Ear Tissue Samples Research Articles

Identification of immune modulating strategies to promote cartilage regeneration.

Abstract The immune response is a key modulator of regeneration. Apoptosis Signal-Regulating Kinase 1 (ASK1) loss can promote cartilage regeneration, thus we investigated how ASK1 loss modulates the immune response. Two mm biopsy punches of ear tissue samples were taken from WT and ASK1 KO mice (n=3) on day 0, 4 and 7 after injury. RNA was extracted and gene expression analyzed using the NanoString nCounter Immunology Gene Panel. Functional enrichments were analyzed using Gene Set Enrichment (GSEA) and Variation (GSVA) Analysis. GSVA showed general apoptosis and p53 pathways were significantly decreased in all KO samples, as expected. The Nanostring curated gene set across all time points and samples showed TLR, NFkB and TNF signaling down regulation. Conversely, MHC class II and cell adhesion were upregulated. GSEA indicated significant differences between cell types involved in the innate and acquired immune response. The most increased GSVA results from the Hallmark gene set was apical junction and most significant decrease was the epithelial-mesenchymal transition (EMT) and IL-6-JAK-STAT3 signaling pathway. GSEA-generated heatmaps of genes that significantly contributed to these pathways give additional insight into how ASK1 loss modulates immune signaling pathways to promote a regenerative environment. This analysis identified signaling pathways that modulate the immune response to support a regenerative environment and insights into how to clinically modulate this process.

A 76-base pair duplication within the enhancer region of the HMX1 gene causes sheep microtia

Sheep congenital microtia is characterized by underdeveloped ears and provides an ideal basis for studying human microtia. This study identified the causal mutation and regulatory mechanisms underlying this disorder. Whole-genome association analysis was conducted using 23 ear tissue samples from sheep with microtia and 28 samples from normal-eared sheep. A significant correlation was found between microtia and a 76-base pair duplication in the enhancer region of the HMX1 gene. Further analysis of offspring phenotypes confirmed an autosomal dominant inheritance pattern. Genotypic analysis showed that individuals that are homozygous for this duplication were earless, heterozygous individuals exhibited shortened ears, and wild-type individuals had normal ears. Moreover, luciferase assays confirmed that this duplication increased HMX1 gene expression, and duplication knock-in mice also exhibited shorter and narrower external ears compared to wild-type mice. Transcriptomic analysis further demonstrated that this duplication enhanced HMX1 gene expression in animal models. This study characterized the causal regulatory mutation underlying sheep microtia.

Genome-Wide Association Study of Milk Composition in Karachai Goats.

This study is first to perform a genome-wide association study (GWAS) to investigate the milk quality traits in Karachai goats. The objective of the study was to identify candidate genes associated with milk composition traits based on the identification and subsequent analysis of all possible SNPs, both genome-wide (high-confidence) and suggestive (subthreshold significance). To estimate the milk components, 22 traits were determined, including several types of fatty acids. DNA was extracted from ear tissue or blood samples. A total of 167 Karachai goats were genotyped using an Illumina GoatSNP53K BeadChip panel (Illumina Inc., San Diego, CA, USA). Overall, we identified 167 highly significant and subthreshold SNPs associated with the milk components of Karachai goats. A total of 10 SNPs were located within protein-coding genes and 33 SNPs in close proximity to them (±0.2 Mb). The largest number of genome-wide significant SNPs was found on chromosomes 2 and 8 and some of them were associated with several traits. The greatest number of genome-wide significant SNPs was identified for crude protein and lactose (6), and the smallest number-only 1 SNP-for freezing point depression. No SNPs were identified for monounsaturated and polyunsaturated fatty acids. Functional annotation of all 43 SNPs allowed us to identify 66 significant candidate genes on chromosomes 1, 2, 3, 4, 5, 8, 10, 13, 16, 18, 21, 23, 25, 26, and 27. We considered these genes potential DNA markers of the fatty acid composition of Karachai goat milk. Also, we found 12 genes that had a polygenic effect: most of them were simultaneously associated with the dry matter content and fatty acids (METTL, SLC1A 8, PHACTR1, FMO2, ECI1, PGP, ABCA3, AMDHD2). Our results suggest that the genes identified in our study affecting the milk components in Karachai goats differed from those identified in other breeds of dairy goats.

"Fading out" - genomic epidemiology of the last persistently infected BVDV cattle in Germany.

Bovine viral diarrhea virus (BVDV) is one of the most important cattle pathogens worldwide, causing major economic losses and animal welfare issues. Disease eradication programs have been implemented in several countries, including Germany where an obligatory nationwide control program is in force since 2011. As molecular epidemiology has become an essential tool to understand the transmission dynamics and evolution of BVDV, 5' untranslated region (UTR) sequences are generated from viruses present in persistently infected animals since the beginning of the BVDV control program. Here, we report the results of the sequence-based subtyping of BVDV strains found from 2018 through 2022 in calves born in Germany. In 2018, 2019 and 2020, BVDV-1d and-1b were the dominant subtypes and cases were spread throughout the area that was not yet officially declared BVDV-free at that time. In addition, BVDV-1a, -1e, -1f and -1h could rarely be detected. From 2021 onwards, subtype 1d clearly took over the dominance, while the other subtypes could be gradually nearly eliminated from the cattle population. The eradication success not only results in a drastic reduction of cases, but also in a marked reduction of strain diversity. Interestingly, before vaccination has been banned in regions and farms with a disease-free status, two live-vaccine virus strains were repeatedly detected in ear tissue samples of newborn calves (n = 14) whose mothers were immunized during gestation. The field-virus sequences are an important basis for molecular tracing and identification of potential relationships between the last outbreaks in the final phase of the German BVDV eradication program, thereby supporting classic epidemiological investigations. Furthermore, the monitoring of the composition of virus subtypes in the cattle population helps to maintain effective diagnostic methods and control measures and is an early warning system for the introduction of new pestiviruses in the naïve cattle population.

National Prevalence of Caprine Prion Protein Genetic Variability at Codons 146, 211, and 222 in Goat Herds in the United States.

Scrapie is a neurodegenerative disease that impacts sheep and goats, characterized by gradual and progressive changes in neurological function. Recent research shows that the scrapie incubation period is significantly influenced by specific variations in amino acids within the prion protein gene (PRNP). The objective of this study was to estimate the national prevalence of caprine PRNP genetic variability at codons 146, 211, and 222 in goat populations across the United States. A total of 3052 blood, ear tissue, and brain tissue samples were collected from goats from 50 states. The participating states were categorized into four Veterinary Service (VS) district regions. The samples underwent DNA extraction, and the PRNP variants corresponding to codons 146, 211, and 222 were amplified and sequenced. The analysis of PRNP variants, when compared to the PRNP reference sequence, revealed seven alleles in twelve genotypes. The homozygous 146NN, 211RR, and 222QQ alleles, which have been linked to an increased risk of scrapie, were found to be the most prevalent among all the goats. The heterozygous 222QK, 211RQ, 146SD, 146ND, and 146NS alleles and the homozygous 222KK, 146SS, and 146DD alleles, known to be associated with reduced scrapie susceptibility and a prolonged incubation period after experimental challenge, were found in 1.098% (222QK), 2.33% (211RQ), 0.58% (146SD), 3.13% (146ND), 20.68% (146NS), 0.005% (222KK), 3.31% (146SS), and 0.67% (146DD) of goats, respectively. The 222QK allele was found most frequently in goats tested from the east (VS District 1, 1.59%) and southwest (VS District 4, 1.08%) regions, whereas the 211RQ allele was found most often in goats tested from the Midwest (VS District 2, 8.03%) and east (VS District 1, 6.53%) regions. The 146NS allele was found most frequently in goats tested from the northwest (VS District 3, 29.02%) and southwest (VS District 4, 20.69%) regions. Our results showed that the prevalence of less susceptible genotypes at PRNP codon 146 may be sufficient to use genetic susceptibility testing in some herds. This may reduce the number of goats removed as part of a herd clean-up plan and may promote the selective breeding goats for less susceptible alleles in high-risk herds at the national level.

A Functional Single Nucleotide Polymorphism in the 3' Untranslated Region of the Porcine JARID2 Gene Is Associated with Aggressive Behavior of Weaned Pigs after Mixing.

In pig production, pigs often show more aggressive behavior after mixing, which adversely affects animal welfare and growth performance. The Jumonji and structural domain-rich AT interaction domain 2 (JARID2) gene plays an important role in neurodevelopment in mice and various psychiatric disorders in humans. The JARID2 gene may impact the aggressive behavior of pigs. By observing the behavior of 500 weaned pigs during the first 72 h after mixing, the ear tissue samples of the 12 most aggressive and 12 least aggressive pigs were selected for DNA resequencing based on the intensity of their aggressive behavior. Large group correlation analysis indicated that the rs3262221458 site located in the 3'-UTR region of the porcine JARID2 gene has a strong relationship with the aggressive behavior of weaned pigs. Pigs with the mutant TT genotype of rs3262221458 have more aggressive behavior than those pigs with the GG and GT genotypes. The dual luciferase assay indicated that the luciferase activity of the plasmids containing the G allele of rs326221458 was significantly less than that of plasmids containing the T allele of rs326221458 and control groups. The binding ability of miR-9828-3p to sequences containing the T allele was less than that of sequences containing the G allele. The overexpression of miR-9828-3p in porcine neuroglial cells (PNGCs) and PK15 cells significantly decreased the mRNA and protein levels of the JARID2 gene. In addition, miR-9828-3p inhibited the proliferation of PNGCs. After inhibiting miR-9828-3p, the mRNA and protein expression levels of JARID2 increased, and the proliferation of PNGCs showed an opposite trend to the cells that forced the expression of miR-9828-3p. In addition, interference with the JARID2 gene by siRNA can effectively inhibit the proliferation of PNGCs. In summary, we found that the rs326221458 locus regulates the expression of the JARID2 gene by affecting the binding of miR-9828-3p and the JARID2 gene, thereby affecting the aggressive behavior of weaned pigs after mixing.

Effect of in vitro Incubation Time and Drying on Genomic DNA Extracted from Bovine Ear Tissue Samples

Background: Use of ear punch samples as a competent source of nucleic acids from bovines, gained momentum around two decades ago, as their collection is quite easy and can be transported with minimal quantities of preservatives at ambient conditions. However, the auricular cartilage makes them more resistant to lysis, resulting in poor genomic DNA yields. The objective of the current study was to investigate whether drying can enhance the gDNA yields from ear tissue samples as a few past experiments gave an unanticipated good gDNA yields from completely dried tissue samples. Methods: Upon receipt, ear tissue samples of the cattle and buffaloes were kept at room temperature for different time intervals (in vitro incubation) and then dried at various temperatures prior to gDNA extraction, with commercially available gDNA extraction kits from two manufacturers (MN and QIAGEN). The extracted gDNA samples were assessed with spectrophotometry, flourometry and agarose gel electrophoresis for concentration, purity and integrity. Result: gDNA yields were gradually increasing in parallel to in vitro incubation time till 250 days and after that there was a decline. Further, gDNA yields were significantly higher for ear tissue samples that were not dried prior to extraction, which did not comply with the past experimental observations. Fluorometry-based quantifications revealed that drying of ear tissue samples at greater than 50°C temperatures reduced double-stranded DNA, however up to 50°C the gDNA yield increased, indicating the tissue samples can be safely transported up to 50°C in the TSUs.

Phylogenetic Analysis of Philippine Native Pigs (Sus scrofa L.) from Batanes, Quezon, and Marinduque Based on Mitochondrial DNA D-loop Markers

By analyzing the mitochondrial DNA (mtDNA) displacement loop (D-loop) region, we aimed to assess the genetic diversity of Philippine native pigs from Batanes, Quezon, and Marinduque and to determine the phylogenetic relationship between these subpopulations and other Asian and European pig breeds. Ear tissue samples were collected from the native pigs in Batanes (n = 17), Quezon (n = 37), and Marinduque (n = 40). Genomic DNA was extracted, and the D-loop sequences were amplified by PCR. The D-loop sequences of 94 Philippine samples were initially analyzed using the Kimura 2-parameter model and neighbor-joining method in MEGA7, identifying a total of 17 haplotypes designated as H1–H17. Notably, only eight haplotypes were specific to individuals coming from a particular province, whereas the remaining haplotypes were detected in samples from different provinces. Additional qualitative trait analysis that the majority of Philippine native pigs had black coat color, a plain coat pattern, short cylindrical snouts, straight head profiles, erect and forward-oriented ears, curly tails, and straight backlines. Phylogenetic analysis of the Philippine samples and 16 exotic pig breeds revealed that Philippine native pigs with H1 and H2 haplotypes formed a distinct clade with Taiwanese Type I Lanyu pig, whereas those with H3–H17 haplotypes clustered with other Asian and European pigs. Therefore, our findings suggest a close and unique relationship between Taiwanese Type I Lanyu and Philippine native pigs. The presence of several haplotypes also implies high genetic variation among Philippine native pigs. Specifically, the native pigs from Quezon and Marinduque are the most closely related, whereas those from Quezon and Batanes are the least closely related.

Characterizing the Inflammatory Microenvironment in K14-HPV16 Transgenic Mice: Mast Cell Infiltration and MicroRNA Expression.

Simple SummaryK14-HPV16 transgenic mice have proved to be a useful model to study the carcinogenic cascade induced by HPV16, the tumor microenvironment and also the epigenetic and genetic factors associated with this type of malignancy. The aim of our study was to evaluate the infiltration of mast cells in two cutaneous regions with different severity of the lesions and to identify potential microRNAs that may regulate mast cell infiltration in this model. We were able to confirm that increased mast cell infiltration is associated with progression of HPV-induced lesions, and that miR-223-3p and miR-125b-5p might be assisting this process via the regulation of mast cell chemotactic proteins.High-risk human papillomavirus (HPV) is the etiologic agent of several types of cancer. Mast cells’ role as either a driving or opposing force for cancer progression remains controversial. MicroRNAs are dysregulated in several HPV-induced cancers, and can influence mast cell biology. The aim of this study was to evaluate mast cell infiltration and to identify microRNAs potentially regulating this process. Transgenic male mice (K14-HPV16; HPV+) and matched wild-type mice (HPV−) received 7,12-Dimethylbenz[a]anthracene (DMBA) (or vehicle) over 17 weeks. Following euthanasia, chest skin and ear tissue samples were collected. Mast cell infiltration was evaluated by immunohistochemistry. MicroRNAs associated with mast cell infiltration were identified using bioinformatic tools. MicroRNA and mRNA relative expression was evaluated by RT-qPCR. Immunohistochemistry showed increased mast cell infiltration in HPV+ mice (p < 0.001). DMBA did not have any statistically significant influence on this distribution. Ear tissue of HPV+ mice showed increased mast cell infiltration (p < 0.01) when compared with chest skin samples. Additionally, reduced relative expression of miR-125b-5p (p = 0.008, 2−ΔΔCt = 2.09) and miR-223-3p (p = 0.013, 2−ΔΔCt = 4.42) seems to be associated with mast cell infiltration and increased expression of target gene Cxcl10. These results indicate that HPV16 may increase mast cell infiltration by down-regulating miR-223-3p and miR-125b-5p.

Borrelia burgdorferi (Spirochaetales: Spirochaetaceae) Infection Prevalence and Host Associations of Ticks Found on Peromyscus spp. in Maryland.

Lyme disease, caused by Borrelia burgdorferi sensu stricto and most commonly transmitted by Ixodes scapularis Say (Ixodida: Ixodidae), is the most common tick-borne disease in Maryland. Because B. burgdorferi s.s. is maintained in enzootic cycles among wild mice (Peromyscus spp) and Ixodes spp ticks, differing patterns of parasitism of ticks on mice could impact the infection prevalence with B. burgdorferi. We determined the infection prevalence of Peromyscus spp as well as questing and partially engorged nymphal ticks collected at six sites on private land in five counties in Maryland from May to August 2020. Questing nymph infection prevalence (NIP) was 14%. We trapped 1258 mice and collected 554 ticks and 413 ear tissue samples. The prevalence of infested Peromyscus spp varied based on host age and sex, with older and male mice more likely to be infested. We detected a significant difference amongst the proportion of attached Ixodes and the location of trapping. Similarly, the prevalence of B. burgdorferi infected Peromyscus spp mice varied between locations (average mouse infection prevalence was 40%), with the highest prevalence in locations where Ixodes were the most commonly found ticks. The B. burgdorferi infection prevalence in partially engorged I. scapularis nymphs retrieved from Peromyscus spp was ~36% which lends further support to the host infection prevalence. Local differences in distribution of infected vectors and reservoirs are important factors to consider when planning interventions to reduce Lyme disease risk.

Immunohistochemical and qPCR Detection of SARS-CoV-2 in the Human Middle Ear Versus the Nasal Cavity: Case Series

Viral infections have already been implicated with otitis media and sudden sensorineural hearing loss. However, the pathophysiology of COVID-19 as it relates to otologic disorders is not well-defined. With the spread of SARS-CoV-2, it is important to evaluate its colonization of middle ear mucosa. Middle ear and nasal tissue samples for quantitative RT-PCR and histologic evaluations were obtained from post-mortem COVID-19 patients and non-diseased control patients. Here we present evidence that SARS-CoV-2 colonizes the middle ear epithelium and co-localizes with the primary viral receptor, angiotensin-converting enzyme 2 (ACE2). Both middle ear and nasal epithelial cells show relatively high expression of ACE2, required for SARS-CoV-2 entry. The epithelial cell adhesion molecule (EpCAM) was use as a biomarker of epithelia. Furthermore, we found that the viral load in the middle ear is lower than that present in the nasal cavity.

Pathogenicity of an African swine fever virus strain isolated in Vietnam and alternative diagnostic specimens for early detection of viral infection

BackgroundAfrican swine fever (ASF), caused by the ASF virus (ASFV), was first reported in Vietnam in 2019 and spread rapidly thereafter. Better insights into ASFV characteristics and early detection by surveillance could help control its spread. However, the pathogenicity and methods for early detection of ASFV isolates from Vietnam have not been established. Therefore, we investigated the pathogenicity of ASFV and explored alternative sampling methods for early detection.ResultsTen pigs were intramuscularly inoculated with an ASFV strain from Vietnam (titer, 103.5 HAD50/mL), and their temperature, clinical signs, and virus excretion patterns were recorded. In addition, herd and environmental samples were collected daily. The pigs died 5–8 days-post-inoculation (dpi), and the incubation period was 3.7 ± 0.5 dpi. ASFV genome was first detected in the blood (2.2 ± 0.8) and then in rectal (3.1 ± 0.7), nasal (3.2 ± 0.4), and oral (3.6 ± 0.7 dpi) swab samples. ASFV was detected in oral fluid samples collected using a chewed rope from 3 dpi. The liver showed the highest viral loads, and ear tissue also exhibited high viral loads among 11 tissues obtained from dead pigs. Overall, ASFV from Vietnam was classified as peracute to acute form. The rope-based oral fluid collection method could be useful for early ASFV detection and allows successful ASF surveillance in large pig farms. Furthermore, ear tissue samples might be a simple alternative specimen for diagnosing ASF infection in dead pigs.ConclusionsOur data provide valuable insights into the characteristics of a typical ASFV strain isolated in Vietnam and suggest an alternative, non-invasive specimen collection strategy for early detection.

Surveillance of Ticks and Tick-Borne Pathogens in Suburban Natural Habitats of Central Maryland.

Lyme and other tick-borne diseases are increasing in the eastern United States and there is a lack of research on integrated strategies to control tick vectors. Here we present results of a study on tick-borne pathogens detected from tick vectors and rodent reservoirs from an ongoing 5-yr tick suppression study in the Lyme disease-endemic state of Maryland, where human-biting tick species, including Ixodes scapularis Say (Acari: Ixodidae) (the primary vector of Lyme disease spirochetes), are abundant. During the 2017 tick season, we collected 207 questing ticks and 602 ticks recovered from 327 mice (Peromyscus spp. (Rodentia: Cricetidae)), together with blood and ear tissue from the mice, at seven suburban parks in Howard County. Ticks were selectively tested for the presence of the causative agents of Lyme disease (Borrelia burgdorferi sensu lato [s.l.]), anaplasmosis (Anaplasma phagocytophilum), babesiosis (Babesia microti), ehrlichiosis (Ehrlichia ewingii, Ehrlichia chaffeensis, and 'Panola Mountain' Ehrlichia) and spotted fever group rickettsiosis (Rickettsia spp.). Peromyscus ear tissue and blood samples were tested for Bo. burgdorferi sensu stricto (s.s), A. phagocytophilum, Ba. microti, and Borrelia miyamotoi. We found 13.6% (15/110) of questing I. scapularis nymphs to be Bo. burgdorferi s.l. positive and 1.8% (2/110) were A. phagocytophilum positive among all sites. Borrelia burgdorferi s.s. was found in 71.1% (54/76) of I. scapularis nymphs removed from mice and 58.8% (194/330) of captured mice. Results from study on tick abundance and pathogen infection status in questing ticks, rodent reservoirs, and ticks feeding on Peromyscus spp. will aid efficacy evaluation of the integrated tick management measures being implemented.

Effect of ESR, FSHB and PRLR Genes on Sperm Traits of Landrace and Yorkshire Boars in the Tropical Environmental Conditions of Vietnam

Background: Fertility traits in pigs made a restricted progress through traditional selection. Applying marker assisted selection could improve these traits. The aim of the study was to investigate the effects of candidate genes Estrogen Receptor (ESR), Follicle Stimulating Hormone Beta (FSHB) and Prolactin Receptor (PRLR) on sperm quality traits of Landrace and Yorkshire boars under tropical conditions in Northern Vietnam.Methods: A total of 6306 ejaculates from 140 boars (92 Landrace and 48 Yorkshire) were collected to estimate sperm ejaculate volume (VOL), spermatozoon motility (MO), sperm concentration (CO) and total number of spermatozoon in ejaculate (NT). Ear tissue samples were collected for genotype identification of SNP present in ESR, FSHB and PRLR genes using PCR-RFLP technique. A mixed model was used to test the effect of different genetic and non-genetic factors on the sperm quality traits.Result: The genotype frequencies of ESR, FSHB and PRLR were in Hardy-Weinberg equilibrium for each breed. Age of boars and month of the year had significant effect on all the sperm quality traits (P less than 0.01) while effect of breed was found to be non-significant on all the sperm quality trait. FSHB gene significantly (P less than 0.05) affected VOL, MO and CO. Boars with BB genotype showed positive effect on VOL but negative effect on MO and CO in comparison with AA genotype boars. ESR gene showed only effect on VOL while PRLR affected only MO. Boars with AA and AB genotypes of ESR gene had a significantly higher VOL than those with BB genotype (P less than 0.05). For PRLR gen, AB genotype was associated with higher MO than AA genotype (P less than 0.05). These results suggest a possibility of using the existing polymorphisms in ESR, FSHB and PRLR genes to improve some sperm traits of Landrace and Yorkshire boars.

Molecular identification of Leishmania in free-ranging black and gold howler monkeys (Alouatta caraya) in northeastern Argentina

Parasitological surveys of non-human primates provides an important opportunity to better understand the epidemiology, transmission dynamics and emergence risk of anthropozoonoses such as leishmaniasis, which affect human populations in several regions accross South America. Our study area, in northeastern Argentina, can be considered a southern marginal region for the presence of leishmaniases and includes the habitat of black and gold howler monkeys, Alouatta caraya. To evaluate if A. caraya serve as potential hosts in the Leishmania cycle, we used molecular methods to examine infection by Leishmania spp. in 109 howler monkeys of different ages captured between July and August 2010. External ear tissue samples were subjected to PCR amplification for the Leishmania ribosomal internal transcribed spacer (ITS-1) and a RFLP assay with the Hae III restriction enzyme, and finally confirmed by sequencing. Nine howler monkeys (8.3%) were infected with Le. braziliensis (2.8%), Le. amazonensis (2.8%) and/or Le. infantum (3.7%). The results also suggest a case of co-infection between Le. braziliensis and Le. amazonensis. Further, we report the first observation of Le. amazonensis in the northeastern region of Argentina. The detection of Leishmania spp. in free-ranging howler monkeys gives rise to questions about the actual prevalence of the parasite in the wild, as well as if the number of infected wild monkeys detected may present a risk of leishmaniasis emergence in surronding human populations. Anyway, the presence of Leishmania spp. in A. caraya suggests the possible importance of these monkeys in the sylvatic and periurban transmission.

Rapid clearance of Borrelia burgdorferi from the blood circulation

BackgroundBorrelia burgdorferi is a tick-borne spirochete that causes Lyme borreliosis (LB). After an initial tick bite, it spreads from the deposition site in the dermis to distant tissues of the host. It is generally believed that this spirochete disseminates via the hematogenous route. Borrelia persica causes relapsing fever and is able to replicate in the blood stream. Currently the exact dissemination pathway of LB pathogens in the host is not known and controversially discussed.MethodsIn this study, we established a strict intravenous infection murine model using host-adapted spirochetes. Survival capacity and infectivity of host-adapted B. burgdorferi sensu stricto (Bbss) were compared to those of B. persica (Bp) after either intradermal (ID) injection into the dorsal skin of immunocompetent mice or strict intravenous (IV) inoculation via the jugular vein. By in vitro culture and PCR, viable spirochetes and their DNA load in peripheral blood were periodically monitored during a 49/50-day course post-injection, as well as in various tissue samples collected at day 49/50. Specific antibodies in individual plasma/serum samples were detected with serological methods.ResultsRegardless of ID or IV injection, DNA of Bp was present in blood samples up to day 24 post-challenge, while no Bbss was detectable in the blood circulation during the complete observation period. In contrast to the brain tropism of Bp, Bbss spirochetes were found in ear, skin, joint, bladder, and heart tissue samples of only ID-inoculated mice. All tested tissues collected from IV-challenged mice were negative for traces of Bbss. ELISA testing of serum samples showed that Bp induced gradually increasing antibody levels after ID or IV inoculation, while Bbss did so only after ID injection but not after IV inoculation.ConclusionsThis study allows us to draw the following conclusions: (i) Bp survives in the blood and disseminates to the host’s brain via the hematogenous route; and (ii) Bbss, in contrast, is cleared rapidly from the blood stream and is a tissue-bound spirochete.

Efficacy of adrenomedullin on cisplatin ototoxicity in rats

Purpose: The aim of this study was to evaluate the efficacy of adrenomedullin on damage associated with cisplatin ototoxicity created in an experimental animal model.Materials and Methods: A total of 32 Wistar Albino rats were separated into 4 groups of 8. To the rats in Group 1 (control), 1% acetic acid was administered intraperitoneally and intratympanically, to Group 2, cisplatin (10mg/kg/2 days) + intratympanic acetic acid, to Group 3, cisplatin + intraperitoneal adrenomedullin (12 µg/kg) and to Group 4, cisplatin +0.1- 0.3ml intratympanic adrenomedullin (12 µg/kg). The drugs were administered on the first 5 days and signal-to-noise ratio (SNR) measurements of the auditory brainstem response (ABR) and distortion product otoacoustic emissions (DPOAE) were made on days 0, 5 and 15. The rats were sacrificed and serum and ear tissue samples were taken for histopathological examination and examination of oxidative stress levels.Results: A statistically significant difference was determined in the cisplatin+intratympanic adrenomedullin group in the Auditory brain-stem response (ABR) measurements on days 0, 5 and 15 . In the Distortion product-evoked otoacoustic emission (DPOAE) measurements, the between-day differences at 16000 frequency in the cisplatin + intraperitoneal adrenomedullin group were accepted as statistically significantConclusion: Adrenomedullin, which is a strong vasodilator and antioxidant, was found to have a protective effect against cisplatin ototoxicity in the cochlea.

Subacute oral administration of folic acid elicits anti-inflammatory response in a mouse model of allergic dermatitis

Folic acid (FA) deficiency is associated with several health problems, including megaloblastic anemia and fetal neural tube defects. Therefore, supplementation with FA is strongly recommended by governments worldwide. Recent published reports indicate that FA functions in immune system maintenance. The main objective of this study is to examine possible anti-inflammatory and antipruritic effects of FA using a mouse model of allergic dermatitis. The mouse model was developed by repetitive sensitization to the Th2-type hapten toluene-2,4-diisocyanate (TDI). During the development of allergic dermatitis, FA was orally administered to the mice at doses of 8, 160, 1000 or 10,000 μg/day for 5 weeks. The ear swelling response and scratching behavior were monitored after the TDI challenge. Serum, ear tissue and auricular lymph node samples were isolated for further analysis 24 h after the TDI challenge. The ear swelling response was reduced in a dose-dependent manner by FA administration, and a significant change was observed at a concentration of 10,000-μg/day group. Comparable results were obtained through histological evaluation and cytokine level measurement in the ear tissue samples. Oral administration of FA exhibited the inhibitory effect on T-cell infiltration and T-cell-related cytokine production in auricular lymph nodes. Scratching behavior was not altered by FA administration. The in vivo evidence was corroborated by in vitro results, which showed that FA treatment significantly interfered with T-cell proliferation in a dose-dependent manner. Our findings imply that subacute oral administration of FA elicits an anti-inflammatory response, mainly through inhibition of T-cell proliferation.

A 2015 outbreak of flea-borne rickettsiosis in San Gabriel Valley, Los Angeles County, California.

Although flea-borne rickettsiosis is endemic in Los Angeles County, outbreaks are rare. In the spring of 2015 three human cases of flea-borne rickettsiosis among residents of a mobile home community (MHC) prompted an investigation. Fleas were ubiquitous in common areas due to presence of flea-infested opossums and overabundant outdoor cats and dogs. The MHC was summarily abated in June 2015, and within five months, flea control and removal of animals significantly reduced the flea population. Two additional epidemiologically-linked human cases of flea-borne rickettsiosis detected at the MHC were suspected to have occurred before control efforts began. Molecular testing of 106 individual and 85 pooled cat fleas, blood and ear tissue samples from three opossums and thirteen feral cats using PCR amplification and DNA sequencing detected rickettsial DNA in 18.8% of the fleas. Seventeen percent of these cat fleas tested positive for R. felis-specific DNA compared to under two (<2) percent for Candidatus R. senegalensis-specific DNA. In addition, serological testing of 13 cats using a group-specific IgG-ELISA detected antibodies against typhus group rickettsiae and spotted fever group rickettsiae in six (46.2%) and one (7.7%) cat, respectively. These results indicate that cats and their fleas may have played an active role in the epidemiology of the typhus group and/or spotted fever group rickettsial disease(s) in this outbreak.

Rickettsia diversity in southern Africa: A small mammal perspective

Worldwide, including Africa, rickettsioses are recognized as emerging or re-emerging infections. To date, little is known about the diversity of Rickettsia species that are naturally associated with small mammals in southern Africa. The aim of the study was to screen a diversity of small mammals for the presence of rickettsial DNA. Animals were trapped at 38 localities in South Africa and Namibia. In total, 1616 ear-tissue samples from 23 species representing 17 genera were tested using real-time (rt)PCR and multi-locus sequence typing (MLST). Of the 1616 samples 251 (15.5%) were positive in an initial rtPCR. In 16 of the 23 investigated animal species rickettsial DNA was detected with an average prevalence of 15.7%. We herein describe for the first time four Rickettsia (R.) species known to be pathogenic for humans in rodents from South Africa, R. conorii, R. massiliae, R. felis and R. helvetica. In addition, by MLST and subsequent phylogenetic analyses so far undescribed Rickettsia species, Candidatus Rickettsia africaustralis, Candidatus Rickettsia rhabdomydis, and Candidatus Rickettsia muridii were confirmed. Further four new genotypes, genotype Rickettsia hofmannii, genotype Rickettsia stutterheimensis, genotype Rickettsia hogsbackensis and genotype Rickettsia kaalplaasensis, respectively, are described. The data indicate a surprisingly high diversity of Rickettsia in small mammals in South Africa and might indicate their possible role as reservoirs for Rickettsia. Ecological questions concerning their natural hosts such as small mammals, but also the role of livestock or pet animals, require further investigation. Particularly, data on the relevance of these rickettsiae for diseases in humans are of further interest.