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This paper reports the use of near infrared and mid-infrared spectroscopy to detect the presence and quantity of eggs of the gastrointestinal nematode Haemonchus contortus in sheep faeces. Haemonchus contortus eggs were quantified in dried, finely ground sheep faeces and in moist, coarsely ground faeces using near infrared and mid-infrared bench top spectrometers and a portable near infrared spectrometer. When Haemonchus contortus eggs were presented without faecal medium, it was found that the wavelength region of 1880–2100 nm was most important for detection. Broad classes of chemical properties found in the near infrared region were identified for dried Haemonchus contortus eggs using a mid-infrared spectrometer. However, when Haemonchus contortus eggs were mixed into the complex matrix of sheep faeces, the development of a robust calibration model for egg detection proved to be challenging (R2

BackgroundExisting diagnostic methods for the parasitic gastrointestinal nematode,Haemonchus contortus, are time consuming and require specialised expertise, limiting their utility in the field. A practical, on-farm diagnostic tool could facilitate timely treatment decisions, thereby preventing losses in production and flock welfare. We previously demonstrated the ability of visible–near-infrared (Vis–NIR) spectroscopy to detect and quantify blood in sheep faeces with high accuracy. Here we report our investigation of whether variation in sheep type and environment affect the prediction accuracy of Vis–NIR spectroscopy in quantifying blood in faeces.MethodsVisible–NIR spectra were obtained from worm-free sheep faeces collected from different environments and sheep types in South Australia (SA) and New South Wales, Australia and spiked with various sheep blood concentrations. Spectra were analysed using principal component analysis (PCA), and calibration models were built around the haemoglobin (Hb) wavelength region (387–609 nm) using partial least squares regression. Models were used to predict Hb concentrations in spiked faeces from SA and naturally infected sheep faeces from Queensland (QLD). Samples from QLD were quantified using Hemastix® test strip and FAMACHA© diagnostic test scores.ResultsPrincipal component analysis showed that location, class of sheep and pooled versus individual samples were factors affecting the Hb predictions. The models successfully differentiated ‘healthy’ SA samples from those requiring anthelmintic treatment with moderate to good prediction accuracy (sensitivity 57–94%, specificity 44–79%). The models were not predictive for blood in the naturally infected QLD samples, which may be due in part to variability of faecal background and blood chemistry between samples, or the difference in validation methods used for blood quantification. PCA of the QLD samples, however, identified a difference between samples containing high and low quantities of blood.ConclusionThis study demonstrates the potential of Vis–NIR spectroscopy for estimating blood concentration in faeces from various types of sheep and environmental backgrounds. However, the calibration models developed here did not capture sufficient environmental variation to accurately predict Hb in faeces collected from environments different to those used in the calibration model. Consequently, it will be necessary to establish models that incorporate samples that are more representative of areas whereH. contortusis endemic.Graphical Abstract

Wolbachia are maternally transmitted intracellular bacteria that naturally infect over 40% of all insect species. Wolbachia have become promising biocontrol agents against mosquito-borne diseases due to their unique effects on mosquito reproduction and immunity. However, the phenotypes mediated by Wolbachia vary between strains and host-pathogen systems. Discovery of novel Wolbachia strains is essential for broadening the application of Wolbachia for mosquito control. In this study, I identified and characterized the anti-pathogen effects of natural Wolbachia infections in Australian mosquitoes. I identified three previously uncharacterized Wolbachia strains based on detection and sequencing of wsp, 16S rRNA and ftsZ genes. Wolbachia sequences were also detected from Coquillettidia xanthogaster, however, it remained unclear if the detection was resulted from a genuine infection. I established colonies of two local species that are naturally infected with Wolbachia; Aedes notoscriptus and Culex sitiens, and discovered a unique pattern of infection rates fluctuating between 15% and 60% in both colonies. Study on the maternal transmission of Wolbachia in Cx. sitiens revealed high transmission rates (99.7%) but low cytoplasmic incompatibility (9.9% mortality). I orally inoculated Wolbachia positive and negative Ae. notoscriptus and Cx. sitiens with the alphavirus Ross River virus (RRV; 103.31 – 6.48 and 105.41 – 6.36 CCID50 per mosquito, respectively). RRV infection rates varied between 17% - 85% for Ae. notoscriptus and 0% - 18% for Cx. sitiens with no significant differences between infection rates for Wol+ and Wol- mosquitoes. However, mean virus load in Wol+ Cx. sitiens was 1000× lower than in Wol- mosquitoes, suggesting that natural Wolbachia infection suppresses virus proliferation in that species. I report on progress towards transinfecting cells (Aag2 and Aa20) and mosquitoes (Aedes aegypti) with Wolbachia, including oral ingestion trials. Inoculations using the Shell vial technique resulted in temporal in vitro infections for up to three passages, however, stable infections were not yet achieved. I observed evidence that Wolbachia infected cells within the epithelial lining of the crop and potentially the midgut by fluorescence microscopy, however, the infection was transient. These discoveries broadened the current understanding on Wolbachia-host-pathogen interactions and shed light on future mosquito control strategies. The behaviour of Wolbachia in new hosts can be difficult to predict, therefore transinfection and further characterization is required to determine if these strains can induce anti-pathogen effects in the context of mosquito control.

Heavy infestations of the Barber’s pole worm, Haemonchus contortus, can cause severe wasting, morbidity and mortality in animals if not promptly treated. The current detection methods for this blood-sucking parasite involve faecal worm egg counts and diagnosis of anaemia, both of which are time consuming and require expertise. As blood is detected in sheep faeces sooner during infection than worm eggs, quantitative evaluation of blood may serve as a sensitive indicator of H. contortus infection. Here we investigated the feasibility of rapid estimation of haemoglobin (Hb) in sheep faeces using visible near-infrared spectroscopy. Haemoglobin (Hb) was assessed at various concentrations in moist sheep faeces using portable visible near-infrared (vis-NIR) spectrometers. Calibration models were developed for the region of 400–600 nm, where Hb absorption bands can be found. Within this wavelength region, Hb in sheep faeces can be estimated with minimal interference from background moisture (970 nm) or chlorophyll (670 nm), suggesting that difference in diets in sheep will have minimal effect on prediction accuracy. Predictions for sheep faeces based on a drenching threshold of 3 µg Hb/mg faeces showed high levels of accuracy with minimal sample preparation (Sensitivity = 89%, specificity = 80%). The success in detecting Hb in sheep faeces indicates the potential of vis-NIR spectroscopy as a rapid, on-farm diagnostic method for predicting blood in sheep faeces, and timing treatment of H. contortus infections.

We report the first use of near-infrared spectroscopy for rapid, low-cost, noninvasive detection of Zika virus in Aedes aegypti., The accelerating global spread of arboviruses, such as Zika virus (ZIKV), highlights the need for more proactive mosquito surveillance. However, a major challenge during arbovirus outbreaks has been the lack of rapid and affordable tests for pathogen detection in mosquitoes. We show for the first time that near-infrared spectroscopy (NIRS) is a rapid, reagent-free, and cost-effective tool that can be used to noninvasively detect ZIKV in heads and thoraces of intact Aedes aegypti mosquitoes with prediction accuracies of 94.2 to 99.3% relative to quantitative reverse transcription polymerase chain reaction (RT-qPCR). NIRS involves simply shining a beam of light on a mosquito to collect a diagnostic spectrum. We estimated in this study that NIRS is 18 times faster and 110 times cheaper than RT-qPCR. We anticipate that NIRS will be expanded upon for identifying potential arbovirus hotspots to guide the spatial prioritization of vector control.

The global approach to mosquito-borne diseases (MBDs) is in need of critical re-evaluation. Although there have been dramatic reductions in malaria incidence since 2000, malaria elimination from high-transmission settings remains problematic. At the same time, arbovirus outbreaks have increased in their frequency and impact. The 2015-2016 Zika virus epidemic exposed the dire state of MBD control in many countries, calling for united global action. Despite international resolve to prevent future epidemics, current practices in MBD control are mostly reactive and of limited efficacy. In this Opinion article, we summarize the views of 25 international mosquito experts about the current state of MBD control and highlight the issues that must be addressed in order to tackle emerging threats on the horizon.