Random forest classification as a tool in epidemiological modelling: Identification of farm-specific characteristics relevant for the occurrence of Fasciola hepatica on German dairy farms.

Fasciola hepatica is an internal parasite of both human and veterinary relevance. In order to control fasciolosis, a multitude of attempts to predict the risk of infection such as risk maps or forecasting models have been developed. These attempts mainly focused on the influence of geo-climatic and meteorological features. Predicting bovine fasciolosis on farm level taking into account farm-specific settings yet remains challenging. In the present study, a new methodology for this purpose, a data-driven machine learning approach using a random forest classification algorithm was applied to a cross-sectional data set of farm characteristics, management regimes, and farmer aspects within two structurally different dairying regions in Germany in order to identify factors relevant for the occurrence of F. hepatica that could predict farm-level bulk tank milk positivity. The resulting models identified farm-specific key aspects in regard to the presence of F. hepatica. In study region North, farm-level production parameters (farm-level milk yield, farm-level milk fat, farm-level milk protein), leg hygiene, body condition (prevalence of overconditioned and underconditioned cows, respectively) and pasture access were identified as features relevant in regard to farm-level F. hepatica positivity. In study region South, pasture access together with farm-level lameness prevalence, farm-level prevalence of hock lesions, herd size, parity, and farm-level milk fat appeared to be important covariates. The stratification of the analysis by study region allows for the extrapolation of the results to similar settings of dairy husbandry. The local, region-specific modelling of F. hepatica presence in this work contributes to the understanding of on-farm aspects of F. hepatica appearance. The applied technique represents a novel approach in this context to model epidemiological data on fasciolosis which allows for the identification of farms at risk and together with additional findings in regard to the epidemiology of fasciolosis, can facilitate risk assessment and deepen our understanding of on-farm drivers of the occurrence of F. hepatica.

Associations of production characteristics with the on-farm presence of Fasciola hepatica in dairy cows vary across production levels and indicate differences between breeds.

Fasciola hepatica is one of the economically most important endoparasites in cattle production. The aim of the present work was to evaluate the relevance of production level on the associations of on-farm presence of F. hepatica with farm-level milk yield, milk fat, and milk protein in Holstein cows, a specialised dairy breed, and in Simmental cows, a dual purpose breed. Furthermore, we investigated whether differential associations were present depending on breed. Data from 560 dairy farms across Germany housing 93,672 cows were analysed. The presence of F. hepatica antibodies was determined via ELISA on bulk tank milk samples. Quantile regression was applied to model the median difference in milk yield, milk fat, and milk protein depending on the interaction of breed and fluke occurrence. Whereas a reduction in milk yield (-1,206 kg, p < 0.001), milk fat (-22.9 kg, p = 0.001), and milk protein (-41.6 kg, p <0.001) was evident on F. hepatica positive German Holstein farms, only milk fat (-33.8 kg, p = 0.01) and milk protein (-22.6 kg, p = 0.03) were affected on F. hepatica positive German Simmental farms. Subsequently, production traits were modelled within each of the two breeds for low, medium, and high producing farms in the presence of F. hepatica antibodies and of confounders. On Holstein farms, the presence of F. hepatica seropositivity was associated with lower production, while on German Simmental farms such an association was less evident. This work demonstrates that production level is relevant when assessing the associations between the exposure to F. hepatica with production characteristics. Moreover, both models indicate a breed dependence. This could point towards a differential F. hepatica resilience of specialised dairy breeds in comparison with dual purpose breeds.

Multinomial logistic regression based on neural networks reveals inherent differences among dairy farms depending on the differential exposure to Fasciola hepatica and Ostertagia ostertagi.

Fasciola hepatica and Ostertagia ostertagi are cattle parasites with worldwide relevance for economic outcome as well as animal health and welfare. The on-farm exposure of cattle to both parasites is a function of host-associated, intrinsic, as well as environmental and farm-specific, extrinsic, factors. Even though knowledge on the biology of both parasites exists, sophisticated and innovative modelling approaches can help to deepen our understanding of key aspects fostering the exposure of dairy cows to these pathogens. In the present study, multiple multinomial logistic regression models were fitted via neural networks to describe the differences among farms where cattle were not exposed to either F. hepatica or O. ostertagi, to one parasite, or to both, respectively. Farm-specific production and management characteristics were used as covariates to portray these differences. This elucidated inherent farm characteristics associated with parasite exposure. In both studied regions, pasture access for cows, farm-level milk yield, and lameness prevalence were identified as relevant factors. In region 'South', adherence to organic farming principles was a further covariate of importance. In region 'North', the prevalence of cows with a low body condition score, herd size, hock lesion prevalence, farm-level somatic cell count, and study year appeared to be of relevance. The present study broadens our understanding of the complex epidemiological scenarios that could predict differential farm-level parasite status. The analyses have revealed the importance of awareness of dissimilarities between farms in regard to the differential exposure to F. hepatica and O. ostertagi. This provides solid evidence that dynamics and relevant factors differ depending on whether or not cows are exposed to F. hepatica, O. ostertagi, or to both.

A machine learning approach using partitioning around medoids clustering and random forest classification to model groups of farms in regard to production parameters and bulk tank milk antibody status of two major internal parasites in dairy cows.

Fasciola hepatica and Ostertagia ostertagi are internal parasites of cattle compromising physiology, productivity, and well-being. Parasites are complex in their effect on hosts, sometimes making it difficult to identify clear directions of associations between infection and production parameters. Therefore, unsupervised approaches not assuming a structure reduce the risk of introducing bias to the analysis. They may provide insights which cannot be obtained with conventional, supervised methodology. An unsupervised, exploratory cluster analysis approach using the k-mode algorithm and partitioning around medoids detected two distinct clusters in a cross-sectional data set of milk yield, milk fat content, milk protein content as well as F. hepatica or O. ostertagi bulk tank milk antibody status from 606 dairy farms in three structurally different dairying regions in Germany. Parasite-positive farms grouped together with their respective production parameters to form separate clusters. A random forests algorithm characterised clusters with regard to external variables. Across all study regions, co-infections with F. hepatica or O. ostertagi, respectively, farming type, and pasture access appeared to be the most important factors discriminating clusters (i.e. farms). Furthermore, farm level lameness prevalence, herd size, BCS, stage of lactation, and somatic cell count were relevant criteria distinguishing clusters. This study is among the first to apply a cluster analysis approach in this context and potentially the first to implement a k-medoids algorithm and partitioning around medoids in the veterinary field. The results demonstrated that biologically relevant patterns of parasite status and milk parameters exist between farms positive for F. hepatica or O. ostertagi, respectively, and negative farms. Moreover, the machine learning approach confirmed results of previous work and shed further light on the complex setting of associations a between parasitic diseases, milk yield and milk constituents, and management practices.

Borrelia Infections in Ageing Ticks: Relationship with Morphometric Age Ratio in Field-Collected Ixodes ricinus Nymphs.

In Europe, Ixodes ricinus plays a major role as a vector of Borrelia burgdorferi sensu lato (s.l.) spirochaetes, the causative agents of Lyme borreliosis, among other pathogens. In unfed ticks, Borrelia spirochaetes experience prolonged nutrient restriction. However, only few studies exist with regard to Borrelia infections in unfed ticks of different physiological ages. Changing body dimensions of unfed ticks, due to the consumption of energy reserves, allow physiological age estimation. The present study investigated the relationship of morphometric age with Borrelia prevalence and spirochaete load in 1882 questing I. ricinus nymphs, collected at two different locations in northern Germany in 2020. In addition, Borrelia species composition was investigated by employing a reverse line blot (RLB) probe panel suitable for the detection of ten different B. burgdorferi s.l. species, as well as the relapsing-fever spirochaete B. miyamotoi. Overall, Borrelia prevalence was 25.8% (485/1882). Whilst there was no statistically significant difference in Borrelia prevalence between the different morphometric age groups, Borrelia infection intensity as determined by probe-based quantitative real-time PCR significantly declined with increasing morphometric age. Borrelia species differentiation by RLB was successful in 29.5% of positive ticks, and revealed B. afzelii as the dominating species (65.0% of the differentiated infections). Additionally, B. garinii, B. valaisiana, B. burgdorferi sensu stricto, B. spielmanii, and B. miyamotoi were detected.

Pasture rewetting in the context of nature conservation shows no long-term impact on endoparasite infections in sheep and cattle.

BACKGROUND: Nature conservation with reduced drainage of pastures has been increasingly promoted in agriculture in recent years. However, moisture on pastures is a crucial factor for the development of free-living stages of many parasite species in ruminants. Hence, for the first time, we conducted a field study between 2015 and 2017 at the German North Sea coast to investigate the long-term effect of pasture rewetting (since 2004) on endoparasite infections in sheep and cattle. METHODS: We examined faecal samples of 474 sheep and 646 cattle from five farms in spring, summer and autumn each year for the presence of endoparasite infections. Animals were kept on conventionally drained, undrained and rewetted pastures. The association between pasture rewetting and endoparasite infection probability was analysed in generalized linear mixed models and including further potential confounders. RESULTS: Infection frequencies for gastrointestinal strongyles, Eimeria spp. and Strongyloides papillosus were significantly higher in sheep (62.9%, 31.7% and 16.7%) than in cattle (39.0%, 19.7% and 2.6%). Fasciola hepatica was detected with a frequency of 13.3% in sheep and 9.8% in cattle, while rumen fluke frequency was significantly higher in cattle (12.7%) than in sheep (3.8%). Nematodirus spp., lungworms (protostrongylids, Dictyocaulus viviparus), Moniezia spp., Trichuris spp. and Dicrocoelium dendriticum were identified in less than 7% of samples. Co-infection with more than three endoparasite taxa was present significantly more often in sheep than in cattle. We identified significant positive correlations above 0.2 for excretion intensities between S. papillosus with strongyles, Eimeria spp. and Nematodirus spp. in sheep and between strongyles and Nematodirus spp. in cattle. Pasture rewetting had no long-term effect on endoparasite infections, neither in sheep nor in cattle. Interestingly, F. hepatica infections decreased significantly in sheep and cattle from 2015 (10.9% and 13.9%) to 2017 (1.4% and 2.1%). CONCLUSIONS: Pasture rewetting for nature conservation did not increase endoparasite infection probability in ruminants in the long term. This finding should be confirmed in ongoing studies aimed at further animal welfare parameters. The rapid decrease in F. hepatica infections over 3 years may suggest climatic impact or competition with rumen flukes in addition to potential anthelmintic treatment after feedback of the results to the farmers.

Seroprevalence of Major Pasture-Borne Parasitoses (Gastrointestinal Nematodes, Liver Flukes and Lungworms) in German Dairy Cattle Herds, Association with Management Factors and Impact on Production Parameters.

Pasture-borne parasites adversely affect bovine health and productivity worldwide. In Europe, gastrointestinal nematodes, especially Ostertagia ostertagi, the liver fluke Fasciola hepatica and the lungworm Dictyocaulus viviparus represent the most important parasites of dairy cattle. The present study assessed exposure towards these parasites among 646 cattle herds in three parts of Germany during 2017-2019 via antibody detection in bulk tank milk (BTM). Overall, O. ostertagi levels indicative of production losses were detected in 41.2% (266/646; 95% confidence interval (CI): 37.4-45.1%) of BTM samples, while F. hepatica seroprevalence amounted to 14.9% (96/646; 95% CI: 12.2-17.9%). Only 2.3% (15/646; 95% CI: 1.4-3.9%) of samples were D. viviparus antibody-positive. Significantly lower O. ostertagi as well as F. hepatica seroprevalence was detected in dual-purpose breeds compared to high-performance breeds from the same region. Management factors related to parasite exposure included access to fresh grass and hay, silage quality and anthelmintic treatment. Furthermore, F. hepatica and O. ostertagi seropositivity was significantly associated with suboptimal herd-level body condition. Interestingly, the relationship between seropositivity and productivity differed between breed types. Negative impacts on milk yield were detected only in high-performance breeds, while O. ostertagi seropositivity was associated with a lower milk fat content in dual-purpose herds.

Dengue emergence in the temperate Argentinian province of Santa Fe, 2009-2020.

Dengue virus (DENV) transmission occurs primarily in tropical and subtropical climates, but within the last decade it has extended to temperate regions. Santa Fe, a temperate province in Argentina, has experienced an increase in dengue cases and virus circulation since 2009, with the recent 2020 outbreak being the largest in the province to date. The aim of this work is to describe spatio-temporal fluctuations of dengue cases from 2009 to 2020 in Santa Fe Province. The data presented in this work provide a detailed description of DENV transmission for Santa Fe Province by department. These data are useful to assist in investigating drivers of dengue emergence in Santa Fe Province and for developing a better understanding of the drivers and the impacts of ongoing dengue emergence in temperate regions across the world. This work provides data useful for future studies including those investigating socio-ecological, climatic, and environmental factors associated with DENV transmission, as well as those investigating other variables related to the biology and the ecology of vector-borne diseases.

A new ELISA and western blot technique based on recombinant TES antigen and/or larval antigen for the detection of toxocariasis in humans.

Serological antibody detection by enzyme-linked immunosorbent assay (ELISA)- and immunoblot-based methods constitutes the best indicator of human Toxocara infection. Nevertheless, the availability of serological tests, particularly western blots (WB), evaluated for sensitivity and specificity is limited. Therefore, an Anti-Toxocara-ELISA immunoglobulin g (IgG) prototype (Proto-ELISA) and an Anti-Toxocara-Westernblot (IgG) prototype (Proto-WB) were evaluated by testing 541 human sera pre-determined for Toxocara infection by an established in-house Anti-Toxocara-ELISA (IH-ELISA). To evaluate sensitivity and specificity of the newly developed ELISA and WB prototypes, results were compared to IH-ELISA and a commercial WB (Com-WB). Compared to the IH-ELISA, a sensitivity of 93.1% (229/246) and a specificity of 94.6% (279/295) of the Proto-ELISA with a Cohen's κ of 0.88 were obtained. The sensitivity of the Proto-WB was 76.7% (240/313) and specificity was 99.6% (227/228) with a Cohen's κ of 0.73 compared to those of Com-WB. A comparison to the IH-ELISA revealed 91.5% (225/246) sensitivity and 94.6% (279/295) specificity of the Proto-WB with a Cohen's κ of 0.86. Cross-reactivity was observed for some samples positive for Ascaris and Trichinella spp. in the Proto-ELISA, Proto-WB and Com-WB. Overall, the evaluated ELISA and WB prototypes showed high sensitivity and specificity, indicating high reliability of these newly developed tests.

Transovarial transmission of Borrelia spp., Rickettsia spp. and Anaplasma phagocytophilum in Ixodes ricinus under field conditions extrapolated from DNA detection in questing larvae.

BACKGROUND: Ixodes ricinus constitutes the main European vector tick for the Lyme borreliosis pathogen Borrelia burgdorferi (sensu lato), the relapsing fever borrelia Borrelia miyamotoi, as well as Anaplasma phagocytophilum and several Rickettsia species. Under laboratory conditions, a transovarial transmission to the next tick generation is described for Rickettsia spp. and Borrelia spp., especially regarding B. miyamotoi, whereas the efficiency of transovarial transfer under field conditions is largely unstudied. METHODS: In order to better estimate the potential infection risk by tick larvae for humans and animals, 1500 I. ricinus larvae from 50 collected "nests" (larvae adhering to the flag in a clumped manner) were individually examined for Borrelia, Rickettsia and A. phagocytophilum DNA using quantitative real-time PCR (qPCR). RESULTS: Thirty-nine of 50 nests each (78.0%, 95% CI: 64.0-88.5%) were positive for Borrelia spp. and Rickettsia spp. DNA, and in three nests (6.0%, 95% CI: 1.3-16.5%) A. phagocytophilum DNA was detected. Overall, DNA from at least one pathogen could be detected in 90.0% (45/50, 95% CI: 78.2-96.7%) of the nests. Of the 1500 larvae, 137 were positive for Borrelia spp. DNA (9.1%, 95% CI: 7.7-10.7%), 341 for Rickettsia spp. DNA (22.7%, 95% CI: 20.6-24.9%) and three for A. phagocytophilum DNA (0.2%, 95% CI: 0-0.6%). Quantity of Borrelia spp. and Anaplasma spp. DNA in positive larvae was low, with 2.7 × 100Borrelia 5S-23S gene copies and 2.4 × 101A. phagocytophilum msp2/p44 gene copies detected on average, while Rickettsia-positive samples contained on average 5.4 × 102gltA gene copies. Coinfections were found in 66.0% (33/50, 95% CI: 51.2-78.8%) of the nests and 8.6% (38/443, 95% CI: 6.1-11.6%) of positive larvae. In fact, larvae had a significantly higher probability of being infected with Borrelia spp. or Rickettsia spp. when both pathogens were present in the nest. CONCLUSIONS: This study provides evidence for transovarial transmission of Rickettsia spp. and Borrelia spp. in I. ricinus under field conditions, possibly facilitating pathogen persistence in the ecosystem and reducing the dependence on the presence of suitable reservoir hosts. Further studies are needed to prove transovarial transmission and to explain the surprisingly high proportion of nests containing Rickettsia and/or Borrelia DNA-positive larvae compared to infection rates in adult ticks commonly reported in other studies.

Shifts in Borrelia burgdorferi (s.l.) geno-species infections in Ixodes ricinus over a 10-year surveillance period in the city of Hanover (Germany) and Borrelia miyamotoi-specific Reverse Line Blot detection.

BACKGROUND: Lyme borreliosis caused by spirochetes of the Borrelia burgdorferi (sensu lato) complex is still the most common tick-borne disease in Europe, posing a considerable threat to public health. The predominant vector in Europe is the widespread hard tick Ixodes ricinus, which also transmits the relapsing fever spirochete B. miyamotoi as well as pathogenic Rickettsiales (Anaplasma phagocytophilum, Rickettsia spp.). To assess the public health risk, a long-term monitoring of tick infection rates with the named pathogens is indispensable. METHODS: The present study is the first German 10-year follow-up monitoring of tick infections with Borrelia spp. and co-infections with Rickettsiales. Furthermore, a specific Reverse Line Blot (RLB) protocol for detection of B. miyamotoi and simultaneous differentiation of B. burgdorferi (s.l.) geno-species was established. RESULTS: Overall, 24.0% (505/2100) of ticks collected in the city of Hanover were infected with Borrelia. In detail, 35.4% (203/573) of adult ticks [38.5% females (111/288) and 32.3% males (92/285)] and 19.8% nymphs (302/1527) were infected, representing consistent infection rates over the 10-year monitoring period. Geno-species differentiation using RLB determined B. miyamotoi in 8.9% (45/505) of positive ticks. Furthermore, a significant decrease in B. afzelii and B. spielmanii infection rates from 2010 to 2015 was observed. Co-infections with Rickettsia spp. and A. phagocytophilum increased between 2010 and 2015 (7.3 vs 10.9% and 0.3 vs 1.1%, respectively). CONCLUSIONS: Long-term monitoring is an essential part of public health risk assessment to capture data on pathogen occurrence over time. Such data will reveal shifts in pathogen geno-species distribution and help to answer the question whether or not climate change influences tick-borne pathogens.

Association of Borrelia and Rickettsia spp. and bacterial loads in Ixodes ricinus ticks.

In recent years, awareness of coinfections has increased as synergistic or antagonistic effects on interacting bacteria have been observed. To date, several reports on coinfections of ticks with Rickettsia and Borrelia spp. are available. However, associations are rarely described and studies are based on rather low sample sizes. In the present study, coinfections of Ixodes ricinus with these pathogens were investigated by determining their association in a meta-analysis. A total of 5079 tick samples examined for Rickettsia and Borrelia spp. via probe-based quantitative real-time PCR in previous prevalence studies or as submitted diagnostic material were included. In Borrelia-positive ticks, genospecies were determined by Reverse Line Blot. Determination of bacterial loads resulted in an increase between developmental tick stages with highest mean bacterial loads in female ticks (7.96×104 in Borrelia single-infected, 4.87×105 in Rickettsia single-infected and 3.22×105 in Borrelia-Rickettsia coinfected females). The determined Borrelia-Rickettsia tick coinfection rate was 12.3% (626/5079) with a significant difference to the expected coinfection rate of 9.0% (457/5079). A significant slight association as well as correlation between Borrelia and Rickettsia were determined. In addition, a significant interrelation of the bacterial load in coinfected ticks was shown. At the level of Borrelia genospecies, significant weak associations with Rickettsia spp. were detected for B. afzelii, B. garinii/bavariensis, B. valaisiana and B. lusitaniae. The positive association provides evidence for interactions between Borrelia and Rickettsia spp. in the tick vector, presumably resulting in higher bacterial replication rates in the tick vector and possibly the reservoir host. However, coinfection may impact the vector negatively as indicated by an absent increase in coinfection rates from nymphs to adults. Future studies are needed to investigate the underlying mechanisms of the positive association in ticks and possible associations in the vertebrate host as well as the potential influence of environmental factors.

Biological function of Dictyocaulus viviparus asparaginyl peptidase legumain-1 and its suitability as a vaccine target.

The present study characterized the biological function of the asparaginyl peptidase legumain-1 (LEG-1) of the bovine lungworm Dictyocaulus viviparus and its suitability as a recombinant vaccine against dictyocaulosis. Quantitative real-time PCR and immunoblot analysis revealed LEG-1 to be almost exclusively transcribed and expressed in parasitic lungworm stages. Immunohistochemistry localized the enzyme in the parasite's gut, which was confirmed by immunoblots detecting LEG-1 in the gut as well as male testes. LEG-1 was recombinantly (rLEG-1) expressed in the yeast Pichia pastoris and subsequently analysed in activity assays for its enzyme functions and substrate specificity. For sufficient functionality, rLEG-1 needed trans-activation through D. viviparus cathepsin L-2, indicating a novel mechanism of legumain activation. After trans-activation, rLEG-1 worked best at pH 5·5 and 35-39 °C and cleaved a legumain-specific artificial substrate as well as the natural substrates bovine collagen types I and II. In a clinical vaccination trial, rLEG-1 did not protect against challenge infection. Results of in vitro characterization, transcription pattern and localization enhance the presumption that LEG-1 participates in digestion processes of D. viviparus. Since rLEG-1 needs trans-activation through a cathepsin, it is probably involved in an enzyme cascade and therefore remains interesting as a candidate in a multi-component vaccine.

Revisited: Borrelia burgdorferi sensu lato infections in hard ticks (Ixodes ricinus) in the city of Hanover (Germany).

BACKGROUND: The present study investigated the prevalence of Borrelia burgdorferi sensu lato (s.l.) genospecies in Ixodes ricinus ticks collected in Hanover, Northern Germany, in 2010. At the same time the study served as fifth-year-follow-up study for data comparison with 2005. METHODS: A total of 2100 questing ticks were collected and analysed by quantitative real-time PCR (qPCR) with subsequent species differentiation via Reverse Line Blot and Sanger sequencing. Simultaneously, results obtained in 2010 were compared to infection rates from 2005 to evaluate the development of B. burgdorferi s.l. infection rates in Hanoverian ticks. RESULTS: Overall, 22.7% (476/2,100) of collected ticks were tested positive for B. burgdorferi s.l. infections. Adult ticks showed an infection rate of 33.3% (124/372), subdivided into 29.6% (58/196) positive males and 37.5% (66/176) positive females. Nymph and larvae infection rates were found to be 20.3% (344/1,697) and 25.8% (8/31), respectively. Species identification was successful for 59.2% (282/476) of positive ticks with B. afzelii as the most frequently detected genospecies, followed by B. garinii (including B. bavariensis) and B. spielmanii. B. burgdorferi sensu stricto (s.s.), B. bissettii, B. valaisiana and B. lusitaniae were also identified. Significant differences concerning seasonal fluctuations as well as local differences were observed. Comparing infection rates of Hanoverian ticks between years, a significant increase (P = 0.002) could be observed for larvae with 1.7% positives (2/60) in 2005 and 25.8% positives (8/31) in 2010. In the latter year, coinfections with Borrelia and Rickettsiales were detected in a total of 7.8% (163/2,100) of collected ticks. Of these, 7.3% (153/2,100) were coinfected with Rickettsia spp., 0.3% (7/2,100) with A. phagocytophilum and 0.1% (3/2,100) were coinfected with all three pathogens. Between years 2005 and 2010, no statistically significant differences in coinfection rates were found. CONCLUSIONS: Comparing B. burgdorferi s.l. infections in Hanoverian I. ricinus ticks in 2010 with data from 2005, a statistically significant increase of infected larvae was noted, whereas the other stages revealed no statistically significant differences. Whether the increased larvae infection rate is an isolated event or results from factual circumstances, e.g. increasing effectiveness of transovarial transmission due to unknown factors, has to be evaluated in further studies.