Gastrointestinal parasite community structure in horses after the introduction of selective anthelmintic treatment strategies.

A relatively new method to study the species richness and diversity of nematode parasites in grazing animals is to perform deep sequencing on composite samples containing a mixture of parasites. In this work, we compared species composition of strongyles in two groups of horses as a function of egg count and age, based on a DNA barcoding approach. Faecal egg counts and larval cultures were obtained from nearly 300 horses, i.e., domestic horses (n = 167) and trotters (n = 130) sampled nationwide. The second internal transcribed spacer region (ITS2) of strongyle nematodes in the larval cultures was first amplified using barcoded universal primers and then sequenced on the PacBio platform. Subsequently, bioinformatic sequence analysis was performed using SCATA to assign operational taxonomic units (OTU). Finally, species occurrence and composition were assessed using R. ITS2 sequences were found in the majority (89%) of larval samples. Sequencing yielded an average of 140 (26 to 503) reads per sample. The OTUs were assigned to 28 different taxa, of which all but three could be identified as species. The average relative abundance of the seven most abundant species (all Cyathostominae) accounted for 87% of the combined data set. The three species with the highest prevalence in both horse groups were Cyathostomum catinatum, Cylicocyclus nassatus and Cylicostephanus calicatus, and they were frequently found in different combinations with other species regardless of horse group. Interestingly, this result is largely consistent with a previous Swedish study based on morphological analysis of adult worms. In addition, two migratory strongylids (Strongylus vulgaris and S. edentatus) occurred in few domestic horses and trotters. Except for C. minutus and C. nassatus, which decreased with age, and C. catinatum and S. vulgaris, which increased, no specific trends were observed with respect to horse age. Taken together, these results are broadly consistent with data obtained before the introduction of selective targeted treatment in Sweden in 2007. All in all, our results suggest that this treatment strategy has not led to a significant change in strongyle nematode community structure in Swedish horses. The study also confirms that nemabiome analysis in combination with diversity index analysis is an objective method to study strongyle communities in horses.

Prevalence and risk factors for hair loss in outdoor-wintered beef cattle under cold weather conditions.

Keeping cattle outdoors year-around is considered an attractive alternative to indoor winter-housing, due to lower investment costs and better welfare. However, hair loss, attributed to lice, may impair cattle's thermal balance during harsh winters. During the winters of 2019-2021, outdoor cattle in Sweden were studied for the prevalence and development of hair loss, while surveys were conducted among the farmers on their perceptions and attitudes around hair loss. Of the 463 groups of cattle from 75 farms enrolled in a welfare control program issued by the Swedish Board of Agriculture, 25.7% (n = 119) had at least one animal with hair loss. When we followed up a subset of animals (n = 3673) which did not receive prophylactic delousing, 15.7% developed hair loss. Hair loss occurrence increased between visits within each winter in these animals, suggesting a contagious etiology. Logistic regression analyses, using the information collected in the control program and the hair loss outcome, showed that preventive delousing before November was effective, alongside keeping animals clean and the group size small. Meanwhile, being older (>2 years) and having access to bedding materials was shown to increase the risk at an animal-level. Some groups (n = 34) had no hair loss despite receiving no prophylactic delousing. Based on the survey conducted among the farmers (n = 15), groups with lower hair loss prevalence belonged to farmers who were more observant of hair loss and gave prompt treatment. This study provides knowledge useful to limit delousing interventions without compromising animal welfare.

First Swedish case of fatal equine parasitic encephalitis by Halicephalobus gingivalis.

BACKGROUND: Halicephalobus gingivalis is a nematode with zoonotic potential which can cause fatal opportunistic infections in various mammals. The parasite has never been diagnosed in Sweden, in any species, prior to the presented case. CASE PRESENTATION: An imported 21-year-old Icelandic mare developed severe neurological signs. The horse was eventually euthanized and submitted for post-mortem examination where severe lesions in the kidneys were noted. Histopathology revealed the presence of H. gingivalis in both kidneys and the brain. Phylogenetic analysis of the parasite determined it to belong to Lineage 1. CONCLUSIONS: With the occurrence of H. gingivalis in Sweden, the disease should be added to the list of differential diagnoses in cases with acute onset of neurological disease in both horses and other mammals including humans.

Selective Anthelmintic Treatment in Horses in Sweden Based on Coprological Analyses: Ten-Year Results.

In Sweden, routine deworming has been used for several decades; however, to slow down the development of anthelmintic resistance, selective treatment is currently recommended. As part of a monitoring programme, equestrian premises submitted faecal samples to the National Veterinary Institute (SVA) twice per year between 2008 and 2017. Analyses for strongyles (small and large), tapeworms and ascarids, followed by premise-specific advice regarding deworming and parasite control strategies, were provided. In total, 43,330 faecal samples, collected from 26,625 horses on 935 premises in springtime (March to June), were analysed by quantitative or semi-quantitative flotation. Moreover, Strongylus vulgaris was detected by larval culture or PCR. Between 4 and 11% of individual horses tested positive for S. vulgaris and 3-10% were shedding tapeworm eggs. There were recurrent high and low egg shedders; 75% of horses with S. vulgaris appeared to have been recently introduced into the herd; the proportion of S. vulgaris-positive premises increased when individual samples rather than pooled samples were used. Based on the results of S. vulgaris diagnostics and strongyle egg-shedding level, 59% of the horses did not need to be dewormed.

Ixodid tick species found in northern Sweden - Data from a frontier area.

Environmental and climatic changes in northern Europe have shaped a geographical area in which new tick species may become established and introduce new tick-borne pathogens. In recent decades, ticks have expanded their latitudinal and altitudinal range limits in northern Sweden. In this study, ticks were collected in 2018 and 2019 in northern Sweden from different hosts, mainly from dogs, cats and humans. The ticks in 2018 (n = 2141, collected from 65 municipalities in 11 provinces) were identified as Ixodes ricinus (n = 2108, 98.5%), Ixodes persulcatus (n = 18, 0.8%), Ixodes trianguliceps (n = 14, 0.7%) and Hyalomma marginatum (n = 1, 0.05%). The ticks collected in 2019 (n = 519, across a smaller area than in 2018, i.e. Sweden's four northernmost provinces) were identified as I. ricinus (n = 242, 46.6%) and I. persulcatus (n = 277, 53.4%). Among those collected in 2019, the majority of I. ricinus (n = 111, 45.9%) were submitted from the province of Västerbotten, while most I. persulcatus (n = 259, 93.5%) were collected in the province of Norrbotten. This study provides updated figures on the geographical distribution of two Ixodes species in northern Sweden. The results confirmed I. ricinus to be the dominant species and that I. persulcatus has enlarged its distributional area compared with previous reports. Updated knowledge of tick distribution is fundamental for the creation of risk maps and will allow relevant advice to be provided to the general public, suggesting measures to prevent tick bites and consequently tick-borne diseases.

Candidatus Rickettsia Vini DNA in Ticks Collected from Nest Burrows of the European Sand Martin (Riparia riparia) in Sweden.

Background: Birds can cross geographical and environmental barriers and thereby facilitate dispersal of tick-borne pathogens both as carriers of infected ticks and as reservoirs of pathogenic microorganisms. Ixodes lividus (Ixodida: Ixodidae) is an endophilic tick in the Palearctic region that is highly specialized on its host, the European sand martin Riparia riparia. The purpose of this study was to determine whether I. lividus ticks sampled from sand martin nests in Sweden carry vector-borne pathogens. Materials and Methods: Fed ticks were collected in the autumns of 2017 and 2019 from the nests of a European sand martin colony in southern Sweden. Ticks were identified morphologically to developmental stage and species and were tested for tick-borne pathogens using PCR-based methods. Results: None of the 41 ticks tested positive for five tick-borne pathogens including Borrelia spp., tick-borne encephalitis virus (TBEV), Neoehrlichia mikurensis, Anaplasma phagocytophilum, and Babesia spp. Thirty-seven (13 females, 23 nymphs and 1 larva) of the 41 ticks tested positive for the gltA gene of Rickettsia spp. The sequences of the 17 kDa and gltA genes were most closely related to Candidatus Rickettsia vini. Conclusion: Our study confirms other reports that I. lividus ticks associated with the European sand martin have high infection prevalence of Ca. R. vini.

A Disease Outbreak in Beef Cattle Associated with Anaplasma and Mycoplasma Infections.

An outbreak of disease in a Swedish beef cattle herd initiated an in-depth study to investigate the presence of bacteria and viruses in the blood of clinically healthy (n = 10) and clinically diseased cattle (n = 20) using whole-genome shotgun sequencing (WGSS). The occurrence of infectious agents was also investigated in ticks found attached to healthy cattle (n = 61) and wild deer (n = 23), and in spleen samples from wild deer (n = 30) and wild boars (n = 10). Moreover, blood samples from 84 clinically healthy young stock were analysed for antibodies against Anaplasma phagocytophilum and Babesia divergens. The WGSS revealed the presence of at least three distinct Mycoplasma variants that were most closely related to Mycoplasma wenyonii. Two of these were very similar to a divergent M. wenyonii variant previously only detected in Mexico. These variants tended to be more common in the diseased cattle than in the healthy cattle but were not detected in the ticks or wild animals. The DNA of A. phagocytophilum was detected in similar proportions in diseased (33%) and healthy (40%) cattle, while 70% of the deer, 8% of ticks collected from the cattle and 19% of the ticks collected from deer were positive. Almost all the isolates from the cattle, deer and ticks belonged to Ecotype 1. Based on sequencing of the groEL-gene, most isolates of A. phagocytophilum from cattle were similar and belonged to a different cluster than the isolates from wild deer. Antibodies against A. phagocytophilum were detected in all the analysed samples. In conclusion, uncommon variants of Mycoplasma were detected, probably associated with the disease outbreak in combination with immune suppression due to granulocytic anaplasmosis. Moreover, A. phagocytophilum was found to be circulating within this cattle population, while circulation between cattle and deer occurred infrequently.

Characterization of the bacterial microbiome of Swedish ticks through 16S rRNA amplicon sequencing of whole ticks and of individual tick organs.

BACKGROUND: The composition of the microbial flora associated with ixodid ticks has been studied in several species, revealing the importance of geographical origin, developmental stage(s) and feeding status of the tick, as well as substantial differences between tissues and organs. Studying the microbiome in the correct context and scale is therefore necessary for understanding the interactions between tick-borne pathogens and other microorganisms as well as other aspects of tick biology. METHODS: In the present study the microbial flora of whole Ixodes ricinus, I. persulcatus and I. trianguliceps ticks were analyzed with 16S rRNA amplicon sequencing. Additionally, tick organs (midguts, Malpighian tubules, ovaries, salivary glands) from flat and engorged I. ricinus female ticks were examined with the same methodology. RESULTS: The most abundant bacteria belonged to the group of Proteobacteria (Cand. Midichloria mitochondrii and Cand. Lariskella). 16S amplicon sequencing of dissected tick organs provided more information on the diversity of I. ricinus-associated microbial flora, especially when organs were collected from engorged ticks. Bacterial genera significantly associated with tick feeding status as well as genera associated with the presence of tick-borne pathogens were identified. CONCLUSIONS: These results contribute to the knowledge of microbial flora associated with ixodid ticks in their northernmost distribution limit in Europe and opens new perspectives for other investigations on the function of these bacteria, including those using other approaches like in vitro cultivation and in vitro models.

A Novel High Discriminatory Protocol for the Detection of Borrelia afzelii, Borrelia burgdorferi Sensu Stricto and Borrelia garinii in Ticks.

Bacteria of the Borrelia burgdorferi sensu lato complex are the causative agents of Lyme borreliosis (LB). Even if the conventional diagnosis of LB does not rely on the species itself, an accurate species identification within the complex will provide a deepened epidemiological scenario, a better diagnosis leading to a more targeted therapeutic approach, as well as promote the general public's awareness. A comparative genomics approach based on the 210 Borrelia spp. genomes available in 2019 were used to set up three species-specific PCR protocols, able to detect and provide species typing of Borrelia afzelii, Borrelia burgdorferi sensu stricto (s.s.) and Borrelia garinii, the three most common and important human pathogenic Lyme Borrelia species in Europe. The species-specificity of these protocols was confirmed on previously identified B. afzelii, B. burgdorferi s.s. and B. garinii specimens detected in Ixodes ricinus samples. In addition, the protocols were validated on 120 DNA samples from ticks collected in Sweden, showing 88% accuracy, 100% precision, 72% sensitivity and 100% specificity. The proposed approach represents an innovative tool in epidemiological studies focused on B. burgdorferi s.l. occurrence in ticks, and future studies could suggest its helpfulness in routine diagnostic tests for health care.

Dirofilaria spp. and Angiostrongylus vasorum: Current Risk of Spreading in Central and Northern Europe.

In the past few decades, the relevance of Dirofilaria immitis and Dirofilaria repens, causing cardiopulmonary and subcutaneous dirofilariosis in dogs and cats, and of Angiostrongylus vasorum, causing canine angiostrongylosis, has steadily increased in Central and Northern Europe. In this review, a summary of published articles and additional reports dealing with imported or autochthonous cases of these parasites is provided for Central (Austria, Czechia, Germany, Hungary, Luxemburg, Poland, Slovakia, Slovenia, and Switzerland) and Northern (Denmark, Finland, Iceland, Norway, and Sweden) Europe. Research efforts focusing on Dirofilaria spp. and A. vasorum have varied by country, and cross-border studies are few. The housing conditions of dogs, pet movements, the spread of competent vectors, and climate change are important factors in the spread of these nematodes. Dogs kept outside overnight are a major factor for the establishment of Dirofilaria spp. However, the establishment of invasive, diurnal, synanthropic, competent mosquito vectors such as Aedes albopictus may also influence the establishment of Dirofilaria spp. The drivers of the spread of A. vasorum remain not fully understood, but it seems to be influenced by habitats shared with wild canids, dog relocation, and possibly climatic changes; its pattern of spreading appears to be similar in different countries. Both Dirofilaria spp. and A. vasorum merit further monitoring and research focus in Europe.

First report on reduced efficacy of ivermectin on Oesophagostomum spp. on Swedish pig farms.

Anthelmintic efficacy was investigated in nine sow herds that had been identified with high faecal egg counts in a prevalence study. Faecal samples were collected from a total of 104 individual sows, and analysed using a centrifugal flotation McMaster technique. Samples positive for strongyle eggs were cultured to third stage larvae (L3) for genus identification and then further identified to Oesophagostomum species by sequencing. Following the initial sample collection, the sows were treated with either fenbendazole (FBZ, n = 5 farms) or ivermectin (IVM, n = 4 farms) at the recommended dosing and sampled again 14 days post treatment. Faecal Egg Count Reduction (FECR) was used to determine the treatment efficacy. With respect to Ascaris suum, the anthelmintic treatment was successful (FECR >90%) on the five farms where this parasite was detected, regardless of what drug had been used. In contrast, 4/9 farms were positive for Oesophagostomum spp. post treatment, out of which three had a FECR of <90%. These three herds had all been treated with injectable IVM. Out of the six farms where treatment showed good efficacy (FECR 95-100%), five herds had used FBZ and one herd IVM. This study is the first to recognise reduced efficacy to IVM on Oesophagostomum spp. in Swedish pigs. Sequencing of the Oesophagostomum L3 showed that both O. dentatum and O. quadrispinulatum were present on 5/9 farms pre-treatment and on 2/9 farms post-treatment, unrelated to what anthelmintic had been used. Given these findings we could not correlate the reduced efficacy by the species of Oesophagostomum present in the herd. Prolonged usage of only one class of anthelmintic may predispose selection of resistance and has been suspected as a cause of treatment failure of porcine Oesophagostomum spp. in other studies. On all three farms showing reduced efficacy, IVM had been used as the sole anthelmintic drug for several years, and two of the farms also used IVM twice or more per year to control sarcoptic mange. A reduced efficacy to the available anthelmintic drugs used in the control of Oesophagostomum spp., may result in a subsequent surge of the possible negative effects caused by this parasite.

Occurrence of Endoparasites in Adult Swedish Dogs: A Coprological Investigation.

The occurrence of endoparasites in Swedish adult dogs (n = 303) was investigated between January and October 2014. Included dogs had to be clinically healthy, older than 1 year and untreated with anthelmintics or endectocides for at least 3 months prior to sampling. They were grouped according to age, category of dog and time since last antiparasitic treatment. Samples were analyzed by flotation to detect parasitic eggs and cysts/oocysts. Among these, 129 (43%) dogs were also analyzed with the Baermann-technique to detect cardiopulmonary larval stages. Parasite dispersal stages were found in 24 (7.9%, CI 95% 4.9-10.1) of the dogs at flotation, while no dog shed cardiopulmonary larval stages. Giardia sp. cysts were observed in 2.6% (n = 8) of dogs examined, cysts of Sarcocystis spp. were observed in 0.6% (n = 2), oocysts of Cystosisopora ohioensis were found in one dog (0.3%). Eggs of Toxocara canis (2.3%, n = 7), Uncinaria stenocephala (1.3%, n = 4) and Trichuris vulpis (0.3%, one dog) were found. None of the dogs were diagnosed with more than one species. Although the occurrence of endoparasites was above the average in dogs ≤ 2 years of age (11.5%), nematodes were more common in older dogs ≥4 years (77.0%). Although the occurrence was lower in working/exhibition dogs (5.9%) than in companion dogs (8.4%) and hunting-dogs (8.6%), these differences were not significant. However, dogs exposed to prey according to the owner had a statistically significant higher prevalence than other dogs (20.5 vs. 5.7%). The Odds Ratio (OR) was 4.0 (CI 95%, 1.58-10.11) for dogs having access to prey, 2.4 (CI 95%, 0.37-8.06) for dogs staying at day-care, and 2 (CI 95%, 0.96-5.96) for bitches. Furthermore, a significant association was observed between infection with nematodes and exposures to prey (p = 0.006). As a reference, data on the endoparasites in canine fecal samples submitted to the National Veterinary Institute (SVA, Uppsala) during 2014 are presented. Overall, this study shows a low occurrence of endoparasites among dogs in Sweden. Any risk-assessment on zoonotic parasites as well as deworming recommendations will take advantage from these updated figures.

Acaricidal activity against Ixodes ricinus nymphs of essential oils from the Libyan plants Artemisia herba alba, Origanum majorana and Juniperus phoenicea.

Ixodes ricinus (L.) (Acari: Ixodidae) is a major vector for the transmission of several important human pathogens. The aim of the present study was to evaluate the in vitro efficacy of different concentrations of essential oils (Eos) on I. ricinus tick nymphs. Oils were obtained from the leaves of three plants native to Libya: white wormwood (Artemisia herba alba Asso), marjoram (Origanum majorana L.) and Arâr (Juniperus phoenicea L., English common name Phoenician juniper). Assays were done using the "open filter paper method". Two concentrations from each oil, 0.5 and 1 µl/cm, were tested. The acaricidal effect was measured in terms of the lethal concentrations (LC50, LC95) and lethal time (LT50, LT95). Mortality rates were obtained by counting the surviving nymphs every 30 min for the first five hours and then at 24, 48 and 72 h. A mortality of 100% was recorded at the higher concentration of oils (1 µl/cm2) from A. herba alba and J. phoenicea at the first 2 h of exposure. Exposure to O. majorana led to 100% mortality on the third day (72 h), and this effect decreased noticeably with 0.5 µl/cm2 oil at the same exposure time. However, 50% of ticks showed a paralysis effect and less movement after 2 h. The LC50 of mortality was reached within the first 24 h of exposure time at 0.5 µl/cm2 of O. majorana, which produced 60% tick's mortality. Chemical composition of the essential oils was elucidated by gas chromatography-mass spectrometry analyses. These results suggest that essential oils deserve further investigation as components of alternative approaches for I. ricinus tick control.

Gastrointestinal parasites in Swedish pigs: Prevalence and associated risk factors for infection in herds where animal welfare standards are improved.

The global pig production has undergone major changes over the past 30 years with larger farms, more intensified production as well as improved hygiene and biosecurity practices. To investigate whether these changes, along with expanded pig welfare, have had an impact on parasite occurrence, a cross-sectional study was conducted in Sweden on farms where the pigs are always loose-housed, floors are solid and bedding material is provided. A total of 1615 faecal samples were collected on 42 conventional indoor farms from a) post-weaning piglets (n = 337); b) growers (n = 345); c) fatteners (n = 308); d) dry sows (n = 277) and e) pre-partum sows (n = 348). Samples were analysed using centrifugal flotation with a saturated glucose-salt solution and a modified McMaster technique, with a lower detection limit of 50 eggs or oocysts per gram. Samples positive for strongyle-type eggs were cultured to third stage larvae for genus identification. Farms also responded to a questionnaire regarding biosecurity, hygienic measures, and other management routines. Risk factors for parasite occurrence were assessed using mixed-effects logistical regression to account for farm-level clustering of samples. Interestingly, the prevalence of Ascaris suum was reduced compared to a similar investigation in the 1980s. In the present study A. suum was detected only in 43 % of the herds, with the highest prevalence in pre-partum sows (37 %) followed by fatteners (25 %). Small sized farms were associated with higher odds of being positive, compared to large sized farms (OR = 159.1, P = 0.010). Oesophagostomum spp. were detected in 64 % of the herds and again mainly in pre-partum sows (63 %). Trichuris suis was detected in 10 % of the herds but only in <1% of the samples. Moreover, Cystoisospora suis and Eimeria spp. were detected on 60 % and 64 % the farms, with the highest prevalence in post-weaning piglets and sows, respectively. Anthelmintic drugs (ivermectin or fenbendazole) were commonly used and administered mainly to pre-partum sows on 93 % of the farms. Toltrazuril against neonatal coccidiosis was administered to piglets on 14 % of the farms. The use of antiparasitic drugs did not significantly affect parasite prevalence. Overall, it appears that the altered farming routines with focus on improved pig welfare have not solely resulted in a higher occurrence of parasites, most likely due to the adequate biosecurity and hygiene practices instituted. Thus, there seems to be no conflict between implementing measures to promote pig welfare and adequately control the more pathogenic and economically important parasites.

Selective flukicide treatment of non-lactating cows and the corresponding production impact of Fasciola hepatica in dairy herds in Sweden.

A control strategy against Fasciola hepatica infection based on selective treatment of non-lactating animals was evaluated in four Swedish dairy herds. The study was conducted over the course of two consecutive seasons in moderately to highly F. hepatica infected herds with robotic milking, where heifers and dry cows received an oral drench with albendazole (10 mg/kg) during three visits in January, February and March in both 2017 and 2018. This resulted in an anthelmintic coverage between 38 % and 58 % of the animals. Furthermore, on each visit, the infection status of all dewormed animals along with 15 randomly selected milking cows were monitored by detection of F. hepatica coproantigens. Individual milk samples were also collected quarterly from the whole herds for measurements of individual antibody levels against the parasite using milk ELISA. In addition, individual data on milk yield and quality were collected on a monthly basis between 2016 and 2018. To further study the impact of the infection on milk production, truly F. hepatica positive and negative cows in the first lactation were identified based on the results from coproantigen and milk ELISA assays. Total F. hepatica coproantigen prevalence in the herds varied between 28 % and 85 % in the first year, and between 27 % and 68 % in the second year of the study. We found that two years of treatments resulted in a significant decrease of coproantigen-positivity especially on the two most heavily infected farms. These results were confirmed by a similar drop in within-herd prevalences obtained by milk ELISA results. The infection had a significant negative impact on milk yields in untreated F. hepatica positive cows. No consistent long-term effect was observed at the herd level probably due to the influx of animals infected before puberty and/or adult animals that were re-infected at dry-off. This is the first study of the effects of F. hepatica infection on milk yield and quality in dairy herds in Sweden.

Morphological and molecular identification of ixodid tick species (Acari: Ixodidae) infesting cattle in Uganda.

In Uganda, the role of ticks in zoonotic disease transmission is not well described, partly, due to limited available information on tick diversity. This study aimed to identify the tick species that infest cattle. Between September and November 2017, ticks (n = 4362) were collected from 5 districts across Uganda (Kasese, Hoima, Gulu, Soroti, and Moroto) and identified morphologically at Uganda Virus Research Institute. Morphological and genetic validation was performed in Germany on representative identified specimens and on all unidentified ticks. Ticks were belonging to 15 species: 8 Rhipicephalus species (Rhipicephalus appendiculatus, Rhipicephalus evertsi evertsi, Rhipicephalus microplus, Rhipicephalus decoloratus, Rhipicephalus afranicus, Rhipicephalus pulchellus, Rhipicephalus simus, and Rhipicephalus sanguineus tropical lineage); 5 Amblyomma species (Amblyomma lepidum, Amblyomma variegatum, Amblyomma cohaerens, Amblyomma gemma, and Amblyomma paulopunctatum); and 2 Hyalomma species (Hyalomma rufipes and Hyalomma truncatum). The most common species were R. appendiculatus (51.8%), A. lepidum (21.0%), A. variegatum (14.3%), R. evertsi evertsi (8.2%), and R. decoloratus (2.4%). R. afranicus is a new species recently described in South Africa and we report its presence in Uganda for the first time. The sequences of R. afranicus were 2.4% divergent from those obtained in Southern Africa. We confirm the presence of the invasive R. microplus in two districts (Soroti and Gulu). Species diversity was highest in Moroto district (p = 0.004) and geographical predominance by specific ticks was observed (p = 0.001). The study expands the knowledge on tick fauna in Uganda and demonstrates that multiple tick species with potential to transmit several tick-borne diseases including zoonotic pathogens are infesting cattle.

First records of adult Hyalomma marginatum and H. rufipes ticks (Acari: Ixodidae) in Sweden.

From July 2018 to January 2019 we recorded 41 specimens of adult Hyalomma ticks, which had been found on horses, cattle or humans in 14 Swedish provinces. In 20 cases we received tick specimens, which were identified morphologically as adults of H. marginatum (n = 11) or H. rufipes (n = 9). These are the first documented records in Sweden of adults of H. marginatum and H. rufipes. Molecular tests for Crimean-Congo haemorrhagic fever virus and piroplasms (Babesia spp. and Theileria spp.) proved negative; 12 out of 20 tested specimens were positive for rickettsiae (R. aeschlimannii was identified in 11 of the ticks). All ticks originated from people or animals that had not been abroad during the previous two months. These data suggest (i) that the adult Hyalomma ticks originated from immature ticks, which had been brought from the south by migratory birds arriving in Sweden during spring or early summer; and that (ii) due to the exceptionally warm summer of 2018 these immature ticks had been able to develop to the adult stage in the summer and/or autumn of the same year. The rapidly changing climate most likely now permits these two Hyalomma species to develop to the adult, reproductive stage in northern Europe. There is consequently a need to revise the risk maps on the potential geographic occurrence of relevant tick species and related tick-borne pathogens in Sweden and in the neighbouring countries.

The repellency and toxicity effects of essential oils from the Libyan plants Salvadora persica and Rosmarinus officinalis against nymphs of Ixodes ricinus.

Essential oils extracted from the leaves of Libyan Rosemary (Rosmarinus officinalis L.), and Miswak (Salvadora persica L.) were evaluated for their acaricidal and repellent effects on Ixodes ricinus L. nymphs (Acari: Ixodidae) using a bioassay based on an 'open filter paper method'. Rosmarinus officinalis leaf essential oil diluted to 0.5 and 1 µl/cm2 in acetone exhibited, respectively, 20 and 100% tick mortality after about 5 h of exposure. A total of 50 and 95% of I. ricinus nymphs were killed by direct contact with the oil when exposed to lethal concentrations (LC) of 0.7 µl/cm2 (LC50) and 0.95 µl/cm2 (LC95), respectively. The LC50 (0.5 µl/cm2) was reached before the end of the first 24 h of exposure time (ET), as tick mortality at 24 h was 60%. Salvadora persica leaf essential oil at 1 µl/cm2 showed a significant repellency effect against I. ricinus nymphs at 1.5 h ET. A 95% repellency was observed at a repellent concentration (RC95) of 1 µl/cm2 of S. persica, but no significant mortality was recorded at this dose of S. persica oil. Gas chromatography-mass spectrometry analyses showed that the main monoterpenes in both oils were 1,8-cineol, α-pinene, and ß-pinene, although in markedly different proportions. These results suggest that essential oils have substantial potential as alternative approaches for I. ricinus tick control.

Acaricidal properties of ylang-ylang oil and star anise oil against nymphs of Ixodes ricinus (Acari: Ixodidae).

Ylang-ylang oil (YYO) from Cananga odorata (Lam.) Hook.f. & Thomson and star anise oil (SAO) from Illicium verum Hook.f. were tested at four concentrations 0.05, 0.1, 0.2, 0.4 µl/cm2. Mortality rates were obtained by counting dead nymphs at 30-min intervals during the first 5 h after the start of exposure and then at 24, 48 and 72 h. Mortality increased with increasing oil concentration and time of exposure. The two highest concentrations of YYO (0.2, 0.4 µl/cm2) gave maximum lethal concentrations (LC) of 50 and 95% mortality after 4.5 h exposure. Mortality of 95% was obtained after 24 h with the next highest dose (0.1 µl/cm2), whereas LC95 required 3 days with the lowest YYO (0.05 µl/cm2). The lethal effect time (LT) was correlated with the duration of exposure, with a significant effect at 0.4 µl YYO/cm2 after 3 h' (LT50 = 3.2 h, LT95 = 4.3 h). In contrast, only the highest concentration of SAO, 0.4 µl SAO/cm2, showed increasing mortality with time of exposure. This reached LT50 after 10 h and LT95 after 24 h. However, with the lower concentration (0.2 µl/cm2) 50% mortality was reached after 24 h and 100% at 72 h. At to the lowest concentration of SAO (0.1 µl/cm2), 67% mortality after 48 h. The study indicates that YYO and SAO exhibit strong acaricidal properties against nymphs of I. ricinus and suggest that both YYO and SAO should be evaluated as potentially useful in the control of ticks.

Exposure to amitraz, fipronil and permethrin affects cell viability and ABC transporter gene expression in an Ixodes ricinus cell line.

BACKGROUND: Over-expression of ATP-binding cassette (ABC) transporter proteins has been implicated in resistance of ticks to acaricides. Tick cell lines are useful for investigating resistance mechanisms, as development of an in vitro model for the study of acaricide resistance would contribute to improving knowledge of the molecular basis behind drug processing and exclusion in ticks. In the present study, cultures of the Ixodes ricinus-derived cell line IRE/CTVM19 were treated with the acaricides amitraz, permethrin or fipronil to determine modulation of ABC transporter gene expression. Cells were treated with different drug concentrations (25, 50, 100, 150 µM) and incubated for ten days. Cell morphology, viability, metabolic activity and relative expression of ABC (B1, B6, B8 and B10) genes were determined at day 10 post-treatment. RESULTS: Cell morphology determined by light microscopy was altered following treatment with all drugs, but only at high concentrations, while total cell numbers decreased with increasing drug dose. Cell viability determined by trypan blue exclusion was not significantly different from untreated controls (P > 0.1) following treatment with amitraz and permethrin, but high concentrations of fipronil caused decrease (up to 37%, P < 0.01) in viability. At all drug concentrations, fipronil and permethrin induced dose-dependent reduction in cell metabolic activity measured by MTT assay (P < 0.01). Quantitative RT-PCR showed that the drugs significantly affected expression of ABC genes. In particular, fipronil treatment downregulated ABCB1 (P < 0.001) and upregulated ABCB6, ABCB8 and ABCB10 (P < 0.01); amitraz treatment down regulated ABCB1 (significant difference between 25 and 150 µM, P < 0.001) and upregulated ABCB8 and ABCB10 at lower concentrations (25 and 50 µM, P < 0.05); and permethrin upregulated ABCB6, ABCB8 and ABCB10 only at 150 µM (P < 0.01). CONCLUSIONS: The adverse effects on cell viability and metabolic activity, and changes in expression of different ABC transporter genes, detected in IRE/CTVM19 cells following treatment with amitraz, permethrin and fipronil, support the proposed application of tick cell lines as in vitro models for the study of resistance to these acaricides in ticks.