Tick Samples Research Articles

Detection of Borrelia burgdorferi Sensu Lato and Relapsing Fever Borrelia in Feeding Ixodes Ticks and Rodents in Sarawak, Malaysia: New Geographical Records of Borrelia yangtzensis and Borrelia miyamotoi.

Members of the Borrelia burgdorferi sensu lato (Bbsl) complex are etiological agents of Lyme disease (LD), and Borrelia miyamotoi is one of the relapsing fever Borrelia (RFB). Despite the serological evidence of LD in Malaysia, there has been no report from Sarawak, Malaysian Borneo. Thus, this study aimed to detect and characterize Borrelia in rodents and Ixodes ticks from primary forests and an oil palm (OP) plantation in Sarawak. Borrelia yangtzensis (a member of the Bbsl complex) was detected in 43.8% (14/32) of Ixodes granulatus; most of the positive ticks were from the OP plantation (13/14). Out of 56 rodents, B. yangtzensis was detected in four Rattus spp. from the OP plantation and B. miyamotoi was detected in one rodent, Sundamys muelleri, from the primary forest. Further, the positive samples of B. yangtzensis were randomly selected for multilocus sequence analysis (MLSA). The MLSA results of successfully amplified tick samples revealed a clustering with the sequences isolated from Japan and China. This study is the first evidence of B. miyamotoi, a known human pathogen in Malaysia, and B. yangtzensis, which is circulating in ticks and rodents in Sarawak, Malaysian Borneo, and presenting a new geographical record of the Borrelia spp.

Prevalence of ticks (Acari: Ixodidae) and Theileria lestoquardi in sheep at El Huda and El Nuhud animals production research stations, Sudan.

A cross-sectional study was performed 200 blood samples and 314 tick samples in El Huda and El Nuhud animals production research stations, Sudan, in May (summer) and December (winter) in 2016, to determine the prevalence of Theileria lestoquardi in sheep and the potential risk factors associated with the infection. A total of 200 blood samples and 314 tick samples were collected from El Huda (n = 103 blood, 97 tick) and El Nuhud (n = 97 blood, 217 tick) stations. Data on the risk factors, such as age, sex, ecotype of sheep, body condition score and seasons were recorded. The overall prevalence of Theileria lestoquardi was 13% (26/200) using PCR. A significant variation in the prevalence of Theileria lestoquardi was observed between the stations and the ecotype of sheep (p ≤ 0.05), whereas the highest prevalence was recorded in El-Huda station (19.4%) as well as in Shugor (22.8%). Other risk factors, like age, sex, body condition, and seasons were not found to be significantly associated with infection. However, the highest prevalence rate was recorded in old animals (21.6%) than the other, in males (17.9%) than females (12.2%), in animals with poor body condition (26.1%) than the other, and in winter (16%) than summer (10%). Four tick species i.e. Rhipicephalus evertsi evertsi (63.1%), Hyalomma anatolicum (13.8%), Hyalomma dromedarii (8.8%), and Hyalomma impeltatum (14.3%) were recorded in El Nuhud station. While in El Huda station, only Rhipicephalus evertsi evertsi (79.4%), Hyalomma anatolicum (20.6%) were recorded. This study revealed that 13% of sheep were suffering from Theileria lestoquardi which is a considerable number at the stations. Therefore, further epidemiological investigations on disease throughout the year are required in order to set a well-coordinated control program.

Microbial communities associated with the camel tick, Hyalomma dromedarii: 16S rRNA gene-based analysis

Hyalomma dromedarii is an important blood-feeding ectoparasite that affects the health of camels. We assessed the profile of bacterial communities associated with H. dromedarii collected from camels in the eastern part of the UAE in 2010 and 2019. A total of 100 partially engorged female ticks were taken from tick samples collected from camels (n = 100; 50/year) and subjected to DNA extraction and sequencing. The 16S rRNA gene was amplified from genomic DNA and sequenced using Illumina MiSeq platform to elucidate the bacterial communities. Principle Coordinates Analysis (PCoA) was conducted to determine patterns of diversity in bacterial communities. In 2010 and 2019, we obtained 899,574 and 781,452 read counts and these formed 371 and 191 operational taxonomic units (OTUs, clustered at 97% similarity), respectively. In both years, twenty-five bacterial families with high relative abundance were detected and the following were the most common: Moraxellaceae, Enterobacteriaceae, Staphylococcaceae, Bacillaceae, Corynebacteriaceae, Flavobacteriaceae, Francisellaceae, Muribaculaceae, Neisseriaceae, and Pseudomonadaceae. Francisellaceae and Enterobacteriaceae coexist in H. dromedarii and we suggest that they thrive under similar conditions and microbial interactions inside the host. Comparisons of diversity indicated that microbial communities differed in terms of richness and evenness between 2010 and 2019, with higher richness but lower evenness in communities in 2010. Principle coordinates analyses showed clear clusters separating microbial communities in 2010 and 2019. The differences in communities suggested that the repertoire of microbial communities have shifted. In particular, the significant increase in dominance of Francisella and the presence of bacterial families containing pathogenic genera shows that H. dromedarii poses a serious health risk to camels and people who interact with them. Thus, it may be wise to introduce active surveillance of key genera that constitute a health hazard in the livestock industry to protect livestock and people.

Deep sequencing and phylogenetic analysis of severe fever with thrombocytopenia syndrome virus from the tick, Haemaphysalis longicornis, in Korea

AbstractThe Asian longhorned tick, Haemaphysalis longicornis, is widely distributed in China, Japan, and Korea and may transmit infectious diseases. Severe fever with thrombocytopenia syndrome (SFTS) is an important tick‐borne disease caused by the SFTS virus (SFTSV). Deep sequencing to confirm the presence of SFTSV in ticks has not been reported in Korea. To detect SFTSV, RNA was extracted from tick samples and analyzed using high‐throughput deep sequencing. Based on BLASTN results, numerous SFTSV reads were identified. Moreover, a nearly complete genome of SFTSV (JNU‐1 isolate) was obtained using Sanger sequencing. The genome of the JNU‐1 isolate includes three segments of 6,286, 3,299 and 1,642 nucleotides (nt) termed large (L), medium (M), and small (S), respectively. Also, phylogenetic and recombination analyses for each segment of SFTSV were performed using the JNU‐1 isolate. The three segments of JNU‐1 isolate were closely related to the genotype B known human‐derived Korean SFTSV isolate; the JNU‐1 isolate showed no recombination sites with other isolates. This study is the first report of detection of SFTSV from ticks using deep sequencing in Korea and provides information on the genetic diversity of SFTSV in East Asia.

Targeting the Exoskeleton Elementome to Track Tick Geographic Origins.

Understanding the origin of ticks is essential for evaluating the risk of tick-borne disease introduction into new territories. However, when collecting engorged ticks from a host, it is virtually impossible to identify the geographical location where this tick was acquired. Recently, the elementome of tick exoskeleton was characterized by using scanning electron microscopy (SEM) and energy dispersive spectroscopy analysis (EDS). The objective of our preliminary proof-of-concept study was to evaluate the use of SEM-EDS for the analysis of tick exoskeleton elementome to gain insight into the tick geographic and host origin. For this preliminary analysis we used 10 samples of engorged ticks (larvae and nymphs of six species from three genera) collected from various resident hosts and locations. The elementome of the tick exoskeleton was characterized in dorsal and ventral parts with three scans on each part using an EDS 80 mm2 detector at 15 kV in a field emission scanning electron microscope. We used principal component analysis (PCA) (varimax rotation) to reduce the redundancy of data under the premise of losing information as little as possible. The PCA was used to test whether the different variables (tick species, stages, hosts, or geographic locations) differ in the composition of exoskeleton elementome (C, O, P, Cl, and Na). Analyses were carried out using SPSS. The PCA analysis explained a high percentage of variance using the first two factors, C and O (86.13%). The first PC (PC-1; 63.12%) was positively related to P, Cl, and Na, and negatively related to C. The second principal component (23.01%) was mainly positively related to C. In the space defined by the two extracted PC (PC-1 and PC-2), the elementome of tick samples was clearly associated with tick species, but not with developmental stages, hosts or geographic locations. A differentiated elementome pattern was observed within Romanian regions (CJ and TL) for the same tick species. The use of the SEM-EDS methodological approach provided additional information about the tick exoskeleton elementome with possible applications to the identification of tick origin host and location.

Molecular detection and identification of spotted fever group rickettsiae in ticks collected from horses in Cuba.

Spotted fever group (SFG) rickettsiae are obligatory intracellular bacteria that cause disease in humans and other animals. Ixodid ticks are the principal vectors of SFG rickettsiae. The present study aimed to determine the prevalence and species identity of SFG rickettsiae in ticks and horses from urban and rural areas of western Cuba using PCR assays. Tick samples, collected from 79 horses, consisted of 14 Amblyomma mixtum adults, 111 Dermacentor nitens adults and 19 pools of D. nitens nymphs (2-5 individuals/pool). The PCR results revealed the presence of Rickettsia spp. in 64% of the A. mixtum adults, 16% of the D. nitens adults, and 11% of the pooled samples of D. nitens nymphs. In contrast, Rickettsia spp. was not detected in any of the 200 horse blood samples included in this study. DNA sequence data of the rickettsial 17 kDa antigen gene showed that Rickettsia amblyommatis was present in A. mixtum; and Rickettsia felis in D. nitens. This is the first report of R. felis in D. nitens in Cuba. The present study extends our knowledge of the potential vector spectrum and distribution of SFG rickettsiae pathogens in western Cuba.

Clinical, Morphological and Molecular Characterization of Canine Babesiosis and its Compatible Tick Vector in Naturally Infected Dogs, in Egypt

The low scientific attention in dealing with dog diseases is still present comparing to other animal species in Egypt in spite of its wide spreading as pets, hunting dogs or for other purposes. Canine babesiosis is a haemoprotozoal parasitic disease inducing significant clinical manifestations on the infected dogs. The present study aimed to accurately identify the causative species of canine babesiosis in naturally infected dogs in Egypt and its compatible tick vector. For this purpose 75 dogs including 19 dogs at Sharkia and 56 dogs at Dakahlia Governorates, Egypt were clinically examined and 20 (26.67%) dogs showed signs of fever, weakness, anorexia, pale mucous membrane and different degrees of anemia. During examination, there were 8 dogs had variant degrees of tick infestation. Further parasitological identification of 50 collected ticks showed that all the collected ticks were belong to Rhipicephalus sanguineus (R. sanguineus) species. Whole blood samples collected from all examined dogs were screened by thin blood film for presence of Babesia species piroplasm. Nine samples (12.0%) showed pyriform shaped protozoa inside red blood cells (RBCs). All blood samples and 5 pooled tick samples (10 ticks/each) were screened against 18s rRNA of Babesia species genome using semi-nested polymerase chain reaction (PCR). Babesia canis vogeli (B. canis vogeli) was the only detected species in fourteen blood samples (18.67%) and all collected tick samples. It is the first study in Egypt to use semi-nested PCR for detection of 18s rRNA of canine babesiosis in ticks collected from naturally diseased dogs.

Lack of Evidence on the Susceptibility of Ticks and Wild Rodent Species to PCV3 Infection.

Porcine circovirus 3 (PCV3) is an emerging virus, first detected in 2016 and widespread in the swine industry. Although not considered a primary pathogen, PCV3 is potentially linked to several clinical conditions that threaten swine farming. Wild boars are considered the main reservoir species for PCV3 infection in the wild, but recent detection in roe deer, chamois and associated ticks has complicated our understanding of its epidemiology. Much emphasis has been placed on ticks, as competent vectors, and wild rodents, which typically feed immature tick stages. The aim of this study was to clarify whether wild rodent species and associated ticks are susceptible to PCV3 infection and involved in its spread. Wild small mammals’ serum samples and hosted ticks were, therefore, collected from areas where no wild boars were present and tested by PCR, targeting the PCV3 rep gene. A total of 90 yellow-necked mice (Apodemus flavicollis), two wood mice (A. sylvaticus), 26 bank voles (Myodes glareolus) and 262 Ixodes spp. ticks were investigated. PCV3 DNA was not detected in serum or in tick samples. These findings support the hypothesis that the investigated species do not have an actual role as PCV3 reservoirs. Further studies would be necessary to state whether these species, or others that we did not test, are involved in PCV3 infection spread—in particular when susceptible species share the same habitat.

Evaluation of un-methylated DNA enrichment in sequencing of African swine fever virus complete genome

African swine fever is a febrile hemorrhagic fever disease that is caused by the African swine fever virus (ASFV) and is lethal for domestic pigs and wild boar. ASFV also infects soft ticks of the genus Ornithodoros, some species of which can act as a vector for ASFV. Whole genome sequencing of ASFV is a challenge because, due to the size difference of the host genome versus the viral genome, the higher proportion of host versus virus DNA fragments renders the virus sequencing poorly efficient. A novel approach of DNA enrichment, based on the separation of methylated and un-methylated DNA, has been reported but without an evaluation of its efficacy. In this study, the efficiency of the un-methylated DNA enrichment protocol was evaluated for pig and tick samples infected by ASFV. As expected, fewer reads corresponding to ASFV were found in the methylated fraction compared to the un-methylated fraction. However, the sequencing coverage of the un-methylated fraction was not improved compared to the untreated DNA. In our hands, the ASFV DNA enrichment was inefficient for tick samples and very limited for pig samples. This enrichment process represents extra work and cost without a significant improvement of ASFV genome coverage. The efficiency of this enrichment approach and the cost/benefit ratio are discussed.

Molecular Characterisation using 16S rRNA and COI Gene Sequences in Hard Ticks of Gwalior, India

Tick infestation in humans and animals represents a global threat for different tick-borne diseases. In the present study, the ticks from the Gwalior region of India have been mapped to create a database of tick diversity. We explored 773 ticks collected from domestic animals and vegetation in Gwalior. Animals were screened visually, and ticks were collected manually, whereas the flag-drag method was used to collect ticks from the vegetation. The 16S rRNA and cytochrome oxidase I (COI) genes of tick samples were amplified and purified for sequencing and respective phylogenetic trees were constructed. The ticks were morphologically identified using taxonomical keys, revealing the presence of five genera in the region: Hyalomma, Haemaphysalis, Rhipicephalus, Boophilus, and Nosomma. Hyalomma spp. (Hy. annatolicum and Hy. marginatum) were the most abundant accounting for 69.598% of the total sample, followed by Rhipicephalus sanguineus (17.335%), Rhipicephalus microplus (7.115%), Haemaphysalis sp. (5.692%), and Nosomma monstrotum (0.258%). The tick sequences were submitted to the GenBank database. Phylogenetic analysis confirmed the morphological identification at the species level. The combination of molecular and morphological analyses of the ticks supported the result obtained with each method, thus providing more reliable estimates for continued surveillance studies.

Identification of tick-borne pathogens in ticks collected from wild animals in Turkey.

Many enzootic life cycles involving wild animals and non-nidicolous ixodids are still unknown. Therefore, the aim of this study was to report the identified tick species collected from seven different animal species (red deer, brown bear, gray wolf, Eurasian lynx, red fox, European hare, and Mediterranean spur-thighed tortoise) living in the wild in Turkey and to investigate the presence of a wide range of tick-borne microorganisms in the tick samples obtained from these animals. The collected ticks (n = 98) were identified as Dermacentor reticulatus, Haemaphysalis parva, Hyalomma aegyptium, Hyalomma excavatum, Hyalomma marginatum, Ixodes ricinus, and Rhipicephalus turanicus. All engorged ticks collected from the wild animals and unfed larvae (n = 30) obtained from a single Rh. turanicus female were also analyzed individually for tick-borne bacterial and protozoan agents via PCR-sequencing. The molecular analyses revealed the presence of Babesia sp. tavsan2, Theileria capreoli, four Hepatozoon spp. (Hep. ursi, Hep. canis, Hep. felis, and Hepatozoon sp.), Hemolivia mauritanica, and three SFG rickettsiae (Candidatus Rickettsia barbariae, Ca. R. goldwasserii, and Rickettsia hoogstraalii) in the collected ticks. This represents the first report of Th. capreoli, Hep. ursi, and Ca. R. barbariae in ticks from Turkey. The evolutionary relationships of microbes in the different host and tick species are also discussed. Multiple novel tick-host associations in the tick life cycle were also revealed.

Large-scale countrywide screening for tick-borne pathogens in field-collected ticks in Latvia during 2017\u20132019

BackgroundTick-borne diseases are of substantial concern worldwide in both humans and animals. Several hard tick species are of medical and veterinary interest in Europe, and changes in the range of tick species can affect the spread of zoonotic pathogens. The aim of the present study was to map the current prevalence and distribution pattern of ticks and related tick-borne pathogens in Latvia, a Baltic state in northern Europe.MethodsNearly 4600 Ixodes ricinus, I. persulcatus and Dermacentor reticulatus tick samples were collected in all regions of Latvia during 2017–2019 and were screened by molecular methods to reveal the prevalence and distribution pattern of a wide spectrum of tick-borne pathogens.ResultsNew localities of D. reticulatus occurrence were found in western and central Latvia, including the Riga region, indicating that the northern border of D. reticulatus in Europe has moved farther to the north. Among the analyzed ticks, 33.42% carried at least one tick-borne pathogen, and 5.55% of tick samples were positive for two or three pathogens. A higher overall prevalence of tick-borne pathogens was observed in I. ricinus (34.92%) and I. persulcatus (31.65%) than in D. reticulatus (24.2%). The molecular analysis revealed the presence of tick-borne encephalitis virus, Babesia spp., Borrelia spp., Anaplasma phagocytophilum and Rickettsia spp. Overall, 15 and 7 tick-borne pathogen species were detected in Ixodes spp. and D. reticulatus ticks, respectively. This is the first report of Borrelia miyamotoi in Latvian field-collected ticks.ConclusionsThis large-scale countrywide study provides a snapshot of the current distribution patterns of Ixodes and Dermacentor ticks in Latvia and gives us a reliable overview of tick-borne pathogens in Latvian field-collected ticks.

Molecular detection of Bartonella in ixodid ticks collected from yaks and plateau pikas (Ochotona curzoniae) in Shiqu County, China

BackgroundBartonella bacteria have been associated with an increasingly wide range of human and animal diseases. These emerging pathogens have been identified as being globally dispersed. Ticks and small rodents are known hosts of Bartonella and play a significant role in the preservation and circulation of Bartonella in nature. This study investigated the occurrence of hoist spp. in ticks (Acari: Ixodidae) and plateau pikas (Ochotona curzoniae) in Shiqu County, which is located on the eastern Qinghai-Tibetan Plateau in China. Shiqu County is spread over approximately 26,000 km2, with an average altitude of above 4200 m and a vast area of pastureland.ResultsA total of 818 ticks (Dermacentor everestianus, 79.0%, 646/818; Haemaphysalis qinghaiensis, 21.0%, 172/818) were collected from yaks in 4 villages of Shiqu County. Only Bartonella melophagi was detected in tick samples, with a total prevalence of 30.1% (246/818). The infection rates of B. melophagi in ticks from Arizha, Maga, Derongma, and Changxgma were 4.8, 76.8, 12.5, and 18.0%, respectively. The infection rate of B. melophagi in Maga was higher (p < 0.01) than those in other villages. Regarding plateau pikas, the total infection rate of Bartonella spp. was 21.7% (62/286), with 16.7% (12/72), 30.9% (25/81), 13.8% (9/65), and 23.5% (16/68) in Arizha, Maga, Derongma, and Changxgma, respectively. Finally, B. queenslandensis and B. grahamii were detected in plateau pika. No significant difference was observed (p > 0.05) in the infection rates between these study sites.ConclusionTo date, only D. everestianus and H. qinghaiensis were found in Shiqu County with high infection of Bartonella spp. in the ticks and plateau pika. The threats of Bartonella species to public health should be closely monitored.

Detection of a putative novel genotype of Anaplasma in gray-brocket deer (Mazama gouazoubira) from Uruguay.

Anaplasmataceae includes the genera Anaplasma, Ehrlichia, Neorickettsia and Wolbachia, comprising a group of obligate intracellular bacteria. The genus Anaplasma has pathogenic species transmitted by ticks of veterinary and human health importance. Wild ungulates such as deer represent important reservoirs and amplifiers of Anaplasmataceae. The interaction between deer and domestic ruminants represents a serious problem due to the transmission of these pathogens through their ectoparasites. In the present study, we investigated the presence of Anaplasmataceae organisms in blood, tissues and tick samples of a gray-brocket deer (Mazama gouazoubira). The specimen was found dead in a farm in northeast Uruguay. PCRs targeting partial regions of 16S rRNA and groESL genes were carried out for Anaplasmataceae DNA detection. Moreover, several ectoparasites were identified: the chewing louse Tricholipeurus albimarginatus, the Neotropical deer louse fly Lipoptena mazamae, and the ticks Haemaphysalis juxtakochi and Rhipicephalus microplus. A consensus sequence of 1274bp of 16S rRNA was generated for Anaplasma sp. from the M. gouazoubira blood sample. All ticks analysed by PCR assays were negative. No band was detected in any of the samples after PCR targeting groESL gene. Phylogenetic analysis using 16S rRNA partial gene sequences, clustered the putative novel genotype sequence obtained in this study, named Anaplasma sp. genotype Mazama-Uruguay, along with Anaplasma sp. detected in Mazama sp., Mazama americana and Mazama bororo, all deer species from Brazil. Furthermore, this cluster showed to be closely related to Anaplasma bovis sequences obtained from various ruminants and other mammals from several parts of the world. The pathogenicity as well as its infecting potential to other cervids or domestic ruminants is currently unknown. Further studies should be performed in order to characterize this novel species, especially targeting other genes.

Molecular detection of Hepatozoon canis in dogs and ticks in Shaanxi province, China

Hepatozoon canis, transmitted by Rhipicephalus sanguineus, is a tick-borne pathogen and causes canine hepatozoonosis. Until now, only limited previous studies were conducted on the molecular detection and characterization of Hepatozoon sp. in dogs in China. Blood samples were collected from 93 sick dogs that were clinically diagnosed as babesiosis but tested negative for Babesia, and 103 apparently healthy dogs, as well as their infesting ticks in Xi’an and Hanzhong cities, Shaanxi province of China. PCR amplifying partial 18S rRNA gene was used to detect the DNA of Hepatozoon sp. Genetic and phylogenetic analysis were performed to determine the Hepatozoon species. Our results demonstrated that H. canis was identified from the sick dogs and the infested ticks in Hanzhong, with no significant differences of prevalence between both genders and ages. No positive blood or tick samples were found in Xi’an. Moreover, all the 18S rRNA gene sequences recovered from both dogs and the infested ticks showed a high genetic similarity with each other, and also presented a close relationship with other known sequences in and outside China. In conclusion, H. canis was identified in babesiosis-suspected dogs and ticks infesting them in Shaanxi, China, although the association between clinical signs and H. canis need further study.

Identifikasi dan Pola Infestasi Caplak pada Biawak Air (Varanus salvator, Byers, d. 2000)

The aim of this study is to identify the type of ticks infestation pattern on water monitor lizard (Varanus salvator, Byers, D. 2000) with regard parameters water monitor lizard species, stadium and region of the water monitor lizard’s body. The total samples of 250 ticks from 30 water monitor lizard (18 male lizards and 12 female lizards) were collected every day starting from noon to evening during July to August 2019 at the monitor slaughtering place in Buduran District, Sidoarjo Regency. The tick samples were identified by the permanent mounting slide method without staining and put in bottles containing 10% KOH for 1-10 hours, then each stadium used a nikon eclipse E100 microscope with a 40x magnification binocular microscope. Data was analyzed using Multiple Correspondance Analytics. The result indicate that 30 V. salvator (100%) are positive infested by tick and the samples obtained consisted of 133 Aponomma sp. and 117 Amblyomma sp. in all regions of the body.

Molecular screening of cattle ticks, tick-borne pathogens and amitraz resistance in ticks of Santo Domingo de los Tsáchilas province in Ecuador

The province of Santo Domingo de los Tsáchilas in Ecuador is a strategic place for cattle production and trade. The lack of knowledge about tick species, tick control and tick-borne diseases in Ecuador prompted this study with the goal of identifying the cattle-tick species and tick-borne agents present in the area and molecularly determining the potential acaricide resistance to amitraz of the major cattle tick species. Eighty-four cattle farms were visited and in 88 % of them, cattle were infested with ticks. Additionally, 24 historical samples from other surrounding Ecuadorian provinces, were screened as well. Besides morphological keys, PCR-RFLP MspI was used to confirm the presence of the Rhipicephalus ticks. The tick samples were also screened for tick-borne agents using PCR-RFLP BseDI and Hhal tests to identify circulating Babesia sp. and Anaplasma spp. Furthermore, the PCR-RFLP EciI technique was used to identify the amitraz resistance gene in populations of Rhipicephalus microplus in the province. Pooled testing was used to determine prevalence at individual-tick level. The presence of R. microplus and Amblyomma cajennense sensu lato (s.l.) ticks was found in 83 % and 21 % of the cattle farms respectively, showing R. microplus is widespread in the province of Santo Domingo de los Tsáchilas. Regarding tick-borne agents, only Anaplasma marginale was observed in 50 % of the visited farms of the province, while about 27 % of the ticks tested positive according to estimations from the data of the tick pools. The presence of Babesia bigemina was only confirmed in samples collected outside the province. The amitraz resistance allele in R. microplus was found in 62 % of the farms, but the percentage of farms with cattle ticks completely resistant to this acaricide was low (2%). The findings of this study should prompt cattle producers and animal health authorities to monitor control strategies, which address the management of resistant tick populations and the epidemiologically-unstable areas of tick-borne diseases.

An outbreak of bovine babesiosis in February, 2019, triggered by above average winter temperatures in southern England and co-infection with Babesia divergens and Anaplasma phagocytophilum

BackgroundBovine babesiosis, commonly known as redwater fever, is a sporadic tick-borne disease in the United Kingdom. Outbreaks occur during the spring, summer and autumn months when ticks are active. This study reports the findings of an investigation of an outbreak of bovine babesiosis during the winter month of February, 2019.MethodsDNA from blood, organ and tick samples taken from affected cattle were tested for the presence of piroplasm and Anaplasma phagocytophilum DNA using PCRs directed to the 18S rRNA gene and msp2 gene respectively. The species of piroplasm was confirmed by sequencing.ResultsBabesia divergens DNA was detected in the blood of five cattle displaying clinical signs of babesiosis within a herd of twenty. This parasite was also detected in three of ten ticks removed from one of the affected cattle. In addition, A. phagocytophilum was detected in three cattle tested and two of ten of the ticks.ConclusionsAn outbreak of bovine babesiosis during February is unusual as the tick vector, Ixodes ricinus, does not generally become active until temperatures rise later in the year. February of 2019 was unusual as average temperatures during the first week of the month reached over 10 °C, well above historical averages that are typically below 5 °C, and a temperature at which ticks can become active. This unusual weather event is likely to have triggered tick questing, that combined with a co-infection with two tick-borne pathogens caused the severe outbreak of disease.

Taxon Appearance From Extraction and Amplification Steps Demonstrates the Value of Multiple Controls in Tick Microbiota Analysis.

BackgroundThe development of high-throughput sequencing technologies has substantially improved analysis of bacterial community diversity, composition, and functions. Over the last decade, high-throughput sequencing has been used extensively to identify the diversity and composition of tick microbial communities. However, a growing number of studies are warning about the impact of contamination brought along the different steps of the analytical process, from DNA extraction to amplification. In low biomass samples, e.g., individual tick samples, these contaminants may represent a large part of the obtained sequences, and thus generate considerable errors in downstream analyses and in the interpretation of results. Most studies of tick microbiota either do not mention the inclusion of controls during the DNA extraction or amplification steps, or consider the lack of an electrophoresis signal as an absence of contamination. In this context, we aimed to assess the proportion of contaminant sequences resulting from these steps. We analyzed the microbiota of individual Ixodes ricinus ticks by including several categories of controls throughout the analytical process: homogenization, DNA extraction, and DNA amplification.ResultsControls yielded a significant number of sequences (1,126–13,198 mean sequences, depending on the control category). Some operational taxonomic units (OTUs) detected in these controls belong to genera reported in previous tick microbiota studies. In this study, these OTUs accounted for 50.9% of the total number of sequences in our samples, and were considered contaminants. Contamination levels (i.e., the percentage of sequences belonging to OTUs identified as contaminants) varied with tick instar and sex: 76.3% of nymphs and 75% of males demonstrated contamination over 50%, while most females (65.7%) had rates lower than 20%. Contamination mainly corresponded to OTUs detected in homogenization and extraction reagent controls, highlighting the importance of carefully controlling these steps.ConclusionHere, we showed that contaminant OTUs from sample laboratory processing steps can represent more than half the total sequence yield in sequencing runs, and lead to unreliable results when characterizing tick microbial communities. We thus strongly advise the routine use of negative controls in tick microbiota studies, and more generally in studies involving low biomass samples.

Identification and distribution of nine tick-borne spotted fever group Rickettsiae in the Country of Georgia

Rickettsial pathogens cause diseases that vary in severity and clinical presentation. Rickettsia species transmitted by ticks are mostly classified within the spotted fever group of rickettsiae (SFGR) and are often associated with febrile diseases. Preliminary studies have detected three human-pathogenic SFGR from ticks in Georgia: Rickettsia aeschlimannii, Rickettsia raoultii, and Rickettsia slovaca. To more broadly assess the presence of tick-borne rickettsiae from Georgia we examined 1594 ticks, representing 18 species from five genera (Ixodes, Hyalomma, Haemaphysalis, Dermacentor, and Rhipicephalus), collected from eight regions of Georgia. A total of 498 tick DNA samples extracted from single ticks or pooled ticks were assessed by molecular methods. Genus-specific Rick17b and species-specific qPCR assays were used to identify six rickettsiae: R. aeschlimannii, R. raoultii, R. slovaca, Rickettsia conorii subsp. conorii, Rickettsia massiliae, and Rickettsia monacensis. Tick samples that were positive for Rickettsia, but not identified by the species-specific assays, were further evaluated by multi-locus sequence typing (MLST) using sequences of four protein-coding genes (gltA, ompA,ompB, sca4). Three additional Rickettsia species were identified by MLST: Candidatus Rickettsia barbariae, Rickettsia helvetica, and Rickettsia hoogstraalii. Overall, nine species of Rickettsia (six human pathogens and three species with unknown pathogenicity) were detected from 12 tick species of five different genera. A distribution map for the tick-borne rickettsiae revealed six newly identified endemic regions in Georgia.