Platelet Lysate for Mesenchymal Stromal Cell Culture in the Canine and Equine Species: Analogous but Not the Same

Platelet lysate (PL) is an attractive platelet-based therapeutic tool and has shown promise as xeno-free replacement for fetal bovine serum (FBS) in human and equine mesenchymal stromal cell (MSC) culture. Here, we established a scalable buffy-coat-based protocol for canine PL (cPL) production (n = 12). The cPL was tested in canine adipose MSC (n = 5) culture compared to FBS. For further comparison, equine adipose MSC (n = 5) were cultured with analogous equine PL (ePL) or FBS. During canine blood processing, platelet and transforming growth factor-β1 concentrations increased (p < 0.05 and p < 0.001), while white blood cell concentrations decreased (p < 0.05). However, while equine MSC showed good results when cultured with 10% ePL, canine MSC cultured with 2.5% or 10% cPL changed their morphology and showed decreased metabolic activity (p < 0.05). Apoptosis and necrosis in canine MSC were increased with 2.5% cPL (p < 0.05). Surprisingly, passage 5 canine MSC showed less genetic aberrations after culture with 10% cPL than with FBS. Our data reveal that using analogous canine and equine biologicals does not entail the same results. The buffy-coat-based cPL was not adequate for canine MSC culture, but may still be useful for therapeutic applications.

GroEL Chaperonin-Based Assay for Early Diagnosis of Scrub Typhus

A point-of-care diagnostic for early and rapid diagnosis of scrub typhus caused by Orientia tsutsugamushi is required for prompt and proper treatment of patients presenting with undifferentiated febrile illnesses. In this study, an immunochromatographic antigen detection test kit (ICT AgTK) that targets the highly conserved O. tsutsugamushi 60 kDa GroEL chaperonin (heat shock protein 60) was developed. E. coli-derived recombinant GroEL expressed from DNA coding for the consensus sequence of 32 GroEL gene sequences extracted from the GenBank database was used to immunize rabbits and mice. Rabbit polyclonal antibodies (pAb) were used for preparing a gold-pAb conjugate, and the rGroEL-specific mouse monoclonal antibody was used as the antigen detection reagent at the ICT test line. In-house validation revealed that the ICT AgTK gave 85, 100 and 95% diagnostic sensitivity, specificity and accuracy, respectively, compared to the combined clinical features and standard IFA when tested on 40 frozen serum samples. The test kits correctly identified 10 scrub typhus samples out of 15 fresh plasma/buffy coat samples of patients with febrile illnesses. For independent laboratory validation, the ICT AgTK was sent to one provincial hospital. The ICT AgTK utilized by the hospital medical technologist correctly identified six scrub typhus samples out of 20 serum samples of patients with fever, as confirmed by specific IgM/IgG detection by IFA. The ICT AgTK is easy to perform with rapid turn-around time. It has the potential to be used as an important tool for on-site and early scrub typhus diagnosis by allowing testing of freshly collected samples (serum, plasma or buffy coat), especially in resource-limited healthcare settings.

Retrospective analysis of canine monocytic ehrlichiosis in Thailand with emphasis on hematological and ultrasonographic changes

Background and Aim: Canine monocytic ehrlichiosis (CME) is a tropical endemic tick-borne disease that causes fatality or chronic infection involving many organs in dogs. This study aimed to examine the prevalence, risk factors, and hematological and ultrasonographic changes in the liver, gallbladder, kidneys, and spleen following CME infection. Materials and Methods: This retrospective study used 30,269 samples collected from dogs at the hematology section of the pathology unit of a university veterinary hospital and 35 samples collected from dogs at the diagnostic imaging unit. CME was determined using the buffy coat smear method. Data were analyzed using descriptive statistics and odds ratios. Results: CCl4 The data revealed that the average yearly prevalence of CME was 1.32%. Risk factors contributing to CME infection were a tick on the body during physical examination, lack of ectoparasite control, and outdoor living. All 148 dogs with CME infection had low platelet counts. The percentages of CME-infected dogs with elevated serum alanine aminotransferase, alkaline phosphatase, and both enzymes above the normal range were 33.6%, 65.9%, and 29.8%, respectively. The rates for elevated serum levels of blood urea nitrogen, creatinine, and both compounds were 33.1%, 19.1%, and 17.3%, respectively. The most common ultrasonographic changes were liver abnormalities (hyperechogenicity or hypoechogenicity, hepatomegaly, and hypoechoic nodules), hyperechogenicity of the kidneys, and an enlarged spleen. These ultrasonographic changes were consistent with the hematology results, which showed a greater elevation of serum liver enzyme levels than renal enzymes. Conclusion: Ultrasonographic changes during CME infection and after treatment with doxycycline can help to monitor and identify persistent pathological changes in the target organs resulting from immune response to CME.

Buffy Coat DNA Methylation Profile Is Representative of Methylation Patterns in White Blood Cell Types in Normal Pregnancy

Background: We aimed to assess the extent to which the buffy coat DNA methylome is representative of methylation patterns in constitutive white blood cell (WBC) types in normal pregnancy.Methods: A comparison of differential methylation of buffy coat DNA vs DNA isolated from polymorphonuclear (PMN) and lymphocytic fractions was performed for each blood sample obtained within 24 h prior to delivery from 29 normotensive pregnant women. Methylation profiles were obtained using an Illumina Human Methylation 450 BeadChip and CHaMP bioinformatics pipeline. A subset of differentially methylated probes (DMPs) showing discordant methylation were further investigated using statistical modeling and enrichment analysis.Results: The smallest number of DMPs was found between the buffy coat and the PMN fraction (2.96%). Pathway enrichment analysis of the DMPs identified biological pathways involved in the particular leukocyte lineage, consistent with perturbations during isolation. The comparisons between the buffy coat and the isolated fractions as a group using linear modeling yielded a small number of probes (∼29,000) with discordant methylation. Demethylation of probes in the buffy coat compared to derived cell lines was more common and was prevalent in shelf and open sea regions.Conclusion: Buffy coat is representative of methylation patterns in WBC types in normal pregnancy. The differential methylations are consistent with perturbations during isolation of constituent cells and likely originate in vitro due to the physical stress during cell separation and are of no physiological relevance. These findings help the interpretation of DNA methylation profiling in pregnancy and numerous other conditions.

Autochthonous Transmission of Angiostrongylus Cantonensis in a Domestic Rabbit (Oryctolagus Cuniculus) in Hawai`i: Detection in Multiple Tissues Using AcanITS1 and AcanR3990 PCR Assays

Background: Hawaii is the hotspot for rat lungworm disease (angiostrongyliasis) caused by the nematode Angiostrongylus cantonensis in the USA. In humans, PCR of the CSF is typically used for diagnosis, however, collection of CSF requires hospitalization. Here, we evaluate the efficacy of two different PCR tests to detect A.cantonensis DNA in multiple tissues including blood from a rabbit presumably infected by eating contaminated lettuce. Methods: Two different PCR assays (AcanR3990, and AcanITS1) were used comparatively to test DNA extracted from slug and rabbit tissues. Assays were conducted using established protocols and were run in triplicate, with negative (dH20) controls included throughout. Results: A juvenile Parmarian martensi (semi-slug) found in local lettuce tested positive for the presence of Angiostrongylus cantonensis DNA. A family and their two domestic rabbits (Oryctolagus cuniculus) consumed this lettuce twice within the five days preceding testing. One rabbit exhibited symptoms consistent with eosinophilic meningitis 3-6 days after being fed the lettuce. Appropriate veterinary treatment was ineffective and the rabbit was subsequently euthanized. This study comparatively applies two different PCR assays to detect A. cantonensis DNA in the peripheral blood, cerebrospinal fluid, brain, heart, and lung tissue of this rabbit, and provides data implicating parasite transmission via contaminated home-grown lettuce. Six of the nine brain DNA samples, as well as the CSF sample, tested positive in replicate or triplicate for A. cantonensis DNA with both PCR assays. The AcanR3990 assay also detected A. cantonensis DNA from the lung, heart septum, all nine samples from the brain, and blood products (plasma, EDTA-treated whole blood, and buffy coat/red blood cells) in replicate or triplicate.

Viremia in Equine Herpes Virus-1 infection and a possible link to Transient Protective Immunity

Equine herpes virus (EHV-1) causes respiratory infections in equine, and results in abortion, paresis, neonatal death, and retinopathy and the virus may become latent following initial infection. Virus entry is via the respiratory route, and the virus replicates in the host in ciliated and non-ciliated epithelial cells of the respiratory tract and in Type 1 and Type 2 pneumocytes in the lung parenchyma. After viral replication in the respiratory system, the virus can become disseminated to other parts of body via viraemic cells. The virus also can cross the placenta which leads to abortion of live or dead fetuses without premonitory signs. Infected horses show transient immunity after natural or experimental infection and immune responses to EHV-1, but the immunoprotective status begins to decline after a few months of active infection. Due to the transient immune response, recovered horses are not immunoprotected and thus are prone to subsequent re-infection. Immunity is not long lived after experimental or natural infection, and as a result the development of an effective vaccine has remained a challenge. In this study viraemic cells were studied in a murine EHV-1 infection model. Mice were infected intranasally and viraemic cells were studied on days three and five which occurs during the peak of the infection. The results of this study may help to identify the nature of viraemic cells and their role in the transient immune response to infection. Buffy coat cells and lungs were removed and stained with a fluorescent antibody test for EHV-1 antigen, and lung specimens were subjected to transmission electron microscopy. Both techniques confirmed the presence of viraemic cells in lung tissues. These viraemic cells were further stained for EHV-1 antigen, and for CD4 or CD8 biomarkers and results are discussed re: pathogenesis of EHV-1 infection, identification of viraemic cells in a murine model and possible link of viraemia to transient immune responses in EHV-1 infection, which demonstrate the validity of this murine model for the investigation of the cytopathologic mechanism and sequelae of EHV manifestation in this model.

Assessment of Collagen-Based Nanostructured Biomimetic Systems with a Co-Culture of Human Bone-Derived Cells

Osteoporosis is a worldwide disease resulting in the increase of bone fragility and enhanced fracture risk in adults. In the context of osteoporotic fractures, bone tissue engineering (BTE), i.e., the use of bone substitutes combining biomaterials, cells, and other factors, is considered a potential alternative to conventional treatments. Innovative scaffolds need to be tested in in vitro systems where the simultaneous presence of osteoblasts (OBs) and osteoclasts (OCs), the two main players of bone remodeling, is required to mimic their crosstalk and molecular cooperation. To this aim, two composite materials were developed, based on type I collagen, and containing either strontium-enriched mesoporous bioactive glasses or rod-like hydroxyapatite nanoparticles. The developed nanostructured systems underwent genipin chemical crosslinking and were then tested with an indirect co-culture of human trabecular bone-derived OBs and buffy coat-derived OC precursors, for 2–3 weeks. The favorable structural and biological properties of the materials proved to successfully support the viability, adhesion, and differentiation of cells, encouraging a further investigation of the developed bioactive systems as biomaterial inks for the 3D printing of more complex scaffolds for BTE.

Free-ranging pigs identified as a multi-reservoir of Trypanosoma brucei and Trypanosoma congolense in the Vavoua area, a historical sleeping sickness focus of Côte d’Ivoire

Background The existence of an animal reservoir of Trypanosoma brucei gambiense (T. b. gambiense), the agent of human African trypanosomiasis (HAT), may compromise the interruption of transmission targeted by World Health Organization. The aim of this study was to investigate the presence of trypanosomes in pigs and people in the Vavoua HAT historical focus where cases were still diagnosed in the early 2010’s. Methods For the human survey, we used the CATT, mini-anion exchange centrifugation technique and immune trypanolysis tests. For the animal survey, the buffy coat technique was also used as well as the PCR using Trypanosoma species specific, including the T. b. gambiense TgsGP detection using single round and nested PCRs, performed from animal blood samples and from strains isolated from subjects positive for parasitological investigations. Results No HAT cases were detected among 345 people tested. A total of 167 pigs were investigated. Free-ranging pigs appeared significantly more infected than pigs in pen. Over 70% of free-ranging pigs were positive for CATT and parasitological investigations and 27–43% were positive to trypanolysis depending on the antigen used. T. brucei was the most prevalent species (57%) followed by T. congolense (24%). Blood sample extracted DNA of T. brucei positive subjects were negative to single round TgsGP PCR. However, 1/22 and 6/22 isolated strains were positive with single round and nested TgsGP PCRs, respectively. Discussion Free-ranging pigs were identified as a multi-reservoir of T. brucei and/or T. congolense with mixed infections of different strains. This trypanosome diversity hinders the easy and direct detection of T. b. gambiense. We highlight the lack of tools to prove or exclude with certainty the presence of T. b. gambiense. This study once more highlights the need of technical improvements to explore the role of animals in the epidemiology of HAT.

Molecular epidemiology of Animal African Trypanosomosis in southwest Burkina Faso

Background: Animal African Trypanosomosis (AAT) is a parasitic disease of livestock that has a major socio-economic impact in the affected areas. It is caused by several species of uniflagellate extracellular protists of the genus Trypanosoma mainly transmitted by tsetse flies: T. congolense, T. vivax and T. brucei brucei . In Burkina Faso, AAT hampers the proper economic development of the southwestern part of the country, which is yet the best watered area particularly conducive to agriculture and animal production. It was therefore important to investigate the extend of the infection in order to better control the disease. The objective of the present study was to assess the prevalence of trypanosome infections and collect data on the presence of tsetse flies. Methods: Buffy coat, Trypanosoma species-specific PCR, Indirect ELISA Trypanosoma sp and trypanolysis techniques were used on 1898 samples collected. An entomological survey was also carried out. Results: The parasitological prevalence of AAT was 1.1%, and all observed parasites were T. vivax . In contrast, the molecular prevalence was 23%, of which T. vivax was predominant (89%) followed by T. congolense (12%) and T. brucei s.l. (7.3%) with a sizable proportion as mixed infections (9.1%). T. brucei gambiense, responsible of sleeping sickness in humans, was not detected. The serological prevalence reached 49%. Once again T. vivax predominated (86.2%), but followed by T. brucei (9.6%) and T. congolense (4.2%), while 34.6% of positive samples tested positive for at least two trypanosome species. Seven samples, from six cattle and one pig, were found positive by trypanolysis. The density per trap of Glossina tachinoides and G. palpalis gambiensis was about three flies. Conclusions/Significance: Overall, our study showed a high prevalence of trypanosome infection in the area, pointing out an ongoing inadequacy of control measures.

Epigenetic Biomarkers as Diagnostic Tools for Neurodegenerative Disorders

Epigenetics is the study of heritable changes in gene expression that occur without alterations to the DNA sequence, linking the genome to its surroundings. The accumulation of epigenetic alterations over the lifespan may contribute to neurodegeneration. The aim of the present study was to identify epigenetic biomarkers for improving diagnostic efficacy in patients with neurodegenerative diseases. We analyzed global DNA methylation, chromatin remodeling/histone modifications, sirtuin (SIRT) expression and activity, and the expression of several important neurodegeneration-related genes. DNA methylation, SIRT expression and activity and neuregulin 1 (NRG1), microtubule-associated protein tau (MAPT) and brain-derived neurotrophic factor (BDNF) expression were reduced in buffy coat samples from patients with neurodegenerative disorders. Our data suggest that these epigenetic biomarkers may be useful in clinical practical for the diagnosis, surveillance, and prognosis of disease activity in patients with neurodegenerative diseases.