Analysis of interaction between the apicomplexan protozoan Toxoplasma gondii and host cells using label-free Raman Spectroscopy

Label-free imaging using Raman micro-spectroscopy (RMS) was used to characterize the spatio-temporal molecular changes of T. gondii tachyzoites and their host cell microenvironment. Raman spectral maps were recorded from isolated T. gondii tachyzoites and T. gondii-infected human retinal cells at 6 hr, 24 hr and 48 hr post-infection. Principal component analysis (PCA) of the Raman spectra of paraformaldehyde-fixed infected cells indicated a significant increase in the amount of lipids and proteins in the T. gondii tachyzoites as the infection progresses within host cells. These results were confirmed by experiments carried out on live T. gondii-infected cells and were correlated with an increase in the concentration of proteins and lipids required for the replication of this intracellular pathogen. These findings demonstrate the potential of RMS to characterize time- and spatially-dependent molecular interactions between intracellular pathogens and the host cells. Such information may be useful for discovery of pharmacological targets or screening compounds with potential neuro-protective activity for eminent effects of changes in brain infection control practices

Assessment of bioaccumulation of cu and Pb in experimentally exposed spiders, Lycosa terrestris and Pardosa birmanica, using different exposure routes

© 2019, Springer-Verlag GmbH Germany, part of Springer Nature. Major concerns exist regarding the environmental and human health risks caused by exposure to heavy metals. Spiders are often used as a model in ecotoxicological studies to assess soil pollution. Here, we measured the bioaccumulation of copper (Cu) and lead (Pb) in spiders, Lycosa terrestris and Pardosa birmanica, by inductively coupled plasma mass spectrometry (ICP-MS). We investigated whether Cu and Pb accumulation differs according to different spider species, single versus combined metal exposure, and routes of exposure. Spiders were exposed to 10mM CuSO4 and 10mM PbCl2 solutions separately or in combination (10mM + 10mM) through different exposure routes (spiked soil and food) for 6weeks. The effect of metals on the survival and body mass of exposed and unexposed (control) spiders was determined. We found that in both spider species, accumulation of metals increased with exposure time. In single metal exposure, Cu accumulation from food was higher than soil exposure in both spider species, whereas the opposite was observed for Pb. The simultaneous uptake of Cu and Pb significantly decreased from food and soil, respectively. Soil exposure caused more accumulation of metals in L. terrestris than P. birmanica. Metal exposure via contaminated food caused higher mortality compared to soil exposure. Body mass of both spider species was significantly decreased and negatively correlated with metal’s concentration. Overall, our results show that bioaccumulation efficiency of Cu and Pb differs significantly in spiders exposed to metal’s mixture compared to single metal exposure and is dependent on the exposure route, the type of metal, and spider species. More understanding of the effects of exposure to metal mixture and exposure routes is essential for designing and supporting risk assessment and ecological monitoring programs

Survey-based pilot study into the chosen therapy and prophylaxis used by UK primary care veterinary surgeons against canine angiostrongylosis

Canine Angiostrongylosis (CA), a gastropod-borne parasitic infection caused by the metastrongyloid nematode Angiostrongylus vasorum, is an important cause of significant morbidity to domestic dogs across the UK as well as in other European countries. This study aimed to ascertain the frequency at which particular drugs were used by primary care practitioners in the UK for therapy against and prophylaxis for CA. Primary care veterinary clinicians were surveyed using an online questionnaire and face-to-face or telephone interviews. Eighty-six veterinary surgeons responded. The majority of practices (n = 52) included lungworm in their standard anthelmintic protocols; moxidectin was the most common drug used for prophylaxis (n = 71). Fenbendazole was the most frequently selected drug, by 45% of vets, for treatment of confirmed cases of CA despite it being unlicensed for this purpose in the UK and the absence of a clear treatment protocol. The results of this pilot study provide an initial insight into the approach taken by primary care practitioners in their approach to CA. This provides an important starting point for future studies investigating the decision-making for CA amongst UK veterinary surgeons, particularly to clarify whether in a larger cohort an unlicensed drug remains the treatment of choice. The absence of a clear protocol for fenbendazole means that treatment of dogs affected by CA may be suboptimal, increasing the risk of morbidity and mortality

Drug discovery against acanthamoeba infections: Present knowledge and unmet needs

Although major strides have been made in developing and testing various antiacanthamoebic drugs, recurrent infections, inadequate treatment outcomes, health complications, and side effects associated with the use of currently available drugs necessitate the development of more effective and safe therapeutic regimens. For any new anti-acanthamoebic drugs to be more effective, they must have either superior potency and safety or at least comparable potency and an improved safety profile compared to the existing drugs. The development of the so-called ‘nextgeneration’ anti-acanthamoebic agents to address this challenge is an active area of research. Here, we review the current status of anti-acanthamoebic drugs and discuss recent progress in identifying novel pharmacological targets and new approaches, such as drug repurposing, development of small interfering RNA (siRNA)-based therapies and testing natural products and their derivatives. Some of the discussed approaches have the potential to change the therapeutic landscape of Acanthamoeba infections

Perception of UK companion animal veterinarians on risk assessment based parasite control

Parasites can pose a risk to companion animals and potentially their owners. Current parasiticide use is possibly impacting the environment, increasing adverse reaction and resistance risk. As such parasiticides should be dispensed by the veterinary team proportional to individual risk, including owners in their approach. A mixed-methods questionnaire was designed and distributed using snowball sampling to ascertain overall awareness, observance, and attitude towards utilising a risk assessment based approach to parasite prophylaxis by UK companion animal veterinarians. 85.7% of veterinarians surveyed reported that they were aware of risk assessment based parasite control whereas only 53.9% said they utilise it always or often. Significant correlations were found between more frequent risk assessment based parasite control utilisation and increased owner involvement (P = 0.0007) and prescription confidence (P = 0.0001). Most attitudes towards adopting risk assessment based parasite control were positive. There was significant association with positive attitude and greater utilisation frequency (P = 0.0010), as well as working in corporate practice (P = 0.0126). Awareness of risk assessment based parasite control has potential to increase responsible utilisation of parasiticides by veterinarians, and therefore mitigate risks associated. Most veterinarians would like to see the profession move towards risk assessment based control use, but institutional changes are required. Further research, and education is also needed

Metabolic footprinting of extracellular metabolites of brain endothelium infected with Neospora caninum in vitro

BACKGROUND: The survival of the intracellular protozoan parasite Neospora caninum depends on its ability to adapt to changing metabolic conditions of the host cell. Thus, defining cellular and metabolic changes in affected target tissues may aid in delineating pathogenetic mechanism. We undertook this study to assess the metabolic response of human brain microvascular endothelial cells (HBMECs) to N. caninum infection in vitro. METHODS: HBMECs were exposed to N. caninum infection and the cytotoxic effects of infection were analyzed by the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazoliumbromidin (MTT) assay and lactate dehydrogenase (LDH) release assay. Metabolic footprinting of the extracellular metabolites of parasite-infected and non-infected culture supernatant was determined by using targeted (Randox RX Imola clinical chemistry analyser) and unbiased RS (Raman microspectroscopy) approaches. RESULTS: The MTT assay did not reveal any cytotoxic effect of N. caninum challenge on host cell viability. Measurement of LDH activity showed that N. caninum significantly induced loss of cell membrane integrity in a time-dependent and dose-dependent manner compared to control cells. Targeted biochemical analysis revealed that beta hydroxybutyrate, pyruvate, ATP, total protein, non-esterified fatty acids, and triglycerides are significantly different in infected cells compared to controls. RS-based footprinting with principal component analysis (PCA) were able to correctly distinguish extracellular metabolites obtained from infected and control cultures, and revealed infection-related spectral signatures at 865 cm(−1), 984 cm(−1), 1046 cm(−1), and 1420 cm(−1), which are attributed to variations in the content of lipids and nucleic acids in infected cultures. CONCLUSIONS: The changing pattern of extracellular metabolites suggests that HBMECs are target of metabolic alterations in N. caninum infection, which seem to reflect the changing metabolic state of infected cells and constitute a level of information exchange that host and parasite use to coordinate activities

First record of besnoitiosis caused by Besnoitia bennetti in donkeys from the UK

Background: The involvement of Besnoitia bennetti in skin pathologies was investigated in a series of 20 donkeys from the Donkey Sanctuary in England, in the 2013-2019 period. Methods: The initial histopathological finding of Besnoitia cysts in skin lumps that were presumed to be sarcoids in 2013 triggered our cognisance of this parasite and resulted in identification of a total of 20 cases. Histopathological examination of surgical biopsy samples collected from 8 live donkeys and tissue specimens from 12 deceased donkeys at post-mortem examination revealed the presence of Besnoitia cysts in all 20 donkeys. The indirect fluorescent antibody test (IFAT) and immunoblotting analysis showed the presence of anti-Besnoitia antibodies in archived serum samples from 4 deceased donkeys. Additionally, infection was evidenced in one live donkey based on IFAT and immunoblot analysis of tissue fluid of a dermal mass containing Besnoitia cysts, and real-time (RT)-PCR analysis and microsatellite genotyping of DNA isolated from the tissue of the same dermal mass confirmed the infection specifically as B. bennetti. Results: Both serological and microsatellite analyses confirmed the aetiology to be B. bennetti. Our findings suggested that in cases of skin masses such as sarcoids, the suspicion of B. bennetti infection should be borne in mind even when clinical and histopathology examination results are negative in order to avoid misdiagnosis. Conclusions: This case series documents, to our knowledge, the first report of B. bennetti infection in donkeys in the UK, indicating that donkey besnoitiosis has become noteworthy in the UK. Further investigations of the occurrence, epidemiological characteristics, and clinical manifestations of B. bennetti infection in donkeys and other equids are warranted.[Figure not available: see fulltext.

Lipidomic analysis of serum from horses with strongyle infection

The development of techniques capable of accurate diagnosis of strongyle infections is at the forefront of research in equine parasitology. The aim of this study was to evaluate the potential, for using lipidomics in the diagnosis of strongyle infection. Blood and fecal samples were collected from 30 horses. Fecal egg count (FEC) results were used to select the serum samples from six uninfected horses (negative controls) and from the five horses with the highest burdens. The lipid portion of serum samples was extracted and analyzed using High Performance Liquid Chromatography-Mass Spectroscopy. The concentrations of 66 lipid metabolites differed between infected and uninfected horses (p<0.025). Database comparison of mass/charge (m/z) ratios and retention times were used to tentatively identify 16 of these metabolites. The roles of these metabolites and reasons for the observable changes were discussed. These results demonstrate the potential for the use of high resolution lipidomic analysis, for the development of a diagnostic technique capable of detecting, and perhaps stratifying, equine strongyle infection

Adverse effects of antipsychotics on micro-vascular endothelial cells of the human blood–brain barrier

Although the mechanisms of action of antipsychotics (APs) on neuronal function are well understood, very little is known about their effects on cells of the blood–brain barrier (BBB); one function of which is to limit the access of these amphiphilic compounds to the central nervous system. To address this question we have investigated the cytological and functional effects of four APs: chlorpromazine (CLP), haloperidol (HAL), risperidone (RIS) and clozapine (CLZ), at concentrations typical of high therapeutic dosage on a human brain microvascular endothelial cell (HBMEC) model of the BBB. At ~10 µM all four APs impaired the ability of HBMECs to reduce MTT which was followed by decreased Trypan blue exclusion and increased Lactate dehydrogenase release. These effects were associated with oxidative stress which was partly reversed by incubation in 10 mM glutathione. At their EC50 concentrations for MTT reduction, all four APs disrupted cellular ultrastructure and morphology. HAL, CPZ and CLZ increased Caspase -3, -8 and -9 activity, chromatin condensation and fragmentation, data indicative of apoptosis. These events were associated with decreased transcytosis of Evans blue and increased transendothelial potential difference and electrical resistance of this BBB model. These findings suggest that at high therapeutic concentrations, CPZ and CLZ are likely to incur cytoxic effects and apoptosis of BBB endothelia with an impairment of barrier functionality. Such events may underlie the aetiology of neuroleptic associated cerebral oedema and neuroleptic malignant syndrome