Ear Biopsies Research Articles

Male C57BL/6J mice have higher presence and abundance of Borrelia burgdorferi in their ventral skin compared to female mice

Borrelia burgdorferi is a tick-borne spirochete that causes Lyme disease in humans. The host immune system controls the abundance of the spirochete in the host tissues. Recent work with immunocompetent Mus musculus mice strain C3H/HeJ found that males had a higher tissue infection prevalence and spirochete load compared to females. The purpose of this study was to determine whether host sex and acquired immunity interact to influence the prevalence and abundance of spirochetes in the tissues of the commonly used mouse strain C57BL/6. Wildtype (WT) mice and their SCID counterparts (C57BL/6) were experimentally infected with B. burgdorferi via tick bite. Ear biopsies were sampled at weeks 4, 8, and 12 post-infection (PI) and five tissues (left ear, ventral skin, heart, tibiotarsal joint of left hind leg, and liver) were collected at necropsy (16 weeks PI). The mean spirochete load in the tissues of the SCID mice was 260.4x higher compared to the WT mice. In WT mice, the infection prevalence in the ventral skin was significantly higher in males (40.0 %) compared to females (0.0 %), and the spirochete load in the rear tibiotarsal joint was significantly higher (4.3x) in males compared to females. In SCID mice, the spirochete load in the ventral skin was 200.0x higher in males compared to females, but there were no significant sex-specific difference in spirochete load in the other tissues (left ear, heart, tibiotarsal joint, or liver). Thus, the absence of acquired immunity greatly amplified the spirochete load in the ventral skin of male mice. It is important to note that the observed sex-specific differences in laboratory mice cannot be extrapolated to humans. Future studies should investigate the mechanisms underlying the male bias in the abundance of B. burgdorferi in the mouse skin.

Proton pump inhibitor alters Th17/Treg balance and induces gut dysbiosis suppressing contact hypersensitivity reaction in mice.

Proton pump inhibitors (PPIs), such as omeprazole, are the most commonly prescribed drugs. Treatment with PPIs alters gut microbiota composition and reduces the production of reactive oxygen (ROS) and proinflammatory IL-1β, IL-6, and TNF-α cytokines. Here, using the T cell-dependent contact hypersensitivity (CHS) response, an animal model of allergic contact dermatitis (ACD) that affects up to 30% of the population, we demonstrated that a two-week omeprazole treatment suppresses the development of CHS. Omeprazole treatment before CHS induction, reduced inflammatory response in ears measured by ear swelling, ear biopsy weight, MPO activity, and proinflammatory cytokine production. These changes were associated with reduced frequency of TCRαβ+ CD4+ IL-17A+ and TCRαβ+ CD8+ IL-17A+ T cells and increased frequency of TCRαβ+ CD4+ CD25+ FoxP3+ Treg, and TCRαβ+ CD4+ IL-10+ Tr1 cells in peripheral lymphoid organs. Omeprazole treatment decreased the production of ROS, TNF-α, and IL-6, which supported Th17 cell induction, and increased the frequency of Clostridium cluster XIVab and Lactobacillus, implicated in Treg cell induction. The fecal microbiota transplantation (FMT) experiment confirmed the role of omeprazole-induced changes in gut microbiota profile in CHS suppression. Our data suggests that omeprazole ameliorates inflammatory response mediated by T-cells.

Optimization of 3D autologous chondrocyte-seeded polyglycolic acid scaffolds to mimic human ear cartilage.

Auricular reconstruction in children with microtia is one of the more complex procedures in plastic surgery. Obtaining sufficient native material to build an ear requires harvesting large fragments of rib cartilage in children. Herein, we investigated how to optimize autologous chondrocyte isolation, expansion and re-implantation using polyglycolic acid (PGA) scaffolds for generating enough cartilage to recapitulate a whole ear starting from a small ear biopsy. Ear chondrocytes isolated from human microtia subjects grew slower than microtia rib or healthy ear chondrocytes and displayed a phenotypic shift due to the passage number. Rabbit ear chondrocytes co-cultured with mesenchymal stem cells (MSC) at a 50 : 50 ratio recapitulated the cartilage biological properties in vitro. However, PGA scaffolds with different proportions of rabbit chondrocytes and MSC did not grow substantially in two months when subcutaneously implanted in immunosuppressed mice. In contrast, rabbit chondrocyte-seeded PGA scaffolds implanted in immunocompetent rabbits formed a cartilage 10 times larger than the original PGA scaffold. This cartilage mimicked the biofunctional and mechanical properties of an ear cartilage. These results indicate that autologous chondrocyte-seeded PGA scaffolds fabricated following our optimized procedure have immense potential as a solution for obtaining enough cartilage for auricular reconstruction and opens new avenues to redefine autologous cartilage replacement.

Assessment of skin sensitization properties of few-layer graphene and graphene oxide through the Local Lymph Node Assay (OECD TG 442B)

Skin contact is one of the most common exposure routes to graphene-based materials (GBMs) during their small-scale and industrial production or their use in technological applications. Nevertheless, toxic effects in humans by cutaneous exposure to GBMs remain largely unexplored, despite skin contact to other related materials has been associated with adverse effects. Hence, this in vivo study was carried out to evaluate the cutaneous effects of two GBMs, focusing on skin sensitization as a possible adverse outcome.Skin sensitization by few-layer graphene (FLG) and graphene oxide (GO) was evaluated following the Organization for Economic Cooperation and Development (OECD) guideline 442B (Local Lymph Node Assay; LLNA) measuring the proliferation of auricular lymph node cells during the induction phase of skin sensitization. Groups of four female CBA/JN mice (8–12 weeks) were daily exposed to FLG or GO through the dorsal skin of each ear (0.4–40 mg/mL, equal to 0.01–1.00 mg/ear) for 3 consecutive days, and proliferation of auricular lymph node cells was evaluated 3 days after the last treatment. During this period, no clinical signs of toxicity and no alterations in body weight and food or water consumptions were observed. In addition, no ear erythema or edema were recorded as signs of irritation or inflammation. Bromo-deoxyuridine (BrdU) incorporation in proliferating lymphocytes from ear lymph nodes (stimulation indexes <1.6) and the histological analysis of ear tissues excluded sensitizing or irritant properties of these materials, while myeloperoxidase activity in ear biopsies confirmed no inflammatory cells infiltrate. On the whole, this study indicates the absence of sensitization and irritant potential of FLG and GO.

The added value of One Health surveillance: data from questing ticks can provide an early signal for anaplasmosis outbreaks in animals and humans

ObjectiveIn 2021, a first outbreak of anaplasmosis occurred in animals and humans in southern Québec, with 64% of confirmed human cases located in Bromont municipality. Ixodes scapularis ticks and Peromyscus mouse ear biopsies collected in Bromont from 2019 to 2021 were analyzed for Anaplasma phagocytophilum (Ap) with the objective of determining whether an early environmental signal could have been detected before the outbreak.MethodsSamples were collected for a concurrent study aiming to reduce Lyme disease risk. Between 2019 and 2021, up to 14 experimental sites were sampled for ticks and capture of small mammals took place on three sites in 2021. Samples were screened for Ap using multiplex real-time PCR, and genetic strains were identified using a single-nucleotide polymorphism assay.ResultsAnalyses showed an increase of 5.7% in Ap prevalence in ticks (CI95: 1.5–9.9) between 2019 and 2020, i.e., one year before the outbreak. A majority of Ap-positive ticks were infected with the zoonotic strain (68.8%; CI95: 50.0–83.9) during the study period. In 2021, 2 of 59 captured Peromycus mice were positive for Ap, for a prevalence of 3.4% (CI95: 0.4–11.7).ConclusionWe conclude that data collected in Bromont could have provided an early signal for an anaplasmosis risk increasing in the targeted region. This is a reminder that integrated surveillance of tick-borne diseases through structured One Health programs, i.e. systematically integrating data from humans, animals and the environment, can provide useful and timely information for better preparedness and response in public health.

A molecular survey of spotted fever group rickettsiae in introduced raccoons (Procyon lotor)

BackgroundThe raccoon Procyon lotor (Linnaeus, 1758) (Carnivora; Procyonidae) is one of the most important and most intensively studied invasive mammal species in Europe. Within the last 30 years the raccoon has spread at an increasing rate, resulting in the establishment of local populations in various regions of Europe. In these newly colonised areas, gaps in knowledge of the raccoon’s biology concern not only most aspects of its ecology in a broad sense, but also its pathogens and parasites. Most micropathogens recorded hitherto in the raccoons that have colonised Europe have documented epizootic and zoonotic potential. Thus, it is considered especially important to investigate the role played by the raccoon in the spread of pathogens through both animal-animal and animal-human pathways.MethodsTissue samples of raccoons from Poland and Germany were examined in this study. In total, 384 tissue samples from 220 raccoons (170 spleen samples, 82 liver biopsies, 132 ear biopsies) were examined using molecular methods. The presence of Rickettsia spp. DNA was screened through amplification of a fragment of the gltA gene. Samples that were PCR positive for gltA were tested for other rickettsial genes, ompB and a 17-kDa antigen. For taxonomic purposes, the obtained sequences were compared with corresponding sequences deposited in GenBank using the Basic Local Alignment Search Tool, and phylogenetic analyses were conducted using Bayesian inference implemented in MrBayes software.ResultsRickettsia DNA was confirmed only in skin biopsies; no isolates from the spleen or liver were positive for Rickettsia DNA. With the exception of one sample from Germany, which was positive for Rickettsia helvetica DNA, all the samples positive for Rickettsia DNA derived from the Polish population of raccoons. DNA of Rickettsia spp. was detected in 25 samples, i.e. 11.4% of the tested raccoons, and R. helvetica was confirmed in 52% of the positive samples. Additionally, single cases of Rickettsia monacensis, Rickettsia raoultii, and Candidatus Rickettsia kotlanii-like were found, and in 32% of all the positive samples similarity was shown to different Rickettsia endosymbionts. Out of the samples that tested positive for gltA, amplicons of ompB and 17 kDa were successfully sequenced from 14 and three samples, respectively.ConclusionsTo the best of our knowledge, this study provides, for the first time, evidence of the occurrence of Rickettsia pathogens and endosymbionts in the European population of raccoons. Further, broader research on different species of wild vertebrates, and ticks, as potential vectors and hosts for tick-borne pathogens, in natural as well as in peri-urban environments, is therefore required.Graphical abstract

Proteome profile of patients with excellent and poor speech intelligibility after cochlear implantation: Can perilymph proteins predict performance?

Modern proteomic analysis and reliable surgical access to gain liquid inner ear biopsies have enabled in depth molecular characterization of the cochlea microenvironment. In order to clarify whether the protein composition of the perilymph can provide new insights into individual hearing performance after cochlear implantation (CI), computational analysis in correlation to clinical performance after CI were performed based on the proteome profile derived from perilymph samples (liquid biopsies). Perilymph samples from cochlear implant recipients have been analyzed by mass spectrometry (MS). The proteins were identified using the shot-gun proteomics method and quantified and analyzed using Max Quant, Perseus and IPA software. A total of 75 perilymph samples from 68 (adults and children) patients were included in the analysis. Speech perception data one year after implantation were available for 45 patients and these were used for subsequent analysis. According to their hearing performance, patients with excellent (n = 22) and poor (n = 14) performance one year after CI were identified and used for further analysis. The protein composition and statistically significant differences in the two groups were detected by relative quantification of the perilymph proteins. With this procedure, a selection of 287 proteins were identified in at least eight samples in both groups. In the perilymph of the patients with excellent and poor performance, five and six significantly elevated proteins were identified respectively. These proteins seem to be involved in different immunological processes in excellent and poor performer. Further analysis on the role of specific proteins as predictors for poor or excellent performance among CI recipients are mandatory. Combinatory analysis of molecular inner ear profiles and clinical performance data using bioinformatics analysis may open up new possibilities for patient stratification. The impact of such prediction algorithms on diagnosis and treatment needs to be established in further studies.

Comprehensive Phytochemical Profiling of Polyherbal Divya-Kayakalp-Vati and Divya-Kayakalp-Oil and Their Combined Efficacy in Mouse Model of Atopic Dermatitis-Like Inflammation Through Regulation of Cytokines.

PurposeAtopic dermatitis (AD) is a chronic inflammatory disease that varies in signs and symptoms in different individuals. General symptoms include dryness of the skin, itching, and development of red to brownish-gray patches. Divya-Kayakalp-Vati (DKV) and -Oil (DKO) are Indian polyherbal compositions prescribed for treating inflammatory skin diseases. In the present study, we evaluated the anti-inflammatory efficacy of DKV and DKO co-treatment (DKV-O) in ameliorating Oxazolone (OXA)-stimulated AD-like inflammation and pro-inflammatory cytokine release in a Swiss albino mouse model.MethodsPhytochemical profiling of the DKV and DKO were done using Liquid Chromatography-Mass Spectroscopy (LC-MS) QToF. Swiss albino mice were sensitized for 7 days and treated with OXA in their ear region. Stimulated and control animals were orally treated with DKV and topically with DKO. Anti-inflammatory efficacy of DKV-O was determined in OXA-treated animals through physiological, histopathological, and biochemical parameter analysis.ResultsDKV and DKO formulations individually contained 39 and 59 phytochemicals, respectively. Many of the phytochemicals have been reported to have anti-inflammatory activities. In the OXA-sensitized Swiss albino mice, combined treatment with DKV-O, and separately with Dexamethasone (positive control) significantly reduced the OXA-stimulated ear edema, biopsy weight, and epidermal thickness. DKV-O further reduced OXA-stimulated induction of inflammatory lesions, neutrophil influx, and release of Interleukin (IL)-1β, IL-6, tumor necrosis factor-α, and myeloperoxidase.ConclusionFinally, DKV-O co-treatment showed good pharmacological effects in ameliorating AD-like inflammation through the modulation of inflammatory cell influx and release of soluble mediators. Therefore, DKV-O treatment can be used as a suitable polyherbal therapeutic against AD-like inflammatory diseases.

Detection of Clinical and Subclinical Lumpy Skin Disease Using Ear Notch Testing and Skin Biopsies.

Lumpy skin disease (LSD) diagnosis is primarily based on clinical surveillance complemented by PCR of lesion crusts or nodule biopsies. Since LSD can be subclinical, the sensitivity of clinical surveillance could be lower than expected. Furthermore, real-time PCR for the detection of LSD viral DNA in blood samples from subclinical animals is only intermittently positive. Therefore, this study aimed to investigate an acceptable, easily applicable and more sensitive testing method for the detection of clinical and subclinical LSD. An animal experiment was conducted to investigate ear notches and biopsies from unaffected skin taken from the neck and dorsal back as alternatives to blood samples. It was concluded that for early LSD confirmation, normal skin biopsies and ear notches are less fit for purpose, as LSDV DNA is only detectable in these samples several days after it is detectable in blood samples. On the other hand, blood samples are less advisable for the detection of subclinical animals, while ear notches and biopsies were positive for LSD viral DNA in all subclinically infected animals by 16 days post infection. In conclusion, ear notches could be used for surveillance to detect subclinical animals after removing the clinical animals from a herd, to regain trade by substantiating the freedom of disease or to support research on LSDV transmission from subclinical animals.

Glycerol Improves Skin Lesion Development in the Imiquimod Mouse Model of Psoriasis: Experimental Confirmation of Anecdotal Reports from Patients with Psoriasis.

Glycerol is used in many skin care products because it improves skin function. Anecdotal reports by patients on the National Psoriasis Foundation website also suggest that glycerol may be helpful for the treatment of psoriasis, although to date no experimental data confirm this idea. Glycerol entry into epidermal keratinocytes is facilitated by aquaglyceroporins like aquaporin-3 (AQP3), and its conversion to phosphatidylglycerol, a lipid messenger that promotes keratinocyte differentiation, requires the lipid-metabolizing enzyme phospholipase-D2 (PLD2). To evaluate whether glycerol inhibits inflammation and psoriasiform lesion development in the imiquimod (IMQ)-induced mouse model of psoriasis, glycerol’s effect on psoriasiform skin lesions was determined in IMQ-treated wild-type and PLD2 knockout mice, with glycerol provided either in drinking water or applied topically. Psoriasis area and severity index, ear thickness and ear biopsy weight, epidermal thickness, and inflammatory markers were quantified. Topical and oral glycerol ameliorated psoriasiform lesion development in wild-type mice. Topical glycerol appeared to act as an emollient to induce beneficial effects, since even in PLD2 knockout mice topical glycerol application improved skin lesions. In contrast, the beneficial effects of oral glycerol required PLD2, with no improvement in psoriasiform lesions observed in PLD2 knockout mice. Our findings suggest that the ability of oral glycerol to improve psoriasiform lesions requires its PLD2-mediated conversion to phosphatidylglycerol, consistent with our previous report that phosphatidylglycerol itself improves psoriasiform lesions in this model. Our data also support anecdotal evidence that glycerol can ameliorate psoriasis symptoms and therefore might be a useful therapy alone or in conjunction with other treatments.

Assessment of the Pathogenicity of Rickettsia amblyommatis, Rickettsia bellii, and Rickettsia montanensis in a Guinea Pig Model.

Members of the genus Rickettsia range from nonpathogenic endosymbionts to virulent pathogens such as Rickettsia rickettsii, the causative agent of Rocky Mountain spotted fever. Many rickettsiae are considered nonpathogenic because they have been isolated from ticks but not vertebrate hosts. We assessed the ability of three presumed endosymbionts: Rickettsia amblyommatis, Rickettsia bellii, and Rickettsia montanensis, to infect a guinea pig animal model. These species were chosen because of their high prevalence in respective tick vectors or published reports suggestive of human or animal pathogenicity. Following intraperitoneal (IP) inoculation of cell culture suspensions of R. rickettsii, R. amblyommatis, R. bellii, or R. montanensis into guinea pigs, animals were monitored for signs of clinical illness for 13 days. Ear biopsies and blood samples were taken at 2- to 3-day intervals for detection of rickettsial DNA by PCR. Animals were necropsied and internal organ samples were also tested using PCR assays. Among the six guinea pigs inoculated with R. amblyommatis, fever, orchitis, and dermatitis were observed in one, one, and three animals respectively. In R. bellii-exposed animals, we noted fever in one of six animals, orchitis in one, and dermatitis in two. No PCR-positive tissues were present in either the R. amblyommatis- or R. bellii-exposed groups. In the R. montanensis-exposed group, two of six animals became febrile, two had orchitis, and three developed dermatitis in ears or footpads. R. montanensis DNA was detected in ear skin biopsies collected on multiple days from three animals. Also, a liver specimen from one animal and spleen specimens of two animals were PCR positive. The course and severity of disease in the three experimental groups were significantly milder than that of R. rickettsii. This study suggests that the three rickettsiae considered nonpathogenic can cause either subclinical or mild infections in guinea pigs when introduced via IP inoculation.

Dietary prebiotics promote intestinal Prevotella in association with a low-responding phenotype in a murine oxazolone-induced model of atopic dermatitis

Atopic dermatitis is a chronic eczema commonly observed among children in Western countries. The gut microbiota is a significant factor in the pathogenesis, and ways to promote intestinal colonizers with anti-inflammatory capabilities are therefore favorable. The present study addressed the effects of a prebiotic, xylooligosaccharide (XOS), on the gut microbiota and ear inflammation in an oxazolone-induced dermatitis model in BALB/c mice. Mice were fed a XOS supplemented or a control diet throughout the experiment. Ear thickness and clinical skin inflammation were scored blindly after three weeks topical challenge with 0.4% oxazolone. The mice were divided into high and low responders to oxazolone-induced dermatitis based on clinical inflammation and histological evaluation of ear biopsies, and significantly fewer high responders were present in the XOS fed group. In addition, XOS fed mice had higher abundance of Prevotella spp. in their gut microbiota compared to the control fed mice. Serum IgE and ear tissue cytokine levels correlated significantly with the clinical scores, and with the abundance of Prevotella spp. The strong association between the low-responding phenotype and high abundance of Prevotella spp., indicates an alleviating effect of this intestinal colonizer in allergic sensitization. Prevotella should be considered as a relevant target for future microbiota-directed treatment strategies in atopic patients.

Identification of novel testing matrices for African swine fever surveillance.

African swine fever (ASF) is a devastating viral disease of pigs and wild boar, and it threatens global food security. We aimed to identify suitable sample matrices for use in ASF surveillance programs. Six pigs inoculated with ASFV were sampled at postmortem. Blood, bone marrow, ear biopsies, and oral, nasal, and rectal swabs were taken from all pigs. All samples were analyzed using 3 real-time PCR (rtPCR) assays and a LAMP assay. ASFV was detected at > 107 genome copies/mL in blood; bone marrow was found to provide the highest viral load. Ct values provided by the rtPCR assays were correlated, and ASFV was detected in all oral, nasal, and rectal swabs and in all ear biopsy samples irrespective of the location from which they were taken. The LAMP assay had lower sensitivity, and detected ASFV in 54 of 66 positive samples, but delivered positive results within 17 min. We identified additional sample matrices that can be considered depending on the sampling situation: bone marrow had a high probability of detection, which could be useful for decomposed carcasses. However, ear biopsies provide an appropriate, high-throughput sample matrix to detect ASFV and may be useful during surveillance programs.

In Vitro and In Vivo Screening of Wild Bitter Melon Leaf for Anti-Inflammatory Activity against Cutibacterium acnes.

Cutibacterium acnes (formerly Propionibacterium acnes) is a key pathogen involved in the development and progression of acne inflammation. The numerous bioactive properties of wild bitter melon (WBM) leaf extract and their medicinal applications have been recognized for many years. In this study, we examined the suppressive effect of a methanolic extract (ME) of WBM leaf and fractionated components thereof on live C. acnes-induced in vitro and in vivo inflammation. Following methanol extraction of WBM leaves, we confirmed anti-inflammatory properties of ME in C. acnes-treated human THP-1 monocyte and mouse ear edema models. Using a bioassay-monitored isolation approach and a combination of liquid–liquid extraction and column chromatography, the ME was then separated into n-hexane, ethyl acetate, n-butanol and water-soluble fractions. The hexane fraction exerted the most potent anti-inflammatory effect, suppressing C. acnes-induced interleukin-8 (IL-8) production by 36%. The ethanol-soluble fraction (ESF), which was separated from the n-hexane fraction, significantly inhibited C. acnes-induced activation of mitogen-activated protein kinase (MAPK)-mediated cellular IL-8 production. Similarly, the ESF protected against C. acnes-stimulated mouse ear swelling, as measured by ear thickness (20%) and biopsy weight (23%). Twenty-four compounds in the ESF were identified using gas chromatograph–mass spectrum (GC/MS) analysis. Using co-cultures of C. acnes and THP-1 cells, β-ionone, a compound of the ESF, reduced the production of IL-1β and IL-8 up to 40% and 18%, respectively. β-ionone also reduced epidermal microabscess, neutrophilic infiltration and IL-1β expression in mouse ear. We also found evidence of the presence of anti-inflammatory substances in an unfractionated phenolic extract of WBM leaf, and demonstrated that the ESF is a potential anti-inflammatory agent for modulating in vitro and in vivo C. acnes-induced inflammatory responses.

Genetic diversity of Borrelia burgdorferi sensu stricto: Novel strains from Mexican wild rodents.

Borrelia burgdorferi s.s. is a Gram-negative spirochaete, the aetiological agent of Lyme disease, the most common vector-borne disease in the Northern hemisphere. Reports on the presence of B.burgdorferi in central Mexico have been strongly criticized, since these were based only on unspecific serological methods. Furthermore, the worldwide genetic diversity of B.burgdorferi s.s. has not been evaluated. For this reason, the aim of the present study was to confirm the presence of B.burgdorferi in the central area of Mexico and to evaluate its relationship with regard to the global genetic diversity of B.burgdorferi s.s. To achieve this, fragments of the flagellin and the outer surface protein A genes were amplified from ear biopsies of the arboreal wild endemic mice Habromys schmidlyi. With these sequences, a concatenated Bayesian analysis was performed to confirm the identity of B.burgdorferi s.s. Afterwards, the global genetic diversity of this bacterial species was evaluated using our sequences and those available in GenBank. A prevalence of 10.4% (5/48) of H.schmidlyi infected with Borrelia sp. was detected, and the phylogenetic analyses confirmed the identity of B.burgdorferi s.s. Using both genes, the genetic diversity was low. However, genetic structuring analyses revealed that populations of western United States and those from Mexico formed slightly different genetic groups, separated from the populations of the rest of the world. Our study not only confirms the presence of this bacterium in central Mexico, but also shows the most southern record of this bacterium so far. It also highlights the importance of H.schmidlyi as a new potential host of this bacterial species. Our study also provides first genetic data on an incipient process of divergence in B.burgdorferi s.s. populations of eastern United States and central Mexico.

Reversal of ochronotic pigmentation in alkaptonuria following nitisinone therapy: Analysis of data from the United Kingdom National Alkaptonuria Centre.

BackgroundIncreased homogentisic acid (HGA) causes ochronosis. Nitisinone decreases HGA. The aim was to study the effect of nitisinone on the ochronosis progression.MethodsPhotographs of the eyes and ears were acquired from patients attending the National Alkaptonuria Centre (NAC) at V‐1 (pre‐baseline visit), V0 (baseline visit when 2 mg nitisinone was commenced), and yearly at V1, V2, and V3 visits. Photographs were inspected for evolution of ochronotic pigment and also scored categorically to derive eye, ear, and combined ochronosis scores. An ear cartilage biopsy was also carried out at V0 and one year after V3 (V4) and ochronotic pigment was assessed and quantitated. Visits were compared for changes in pigment. Fasting blood and 24‐hour urine samples were collected for measurement of HGA.ResultsThere were 80 AKU patients at V0, and 52, 47, and 40 at V1, V2, and V3 in the group with variable numbers (VAR Group) respectively; 23 patients attended once before V0, in the V‐1 visit. Photographs of patients show increase in eye pigment between V‐1 and V0, followed by decrease post‐nitisinone at V1, V2, and V3. Ear and combined ochronosis semiquantitative scoring showed an increase between V‐1 and V0 (P < .01), followed by a decrease at V1, V2, and V3, in the VAR group (P < .01). Ochronotic pigment in ear biopsy between V0 and V4 showed a 19.1% decrease (P < .05).ConclusionsNitisinone decreases HGA and partially reverses ochronosis.

Circulation of Rickettsia species and rickettsial endosymbionts among small mammals and their ectoparasites in Eastern Slovakia.

Bacteria belonging to the genus Rickettsia are known as causative agents of vector-borne zoonotic diseases, such as spotted fevers, epidemic typhus and endemic typhus. Different species of ticks, mites and fleas could act as reservoirs and arthropod vectors of different pathogenic Rickettsia species. The aim of this work was to establish active surveillance of Rickettsia spp. in mites, ticks and fleas collected from small mammals (rodents and shrews) in Eastern Slovakia. A total of 964 animal ear biopsies, 871 mites, 667 ticks and 743 fleas were collected from small mammals in the Košice region, Eastern Slovakia. All specimens were identified using specialized taxonomic keys, and were conserved in ethanol until DNA extraction was performed. After DNA extraction, identification of Rickettsia species was performed by PCR-based methods. The total prevalence of rickettsiae from ear biopsies was 4.6% (95% CI, 3.2-5.9), in tested mites 9.3% (95% CI, 7.4-11.2), 17.2% (95% CI, 14.3-20.1) in I. ricinus ticks and 3.5% (95% CI, 2.2-4.8) in fleas. Sequence analysis of the partial gltA gene and Rickettsia helvetica-, Rickettsia slovaca-, Rickettsia raoultii- species specific real-time PCR tests revealed the presence of R. helvetica, R. slovaca, unidentified Rickettsia and rickettsial endosymbionts. These pathogenic and symbiotic species were confirmed in the following ectoparasite species-Laelaps jettmari, Haemogamasus nidi, Laelaps agilis and Eulaelaps stabularis mites, Ixodes ricinus ticks, Ctenophthalmus solutus, C. assimilis and Megabothris turbidus fleas infesting host-Apodemus agrarius, A. flavicollis, Microtus arvalis and Myodes glareolus small mammals. These results confirm the circulation of R. helvetica, R. slovaca, unidentified Rickettsia and rickettsial endosymbionts in mites, ticks and fleas collected on small mammals in the Košice region, Eastern Slovakia.

Feeding behavior and activity of Phlebotomus pedifer and potential reservoir hosts of Leishmania aethiopica in southwestern Ethiopia.

Cutaneous leishmaniasis (CL) is a major public health concern in Ethiopia. However, knowledge about the complex zoonotic transmission cycle is limited, hampering implementation of control strategies. We explored the feeding behavior and activity of the vector (Phlebotomus pedifer) and studied the role of livestock in CL transmission in southwestern Ethiopia. Blood meal origins of engorged sand flies were determined by sequencing host DNA. A host choice experiment was performed to assess the feeding preference of P. pedifer when humans and hyraxes are equally accessible. Ear and nose biopsies from livestock were screened for the presence of Leishmania parasites. Sand flies were captured indoor and outdoor with human landing catches and CDC light traps to determine at which time and where P. pedifer is mostly active. A total of 180 P. pedifer sand flies were found to bite hosts of 12 genera. Humans were the predominant blood meal source indoors (65.9%, p < 0.001), while no significant differences were determined outdoors and in caves. In caves, hyraxes were represented in blood meals equally as humans (45.5% and 42.4%, respectively), but the host choice experiment revealed that sand flies have a significant preference for feeding on hyraxes (p = 0.009). Only a single goat nose biopsy from 412 animal samples was found with Leishmania RNA. We found that P. pedifer is predominantly endophagic (p = 0.003), but occurs both indoors and outdoors. A substantial number of sand flies was active in the early evening, which increased over time reaching its maximum around midnight. In contrast to earlier suggestions of exclusive zoonotic Leishmania transmission, we propose that there is also human-to-human transmission of CL in southwestern Ethiopia. Livestock does not play a role in CL transmission and combined indoor and outdoor vector control measures at night are required for efficient vector control.

Effect of rodent density on tick and tick-borne pathogen populations: consequences for infectious disease risk

BackgroundRodents are considered to contribute strongly to the risk of tick-borne diseases by feeding Ixodes ricinus larvae and by acting as amplifying hosts for pathogens. Here, we tested to what extent these two processes depend on rodent density, and for which pathogen species rodents synergistically contribute to the local disease risk, i.e. the density of infected nymphs (DIN).MethodsIn a natural woodland, we manipulated rodent densities in plots of 2500 m2 by either supplementing a critical food source (acorns) or by removing rodents during two years. Untreated plots were used as controls. Collected nymphs and rodent ear biopsies were tested for the presence of seven tick-borne microorganisms. Linear models were used to capture associations between rodents, nymphs, and pathogens.ResultsInvestigation of data from all plots, irrespective of the treatment, revealed a strong positive association between rodent density and nymphal density, nymphal infection prevalence (NIP) with Borrelia afzelii and Neoehrlichia mikurensis, and hence DIN’s of these pathogens in the following year. The NIP, but not the DIN, of the bird-associated Borrelia garinii, decreased with increasing rodent density. The NIPs of Borrelia miyamotoi and Rickettsia helvetica were independent of rodent density, and increasing rodent density moderately increased the DINs. In addition, NIPs of Babesia microti and Spiroplasma ixodetis decreased with increasing rodent density, which had a non-linear association with DINs of these microorganisms.ConclusionsA positive density dependence for all rodent- and tick-associated tick-borne pathogens was found, despite the observation that some of them decreased in prevalence. The effects on the DINs were variable among microorganisms, more than likely due to contrasts in their biology (including transmission modes, host specificity and transmission efficiency). The strongest associations were found in rodent-associated pathogens that most heavily rely on horizontal transmission. Our results draw attention to the importance of considering transmission mode of a pathogen while developing preventative measures to successfully reduce the burden of disease.

Generation of inner ear organoids from human pluripotent stem cells.

The sensory epithelia of the inner ear contain mechanosensitive hair cells that transmit sound, gravity and head motion signals. This protocol describes an in vitro 3D differentiation method, by which the inner ear sensory epithelium harboring hair cells are derived from human pluripotent stem cells (hPSCs). To begin the differentiation, hPSCs are aggregated in low-binding 96-well plates and treated with extracellular matrix proteins to promote epithelialization. By recapitulating signaling pathway activation and attenuation during in vivo inner ear development, the aggregates are treated with small molecules and recombinant proteins that modulate signaling pathways such as BMP, FGF and WNT in a stepwise manner. These treatments induce sequential formation of non-neural ectoderm (NNE), otic-epibranchial progenitor domain (OEPD), and otic placodes. The otic placodes subsequently undergo self-guided morphogenesis to form otic vesicles, which eventually give rise to sensory epithelia containing inner ear hair cells and supporting cells, as well as neurons forming synapses with the hair cells. These hPSC-derived inner ear sensory structures are designated human inner ear organoids. As human inner ear biopsies are nearly impossible to obtain without causing severe injuries to the auditory system of the patients, the human inner ear organoid system provides a powerful in vitro platform for studying human inner ear disease and development.