Publications by authors named "Frans Jongejan"

97 Publications

Unique Mitochondrial Single Nucleotide Polymorphisms Demonstrate Resolution Potential to Discriminate Vaccine and Buffalo-Derived Strains.

Life (Basel) 2020 Dec 8;10(12). Epub 2020 Dec 8.

Institute for Parasitology and Tropical Veterinary Medicine, Freie Universität Berlin, Robert-von-Ostertag-Str. 7-13, 14163 Berlin, Germany.

Distinct pathogenic and epidemiological features underlie different strains resulting in different clinical manifestations of East Coast Fever and Corridor Disease in susceptible cattle. Unclear delineation of these strains limits the control of these diseases in endemic areas. Hence, an accurate characterization of strains can improve the treatment and prevention approaches as well as investigate their origin. Here, we describe a set of single nucleotide polymorphisms (SNPs) based on 13 near-complete mitogenomes of strains originating from East and Southern Africa, including the live vaccine stock strains. We identified 11 SNPs that are non-preferentially distributed within the coding and non-coding regions, all of which are synonymous except for two within the gene of buffalo-derived strains. Our analysis ascertains haplotype-specific mutations that segregate the different vaccine and the buffalo-derived strains except Muguga and Serengeti-transformed strains suggesting a shared lineage between the latter two vaccine strains. Phylogenetic analyses including the mitogenomes of other species: , , and , with the latter two sequenced in this study for the first time, were congruent with nuclear-encoded genes. Importantly, we describe seven haplotypes characterized by synonymous SNPs and parsimony-informative characters with the other three transforming species mitogenomes. We anticipate that tracking mitochondrial haplotypes from this study will provide insight into the parasite's epidemiological dynamics and underpin current control efforts.
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http://dx.doi.org/10.3390/life10120334DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7764068PMC
December 2020

Parasites and vector-borne diseases disseminated by rehomed dogs.

Parasit Vectors 2020 Nov 10;13(1):546. Epub 2020 Nov 10.

Institute of Animal Hygiene and Veterinary Public Health, University of Leipzig, Leipzig, Germany.

The Companion Vector-Borne Diseases (CVBD) World Forum is a working group of leading international experts who meet annually to evaluate current scientific findings and future trends concerning the distribution, pathogenesis, clinical presentation, diagnosis and prevention of vector-borne infections of dogs and cats. At the 14th Symposium of the CVBD World Forum in Trieste, Italy (March 25-28, 2019), we identified the need to (i) bring attention to the potential spread of parasites and vectors with relocated dogs, and (ii) provide advice to the veterinary profession regarding the importance of surveillance and treatment for parasites and vector-borne infections when rehoming dogs. This letter shares a consensus statement from the CVBD World Forum as well as a summary of the problem faced, including the role of veterinary professionals in parasite surveillance, causal issues, and the importance of interdisciplinary cooperation in addressing the problem. To limit opportunities for dissemination of parasites and vectors, whenever possible, underlying problems creating the need for dog rehoming should be addressed. However, when it is necessary to rehome dogs, this should ideally take place in the country and national region of origin. When geographically distant relocation occurs, veterinary professionals have a vital role to play in public education, vigilance for detection of exotic vectors and infections, and alerting the medical community to the risk(s) for pathogen spread. With appropriate veterinary intervention, dog welfare needs can be met without inadvertently allowing global spread of parasites and their vectors.
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http://dx.doi.org/10.1186/s13071-020-04407-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7653694PMC
November 2020

Interrupted Blood Feeding in Ticks: Causes and Consequences.

Microorganisms 2020 Jun 16;8(6). Epub 2020 Jun 16.

Ceva Santé Animale, 10 Avenue de la Ballastière, 33500, Libourne, France.

Ticks are obligate hematophagous arthropods and act as vectors for a great variety of pathogens, including viruses, bacteria, protozoa, and helminths. Some tick-borne viruses, such as Powassan virus and tick-borne encephalitis virus, are transmissible within 15-60 min after tick attachment. However, a minimum of 3-24 h of tick attachment is necessary to effectively transmit bacterial agents such as spp., spp., and spp. to a new host. Longer transmission periods were reported for spp. and protozoans such as spp., which require a minimum duration of 24-48 h of tick attachment for maturation and migration of the pathogen. Laboratory observations indicate that the probability of transmission of tick-borne pathogens increases with the duration an infected tick is allowed to remain attached to the host. However, the transmission time may be shortened when partially fed infected ticks detach from their initial host and reattach to a new host, on which they complete their engorgement. For example, early transmission of tick-borne pathogens (e.g., , , and ) and a significantly shorter transmission time were demonstrated in laboratory experiments by interrupted blood feeding. The relevance of such situations under field conditions remains poorly documented. In this review, we explore parameters of, and causes leading to, spontaneous interrupted feeding in nature, as well as the effects of this behavior on the minimum time required for transmission of tick-borne pathogens.
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http://dx.doi.org/10.3390/microorganisms8060910DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7355616PMC
June 2020

Amblyomma hebraeum is the predominant tick species on goats in the Mnisi Community Area of Mpumalanga Province South Africa and is co-infected with Ehrlichia ruminantium and Rickettsia africae.

Parasit Vectors 2020 Apr 21;13(1):172. Epub 2020 Apr 21.

Vectors and Vector-borne Diseases Research Programme, Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort, 0110, Republic of South Africa.

Background: In sub-Saharan Africa, Amblyomma ticks are vectors of heartwater disease in domestic ruminants, caused by the rickettsial pathogen Ehrlichia ruminantium. Immature tick stages often bite humans, whereby they act as vectors of tick-bite fever caused by Rickettsia africae. Moreover, Amblyomma ticks cause damage to livestock due to their feeding behaviour. In South Africa, we studied the abundance of Amblyomma hebraeum ticks on goats of emerging farmers in Mpumalanga Province. A selected number of A. hebraeum nymphs and adult ticks was tested for co-infection with E. ruminantium and R. africae.

Methods: A total of 630 indigenous goats, belonging to farmers in the Mnisi Community area, were examined for ticks in 2013 and 2014. All ticks were identified, and a selected number was tested by PCR with reverse line blot hybridisation.

Results: In total, 13,132 ticks were collected from goats distributed over 17 different households. Amblyomma hebraeum was the predominant species, followed by R. microplus. Rhipicephalus appendiculatus, R. simus and R. zambeziensis were also identified. Amblyomma hebraeum was present throughout the year, with peak activity of adults in summer (November) and nymphs in winter (July). The ratio between adults and nymphs ranged from 1:2.7 in summer to 1:55.1 in winter. The mean prevalence of infection for E. ruminantium by PCR/RLB in adult ticks was 17.4% (31/178), whereas 15.7% (28/178) were infected with R. africae. In pooled nymphs, 28.4% were infected with E. ruminantium and 38.8% carried R. africae infection. Co-infections of E. ruminantium and R. africae in adult and pooled nymphal ticks were 3.9% (7/178) and 10% (14.9), respectively. Lameness of goats due to predilection of ticks for the interdigital space of their feet was observed in 89% of the households.

Conclusions: Goats act as important alternative hosts for cattle ticks, which underscored the necessity to include goats in control programs. It is suggested to use acaricide-impregnated leg-bands as a sustainable method to kill ticks and prevent lameness in goats. The challenge of goats by considerable numbers of E. ruminantium-infected ticks is a major obstacle for upgrading the indigenous goat breeds. Humans may be at risk to contract tick-bite fever in this area.
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http://dx.doi.org/10.1186/s13071-020-04059-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7171862PMC
April 2020

Invasive cattle ticks in East Africa: morphological and molecular confirmation of the presence of Rhipicephalus microplus in south-eastern Uganda.

Parasit Vectors 2020 Apr 3;13(1):165. Epub 2020 Apr 3.

Department of Veterinary Tropical Diseases, Faculty of Veterinary Science, University of Pretoria, Private Bag X04, Onderstepoort, 0110, South Africa.

Background: Rhipicephalus microplus, an invasive tick species of Asian origin and the main vector of Babesia species, is considered one of the most widespread ectoparasites of livestock. The tick has spread from its native habitats on translocated livestock to large parts of the tropical world, where it has replaced some of the local populations of Rhipicephalus decoloratus ticks. Although the tick was reported in Uganda 70 years ago, it has not been found in any subsequent surveys. This study was carried out to update the national tick species distribution on livestock in Uganda as a basis for tick and tick-borne disease control, with particular reference to R. microplus.

Methods: The study was carried out in Kadungulu, Serere district, south-eastern Uganda, which is dominated by small scale livestock producers. All the ticks collected from 240 cattle from six villages were identified microscopically. Five R. microplus specimens were further processed for phylogenetic analysis and species confirmation.

Results: The predominant tick species found on cattle was Rhipicephalus appendiculatus (86.9 %; n = 16,509). Other species found were Amblyomma variegatum (7.2 %; n = 1377), Rhipicephalus evertsi (2.3 %; n = 434) and R. microplus (3.6 %; n = 687). Phylogenetic analysis of the 12S rRNA, 16S rRNA and ITS2 gene sequences of R. microplus confirmed the morphological identification.

Conclusions: It is concluded that R. microplus has replaced R. decoloratus in the sampled villages in Kadungulu sub-county, since the latter was not any longer found in this area. There is currently no livestock movement policy in force in Uganda, which could possibly limit the further spread of R. microplus ticks. Future surveys, but also retrospective surveys of museum specimens, will reveal the extent of distribution of R. microplus in Uganda and also for how long this tick has been present on livestock without being noticed.
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http://dx.doi.org/10.1186/s13071-020-04043-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7118885PMC
April 2020

Cutaneous Immunoprofiles of Three Spotted Fever Group Rickettsia Cases.

Infect Immun 2020 03 23;88(4). Epub 2020 Mar 23.

State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, People's Republic of China

Spotted fever group rickettsia (SFGR) can cause mild to fatal illness. The early interaction between the host and rickettsia in skin is largely unknown, and the pathogenesis of severe rickettsiosis remains an important topic. A surveillance of SFGR infection by PCR of blood and skin biopsy specimens followed by sequencing and immunohistochemical (IHC) detection was performed on patients with a recent tick bite between 2013 and 2016. Humoral and cutaneous immunoprofiles were evaluated in different SFGR cases by serum cytokine and chemokine detection, skin IHC staining, and transcriptome sequencing (RNA-seq). A total of 111 SFGR cases were identified, including 79 " Rickettsia tarasevichiae," 22 , 8 , and 2 cases. The sensitivity to detect SFGR in skin biopsy specimens (9/24, 37.5%) was significantly higher than that in blood samples (105/2,671, 3.9%) ( < 0.05). As early as 1 day after the tick bite, rickettsiae could be detected in the skin. infection was more severe than " Rickettsia" and infections. Increased levels of serum interleukin-18 (IL-18), IP10, and monokine induced by gamma interferon (MIG) and decreased levels of IL-2 were observed in febrile patients infected with compared to those infected with " Rickettsia." RNA-seq and IHC staining could not discriminate between SFGR-infected and uninfected tick bite skin lesions. However, the type I interferon (IFN) response was differently expressed between and infections at the cutaneous interface. It is concluded that skin biopsy specimens were more reliable for the detection of SFGR infection in human patients although the immunoprofile may be complicated by immunomodulators induced by the tick bite.
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http://dx.doi.org/10.1128/IAI.00686-19DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7093127PMC
March 2020

Canine Leishmaniasis Control in the Context of One Health.

Emerg Infect Dis 2019 12;25(12):1-4

Dogs are the main reservoir of Leishmania infantum and in some countries have been regularly culled as part of government policy to control visceral leishmaniasis. At the 13th Symposium of the Companion Vector-Borne Diseases World Forum in Windsor, UK, March 19-22, 2018, we consolidated a consensus statement regarding the usefulness of dog culling as a means of controlling visceral leishmaniasis. The statement highlighted the futility of culling infected dogs, whether healthy or sick, as a measure to control the domestic reservoir of L. infantum and reduce the risk for visceral leishmaniasis.
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http://dx.doi.org/10.3201/eid2512.190164DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6874277PMC
December 2019

"Tekenscanner": a novel smartphone application for companion animal owners and veterinarians to engage in tick and tick-borne pathogen surveillance in the Netherlands.

Parasit Vectors 2019 Mar 26;12(1):116. Epub 2019 Mar 26.

Utrecht Centre for Tick-borne Diseases (UCTD), FAO Reference Centre for Ticks and Tick-borne Diseases, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584 CL, Utrecht, The Netherlands.

Background: The engagement of companion animal owners into the process of collecting epidemiological data can be facilitated through smartphone applications. In April 2018, the "tekenscanner" (Dutch for tick scanner) app was launched with the aim of engaging pet owners and veterinarians to record ticks removed from their pets and submit these ticks for identification and pathogen testing. Tick-borne pathogens identified in ticks removed from dogs and cats during the first 6 months after the app was launched in the Netherlands are reported.

Methods: The tekenscanner app was used to record the geographical coordinates of ticks removed from dogs or cats onto a map of the Netherlands. A barcode was assigned to each tick for the easy tracking of each submission to our laboratory for taxonomic identification. Thereafter, DNA extracted from the ticks was PCR amplified, subjected to reverse line blot hybridization (RLB) and screened for a broad range of tick-borne pathogens. Results were added to the same app, usually within 2 weeks after the submission of each tick.

Results: The app was downloaded 5591 times and resulted in the collection of 1273 georeferenced and barcoded ticks, with a peak submission in May and June of 2018. There were 1005 ticks collected from 406 dogs and 268 ticks collected from 111 cats. Ixodes ricinus was the predominant species (90.0%), with all stages found on dogs as well as on cats. Ixodes hexagonus (7.3%) female and nymphal ticks were also identified on both hosts, whereas adults of Dermacentor reticulatus (2.4%) and Rhipicephalus sanguineus (0.2%) were exclusively found on dogs. Nearly 15% of the ticks recovered from dogs carried one or more pathogens, whereas 13.8% of the ticks removed from cats were infected. Ixodes ricinus collected from dogs contained Borrelia spp. (1.9%), Babesia spp. (0.7%), Anaplasma phagocytophilum (1.3%), "Candidatus Neoehrlichia mikurensis" (2.9%) and Rickettsia helvetica (7.3%). Ixodes ricinus recovered from cats were infected with Borrelia spp. (1.9%), Babesia spp. (0.4%), A. phagocytophilum (1.9%), "Ca. Neoehrlichia mikurensis" (2.6%) and R. helvetica (6.7%). Ixodes hexagonus ticks (n = 93) were not infected. Dermacentor reticulatus ticks, found only in autumn, were infected with Rickettsia raoultii (16 %) and A. phagocytophilum. Three R. sanguineus, on dogs from France and the USA imported into the Netherlands, were all negative.

Conclusions: The tekenscanner app is a versatile tool to use for submission of ticks and facilitated the fast feedback of test results. Community engagement through the app is suitable for identifying hotspots for ticks and tick-borne pathogens and provided an early warning system for exotic ticks invading the Netherlands.
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http://dx.doi.org/10.1186/s13071-019-3373-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6434828PMC
March 2019

Recombination and purifying and balancing selection determine the evolution of major antigenic protein 1 (map 1) family genes in Ehrlichia ruminantium.

Gene 2019 Jan 11;683:216-224. Epub 2018 Oct 11.

Laboratory of Parasitology, Department of Disease Control, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan. Electronic address:

Heartwater is an economically important disease of ruminants caused by the tick-borne bacterium Ehrlichia ruminantium. The disease is present throughout sub-Saharan Africa as well as on several islands in the Caribbean, where it poses a risk of spreading onto the American mainland. The dominant immune response of infected animals is directed against the variable outer membrane proteins of E. ruminantium encoded by a polymorphic multigene family. Here, we examined the full-length sequence of the major antigenic protein 1 (map1) family genes in multiple E. ruminantium isolates from different African countries and the Caribbean, collected at different time points to infer the possible role of recombination breakpoint and natural selection. A high level of recombination was found particularly in map1 and map1-2. Evidence of strong negative purifying selection in map1 and balancing selection to maintain genetic variation across these samples from geographically distinct countries suggests host-pathogen co-evolution. This co-evolution between the host and pathogen results in balancing selection by maintaining genetic diversity that could be explained by the demographic history of long-term pathogen pressure. This signifies the adaptive role and the molecular evolutionary forces underpinning E. ruminantium map1 multigene family antigenicity.
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http://dx.doi.org/10.1016/j.gene.2018.10.028DOI Listing
January 2019

Transmission of Rickettsia raoultii and Rickettsia massiliae DNA by Dermacentor reticulatus and Rhipicephalus sanguineus (s.l.) ticks during artificial feeding.

Parasit Vectors 2018 Sep 3;11(1):494. Epub 2018 Sep 3.

Utrecht Centre for Tick-borne Diseases, FAO Reference Centre for Ticks and Tick-borne Diseases, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584 CL, Utrecht, The Netherlands.

Background: Tick-borne rickettsial pathogens are emerging worldwide and pose an increased health risk to both humans and animals. A plethora of rickettsial species has been identified in ticks recovered from human and animal patients. However, the detection of rickettsial DNA in ticks does not necessarily mean that these ticks can act as vectors for these pathogens. Here, we used artificial feeding of ticks to confirm transmission of Rickettsia massiliae and Rickettsia raoultii by Rhipicephalus sanguineus (sensu lato) and Dermacentor reticulatus ticks, respectively. The speed of transmission was also determined.

Methods: An artificial feeding system based on silicone membranes were used to feed adult R. sanguineus (s.l.) and D. reticulatus ticks. Blood samples from in vitro feeding units were analysed for the presence of rickettsial DNA using PCR and reverse line blot hybridisation.

Results: The attachment rate of R. sanguineus (s.l.) ticks were 40.4% at 8 h post-application, increasing to 70.2% at 72 h. Rickettsia massiliae was detected in blood samples collected 8 h after the R. sanguineus (s.l.) ticks were placed into the in vitro feeding units. D. reticulatus ticks were pre-fed on sheep and subsequently transferred to the in vitro feeding system. The attachment rate was 29.1 % at 24 h post-application, increasing to 43.6 % at 96 h. Rickettsia raoultii was detected in blood collected 24 h after D. reticulatus was placed into the feeding units.

Conclusions: Rhipicephalus sanguineus (s.l.) and D. reticulatus ticks are vectors of R. massiliae and R. raoultii, respectively. The transmission of R. massiliae as early as 8 h after tick attachment emphasises the importance of removing ticks as soon as possible to minimise transmission. This study highlights the relevance of in vitro feeding systems to provide insight into the vectorial capacity of ticks and the dynamics of tick-borne pathogen transmission.
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http://dx.doi.org/10.1186/s13071-018-3075-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6122679PMC
September 2018

Investigation on Ehrlichia Infection in Small Mammals and Ticks from Tengchong, Yunnan Province, Southern China.

Vector Borne Zoonotic Dis 2018 10 13;18(10):563-566. Epub 2018 Jun 13.

2 State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology , Beijing, P.R. China .

Background: Rare investigation on tick-borne pathogens was carried out in Yunnan, China. In this study, we did a survey on Ehrlichia infection in small mammals and ticks. A total of 40 small mammals and 49 ticks were collected from Tengchong, Yunnan province. PCR targeting 16S ribosomal RNA (rRNA), citrate synthase, GroEL heat-shock protein operon, and major outer membrane protein genes was performed and positive amplicons were sequenced.

Results: The 40 small mammals were identified as 10 species, 2 (5.0%) of which were infected with Ehrlichia, 4 (10.0%) were infected with Anaplasma phagocytophilum and another 2 (5.0%) were infected with Candidatus Neoehrlichia mikurensis. Six (12.2%) ticks were positive for Ehrlichia and another two (4.1%) were infected with A. phagocytophilum. Neither small mammals nor ticks had coinfection. The detected Ehrlichia was named as Ehrlichia sp. YN04, which was in the same clade of Ehrlichia sp. 360 by phylogenetic analysis. The sequences of the pathogen recovered from small mammals and ticks were identical with each other.

Conclusion: The study reports one Ehrlichia species first detected from small mammals and ticks in mainland China. As Yunnan is a famous "Global Biodiversity Hotspot" in the world, we may expect much more tick-borne infectious pathogens existing and declare more public health attention in this region.
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http://dx.doi.org/10.1089/vbz.2017.2205DOI Listing
October 2018

Molecular evidence for the transovarial passage of Babesia gibsoni in Haemaphysalis hystricis (Acari: Ixodidae) ticks from Taiwan: a novel vector for canine babesiosis.

Parasit Vectors 2018 03 20;11(1):134. Epub 2018 Mar 20.

Utrecht Centre for Tick-borne Diseases (UCTD), FAO Reference Centre for Ticks and Tick-borne Diseases, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, 3584, CL, Utrecht, The Netherlands.

Background: Babesia gibsoni is the predominant tick-borne protozoan blood parasite affecting dogs throughout the Oriental region. Babesia gibsoni is transmitted by Haemaphysalis longicornis, whereas a similar role has been suggested for Rhipicephalus sanguineus. Haemaphysalis longicornis does not occur in Taiwan, but R. sanguineus is widely distributed on dogs. However, clinical cases of babesiosis are mainly restricted to the northern part of the island. The discrepancy between tick distribution and clinical cases stimulated us to investigate the tick species distribution on dogs in northern Taiwan, with the aim to identify the local vector for canine babesiosis.

Methods: Ticks were collected from stray dogs or free ranging pet dogs in northern Taiwan between 2015 and 2017 and, after identification, were tested for the presence of tick-borne Babesia parasites using PCR and reverse line blot (RLB) hybridisation. Moreover, engorged ticks collected from the dogs were incubated at 28 °C to allow them to oviposit. Their subsequent larval progeny was also examined by PCR/RLB.

Results: A total of 1085 ticks collected from 144 stray dogs at different residential areas consisted of 5 different species: H. hystricis (n = 435), R. sanguineus (n = 582), R. haemaphysaloides (n = 43), Amblyomma testudinarium (n = 14) and Ixodes ovatus (n = 11) were identified. Babesia gibsoni DNA was detected in H. hystricis females (10.3%), males (7.0%) and in 2.6% of the nymphs. One R. sanguineus female and one A. testudinarium female tick also carried B. gibsoni DNA. DNA of B. gibsoni was demonstrated in 11 out of 68 (16.2%) batches of larval ticks derived from engorged H. hystricus ticks only. Babesia vogeli DNA was detected only in R. sanguineus females (2.6%) and males (2.4%). DNA of B. vogeli was detected in 13 out of 95 (13.7%) batches of larval ticks derived from engorged R.sanguineus females.

Conclusions: Babesia gibsoni DNA was detected in the larval progeny of H. hystricis ticks only, whereas B. vogeli was restricted to the larvae of R. sanguineus. This provides evidence for transovarial passage of B. gibsoni in H. hystricis and evidence that this tick does act as the local vector for this parasite on dogs in northern Taiwan where most cases of babesiosis are reported. The vectorial capacity of R. sanguineus for babesiosis is probably restricted to the transmission of B. vogeli only.
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http://dx.doi.org/10.1186/s13071-018-2722-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5859485PMC
March 2018

A glycosylated recombinant subunit candidate vaccine consisting of Ehrlichia ruminantium major antigenic protein1 induces specific humoral and Th1 type cell responses in sheep.

PLoS One 2017 28;12(9):e0185495. Epub 2017 Sep 28.

Utrecht Centre for Tick-Borne Diseases, FAO Reference Centre for Ticks and Tick-Borne Diseases, Faculty of Veterinary Medicine, Utrecht University, Yalelaan 1, Utrecht, The Netherlands.

Heartwater, or cowdriosis, is a tick-borne disease of domestic and wild ruminants that is endemic in the Caribbean and sub-Saharan Africa. The disease is caused by an intracellular pathogen, Ehrlichia ruminantium and may be fatal within days of the onset of clinical signs with mortality rates of up to 90% in susceptible hosts. Due to the presence of competent tick vectors in North America, there is substantial risk of introduction of heartwater with potentially devastating consequences to the domestic livestock industry. There is currently no reliable or safe vaccine for use globally. To develop a protective DIVA (differentiate infected from vaccinated animals) subunit vaccine for heartwater, we targeted the E. ruminantium immunodominant major antigenic protein1 (MAP1) with the hypothesis that MAP1 is a glycosylated protein and glycans contained in the antigenic protein are important epitope determinants. Using a eukaryotic recombinant baculovirus expression system, we expressed and characterized, for the first time, a glycoform profile of MAP1 of two Caribbean E. ruminantium isolates, Antigua and Gardel. We have shown that the 37-38 kDa protein corresponded to a glycosylated form of the MAP1 protein, whereas the 31-32 kDa molecular weight band represented the non-glycosylated form of the protein frequently reported in scientific literature. Three groups of sheep (n = 3-6) were vaccinated with increasing doses of a bivalent (Antigua and Gardel MAP1) rMAP1 vaccine cocktail formulation with montanide ISA25 as an adjuvant. The glycosylated recombinant subunit vaccine induced E. ruminantium-specific humoral and Th1 type T cell responses, which are critical for controlling intracellular pathogens, including E. ruminantium, in infected hosts. These results provide an important basis for development of a subunit vaccine as a novel strategy to protect susceptible livestock against heartwater in non-endemic and endemic areas.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0185495PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5619772PMC
October 2017

Cultivation of Anaplasma ovis in the HL-60 human promyelocytic leukemia cell line.

Emerg Microbes Infect 2017 Sep 20;6(9):e83. Epub 2017 Sep 20.

State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing 100071, China.

The tick-borne bacterium Anaplasma ovis is a widely distributed pathogen affecting sheep, goats and wild ruminants. Here, the HL-60 human promyelocytic leukemia cell line was used to isolate A. ovis from PCR-positive sheep and goats in Heilongjiang Province, China. Two weeks after inoculation, morulae were observed in cytoplasmic vacuoles in four different HL-60 cultures. Confocal microscopy using a Cy3-labeled A. ovis-specific probe confirmed that the HL-60 cells were infected with A. ovis. Cells from the 6th HL-60 subculture displayed positive fluorescence when incubated with A. ovis antiserum in the indirect fluorescent antibody assay. PCR amplification and sequencing of 16S rRNA, groEL, gltA, msp2 and msp4 Anaplasma genes revealed that the four A. ovis culture isolates were identical. Phylogenetic analysis showed that the sequences clustered with other A. ovis strains but could clearly be distinguished from other Anaplasma species. When the 18th subculture of infected HL-60 cells was examined by electron microscopy, lysosomes were often observed near the vacuoles. After the 24th subculture, Giemsa staining and PCR indicated that the HL-60 cells were negative for A. ovis. Although A. ovis can infect HL-60 cells for only four months, the ability of the organism to infect and multiply in HL-60 cells provides a tool to study intra-erythrocytic Anaplasma and host cell interactions.
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http://dx.doi.org/10.1038/emi.2017.70DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5625320PMC
September 2017

Influence of the spatial heterogeneity in tick abundance in the modeling of the seasonal activity of Ixodes ricinus nymphs in Western Europe.

Exp Appl Acarol 2017 Feb 26;71(2):115-130. Epub 2017 Jan 26.

EPIA, INRA, 63122, Saint Genès Champanelle, France.

The seasonal weather-driven activity of the tick Ixodes ricinus is frequently explored using multisite surveys. This study aimed to investigate the statistical modeling of seasonal trends in the activity of I. ricinus nymphs when both the influence of abiotic factors and spatial heterogeneity were taken into account. Time series data of abiotic covariates (temperature, relative humidity, rainfall and photoperiod) and nymphal tick counts were recorded on several sites in The Netherlands, Belgium and in France in 2008 and 2009. The sites were divided into two subsets which were used for model construction or model validation. A generalized linear mixed model was set up, with aggregated abiotic covariates as fixed effects, and the collection site as a random effect to account for the site-varying density in nymphs. A linear regression model was developed to estimate the site effect against the observed local abundance on each site. The activity patterns simulated from the weather and photoperiod covariates realistically reproduced the observed seasonal trends in nymphal tick activity. The fit between observed and simulated nymphal count time series was greatly improved when the site-specific local abundance in nymphs was included. Our modeling approach allows indicators of local tick abundance and the temporal modeling of I. ricinus activity to be combined. The model presented here can also be used to study scenarios on the temporal patterns of I. ricinus activity in the present and in the context of climate change.
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http://dx.doi.org/10.1007/s10493-016-0099-1DOI Listing
February 2017

Canine and ovine tick-borne pathogens in camels, Nigeria.

Vet Parasitol 2016 Sep 20;228:90-92. Epub 2016 Aug 20.

Division of Infection and Pathway Medicine, Edinburgh Medical School: Biomedical Sciences, The University of Edinburgh, Edinburgh, United Kingdom. Electronic address:

In April 2008, whole blood samples were collected from 36 dromedary camels in Sokoto, North-western Nigeria. Following PCR and reverse line blotting, twenty-two samples (61%) resulted positive for Ehrlichia/Anaplasma spp. and three (8%) for Theileria/Babesia spp., with three (8%) cases of co-infections being found. Both sequence and BLAST analyses identified Ehrlichia/Anaplasma spp. and Theileria/Babesia spp. positive cases as Anaplasma platys and Theileria ovis, respectively. This is the first report of the detection of A. platys and T. ovis in camels from sub-Saharan Africa. The epidemiological relevance of this finding is enhanced by the close living of these animals with both dogs and small ruminants. The high prevalence detected for A. platys suggests a possible role of camels as carriers of this infection.
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http://dx.doi.org/10.1016/j.vetpar.2016.08.014DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5061551PMC
September 2016

Comparative efficacy of oral administrated afoxolaner (NexGard™) and fluralaner (Bravecto™) with topically applied permethrin/imidacloprid (Advantix(®)) against transmission of Ehrlichia canis by infected Rhipicephalus sanguineus ticks to dogs.

Parasit Vectors 2016 06 17;9(1):348. Epub 2016 Jun 17.

Bayer Animal Health GmbH, Monheim, Germany.

Background: The ability of the topical spot-on Advantix(®) (50 % permethrin/10 % imidacloprid) to prevent transmission of Ehrlichia canis by infected Rhipicephalus sanguineus ticks to dogs has previously been reported. The recent market introduction of chewable tablets containing the novel compounds, afoxolaner (NexGard™) and fluralaner (Bravecto™) enabled us to conduct a comparative efficacy study with respect to the ability of these three products to block transmission of E. canis by ticks to dogs. The speed of kill, immediate drop-off rate and anti-attachment efficacy of the respective products were also studied.

Methods: The study was a blinded parallel group design, wherein 32 dogs were randomised into four different groups of eight dogs. Group 1 served as negative placebo control, group 2 and 3 were treated on Days 0, 28 and 56 with NexGard™ and Advantix(®), respectively. Group 4 was dosed once on Day 0 with Bravecto™. For tick efficacy assessments 50 non-infected ticks were placed onto the dogs on Days 30, 35, 42, 49, 56, 63, 70, 77 and 84 and on animal tick counts were performed at 3 h, 6 h and 12 h after infestation. To evaluate the ability to block transmission of E. canis, each dog was challenged by releasing 80 adult E. canis-infected R. sanguineus ticks into their sleeping kennels on Days 31, 38, 45 and 52. The animals were monitored for clinical signs of monocytic ehrlichiosis (pyrexia and thrombocytopenia) and were tested for E. canis DNA by PCR and for specific antibodies using IFA. A dog was considered infected with E. canis if both PCR and IFA yielded positive test results up to Day 84.

Results: Mean arithmetic tick counts on dogs treated with the Advantix(®) spot-on were significantly (P < 0.0005) lower throughout the study as compared with the negative controls and was, with respect to the speed of kill and resulting onset of acaricidal efficacy, superior over NexGard™ and Bravecto™ at all time points in the 12 h period observed (3 h, 6 h and 12 h). None of the dogs treated with the Advantix(®) spot-on became infected with E. canis, whereas six out of eight untreated control dogs acquired the infection. Furthermore, E. canis infection was diagnosed in four out of eight dogs treated with NexGard™ and in two out of eight dogs treated with Bravecto™.

Conclusions: The speed of kill of the two recently registered systemic compounds against R. sanguineus was not sufficiently fast to prevent transmission of E. canis and resulted in only low partial blocking and protection capacity while Advantix(®) effectively blocked transmission of E. canis to dogs in the challenge period and thus provided adequate protection for dogs against monocytic ehrlichiosis.
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http://dx.doi.org/10.1186/s13071-016-1636-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4912781PMC
June 2016

Draft Genome Sequences of Three Strains of Ehrlichia ruminantium, a Tick-Borne Pathogen of Ruminants, Isolated from Zimbabwe, The Gambia, and Ghana.

Genome Announc 2016 Jun 16;4(3). Epub 2016 Jun 16.

Division of Collaboration and Education, Hokkaido University Research Center for Zoonosis Control, Sapporo, Hokkaido, Japan Global Institution for Collaborative Research and Education, Hokkaido University, Sapporo, Hokkaido, Japan Department of Disease Control, School of Veterinary Medicine, University of Zambia, Lusaka, Zambia

The rickettsial bacterium Ehrlichia ruminantium is the causative pathogen of heartwater in ruminants. Here, we report the draft genome sequences of three strains of E. ruminantium, namely, the Crystal Springs strain from Zimbabwe, the Kerr Seringe strain from The Gambia, and the Sankat 430 strain from Ghana.
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http://dx.doi.org/10.1128/genomeA.00453-16DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4911466PMC
June 2016

Tick-borne pathogens of zoonotic and veterinary importance in Nigerian cattle.

Parasit Vectors 2016 Apr 18;9:217. Epub 2016 Apr 18.

Division of Infection and Pathway Medicine, School of Biomedical Sciences, The University of Edinburgh, Edinburgh, UK.

Background: Ticks and tick-borne diseases undermine cattle fitness and productivity in the whole of sub-Saharan Africa, including Nigeria. In this West African country, cattle are challenged by numerous tick species, especially during the wet season. Consequently, several TBDs are known to be endemic in Nigerian cattle, including anaplasmosis, babesiosis, cowdriosis and theilerioris (by Theileria mutans and Theileria velifera). To date, all investigations on cattle TBDs in Nigeria have been based on cytological examinations and/or on serological methods. This study aimed to ascertain the occurrence of tick-borne pathogens of veterinary and zoonotic importance in cattle in Nigeria using molecular approaches.

Methods: In October 2008, 704 whole blood samples were collected from indigenous cattle in the Plateau State, Nigeria. Analysis for tick-borne pathogens was conducted by means of PCR-based reverse line blotting (RLB) and sequencing targeting a panel of five genera of microorganisms (i.e. Babesia, Theileria, Anaplasma, Ehrlichia and Rickettsia spp.).

Results: In total, 561/704 (82.6%) animals were found infected, with 465 (69.6%) of them being infected by two or more microorganisms, with up to 77 possible combinations of pathogens detected. Theileria mutans was the most prevalent microorganism (66.3%), followed by Theileria velifera (52.4%), Theileria taurotragi (39.5%), Anaplasma marginale (39.1%), Anaplasma sp. (Omatjenne) (34.7%), Babesia bigemina (7.9%), Anaplasma centrale (6.3%), Anaplasma platys (3.9%), Rickettsia massiliae (3.5%), Babesia bovis (2.0%) and Ehrlichia ruminantium (1.1%). Calves were found significantly less infected than juvenile and adult cattle.

Conclusions: This study provides updated, molecular-based information on cattle TBDs in Nigeria. The molecular approach employed allowed the diagnosis of numerous positive cases including carrier statuses, multiple infections and novel pathogen detections within the indigenous cattle population. Moreover, the RLB method here described enabled the detection of veterinary agents not only pertaining to bovine health, including also those of zoonotic importance. The high prevalence recorded for T. mutans, T. velifera, A. marginale, T. taurotragi and Anaplasma sp. (Omatjenne), suggests they may be endemically established in Nigeria, whereas the lower prevalence recorded for other microorganisms (i.e. A. centrale and B. bovis) highlights a less stable epidemiological scenario, requiring further investigations.
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http://dx.doi.org/10.1186/s13071-016-1504-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4836144PMC
April 2016

A novel combination of fipronil and permethrin (Frontline Tri-Act®/Frontect®) reduces risk of transmission of Babesia canis by Dermacentor reticulatus and of Ehrlichia canis by Rhipicephalus sanguineus ticks to dogs.

Parasit Vectors 2015 Nov 19;8:602. Epub 2015 Nov 19.

Merial S.A.S., 29 Av Tony Garnier, 69007, Lyon, France.

Background: The ability of Frontline Tri-Act®/Frontect®, a topical ectoparasiticide containing fipronil and permethrin for dogs, to prevent the transmission of Babesia canis as well as Ehrlichia canis was evaluated by infesting dogs with infected vector ticks.

Methods: For the Babesia canis study, 16 dogs were randomly allocated to two groups. Eight dogs were treated on day 0 with a topical spot-on formulation containing 6.76 % w/v fipronil plus 50.48 % w/v permethrin and eight dogs served as the untreated control group. Dermacentor reticulatus ticks, with a B. canis infection rate ranging between 2 and 10 %, were placed onto dogs on days 7, 14, 21 and 28. In situ tick counts were performed on Days 9, 16 and 23. Ticks were counted and removed on Day 30. Infection of the dogs with B. canis was monitored by rectal temperature readings, clinical examinations and blood smears as well as PCR and IFA (indirect fluorescent antibody assay). For the Ehrlichia canis study, another 16 dogs were allocated to two groups. Eight dogs were treated with the fipronil and permethrin combination on days 0 and 28 and eight dogs served as untreated controls. Rhipicephalus sanguineus ticks, carrying an infection rate of 13 % for E. canis, were released in the sleeping kennels of the dogs on days 7, 14, 21, 28, 35, 42, 49 and 56. Ticks were counted in situ on the dogs on a weekly basis. All ticks were removed and counted on the final assessment day 58. Infection of the dogs with E. canis was monitored by rectal temperature, clinical examinations, and testing of blood samples by PCR, IFA and platelet counts.

Results: B. canis was transmitted by D. reticulatus ticks to all eight untreated control dogs and to one treated dog, which was confirmed by blood smears, PCR and IFA. E.canis was transmitted by R. sanguineus ticks to all eight untreated control dogs. Two of the dogs in the treated group were found positive based on PCR and/or IFA.

Conclusions: Frontline Tri-Act®/Frontect® significantly lowered the risk for dogs to acquire a B. canis infection by 87.5 % over a challenge period of 28 days. The risk for dogs to acquire E. canis was reduced by 75 % over a period of 56 days.
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http://dx.doi.org/10.1186/s13071-015-1207-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4653891PMC
November 2015

Development of a generic Ehrlichia FRET-qPCR and investigation of ehrlichioses in domestic ruminants on five Caribbean islands.

Parasit Vectors 2015 Oct 6;8:506. Epub 2015 Oct 6.

Jiangsu Co-innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University College of Veterinary Medicine, Yangzhou, Jiangsu, 225009, P. R. China.

Background: The Ehrlichia are obligate intracellular Gram-negative tick-borne bacteria that are important human and animal pathogens. There is a need for assays to rapidly and reliably detect and differentiate the five generally recognized species into groups in a single reaction: E. canis, E. chaffeensis, E. ewingii, E. muris and E. ruminantium.

Methods: We developed primers and probes against the 16S rRNA gene to enable us to reliably detect the five major Ehrlichia spp. in a single FRET-qPCR. We tested the Ehrlichia FRET-qPCR on reference strains and on DNA from the blood of domestic ruminants from five Caribbean islands. The Ehrlichia present were determined using melting point analysis and by sequencing the Ehrlichia FRET-qPCR products as well as those of a nested PCR against the citrate synthase gene (gltA).

Results: Our Ehrlichia FRET-qPCR was negative for the closely related Anaplasma marginale and A. phagocytophilum but gave positive reactions with reference strains of the most generally recognized species and with other less characterized Ehrlichia of domestic ruminants, mainly E. ovina, the Panola Mountain Ehrlichia, and Ehrlichia sp. BOV2010. Melting point analysis revealed 4 distinct groups: E. ruminantium (T m ~55.8 °C); E. chaffeensis and E. ewingii (T m ~57.7 °C); E. canis, E. muris, E. ovina and Ehrlichia sp. BOV 2010 (T m ~62.0 °C); and the Panola Mountain Ehrlichia (T m ~65.5 °C). The detection limit of the FRET-qPCR was ~ 5 gene copies in a reaction and the sequences of the FRET-qPCR products were as expected. With DNA from domestic ruminants from the Caribbean we found 12.2 % (134/1,101) positive: cattle (76/385; 19.7 %), sheep (45/340; 13.2 %) and goats (13/376; 3.5 %). Melting point analysis and sequencing of the FRET-qPCR and nested PCR gltA products showed the Ehrlichia we detected were E. canis or very closely related organisms.

Conclusions: In a single reaction, our Ehrlichia FRET-qPCR can detect the Ehrlichia spp. we studied and differentiate them into four groups. Domestic ruminants in the Caribbean are not uncommonly exposed to Ehrlichia, possibly E. canis or very closely related organisms.
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http://dx.doi.org/10.1186/s13071-015-1118-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4595018PMC
October 2015

Novel foci of Dermacentor reticulatus ticks infected with Babesia canis and Babesia caballi in the Netherlands and in Belgium.

Parasit Vectors 2015 Apr 17;8:232. Epub 2015 Apr 17.

Unit of Veterinary Entomology, Department of Biomedical Sciences, Institute of Tropical Medicine, Nationalestraat 155, B2000, Antwerp, Belgium.

Background: Autochthonous populations of Dermacentor reticulatus ticks in the Netherlands were discovered after fatal cases of babesiosis occurred in resident dogs in 2004. The presence of D. reticulatus in the Netherlands has also linked with the emergence of piroplasmosis in the resident horse population. The aim of this study was to put together results of continued surveillance of field sites and hosts for this tick in the Netherlands and also in Belgium and determine their infection status for Babesia and Theileria species.

Methods: Ticks were collected from the vegetation at 11 locations between 2011 and 2013. D. reticulatus ticks were also collected from different hosts between 2007 and 2013. Ticks were screened by PCR and reverse line blot (RLB).

Results: A total of 1368 D. reticulatus ticks were collected from 4 previously known field locations and from 5 new locations in the Netherlands and from 2 sites in Belgium (one old and one new location). A total of 855 ticks collected from 8 locations in the Netherlands and 2 locations in Belgium were tested. Fourteen ticks (1,64%) collected at 4 field locations (Dintelse Gorzen, Rozenburg, Slikken van de Heen and St. Philipsland) were positive for Babesia canis, whereas two ticks were positive for Babesia caballi, one tick in the Dintelse Gorzen in the Netherlands and one tick was found positive in De Panne in Belgium. A further 1092 D. reticulatus ticks were collected between 2007 and 2013 from 40 dogs (132 ticks), two ticks from two humans, 51 ticks from 15 horses, two ticks from two cats, one tick from a roe deer, whereas most ticks (904) were collected from cattle (n = 25). Ticks were found throughout the year on dogs in nearly all provinces of the Netherlands. None of the ticks collected from these hosts were infected.

Conclusions: D. reticulatus is continuing its spread into novel areas. The finding that some autochthonous ticks are infected with B. canis and B. caballi poses a threat to the resident dog and horse population and justifies year-round tick control measures.
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http://dx.doi.org/10.1186/s13071-015-0841-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4404102PMC
April 2015

No evidence of African swine fever virus replication in hard ticks.

Ticks Tick Borne Dis 2014 Sep 26;5(5):582-9. Epub 2014 Jun 26.

Virology Department, Central Veterinary Institute, Part of Wageningen UR (CVI), P.O. Box 65, 8200 AB Lelystad, The Netherlands. Electronic address:

African swine fever (ASF) is caused by African swine fever virus (ASFV), a tick-borne DNA virus. Soft ticks of the genus Ornithodoros are the only biological vectors of ASFV recognized so far. Although other hard ticks have been tested for vector competence, two commonly found tick species in Europe, Ixodes ricinus and Dermacentor reticulatus, have not been assessed for their vector competence for ASFV. In this study, we aimed to determine whether virus replication can occur in any of these two hard tick species (I. ricinus and/or D. reticulatus), in comparison with O. moubata (the confirmed vector), after feeding them blood containing different ASFV isolates using an improved in vitro system. DNA quantities of ASFV in these infected hard ticks were measured systematically, for 6 weeks in I. ricinus, and up to 8 weeks in D. reticulatus, and the results were compared to those obtained from O. moubata. There was evidence of virus replication in the O. moubata ticks. However, there was no evidence of virus replication in I. ricinus or D. reticulatus, even though viral DNA could be detected for up to 8 weeks after feeding in some cases. This study presents the first results on the possible vector competence of European hard (ixodid) ticks for ASFV, in a validated in vitro feeding setup. In conclusion, given the lack of evidence for virus replication under in vitro conditions, D. reticulatus and I. ricinus are unlikely to be relevant biological vectors of ASFV.
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http://dx.doi.org/10.1016/j.ttbdis.2013.12.012DOI Listing
September 2014

The efficacy of Advantix® to prevent transmission of Ehrlichia canis to dogs by Rhipicephalus sanguineus ticks.

Parasite 2013 21;20:36. Epub 2013 Oct 21.

ClinVet International (Pty) Ltd, PO Box 11186, Universitas, Bloemfontein 9321, South Africa.

The capacity of a topical combination of imidacloprid and permethrin (Advantix(®)) to prevent transmission of Ehrlichia canis was studied in two groups of six dogs. One group served as controls, whereas the other group was treated. All dogs were exposed to E. canis-infected Rhipicephalus sanguineus ticks on Days 7, 14, 21 and Day 28 post acaricidal treatment. The adult R. sanguineus ticks were released into the individual kennels of the dogs to simulate natural tick exposure. In situ tick counts were conducted on Day 9, 16 and 23 and any remaining ticks were counted and removed on Day 30. The efficacy of the acaricidal treatment against R. sanguineus ranged between 96.1% and 98.9% at 48 h post-application and lasted up to 4 weeks. Four out of six control dogs became infected with E. canis, as demonstrated by the presence of specific E. canis antibodies and the detection by PCR of E. canis DNA in blood samples. These dogs became thrombocytopenic and displayed fever and were consecutively rescue-treated by doxycycline. None of the six treated dogs became infected with E. canis, as confirmed by the lack of specific antibodies and absence of E. canis DNA in blood samples. Advantix(®) prevented transmission of E. canis and provided protection against monocytic ehrlichiosis for 4 weeks post acaricidal treatment.
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http://dx.doi.org/10.1051/parasite/2013037DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3798889PMC
June 2015

Transmission of Ehrlichia canis by Rhipicephalus sanguineus ticks feeding on dogs and on artificial membranes.

Vet Parasitol 2013 Nov 3;197(3-4):595-603. Epub 2013 Aug 3.

ClinVet International (Pty) Ltd, P.O. Box 11186, Universitas, Bloemfontein 9321, South Africa. Electronic address:

A South African strain of Ehrlichia canis was isolated and used to infect a laboratory-bred Beagle dog. Rhipicephalus sanguineus nymphs, which fed on this dog, moulted to adult ticks which carried infection rates of E. canis between 12% and 19% and were used in a series of in vivo and in vitro experiments. Five groups of 6 dogs were challenged with the infected R. sanguineus ticks, which were removed 24h, 12h, 6h or 3h after the ticks had been released onto the dogs. The animals were monitored for fever and thrombocytopenia and were considered infected if they became serologically positive for E. canis antibodies as well as PCR positive for E. canis DNA. Seven dogs became infected with E. canis in the following groups: Group 1 (24h tick challenge) 1 out of 6; Group 2 (12h) 1 of 6; Group 3 (6h) 2 of 6; Group 4 (6h) 2 of 6 and Group 5 (3h) 1 out of 6. Six of those 7 infected dogs developed fever and a significant thrombocytopenia. One dog did not show any symptoms, but seroconverted and was found PCR positive on several occasions. Five additional dogs were PCR positive on one test sample only but were not considered infected because they did not develop any specific E. canis antibodies. In vitro, R. sanguineus ticks attached and fed on bovine blood through silicone membranes with attachment rates up to 72.5% after 24h increasing to 84.2% at 72 h. The ticks transmitted E. canis as soon as 8h post application as demonstrated by E. canis DNA found in the nutritive blood medium. In conclusion, transmission of E. canis by R. sanguineus ticks starts within a few hours after attachment, which is earlier than previously thought. These findings underpin the need for acaricides to provide either a repellent, an anti-attachment and/or a rapid killing effect against ticks in order to decrease the risk of transmission of E. canis.
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http://dx.doi.org/10.1016/j.vetpar.2013.07.026DOI Listing
November 2013

Molecular evidence of Ehrlichia canis and Rickettsia massiliae in ixodid ticks of carnivores from South Hungary.

Acta Vet Hung 2013 Mar;61(1):42-50

Szent István University Department of Parasitology and Zoology, Faculty of Veterinary Science István u. 2 H-1078 Budapest Hungary.

To monitor the emergence of thermophilic, Mediterranean ixodid tick species and tick-borne pathogens in southern Hungary, 348 ticks were collected from shepherd dogs, red foxes and golden jackals during the summer of 2011. Golden jackals shared tick species with both the dog and the red fox in the region. Dermacentor nymphs were collected exclusively from dogs, and the sequence identification of these ticks indicated that dogs are preferred hosts of both D. reticulatus and D. marginatus nymphs, unlike previously reported. Subadults of three ixodid species were selected for reverse line blot hybridisation (RLB) analysis to screen their vector potential for 40 pathogens/groups. Results were negative for Anaplasma, Babesia and Theileria spp. Investigation of D. marginatus nymphs revealed the presence of Ehrlichia canis, Rickettsia massiliae and Borrelia afzelii for the first time in this tick species. These findings broaden the range of those tick-borne agents, which are typically transmitted by Rhipicephalus sanguineus, but may also have Dermacentor spp. as potential or alternative vectors. Ehrlichiacanis was also newly detected in Ixodes canisuga larvae from red foxes. In absence of transovarial transmission in ticks this implies that Eurasian red foxes may play a reservoir role in the epidemiology of canine ehrlichiosis.
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http://dx.doi.org/10.1556/AVet.2012.050DOI Listing
March 2013

Whole-genome sequencing of Theileria parva strains provides insight into parasite migration and diversification in the African continent.

DNA Res 2013 Jun 12;20(3):209-20. Epub 2013 Feb 12.

Division of Collaboration and Education, Research Center for Zoonosis Control, Hokkaido University, Sapporo-shi, Hokkaido 001-0020, Japan.

The disease caused by the apicomplexan protozoan parasite Theileria parva, known as East Coast fever or Corridor disease, is one of the most serious cattle diseases in Eastern, Central, and Southern Africa. We performed whole-genome sequencing of nine T. parva strains, including one of the vaccine strains (Kiambu 5), field isolates from Zambia, Uganda, Tanzania, or Rwanda, and two buffalo-derived strains. Comparison with the reference Muguga genome sequence revealed 34 814-121 545 single nucleotide polymorphisms (SNPs) that were more abundant in buffalo-derived strains. High-resolution phylogenetic trees were constructed with selected informative SNPs that allowed the investigation of possible complex recombination events among ancestors of the extant strains. We further analysed the dN/dS ratio (non-synonymous substitutions per non-synonymous site divided by synonymous substitutions per synonymous site) for 4011 coding genes to estimate potential selective pressure. Genes under possible positive selection were identified that may, in turn, assist in the identification of immunogenic proteins or vaccine candidates. This study elucidated the phylogeny of T. parva strains based on genome-wide SNPs analysis with prediction of possible past recombination events, providing insight into the migration, diversification, and evolution of this parasite species in the African continent.
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http://dx.doi.org/10.1093/dnares/dst003DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3686427PMC
June 2013

A novel approach, based on BLSOMs (Batch Learning Self-Organizing Maps), to the microbiome analysis of ticks.

ISME J 2013 May 10;7(5):1003-15. Epub 2013 Jan 10.

Division of Collaboration and Education, Research Center for Zoonosis Control, Hokkaido University, Sapporo, Hokkaido, Japan.

Ticks transmit a variety of viral, bacterial and protozoal pathogens, which are often zoonotic. The aim of this study was to identify diverse tick microbiomes, which may contain as-yet unidentified pathogens, using a metagenomic approach. DNA prepared from bacteria/archaea-enriched fractions obtained from seven tick species, namely Amblyomma testudinarium, Amblyomma variegatum, Haemaphysalis formosensis, Haemaphysalis longicornis, Ixodes ovatus, Ixodes persulcatus and Ixodes ricinus, was subjected to pyrosequencing after whole-genome amplification. The resulting sequence reads were phylotyped using a Batch Learning Self-Organizing Map (BLSOM) program, which allowed phylogenetic estimation based on similarity of oligonucleotide frequencies, and functional annotation by BLASTX similarity searches. In addition to bacteria previously associated with human/animal diseases, such as Anaplasma, Bartonella, Borrelia, Ehrlichia, Francisella and Rickettsia, BLSOM analysis detected microorganisms belonging to the phylum Chlamydiae in some tick species. This was confirmed by pan-Chlamydia PCR and sequencing analysis. Gene sequences associated with bacterial pathogenesis were also identified, some of which were suspected to originate from horizontal gene transfer. These efforts to construct a database of tick microbes may lead to the ability to predict emerging tick-borne diseases. Furthermore, a comprehensive understanding of tick microbiomes will be useful for understanding tick biology, including vector competency and interactions with pathogens and symbionts.
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http://dx.doi.org/10.1038/ismej.2012.171DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3635243PMC
May 2013

Prevention of transmission of Ehrlichia canis by Rhipicephalus sanguineus ticks to dogs treated with a combination of fipronil, amitraz and (S)-methoprene (CERTIFECT®).

Vet Parasitol 2013 Mar 20;193(1-3):223-8. Epub 2012 Dec 20.

ClinVet International (Pty) Ltd., P.O. Box 11186, Universitas, Bloemfontein 9321, South Africa.

The ability of CERTIFECT(®) (a combination of fipronil, amitraz and (S)-methoprene) to prevent transmission of Ehrlichia canis was studied in two groups of eight dogs. One group was treated with CERTIFECT while the other group remained untreated. All dogs were exposed to E. canis-infected Rhipicephalus sanguineus ticks on Days 7, 14, 21 and again on day 28 post-treatment by releasing ticks into the kennels of the dogs to simulate the natural way of infestation. Animals were examined in situ for ticks on Days 9, 16 and 23 and any ticks present were counted and removed on Day 30. The efficacy of CERTIFECT against R. sanguineus was 100%, since no ticks were found on the treated dogs at any time. Clinical examinations (including monitoring body temperature and blood collections for PCR analysis and serology) were performed at intervals throughout the study until Day 56. Five out of 8 untreated control dogs (62.5%) became infected with E. canis, as demonstrated by detection of specific E. canis antibodies and the presence of E. canis DNA in blood samples by PCR. These dogs displayed fever and thrombocytopenia and were rescue-treated with doxycline. None of the 8 dogs treated with CERTIFECT became infected with E. canis, in comparison to the 5/8 control dogs, as confirmed by the lack of specific antibodies and absence of any ehrlichial DNA in blood samples by PCR. CERTIFECT prevented transmission of E. canis and effectively provided protection against monocytic ehrlichiose for at least 4 weeks post treatment.
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http://dx.doi.org/10.1016/j.vetpar.2012.12.009DOI Listing
March 2013

Prevention of transmission of Babesia canis by Dermacentor reticulatus ticks to dogs treated with an imidacloprid/flumethrin collar.

Vet Parasitol 2013 Feb 26;192(1-3):273-8. Epub 2012 Oct 26.

ClinVet International (Pty) Ltd., P.O. Box 11186, Universitas, Bloemfontein 9321, South Africa.

A group of 8 dogs was treated with an imidacloprid/flumethrin collar (Seresto(®)) 28 days prior to infestation with adult Dermacentor reticulatus ticks, infected with Babesia canis. The ability of the collar to prevent transmission of B. canis in the treated group was compared to an untreated control group. All 8 dogs in the untreated control group became infected with B. canis parasites, which were detected in blood smears as early as day 6 post tick-application. All control dogs developed clinical signs of babesiosis and were rescue-treated with imidocarb dipropionate. These dogs also developed specific B. canis antibodies as identified by serology (IFA test) and were confirmed PCR/RLB positive. None of the 8 dogs treated with the imidacloprid/flumethrin collar became infected with B. canis, which was confirmed by the absence of specific B. canis antibodies and babesial DNA as confirmed by PCR/RLB. The collar caused 96.02% of the ticks to die within 48h post challenge and this increased to 100% within 4 days. Although a high percentage of 44% of the Dermacentor ticks were infected with B. canis, they were unable to transmit the infection to the treated group. Hence, the imidacloprid/flumethrin collar effectively prevented transmission of B. canis 1 month after application onto the dogs.
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http://dx.doi.org/10.1016/j.vetpar.2012.10.017DOI Listing
February 2013