Long-wave infrared pulsed external-cavity QCL spectrometer using a hollow waveguide gas cell.

A spectrometer built using an external cavity pulsed quantum cascade laser is described. The spectrometer has a tuning range from 10 - 13 µm (1,000 - 769 cm-1) and is designed to target volatile organic compounds (VOCs) which often exhibit water-free molecular absorption within the region. The spectrometer utilizes a hollow silica waveguide gas cell which has an internal volume of a few millilitres, a fast response time (∼1 s), and is advantageous when only low sample volumes, similar to the cell volume, are available. Propane is used as a test gas because it is easy to handle, and its spectral profile is comparable to VOCs of interest. Its absorption in the region is primarily within the ν21 band which spans from 10.55 - 11.16 µm (948 - 896 cm-1). Spectral measurements at a range of concentrations show good linearity and an Allan deviation of absorbance values recorded over a 100-minute period indicates a minimum detectable absorbance of 3.5×10-5 at an integration time of 75 s.

BEVA primary care clinical guidelines: Equine parasite control.

BACKGROUND: There is a lack of consensus on how best to balance our need to minimise the risk of parasite-associated disease in the individual horse, with the need to limit the use of anthelmintics in the population to preserve their efficacy through delaying further development of resistance. OBJECTIVES: To develop evidence-based guidelines utilising a modified GRADE framework. METHODS: A panel of veterinary scientists with relevant expertise and experience was convened. Relevant research questions were identified and developed with associated search terms being defined. Evidence in the veterinary literature was evaluated using the GRADE evidence-to-decision framework. Literature searches were performed utilising CAB abstracts and PubMed. Where there was insufficient evidence to answer the research question the panel developed practical guidance based on their collective knowledge and experience. RESULTS: Search results are presented, and recommendation or practical guidance were made in response to 37 clinically relevant questions relating to the use of anthelmintics in horses. MAIN LIMITATIONS: There was insufficient evidence to answer many of the questions with any degree of certainty and practical guidance frequently had to be based upon extrapolation of relevant information and the panel members' collective experience and opinions. CONCLUSIONS: Equine parasite control practices and current recommendations have a weak evidence base. These guidelines highlight changes in equine parasite control that should be considered to reduce the threat of parasite-associated disease and delay the development of further anthelmintic resistance.

Efficacy of flukicides against Fasciola hepatica and first report of triclabendazole resistance on German sheep farms.

Fasciola hepatica infections lead to severe health problems and production losses in sheep farming, if not treated effectively. Triclabendazole has been used extensively over decades due to its unique efficacy range against all definitive hostfluke stages but published data about the susceptibility of F. hepatica to anthelmintics in Germany are lacking. This study aimed to identify current F. hepatica infections in German sheep flocks by coproscopic examinations and to evaluate the efficacy of anthelmintics with a focus on triclabendazole in a field study conducted from 2020 to 2022. Initial screening included 71 sheep farms, many of them with known history of fasciolosis. In this highly biased sample set, the frequency of F. hepatica infection at individual sheep and farm level were 12.8% and 35.2%, respectively. Additionally, eggs of Paramphistominae were found at frequencies of 4.8% and 15.5% at individual sheep and farm level, respectively. Due to low egg shedding intensity, faecal egg count reduction (FECR) tests could only be conducted on a few farms. The efficacy of triclabendazole was tested on 11 farms and albendazole on one farm, including 3-53 sheep/farm. Individual faecal samples were collected before and two weeks after treatment to evaluate the FECR using the sedimentation or FLUKEFINDER® or a modified FLUKEFINDER® method. On all farms a coproantigen reduction test was conducted in parallel. Lacking efficacy of triclabendazole even at double dosage was shown on one farm associated with a high number of animal losses due to acute fasciolosis. On this farm, the Fasciola miracidium development test was additionally performed, revealing a high in vitro ovicidal activity of albendazole while closantel was effective in vivo. On all other farms, sufficient efficacy of triclabendazole was observed. In conclusion, triclabendazole resistance appears not to be widespread on German sheep farms but, when present, can have serious effects on animal health.

Coproscopical diagnosis of patent Fasciola hepatica infections in sheep - A comparison between standard sedimentation, FLUKEFINDER® and a combination of both.

The liver fluke Fasciola hepatica is a highly pathogenic and zoonotic trematode with a cosmopolitan distribution. In livestock, infections may lead to significant economic losses if not diagnosed promptly and treated effectively. Particularly for small ruminants, the standard method for the detection of fluke infection is based on coproscopical methods such as the sedimentation method, which detects F. hepatica eggs in faecal samples. In this respect a recent innovative coproscopical approach to diagnose patent infections is the FLUKEFINDER® method, which relies on differential sieving before sedimentation. These two methods and a combination of both methods that allows larger amounts of faeces to be processed with the FLUKEFINDER® apparatus were compared, to assess which method is most appropriate to determine the prevalence and intensity of F. hepatica egg shedding. The methods were compared for their ability to recover eggs from ovine faecal samples containing different numbers of fluke eggs per gram (EPG) of faeces and diluting the samples further by mixing with faeces from uninfected sheep. To compare the specificity of the test procedures, positive and negative samples with a low EPG were analysed in parallel by an investigator blinded to the nature of the samples. Significant differences concerning the EPG outcome were found: The FLUKEFINDER® method demonstrated the highest EPG values (p < 0.001) in the undiluted samples as well as in all mixing levels, followed by the modified FLUKEFINDER® method. The standard sedimentation showed the lowest EPG values and the highest variability between technical replicates. The precision of the FLUKEFINDER® method and the modified FLUKEFINDER® method were significantly higher than the precision of the standard sedimentation as determined by comparison of variability between technical replicates. The highest raw egg counts were detected using the modified FLUKEFINDER® method. The FLUKEFINDER® method and the combined method showed a sensitivity of 100 % even at the lowest egg concentrations, whereas the sensitivity of the standard sedimentation was 98.1 % for the same set of samples (i.e. one false negative sample). In a separate investigation aiming to estimate the specificity no differences were found between the three methods: all protocols showed 100 % specificity and were able to correctly distinguish between truly positive and truly negative samples without any evidence of cross-contamination between positive and negative samples processed in parallel.

A major locus confers triclabendazole resistance in Fasciola hepatica and shows dominant inheritance.

Fasciola hepatica infection is responsible for substantial economic losses in livestock worldwide and poses a threat to human health in endemic areas. The mainstay of control in livestock and the only drug licenced for use in humans is triclabendazole (TCBZ). TCBZ resistance has been reported on every continent and threatens effective control of fasciolosis in many parts of the world. To date, understanding the genetic mechanisms underlying TCBZ resistance has been limited to studies of candidate genes, based on assumptions of their role in drug action. Taking an alternative approach, we combined a genetic cross with whole-genome sequencing to localise a ~3.2Mbp locus within the 1.2Gbp F. hepatica genome that confers TCBZ resistance. We validated this locus independently using bulk segregant analysis of F. hepatica populations and showed that it is the target of drug selection in the field. We genotyped individual parasites and tracked segregation and reassortment of SNPs to show that TCBZ resistance exhibits Mendelian inheritance and is conferred by a dominant allele. We defined gene content within this locus to pinpoint genes involved in membrane transport, (e.g. ATP-binding cassette family B, ABCB1), transmembrane signalling and signal transduction (e.g. GTP-Ras-adenylyl cyclase and EGF-like protein), DNA/RNA binding and transcriptional regulation (e.g. SANT/Myb-like DNA-binding domain protein) and drug storage and sequestration (e.g. fatty acid binding protein, FABP) as prime candidates for conferring TCBZ resistance. This study constitutes the first experimental cross and genome-wide approach for any heritable trait in F. hepatica and is key to understanding the evolution of drug resistance in Fasciola spp. to inform deployment of efficacious anthelmintic treatments in the field.

A survey of sheep and/or cattle farmers in the UK shows confusion over the diagnosis and control of rumen fluke and liver fluke.

Calicophoron daubneyi (rumen fluke) is an emerging parasitic infection of livestock across Europe. Despite increasing in prevalence, little is known about the level of awareness of rumen fluke or current control practices used by UK farmers. Fasciola hepatica (liver fluke) is a common parasitic infection of cattle and sheep in the UK. Co-infections with these parasites can present in sheep and cattle, but the only drug with reported efficacy against rumen fluke is oxyclozanide. Between December 2019 and March 2020, 451 sheep and/or cattle farmers completed an online questionnaire, capturing their awareness and current means of control for liver fluke and rumen fluke. Most respondents (70%) were aware of rumen fluke, with 14% recording its presence on their farms and 18% having previously treated for rumen fluke. Almost all respondents (99%) were aware of liver fluke and higher numbers of respondents reported its presence on farm (67%) with 88% having previously treated for liver fluke. Respondents who were aware of rumen fluke said they were concerned about the parasite (81%), although rumen fluke was less of a concern than liver fluke (p < 0.05). Of respondents who reported rumen fluke presence on their farm, 42% cited incorrect diagnostic methods, including those traditionally used to detect liver fluke. Respondents were more likely to treat annually for liver fluke, as opposed to rumen fluke (p < 0.05). The most frequently used drug for the treatment of liver fluke infection was triclabendazole (53% sheep treatments, 34% cattle treatments) and only a minority of farmers treated with a product effective against rumen fluke (oxyclozanide; 42% cattle treatments, 35% sheep treatments). A small proportion of farmers stated that they used a non-flukicide drug to treat sheep for liver fluke infection (1.6% sheep treatments). These results demonstrate a broad awareness of liver and rumen fluke in sheep and cattle, but reveal confusion amongst farmers about their diagnosis and treatment, highlighting the need to provide best practice advice to the livestock industry for the control of both parasites.

Co-infection with Trypanosoma congolense and Trypanosoma brucei is a significant risk factor for cerebral trypanosomosis in the equid population of the Gambia.

Trypanosomosis is a major cause of morbidity and mortality in working equids in The Gambia. Recently, a progressive, severe neurological syndrome characterised by a diffuse lymphoplasmacytic meningoencephalitis has been identified and associated with Trypanosoma brucei infection of the central nervous system. The pathogenesis of cerebral trypanosomosis is unclear and the clinical syndrome not well described. This observational cross-sectional study aimed to identify host and parasite related risk factors associated with the development of cerebral trypanosomosis and to describe the neurological syndrome associated with cerebral trypanosomosis. History, signalment, clinical and laboratory parameters were collected from 326 horses and donkeys presented to The Gambia Horse and Donkey Trust. Neurological derangements in affected animals were described. Species-specific polymerase chain reaction (PCR) for Trypanosoma congolense, Trypanosoma vivax and Trypanosoma brucei was performed. The associations between signalment, clinical and laboratory parameters and PCR results were assessed using multivariable logistic regression. The overall prevalence of trypanosomosis was 50 %, with infections dominated by T. congolense (44.1 %) and a lower intensity of T. brucei (7.4 %) and T. vivax (6.5 %). Overall, 54.8 % of neurological cases were PCR positive for trypanosomosis. Within the neurological sub-population prevalence remained similar to the whole population for T. congolense (48.4 %) and T. vivax (6.5 %); whilst the prevalence increased markedly for T. brucei (32.3 %). Co-infections were identified in 32.3 % of neurological cases. Donkeys typically presented with progressive cerebral dysfunction and cranial nerve deficits, whereas in horses a progressive spinal ataxia was predominant. Mortality in affected animals was high (82.4 %). The final multivariable model identified a significant association between body condition score ≤2 (OR 11.4; 95 % CI 4.6-27.9; P = <0.001), and T. congolense and T. brucei. coinfection (OR 20.6; 95 % CI 1.71-244.1; P = 0.016) with the presence of neurological deficits. This study has provided clinically relevant information confirming the link between T. brucei and neurological disease outbreak in the equid population of The Gambia, and crucially identified co-infection with T. brucei and T. congolense as a major risk factor for the development of neurological trypanosomosis. Further research is required to identify the epidemiology of co-infection in equidae of The Gambia, so that cerebral trypanosomosis can be better prevented in this vulnerable population.

A Comparison of the Colonic Microbiome and Volatile Organic Compound Metabolome of Anoplocephala perfoliata Infected and Non-Infected Horses: A Pilot Study.

Anoplocephala perfoliata is a common equine tapeworm associated with an increased risk of colic (abdominal pain) in horses. Identification of parasite and intestinal microbiota interactions have consequences for understanding the mechanisms behind parasite-associated colic and potential new methods for parasite control. A. perfoliata was diagnosed by counting of worms in the caecum post-mortem. Bacterial DNA was extracted from colonic contents and sequenced targeting of the 16S rRNA gene (V4 region). The volatile organic compound (VOC) metabolome of colonic contents was characterised using gas chromatography mass spectrometry. Bacterial diversity (alpha and beta) was similar between tapeworm infected and non-infected controls. Some compositional differences were apparent with down-regulation of operational taxonomic units (OTUs) belonging to the symbiotic families of Ruminococcaceae and Lachnospiraceae in the tapeworm-infected group. Overall tapeworm burden accounted for 7-8% of variation in the VOC profile (permutational multivariate analysis of variance). Integration of bacterial OTUs and VOCs demonstrated moderate to strong correlations indicating the potential of VOCs as markers for bacterial OTUs in equine colonic contents. This study has shown potential differences in the intestinal microbiome and metabolome of A. perfoliata infected and non-infected horses. This pilot study did not control for extrinsic factors including diet, disease history and stage of infection.

A host-independent role for Fasciola hepatica transforming growth factor-like molecule in parasite development.

The trematode parasite Fasciola hepatica causes chronic infection in hosts, enabled by an immunosuppressed environment. Both host and parasite factors are known to contribute to this suggesting that avoidance of immunopathology is beneficial to both parties. We have previously characterised a parasite transforming growth factor (TGF)-like molecule, FhTLM, that interacts with host macrophages to prevent antibody-dependent cell cytotoxicity (ADCC). FhTLM is one of many described helminth TGF homologues and multiple helminths are now known to utilise host immune responses as developmental cues. To test whether, or how, F. hepatica uses FhTLM to manipulate host immunity, we initially examined its effects on the CD4 T-cell phenotype. Despite inducing IL-10, there was no induction of FoxP3 within the CD4 T-cell compartment. In addition to inducing IL-10, a wide range of chemokines were elicited from both CD4 T-cells and macrophages. However, no growth or survival advantage was conferred on F. hepatica in our co-culture system when CD4 T-cells, macrophages, or eosinophils were tested. Finally, using RNA interference we were able to verify a host-independent role for FhTLM in parasite growth. Despite the similarities of FhTLM with other described helminth TGF homologues, here we demonstrate species-specific divergence.

Evidence of population structuring following population genetic analyses of Fasciola hepatica from Argentina.

Fasciola hepatica, the liver fluke, is a trematode parasite that causes disease of economic importance in livestock. As a zoonosis this parasite also poses a risk to human health in areas where it is endemic. Population genetic studies can reveal the mechanisms responsible for genetic structuring (non-panmixia) within parasite populations and provide valuable insights into population dynamics, which in turn enables theoretical predictions of evolutionary dynamics such as the evolution of drug resistance. Here we genotyped 320 F. hepatica collected from 14 definitive hosts from four provinces in Argentina. STRUCTURE analysis indicated three population clusters, and principal coordinate analysis confirmed this, showing population clustering across provinces. Similarly, pairwise FST values amongst all four provinces were significant, with standardised pairwise FST (F'ST) ranging from 0.0754 to 0.6327. Therefore, population genetic structure was evident across these four provinces in Argentina. However, there was no evidence of deviation from Hardy-Weinberg equilibrium, so it appears that within these sub-populations there is largely random mating. We identified 263 unique genotypes, which gave a clonal diversity of 82%. Parasites with identical genotypes, clones, accounted for 26.6% of the parasites studied and were found in 12 of the 14 hosts studied, suggesting some clonemate transmission.

Differences in the antibody response to adult Fasciola hepatica excretory/secretory products in experimentally and naturally infected cattle and sheep.

Fasciola hepatica (the liver fluke) is a common, global parasite of livestock. It can be highly pathogenic and has health and welfare implications for infected individuals. Typically, in ruminants, infections are sub-clinical, but if undiagnosed, they can lead to significant production losses. Accurate diagnosis is crucial to identify infection. Antibody detection ELISAs are commonly used to diagnose infection due to their high sensitivity and specificity and are typically based on native fluke excretory/secretory (ES) products or cathepsin L1 (CL1), the immunodominant antigen within ES products. These tests have been developed based on the antibody response of experimentally infected animals; however, this response has not been well characterised in naturally infected animals. We compared the antibody recognition of a recombinant CL1 (rCL1) antigen and native adult fluke ES products. Whilst samples from experimentally infected animals showed strong recognition of rCL1, serum antibodies from naturally infected animals did not. These results were confirmed by peptide array. Immunoblotting sera against ES products showed that experimentally infected animals had a strong, specific response to CL1/CL2 proteins whilst antibodies from naturally infected animals recognised multiple proteins and had a variable response to CL1/CL2. Mass spectrometry of proteins separated by 2D SDS PAGE, identified several antigens recognised by serum antibodies from a naturally infected cow, including cathepsins L1, L2 and L5, glutathione S-transferase and a dihydrolipoyl dehydrogenase. Overall, these results show that the antibody response in naturally infected animals to adult fluke ES products is qualitatively different to experimentally infected animals. This suggests that a diagnostic test based on CL1 alone may not be appropriate for diagnosis of natural F. hepatica infections in sheep and cattle.

No Worm Is an Island; The Influence of Commensal Gut Microbiota on Cyathostomin Infections.

The importance of the gut microbiome for host health has been the subject of intense research over the last decade. In particular, there is overwhelming evidence for the influence of resident microbiota on gut mucosal and systemic immunity; with significant implications for the outcome of gastrointestinal (GI) infections, such as parasitic helminths. The horse is a species that relies heavily on its gut microbiota for GI and overall health, and disturbances in this complex ecosystem are often associated with life-threatening disease. In turn, nearly all horses harbour parasitic helminths from a young age, the most prevalent of which are the small strongyles, or cyathostomins. Research describing the relationship between gut microbiota and cyathostomin infection is in its infancy, however, to date there is evidence of meaningful interactions between these two groups of organisms which not only influence the outcome of cyathostomin infection but have long term consequences for equine host health. Here, we describe these interactions alongside supportive evidence from other species and suggest novel theories and avenues for research which have the potential to revolutionize our approach to cyathostomin prevention and control in the future.

Anaerobic fermentation results in loss of viability of Fasciola hepatica metacercariae in grass silage.

The parasitic liver fluke, Fasciola hepatica, has a detrimental impact on food security and poses a welfare concern to ruminant livestock. F. hepatica metacercariae, shed from an intermediate mud snail host, encyst on vegetation and present a source of infection to grazing livestock. Feeding grass silage to ruminants is a common practice, however the role it plays in the transmission of F. hepatica remains largely unknown. Our current understanding relies on historical studies that are not representative of current silage production and did not apply molecular methods to detect F. hepatica DNA persistence within silages. This study determined the impact of specific fermentation factors, including grass dry matter (DM) content (20, 30 & 40 %), length of ensiling period and maintaining an anaerobic environment on F. hepatica metacercariae viability. In vitro excystment assays demonstrated that regardless of grass DM content, metacercariae ensiled under anaerobic conditions were not viable from two weeks post-sealing. Metacercariae recovered from ensiled grass of 20 % DM content subjected to aerobic spoilage, remained viable for up to 10 weeks. DNA of F. hepatica remained detectable for up to 10 weeks in both anaerobic and spoiled silages. This study highlights i) the importance of maintaining an anaerobic ensiling environment to eliminate the risk of F. hepatica transmission from silage and ii) an inverse relationship between grass DM content and duration of metacercariae survival within spoiled silages. Improving our understanding of trematode metacercariae survival rates within silages, especially of highly pathogenic species such as F. hepatica, allows farmers to make informed decisions regarding on-farm parasite control.

Instrumentation for quantitative analysis of volatile compounds emission at elevated temperatures. Part 2: Analysis of carbon fibre reinforced epoxy composite.

We have investigated the release of gases and volatile organic compounds (VOCs) from a carbon fibre reinforced epoxy composite matrix used in aircraft structural components. Analysis was performed at several temperatures both up to and above the recommended operating temperature (121 °C) for the material, to a maximum of 250 °C. Gas chromatography-mass spectrometry (GC-MS) combined with thermal desorption (TD-GC-MS) was used to identify and quantify VOCs, and in parallel real-time gas detection with commercial off-the-shelf (COTS) gas sensors. Under hydrocarbon free air, CO, SO2, NO, NO2 and VOCs (mainly aldehydes, ketones and a carboxylic acid) were detected as the gaseous products released during the thermal exposure of the material up to 250 °C, accompanied by increased relative humidity (4%). At temperatures up to 150 °C, gas and volatile emission was limited.

Instrumentation for quantitative analysis of volatile compounds emission at elevated temperatures. Part 1: Design and implementation.

A novel suite of instrumentation for the characterisation of materials held inside an air-tight tube furnace operated up to 250 °C has been developed. Real-time detection of released gases (volatile organic compounds (VOCs), CO2, NO, NO2, SO2, CO and O2) was achieved combining commercial off-the-shelf (COTS) gas sensors and sorbent tubes for further qualitative and semi-quantitative analysis by gas chromatography-mass spectrometry coupled to thermal desorption (TD-GC-MS). The test system was designed to provide a controlled flow (1000 cm3 min-1) of hydrocarbon free air through the furnace. The furnace temperature ramp was set at a rate of 5 °C min-1 with 10 min dwell points at 70 °C, 150 °C, 200 °C and 250 °C to allow time for stabilisation and further headspace sampling onto sorbent tubes. Experimental design of the instrumentation is described here and an example data set upon exposure to a gas sample is presented.

A survey of the level of horse owner uptake of evidence-based anthelmintic treatment protocols for equine helminth control in the UK.

Interval treatment control programmes used widely in equine helminth control have favoured the development of anthelmintic resistance worldwide. Best practice guidelines have been designed to address resistance and include the requirement for improved pasture hygiene to break helminth transmission cycles, along with anthelmintic application informed by the results of diagnostic tests to reduce selection pressure for resistance. Using an online questionnaire, this study examined uptake of measures recommended in these guidelines by UK horse owners. The survey comprised 58 questions spanning grazing management, anthelmintic use and use of faecal egg count (FEC) testing to inform treatment decisions. Analysis was carried out using a combination of Chi-square and Mann-Whitney tests. In total, 705 owners responded and, following specific exclusion criteria, the responses of 652 individuals were analysed. The majority of the respondents owned <20 horses on private premises or livery yards in England. The main outputs of the survey were as follows. Overall, 60.9% of respondents used FEC tests to inform the requirement to administer anthelmintics, with macrocyclic lactones the most frequently-used anthelmintics. Of the respondents, 38% obtained advice on anthelmintic choice from their veterinarians; however, many respondents (43.8%) purchased anthelmintics via the internet. Encouragingly, 74.4% of respondents stated that they practiced good pasture hygiene by removing dung from pasture. Generally, there were differences between the responses of participants who based anthelmintic treatments on FEC testing (targeted treatments; TT) and those who practiced calendar-based anthelmintic treatments (interval treatments; IT). Briefly, the "key" findings from the Chi-square analysis included higher levels of satisfaction with the level of knowledge about equine parasites/parasitic diseases and higher levels of concern about anthelmintic resistance from TT-respondents compared to IT-participants. Confusion on the interpretation of quarantine recommendations was identified in this study group and there was poor uptake of testing for anthelmintic effectiveness. Overall, compared to previous reports, this study indicated improved engagement of UK horse owners with some helminth control practices recommended to reduce the spread of anthelmintic resistance. However, a proportion of respondents did not utilise these practices and there were still important gaps in the use of appropriate quarantine and efficacy testing. These identified gaps must be taken into consideration in knowledge dissemination activities in the future.