposi ocular inicial

ABSTRACT We present an interesting case of a 62-year-old black female, presented to the ophthalmological hospital with a little "nevus" on the left eye previously visualized at the mirror, with one month of development. Physical examination with slit lamp (biomicroscopy) showed a group of painless veins, with vascular redness, and a mass nodular aspect in the mid temporal bulbar conjunctiva, of approximately 2mmx4mm.

RESUMO Apresentamos o interessante caso de uma mulher negra de 62 anos, que deu entrada no hospital oftalmológico com um pequeno nevo no olho esquerdo previamente visualizado ao espelho, com 1 mês de evolução. O exame físico com lâmpada de fenda (biomicroscopia) mostrou um grupo de veias indolor e vermelhidão vascular, com uma massa de aspecto nodular na conjuntiva bulbar temporal média, de aproximadamente 2mmx4mm.

A filter-assisted culture method for isolation of Treponema spp. from bovine digital dermatitis and their identification by MALDI-TOF MS.

Digital dermatitis (DD) is a major infectious foot disease of cattle worldwide. Some DD stages are associated with lameness, and the disease has significant economic and animal welfare consequences. The pathogenesis of the disease is not yet fully understood, but Treponema spp. have been associated consistently with clinical cases. Isolation of these fastidious bacteria is difficult and cumbersome. We describe an improved method enabling the culturing of the 3 Treponema spp. (T. pedis, T. phagedenis, and T. medium) from bovine foot specimens derived from DD lesions, using a combination of membrane filtering and subsequent growth on selective agar media. The entire procedure from sampling to verification of individual Treponema spp. takes up to 24 d. In addition, we established a MALDI-TOF MS-based identification method to be applied for confirmation of the different Treponema spp. This scheme provides an unambiguous, simple, and straightforward identification procedure for DD-associated Treponema spp.

Effects of different culture media on growth of Treponema spp. isolated from digital dermatitis.

Digital dermatitis (DD) lesions in cattle are characterized by the presence of multiple Treponema species. Current culture media for isolating treponemes generally uses serum supplementation from different animals to target particular Treponema sp.; however, their suitability for DD Treponema isolation has not been fully determined. We studied the effect of culture media (OTEB, NOS and TYGV) and serum supplementation on mixed Treponema spp. dynamics. Bacterial growth was evaluated by direct microscopic count, optical density, wet weight and a species-specific qPCR and the correlations between these independent methods were calculated. Wet weight, optical density and bacterial count correlated best with each other. Different Treponema species performed differently under the tested culture media. T. phagedenis growth was enhanced in OTEB media supplemented with bovine fetal serum (BFS) or horse serum (HS). T. medium had lower generation time when culture media were supplemented with rabbit serum (RS). Lowest generation time for T. pedis and T. denticola were obtained in NOS media supplemented with HS and OTEB media supplemented with BFS, respectively. Detection of cystic forms observed after 5 days of culture did not differ among the culture media. Correlation between different Treponema spp. growth quantification techniques indicated that alternative quantification methods such as qPCR and wet weight could be used depending on the purpose. We conclude that effects of culture media and serum supplementation on mixed Treponema spp. communities should be taken into account when isolating a specific Treponema species.

Short communication: Informative value of an ELISA applied to bulk tank milk to assess within-herd prevalence of digital dermatitis in dairy herds.

The objective of this study was to assess whether an antibody ELISA applied to bulk tank milk (BTM) could be used to accurately estimate within-herd prevalence of digital dermatitis (DD). The ELISA was designed for the detection of antibodies against Treponema phagedenis-like strain V1 (PrrA antigen). The hind feet of all lactating cows from 40 commercial French dairy herds with a history of DD were scored by an observer in the milking parlor, using the 4 M-stage system. After milking, a BTM sample was collected and tested for anti-Treponema phagedenis-like antibodies using the antibody ELISA. Within-herd DD prevalence at the cow level was determined using 2 different approaches: (1) having DD lesion on at least 1 hind foot (Prev; prevalence of affected cows), and (2) having an M1 or M2 lesion on at least 1 hind foot (PrevA; prevalence of cows affected by DD in an active stage). Receiver operating characteristic analysis was used to determine both optimal within-herd DD prevalence and BTM sample to positive (S/P) ratio cut-off values. Two optimal cut-off values were identified. Herds with an S/P ratio of BTM ≤0.2 had a Prev ≤10% (sensitivity = 0.97, specificity = 1), whereas herds with an S/P ratio of BTM >0.38 had a Prev >40% (sensitivity = 0.94, specificity = 0.86). In the same way but with a slightly lower specificity, an S/P ratio >0.38 corresponds also to a PrevA >18% (sensitivity = 0.92, specificity = 0.70). The BTM antibody ELISA shows great promise for screening purposes during DD management programs.

Yaws, Haemophilus ducreyi, and Other Bacterial Causes of Cutaneous Ulcer Disease in the South Pacific Islands.

Cutaneous ulcers in the tropics are a painful and debilitating condition that anchors people into poverty. In rural regions of the South Pacific, infectious cutaneous ulcers are caused mainly by bacteria, including Treponema pallidum pertenue (yaws), Haemophilus ducreyi, and polymicrobial ulcers. For this group of infections the term cutaneous ulcer disease (CUD) is proposed. Some infections can cause malformations on the bone that have a permanent impact on lives in endemic communities. Better characterization of CUD may help design diagnostic tools and more effective antimicrobial therapies. This review updates the knowledge of CUD and discusses optimized terminology and syndromic management.

Passive Filtration, Rapid Scanning Electron Microscopy, and Matrix-Assisted Laser Desorption Ionization-Time of Flight Mass Spectrometry for Treponema Culture and Identification from the Oral Cavity.

We present here a new passive-filtration-based culture device combined with rapid identification with a new electron microscope (Hitachi TM4000) for the detection and culture of Treponema species from the human oral cavity. Of the 44 oral samples cultivated, 15 (34%) were found to be positive for Treponema using electron microscopy and were also culture positive. All were subcultured on agar plates; based on genome sequencing and analyses, 10 were strains of Treponema pectinovorum and 5 were strains of Treponema denticola The 29 samples that were negative for Treponema remained culture negative. In addition, 14 Treponema species ordered from the DSMZ collection were cultured in the T-Raoult culture medium optimized here. Finally, matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) was used and 30 novel spectra were added to the MALDI-TOF MS database. We have successfully developed a new and effective method for treponemal detection, culture, and identification.

Detection of Treponema phagedenis-like antibodies in serum and bulk milk from cows with and without digital dermatitis.

Digital dermatitis (DD) in dairy cows is a widespread disease linked to infection with Treponema. The traditional diagnostic method is clinical inspection, which is subjective and laborious. We explored the performance of 4 different immunogenic proteins from Treponema phagedenis in a new antibody ELISA for analysis of serum or milk. Analysis of samples from 390 cows in 25 herds showed that the ELISA could distinguish the majority of cows with DD from healthy cows. By changing the cutoff and applying parallel or serial testing, high sensitivity or specificity could be achieved. The investigation indicated that aggregated test results can be useful in the assessment of a herd's DD status. In addition, analysis of bulk tank milk samples showed good agreement with results from individual cows. The test system could be useful in research on the epidemiology and immunology of DD.

Development of real-time PCR and loop-mediated isothermal amplification (LAMP) assays for the differential detection of digital dermatitis associated treponemes.

Bovine digital dermatitis (DD) is a severe infectious cause of lameness in cattle worldwide, with important economic and welfare consequences. There are three treponeme phylogroups (T. pedis, T. phagedenis, and T. medium) that are implicated in playing an important causative role in DD. This study was conducted to develop real-time PCR and loop-mediated isothermal amplification (LAMP) assays for the detection and differentiation of the three treponeme phylogroups associated with DD. The real-time PCR treponeme phylogroup assays targeted the 16S-23S rDNA intergenic space (ITS) for T. pedis and T. phagedenis, and the flagellin gene (flaB2) for T. medium. The 3 treponeme phylogroup LAMP assays targeted the flagellin gene (flaB2) and the 16S rRNA was targeted for the Treponeme ssp. LAMP assay. The real-time PCR and LAMP assays correctly detected the target sequence of all control strains examined, and no cross-reactions were observed, representing 100% specificity. The limit of detection for each of the three treponeme phylogroup real-time PCR and LAMP assays was ≤ 70 fg/µl. The detection limit for the Treponema spp. LAMP assay ranged from 7-690 fg/µl depending on phylogroup. Treponemes were isolated from 40 DD lesion biopsies using an immunomagnetic separation culture method. The treponeme isolation samples were then subjected to the real-time PCR and LAMP assays for analysis. The treponeme phylogroup real-time PCR and LAMP assay results had 100% agreement, matching on all isolation samples. These results indicate that the developed assays are a sensitive and specific test for the detection and differentiation of the three main treponeme phylogroups implicated in DD.

[Nonviral sexually transmitted infections-epidemiology, clinical manifestations, diagnostic workup, therapy : Part 3: Treponemes, Gardnerella and trichomonads]. / Nichtvirale sexuell übertragene Infektionen ­ Epidemiologie, Klinik, Labordiagnostik und Therapie : Teil 3: Treponemen, Gardnerella und Trichomonaden.

In Germany, the reported syphilis prevalence has increased continuously since 2010, with a total of 6834 syphilis cases being reported in 2015. The largest increase of reported syphilis occurred in men who have sex with men (MSM). The antibiotic agent of choice for treatment of syphilis is still penicillin. There are no penicillin-resistant Treponema pallidum strains. Alternatives are ceftriaxone and doxycycline. In Germany, azithromycin is not approved for treatment of syphilis; however, therapy failures are increasingly reported. Bacterial vaginosis is accompanied by vaginal discharge. The vaginal secretion exhibits an increased pH value higher than 4.5. Clinical symptoms are pruritus, burning, and the characteristic amine odor. The probability for bacterial vaginosis is highest in women with higher numbers of sexual partners, unmarried women, early first sexual intercourse, in commercial female sex workers, and those women who regularly apply vaginal douches. The main pathogen of bacterial vaginosis is Gardnerella vaginalis. For oral therapy metronidazole is given, alternatively clindamycin; the latter should be applied additionally as topical agent. Trichomoniasis is considered as the nonviral sexually transmitted infection with the highest prevalence worldwide. Other than direct microscopic detection of the protozoa (trophozoites) in vaginal secretion or urine, PCR has been approved as the diagnostic method with the highest sensitivity. Oral metronidazole represents the therapy of choice in trichomoniasis.

A randomized trial to evaluate the effect of a trace mineral premix on the incidence of active digital dermatitis lesions in cattle.

A balanced, parallel-group, single-blinded randomized efficacy study divided into 2 periods was conducted to evaluate the effect of a premix containing higher than typically recommended levels of organic trace minerals and iodine (HOTMI) in reducing the incidence of active digital dermatitis (DD) lesions acquired naturally and induced by an experimental infection challenge model. For the natural exposure phase of the study, 120 healthy Holstein steers 5 to 7 mo of age without signs of hoof disease were randomized into 2 groups of 60 animals. The control group was fed a standard trace mineral supplement and the treatment group was fed the HOTMI premix, both for a period of 60 d. On d 60, 15 steers free of macroscopic DD lesions were randomly selected from each group for the challenge phase and transported to an experimental facility, where they were acclimated and then challenged within a DD infection model. The same diet group allocation was maintained during the 60 d of the challenge phase. The primary outcome measured was the development of an active DD lesion greater than 20mm in diameter across its largest dimension. No lesions were identified during the natural exposure phase. During the challenge phase, 55% (11/20) and 30% (6/20) of feet were diagnosed with an active DD lesion in the control and treatment groups, respectively. Diagnosis of DD was confirmed by histopathologic demonstration of invasive Treponema spp. within eroded and hyperplastic epidermis and ulcerated papillary dermis. All DD confirmed lesions had dark-field microscopic features compatible with DD and were positive for Treponema spp. by PCR. As a secondary outcome, the average DD lesion size observed in all feet was also evaluated. Overall mean (standard deviation) lesion size was 17.1 (2.36) mm and 11.1 (3.33) mm for the control and treatment groups, respectively, with this difference being driven by acute DD lesions >20mm. A trend existed for the HOTMI premix to reduce the total DD infection rate and the average size of the experimentally induced lesions. Further research is needed to validate the effect of this intervention strategy in the field and to generate prevention and control measures aimed at optimizing claw health based on nutritional programs.

Immune response against Treponema spp. and ELISA detection of digital dermatitis.

The objective of this longitudinal study was to evaluate the immune response against Treponema spp. infection in dairy heifers affected with digital dermatitis (DD). In addition, the accuracy of an indirect ELISA detecting anti-Treponema IgG antibodies in identifying clinical DD status has been assessed. A cohort of 688 pregnant Holstein heifers was evaluated at least 3 times before calving during a period of 6 mo. Complete clinical assessment of DD presence on the back feet of each heifer and blood extraction were performed in a stand-up chute. Digital dermatitis cases were characterized by the M-stage classification system and size and level of skin proliferation. An ELISA was performed on blood serum samples obtained from a subcohort of 130 heifers. For description purposes, the animals were classified by the number of clinical cases experienced during the study period as type I (no clinical cases were observed), type II (only 1 acute clinical case diagnosed), and type III (at least 2 acute clinical cases diagnosed). Multivariable repeated-measures models were used to evaluate the immune response against Treponema spp. infection. A binormal Bayesian model for the ELISA data without cut-point values was used to assess the accuracy of the ELISA as a diagnostic tool. Animals that never experienced a DD event throughout the study kept a constant low level of antibody titer. A 56% increase in mean ELISA titer was observed in heifers upon a first clinical DD case diagnosis. After topical treatment of an acute DD case with oxytetracycline, the antibody titer decreased progressively in type II heifers, achieving mean levels of those observed in healthy cows after a mean of 223 d. Surprisingly, antibody titer was not increased in the presence of M1 (DD lesion <20mm in diameter surrounded by healthy skin) and M4.1 (DD lesion <20mm in diameter embedded in a circumscribed dyskeratotic or proliferative skin alteration) DD stages. Type III cows showed a slight increase in antibody levels. The presence of skin proliferation at first DD diagnosis was found to be associated with an odds ratio of 2.04 of becoming a type III heifer in relation to heifers presenting first lesions without skin proliferation. The ELISA validity was estimated by an area under the curve of 0.88. Predicted probabilities of infection are provided for a range of ELISA values and prevalence of infection. Early detection and treatment is essential to control DD and the ELISA can be used in understanding the immunopathology of DD and shows great promise for prescreening purposes during DD management programs in combination with traditional clinical inspection.

Identification of Treponema pedis as the predominant Treponema species in porcine skin ulcers by fluorescence in situ hybridization and high-throughput sequencing.

Skin lesions often seen in pig production are of great animal welfare concern. To study the potential role of Treponema bacteria in porcine skin ulcers, we investigated the presence and distribution of these organisms in decubital shoulder ulcers (n=51) and ear necroses (n=54) by fluorescence in situ hybridization (FISH) and high-throughput sequencing. In addition, two cases of facial ulcers and five cases of other skin ulcers were included in the study. Samples from all 112 skin lesions and intact skin from pigs without skin ulcers (n=14) were screened by FISH. Three different oligonucleotide probes targeting 16S rRNA were used, specific for domain bacterium, Treponema spp. and species T. pedis. Screening showed that two cases each of facial and other ulcers, 35 (69%) of shoulder ulcers and 32 (59%) of ear necroses were positive for Treponema spp. T. pedis was the unequivocally, predominant species typically constituting more than 90% of the treponemes in a lesion, assessed visually by microscopy. Altogether, T. pedis was demonstrated in 69 of the 71 Treponema spp. positive lesions. We conclude that Treponema spp. are frequently present and abundant in various skin ulcers of pigs. The results from this study point toward an important role of T. pedis as a secondary bacterial infection in porcine skin ulcers, especially in severe and chronic lesions.

The endemic treponematoses.

The agents of human treponematoses include four closely related members of the genus Treponema: three subspecies of Treponema pallidum plus Treponema carateum. T. pallidum subsp. pallidum causes venereal syphilis, while T. pallidum subsp. pertenue, T. pallidum subsp. endemicum, and T. carateum are the agents of the endemic treponematoses yaws, bejel (or endemic syphilis), and pinta, respectively. All human treponematoses share remarkable similarities in pathogenesis and clinical manifestations, consistent with the high genetic and antigenic relatedness of their etiological agents. Distinctive features have been identified in terms of age of acquisition, most common mode of transmission, and capacity for invasion of the central nervous system and fetus, although the accuracy of these purported differences is debated among investigators and no biological basis for these differences has been identified to date. In 2012, the World Health Organization (WHO) officially set a goal for yaws eradication by 2020. This challenging but potentially feasible endeavor is favored by the adoption of oral azithromycin for mass treatment and the currently focused distribution of yaws and endemic treponematoses and has revived global interest in these fascinating diseases and their causative agents.

Advances in the diagnosis of endemic treponematoses: yaws, bejel, and pinta.

Improved understanding of the differential diagnosis of endemic treponematoses is needed to inform clinical practice and to ensure the best outcome for a new global initiative for the eradication of yaws, bejel, and pinta. Traditionally, the human treponematoses have been differentiated based upon their clinical manifestations and epidemiologic characteristics because the etiologic agents are indistinguishable in the laboratory. Serological tests are still considered standard laboratory methods for the diagnosis of endemic treponematoses and new rapid point-of-care treponemal tests have become available which are extremely useful in low-resource settings. In the past ten years, there has been an increasing effort to apply polymerase chain reaction to treponematoses and whole genome fingerprinting techniques have identified genetic signatures that can differentiate the existing treponemal strains; however, definitive diagnosis is also hampered by widespread unavailability of molecular diagnostics. We review the dilemmas in the diagnosis of endemic treponematoses, and advances in the discovery of new diagnostic tools.

Periodontopathogen profile of healthy and oral lichen planus patients with gingivitis or periodontitis.

Oral lichen planus (OLP) is a chronic inflammatory disease that is frequently detected in oral tissues. The aim of our study was to identify the prevalence of the detection of periodontopathogenic microorganisms (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia and Treponema denticola in OLP patients and to compare with this prevalence of periodontopathogenic microorganisms in healthy non-OLP patients. Our study included 27 (18 chronic periodontitis (OLPP) and 9 gingivitis (OLPG)) patients diagnosed with OLP along with 26 (13 chronic periodontitis (HP) and 13 gingivitis (HG)) healthy non-OLP patients. The multiplex polymerase chain reaction (PCR) with subsequent reverse hybridization method (micro-IDent) was used for identifying periodontopathogenic microorganisms present in subgingival plaque samples. The percentages of detection for A. actinomycetemcomitans, P. gingivalis, P. intermedia, T. forsythia and T. denticola in subgingival plaque samples taken from OLP patients (OLPG and OLPP) were 18.5%, 85.1%, 81.4%, 88.8% and 74%, respectively. Meanwhile, in the non-OLP patients (HG and HP), these values were 7.6%, 50%, 46.1%, 73% and 57.7%, respectively. Thus, comparing the non-OLP groups with the OLP groups, the periodontopathogens' percentages of detection in the OLP groups were higher than those in the non-OLP groups. According to our study results, OLP patients have higher levels of infection with A. actinomycetemcomitans, P. gingivalis, P. intermedia, T. forsythia and T. denticola than non-OLP patients. We argue that the high percentages in patients with OLP may help identify the importance of periodontopathogenic microorganisms in the progress of periodontal diseases of OLP.

Periodontal conditions during the pregnancy associated with periodontal pathogens.

AIM: To describe the bacterial associations in the periodontal pockets of pregnant women and to correlate the presence of Prevotella intermedia, Tannerella forsythia (T. forsythia), Treponema denticola (T. denticola), Aggregatibacter actinomycetemcomitans, and Porphyromona gingivalis (P. gingivalis) with periodontal parameters of severity. METHODS: The analysis was performed with 150 pregnant women. The examination consisted of an evaluation of bleeding, suppuration, probing depths, clinical attachment levels, hypermobility scores, the Silness and Löe Plaque Index, and the Löe and the Silness Gingival Index. Each periodontal pathogen was identified by polymerase chain reaction. RESULTS: A statistically-significant association was observed (P < 0.01) between P. gingivalis and T. forsythia, between P. gingivalis and T. denticola, and between T. forsythia and T. denticola. Age was observed to be a risk factor in the development of moderate periodontitis (odds ratio [OR] = 4.92, 95% confidence interval [CI] = 1.1-21.3, P = 0.0328). Age was significantly associated with increased pocket depth and plaque index (OR = 6.36, 95% CI = 1.8-22.2, P = 0.0037). In pregnant women, the presence of P. gingivalis was found to increase the risk of developing a clinical attachment level ≥ 5 mm. CONCLUSION: A high prevalence of P. gingivalis in pregnant women, especially in combination with T. forsythia and T. denticola, was associated with an increased risk of developing moderate periodontitis, and that association was more marked in pregnant women aged 30 years or older.