Species identification of adult ixodid ticks by Raman spectroscopy of their feces.

BACKGROUND: Ticks and tick-borne diseases pose significant challenges to cattle production, thus the species identification of ticks and knowledge on their presence, abundance, and dispersal are necessary for the development of effective control measures. The standard method of inspection for the presence of ticks is the visual and physical examination of restrained animals, but the limitations of human sight and touch can allow larval, nymphal, and unfed adult ticks to remain undetected due to their small size and site of attachment. However, Raman spectroscopy, an analytical tool widely used in agriculture and other sectors, shows promise for the identification of tick species in infested cattle. Raman spectroscopy is a non-invasive and efficient method that employs the interaction between molecules and light for the identification of the molecular constituents of specimens. METHODS: Raman spectroscopy was employed to analyze the structure and composition of tick feces deposited on host skin and hair during blood-feeding. Feces of 12 species from a total of five genera and one subgenus of ixodid ticks were examined. Spectral data were subjected to partial least squares discriminant analysis, a machine-learning model. We also used Raman spectroscopy and the same analytical procedures to compare and evaluate feces of the horn fly Haematobia irritans after it fed on cattle. RESULTS: Five genera and one sub-genus at overall true prediction rates ranging from 92.3 to 100% were identified from the Raman spectroscopy data of the tick feces. At the species level, Dermacentor albipictus, Dermacentor andersoni and Dermacentor variabilis at overall true prediction rates of 100, 99.3 and 100%, respectively, were identified. There were distinct differences between horn fly and tick feces with respect to blood and guanine vibrational frequencies. The overall true prediction rate for the separation of tick and horn fly feces was 98%. CONCLUSIONS: Our findings highlight the utility of Raman spectroscopy for the reliable identification of tick species from their feces, and its potential application for the identification of ticks from infested cattle in the field.

Detection of winter tick, Dermacentor albipictus, infestations using near infrared reflectance spectroscopy of bovine feces.

The objective of this study was to determine whether artificial infestations of D. albipictus could be detected in cattle using near infrared reflectance spectroscopy of bovine feces (fNIRS) and if detection capability was sensitive to size of tick infestation and phase of on-host stage-specific tick development. Fecal samples were collected daily from six non-infested then later tick-infested Bos taurus yearling heifers who each served as their own control. Cattle with D. albipictus infestations arising from as few as 1000 larvae were identified by fecal chemistry changes using fNIRS technology. In two separate trials, three animal pairs were infested with one of three treatment levels (low: ∼ 1000, medium: ∼ 4000, and high: ∼ 8000) of D. albipictus larvae in a repeated measures experimental design. Trial 1 consisted of tick naïve cattle while Trial 2 consisted of prior tick exposed cattle. Date of drop and daily sum of engorged female ticks were tabulated to characterize each infestation. Cluster, common factor, principal component and MANOVA analyses were used to define and assess fecal spectra changes associated with experimental stages of infestation. Cluster analyses found significant differences in fecal samples for heifer pairs in each treatment level group (low, medium, and high) in Trial 1 and then in Trial 2 from two pre-infestation control periods (outside and inside), three stages of tick development (larval feeding, nymphal feeding, adult feeding), and post-tick recovery periods. Five shifts in fecal chemistry of non-infested and tick-infested periods were identified by six clusters of NIRS fecal spectra measured between 576 and 1126 nm. The PCA's resulted in 97.56% and 97.77% for Trials 1 and 2 respectively of the total variation in the 1050 frequencies being explained by the first three principal components (P1, P2, P3). Results from the MANOVA and the Wilk's Lambda test for both trials showed highly significant evidence (p-values < 0.0001) of a difference in the means of the three principal components across the six Stages. There was significant evidence in Trial 1 (p-values = 0.0067) and Trial 2 (p-values < 0.0001) of a difference between the means of the three principal components across the three levels of tick infestation. These significant pair-wise comparisons reflect developmental phases of tick attachment and blood-feeding that define periods of increasing, peak and declining stress identified in five fecal chemistry shifts defined by six fecal spectral clusters.

Raman-based identification of tick species (Ixodidae) by spectroscopic analysis of their feces.

Ticks are blood-feeding parasites that vector a large number of pathogens of medical and veterinary importance. There are strong connections between tick and pathogen species. Timely detection of certain tick species on cattle can cease the spread of numerous devastating diseases such as Bovine babiesiosis and anaplasmosis. Detection of ticks is currently performed by slow and laborious scout-based inspection of cattle. In this study, we investigated the possibility of identification of tick species (Ixodidae) based on spectroscopic signatures of their feces. We collected Raman spectra from individual grains of feces of seven different species of ticks. Our results show that Raman spectroscopy (RS) allows for highly accurate (above 90%) differentiation between tick species. Furthermore, RS can be used to predict the tick developmental stage and differentiate between nymphs, meta-nymphs and adult ticks. We have also demonstrated that diagnostics of tick species present on cattle can be achieved using a hand-held Raman spectrometer. These findings show that RS can be used for non-invasive, non-destructive and confirmatory on-site analysis of tick species present on cattle.