Molecular evidence of vector-borne pathogens coinfecting dogs from Poland

Ticks of the genus Ixodes are vectors for many pathogens, including Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum and Rickettsia spp., and may also serve as vectors for Bartonella spp. However, the role of ticks in Bartonella transmission requires additional studies. The aim of this study was to investigate whether coinfection with two or more vector-borne pathogens can occur in the following three groups of dogs: I - dogs with suspected borreliosis (N = 92), II - dogs considered healthy (N = 100), and III - dogs with diagnosed babesiosis (N = 50). Polymerase chain reactions were performed to detect DNA of Anaplasma phagocytophilum, Rickettsia spp. and Bartonella spp. in the blood of dogs. In dogs of Group I, the DNA of both A. phagocytophilum and Bartonella sp. was detected (14% and 1%, respectively). In eight dogs, coinfection was indicated: A. phagocytophilum or Bartonella sp. with B. burgdorferi s.l. (the presence of antibodies against and/or DNA B. burgdorferi s.l.). In the case of five dogs positive for A. phagocytophilum DNA, no coinfection with B. burgdorferi s.l. was shown. In Group II, the DNA of A. phagocytophilum was detected in four dogs. In Group III, no pathogenic agents possibly transmitted by ticks were confirmed. No DNA of R. helvetica was detected in any of the groups studied.     

Prevalence of Babesia canis, Borrelia afzelii, and Anaplasma phagocytophilum infection in hard ticks removed from dogs in Warsaw (central Poland)

The purposes of this study were to specify the occurrence and prevalence of Babesia canis, Borrelia burgdorferi sensu lato, and Anaplasma phagocytophilum in ticks removed from dogs in Warsaw, and to determine the Borrelia species occurring in Ixodes ricinus ticks. Among 590 collected ticks, 209 were identified as I. ricinus, and 381 as Dermacentor reticulatus. DNA of B. canis was detected in 11% of D. reticulatus ticks. We found that 6.2% of I. ricinus ticks harbored B. burgdorferi s.l. specific DNA and 2.9% harbored A. phagocytophilum DNA. In these samples sequencing of the detected Borrelia amplicon confirmed infection with Borrelia afzelii genospecies. New sequences were submitted to the GenBank database (accession no. EU152128, EU152127, EU152126). This work is the first detection of B. afzelii and A. phagocytophilum in ticks from Warsaw, and the first survey for the prevalence of B. canis, B. afzelii, and A. phagocytophilum in ticks in central Poland.     

Broad-range PCR-TTGE for the first-line detection of bacterial pathogen DNA in ticks

Ticks are known or suspected vectors for a wide range of bacterial pathogens. One of the first steps for tick-borne risk assessment is the detection of these pathogens in their vectors. In the present study, a broad-range PCR amplification of the eubacterial gene encoding the 16S rRNA gene combined with Temporal Temperature Gradient gel Electrophoresis (TTGE) was evaluated as a method allowing the one-step detection of bacterial pathogen DNA in ticks. Firstly, DNA extracts from bacteria known to be tick-borne pathogens, i.e., Borrelia burgdorferi lato sensu, Anaplasma phagocytophilum, Spotted Fever Group (SFG) Rickettsia spp., were used to establish a TTGE pathogen DNA reference marker. Secondly, we used broad-range PCR-TTGE to detect the presence of DNA from these three pathogens in 55 DNA extracts from pools of 10 nymphal Ixodes ricinus ticks, which have been previously shown to carry DNA from at least one of those bacteria by specific PCR. Among the 20 B. burgdorferi specific-PCR samples, 15 (75%) were also found to be positive using PCR-TTGE. Sixteen of the seventeen (94%) Rickettsia spp. PCR-specific samples were positive using PCR-TTGE detection and all PCR-specific positive extracts (11/11, 100%) for A. phagocytophilum were also positive using PCR-TTGE. Moreover, we identified unexpected bacterial sequences that were not related to any of the three pathogens such as a sequence related to Spiroplasma sp. Thus, broad-range PCR-TTGE allowed the single step detection of DNA from up to 3 pathogens in the same co-infected samples as well as detection of DNA from unexpected bacteria.     

Ixodes frontalis and avian tick-related syndrome in the United Kingdom

OBJECTIVES: This study aimed to characterise tick species responsible for avian tick infestations in the UK, to analyse various risk factors for tick-related syndrome in tick-infested birds and to test samples for the presence of certain tick-transmitted pathogens. METHODS: Ticks, blood, splenic tissue and tick attachment site tissue from birds with attached ticks were requested from veterinarians and wildlife sanctuaries around the UK. Ticks were identified according to standard keys, and samples were analysed via DNA PCR test for Borrelia burgdorferi sensu lato, Babesia species, Bartonella species and Ehrlichia species. RESULTS: Ixodes frontalis was the most commonly identified tick, and an association of adult female I frontalis with tick-related syndrome in birds was demonstrated. Tick infestation was markedly seasonal. I frontalis was found on 32 species of birds. DNA PCR testing was uniformly negative. Of the birds known to have been treated, 75 per cent (nine of 12) survived. CLINICAL SIGNIFICANCE: Tick-related syndrome is a poorly understood syndrome, with sporadic distribution, both geographically and seasonally. This study confirms I frontalis as the most common cause of this syndrome in the UK and identifies some features of the tick life cycle in this country. The benefit of treatment in affected birds is highlighted. Risk factors for tick-related syndrome are examined and preventive strategies discussed.     

Borrelia burgdorferi sensu lato and Rickettsia spp. infections in hard ticks (Ixodes ricinus) in the region of Hanover (Germany)

In a total of 605 Ixodes (I.) ricinus ticks collected in the spring-months March, April and May 2005, quantitative real time PCR (qPCR) revealed 26.6% Borrelia (B.) burgdorferi sensu lato (sl)-positive ticks, i. e. divided by sex and stage into 31.9% positive adults (34.8% females and 29.0% males) and 18.5% positive nymphs. Mono-infections with genospecies from the B. burgdorferi sl-complex were found in over two thirds of the positive individuals, whereas almost one third showed double- or even triple-infections. Genospecies-specific conventional PCR determined B. afzelii as the most frequent genospecies followed by B. garinii, B. spielmanii, B. valaisiana and B. burgdorferi sensu stricto (ss). Rickettsia spp. were found in 34.2% of the collected ticks, divided into 37.6% adults (42.5% females and 32.8% males) and 29.0% nymphs. Co-infections of Rickettsia-positive ticks with B. burgdorferi sl spirochaetes were present in 10.1% of the ticks. Thereby, adult ticks exhibited a co-infection rate of 13.4% (15.5% females and 11.3% males) and nymphs of 5.0%. Independently of the above mentioned study, 3939 Ixodes ticks, sent in between 2006 and 2010 for B. burgdorferi sl-diagnostic, were examined by qPCR exclusively for B. burgdorferi sl. The resulting B. burgdorferi sl prevalence was 23.1% and 24.4% in 2006 and 2007, respectively, followed by a continuous decrease to 12.8% in 2010. To analyse whether this observed decrease in infection frequency is due to sampling bias, in a current study randomly sampled ticks collected from defined sites equally distributed over the city of Hanover are investigated in a statistically relevant sample size.     

Prevalence of Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum in questing Ixodes ricinus ticks in relation to the density of wild cervids

Background

Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum have been considered as pathogens in animals and humans. The role of wild cervids in the epidemiology is not clear. We analyzed questing Ixodes ricinus ticks collected in spring for these pathogens from sites with high (Fjelløyvær and Strøm) and low density (Tjore, Hinnebu and Jomfruland) of wild cervids to study the spread of the pathogens in questing ticks.

Methods

For detection of Anaplasma phagocytophilum a 77-bp fragment in the msp2 gene was used. Detection of Borrelia burgdorferi sensu lato was performed using the FL6 and FL7 primers according to sequences of conserved regions of the fla gene. The OspA gene located on the linear 49-kb plasmid was used as target in multiplex PCR for genotyping. Genospecies-specific primers were used in the PCR for Borrelia burgdorferi sensu stricto, B. afzelii and B. garinii.

Results

Infection rates with Borrelia spp. were significantly lower at Fjelløyvær and Strøm compared to Tjore and Hinnebu; Fjelløyvær vs. Tjore (χ² = 20.27, p < 0.0001); Fjelløyvær vs. Hinnebu (χ² = 24.04, p < 0.0001); Strøm vs. Tjore (χ² = 11.47, p = 0.0007) and Strøm vs. Hinnebu (χ² = 16.63, p < 0.0001). The Borrelia genospecies were dominated by. B. afzelii (82%) followed by B. garinii (9.7%) and B. burgdorferi sensu stricto (6.9%). B. burgdorferi s.s. was only found on the island of Jomfruland. The infection rate of Anaplasma phagocytophilum showed the following figures; Fjelløyvær vs Hinnebu (χ² = 16.27, p = 0.0001); Strøm vs. Tjore (χ² = 13.16, p = 0.0003); Strøm vs. Hinnebu (χ² = 34.71, p < 0.0001); Fjelløyvær vs. Tjore (χ² = 3.19, p = 0.0742) and Fjelløyvær vs. Støm (χ² = 5.06, p = 0.0245). Wild cervids may serve as a reservoir for A. phagocytophilum. Jomfruland, with no wild cervids but high levels of migrating birds and rodents, harboured both B. burgdorferi s.l. and A. phagocytophilum in questing I. ricinus ticks. Birds and rodents may play an important role in maintaining the pathogens on Jomfruland.

Conclusion

The high abundance of roe deer and red deer on the Norwegian islands of Fjelløyvær and Strøm may reduce the infection rate of Borrelia burgdorferi sensu lato in host seeking Ixodes ricinus, in contrast to mainland sites at Hinnebu and Tjore with moderate abundance of wild cervids. The infection rate of Anaplasma phagocytophilum showed the opposite result with a high prevalence in questing ticks in localities with a high density of wild cervids compared to localities with lower density.     

Identification of Borrelia burgdorferi genospecies inducing Lyme disease in dogs from Western Poland

Canine Lyme borreliosis may be caused by three Borrelia burgdorferi sensu lato genospecies. The prevalence of infection by Borrelia species was determined by nested polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) with the enzyme Fsp4H I in the blood of dogs naturally infested by ticks in an endemic region of Poland. Blood samples were collected from 98 dogs of various breeds, delivered to the Veterinary Clinic in Szczecin (northwestern Poland) for various reasons. Nested PCR revealed the presence of DNA characteristic of only 1 genospecies, i.e. B. burgdorferi sensu stricto (s.s.), in all PCR-positive samples. Digestion of PCR products from a fragment of the fla gene amplified with primers FLA1 and FLA2 gave only one band pattern consistent with the pattern obtained from sequence analysis of the fla gene from a reference isolate of B. burgdorferi s.s. GeHo (X15660) from GenBank.     

Detection of Borrelia garinii, Borrelia tanukii and Borrelia sp. closely related to Borrelia valaisiana in Ixodes ticks removed from dogs and cats in Japan

Ticks removed from 1136 dogs and 134 cats all over Japan were examined for Borrelia infection by PCR and sequencing. The 5S-23S rDNA intergenic spacer of Borrelia was detected from two Ixodes persulcatus ticks from two dogs and two unidentified Ixodes spp. from another two dogs in Hokkaido, and two Ixodes granulatus ticks from two cats in Okinawa. Additional 2 I. granulatus from the same cats also showed positive. Sequence analysis of the PCR products revealed that the one from Hokkaido was similar to B. garinii, the three from Hokkaido to B. tanukii, and the four from Okinawa to a novel Borrelia sp. closely related to B. valaisiana. The data was confirmed by analysis of the flagellin gene sequence. Infected ticks carried by companion animals can be introduced into the human environment.     

Tick infestation and the prevalence of Borrelia burgdorferi and Babesia divergens in cattle in Bavaria

During the grazing period 2002 319 cattle from 31 farms located in 6 districts of southern Bavaria were examined for the presence of ticks in 4- to 5-week intervals, and 287 serum samples were tested for the presence of antibodies against Borrelia burgdorferi and Babesia divergens. Ticks were detected in all 31 farms with a mean prevalence of 69%. 3218 out of 3453 collected ticks were Ixodes ricinus; 139 nymphs, 19 larvae and 77 damaged adult specimens could only be determined to the Genus level (Ixodes).The seasonal pattern revealed the highest frequencies of ticks in May/June and September.The intensity of tick infestation of positive animals was generally low. 76.5% of parasitized cattle had 1-6 ticks per day of investigation. Individual cattle showed up to 250 ticks per day. The percentage of infested animals in each herd varied within the period between 0-100%. The examination of serum samples by immunofluorescence technique (IFAT) revealed positive anti-Borrelia antibody titers (> or = 1:64) for 45.6% of the animals. The within-farm seroprevalence of borreliosis ranged from 20 to 100% in 27 of the 31 farms. A significant correlation could be detected between the number of ticks/cattle and the anti-Borrelia burgdorferi IgG-titer. By contrast, there was no significant correlation between the age of the animals and anti-Borrelia serum titers. For comparative reasons, 64 IFAT-positive serum samples were tested by Western blot techniques for the presence of antibodies cross-reacting with Borrelia garinii antigen. These analyses revealed that 69% of the samples reacted positively, 28% were unclear and 3% were negative. Examinations of the 287 serum samples for the presence of anti-Babesia divergens antibodies revealed one positive animal with a titer of 1:16.     

Comparison of direct fluorescent antibody staining and real-time polymerase chain reaction for the detection of Borrelia burgdorferi in Ixodes scapularis ticks.

Borrelia burgdorferi, the agent responsible for causing Lyme disease in humans and animals, is transmitted via the bite of infected Ixodes spp. ticks. Ticks removed from humans and animals are routinely tested by diagnostic laboratories to determine if they are infected with these bacteria. The objective of this study was to compare the efficacy of 2 commonly used methods, direct fluorescent antibody staining and real-time polymerase chain reaction (PCR), for the detection of B. burgdorferi in Ixodes scapularis ticks. One hundred and twenty-seven adult I. scapularis ticks collected in Connecticut, a Lyme disease endemic area, were tested, and results were compared. Results showed 24.8% ticks tested positive for Borrelia spp. by fluorescent antibody testing and 32.5% ticks were positive for B. burgdorferi by real-time PCR testing. When ticks were grouped into categories by level of engorgement (unengorged, partially engorged, and fully engorged), 95% of unengorged ticks, 90.5% of partially engorged, and 86.8% of engorged ticks tested were in agreement. Ten of the 127 ticks examined were too dehydrated to be tested by the fluorescent antibody technique; half of these tested positive by PCR. Real-time PCR appears to be the better of these 2 methods for the diagnosis of this bacterial infection in I. scapularis ticks.     

Epidemiological studies of the infection of ticks with borreliosis agents in small mammals from north Germany

In a two years study into the infestation of ticks with Borrelia burgdorferi from mice in North Germany 1330 mice out of 11 species could be examined. Altogether 508 mice showed to be parasitized by 1445 ticks belonging to three species of Ixodes. 777 I. ricinus from 334 mice could be tested for B. burgdorferi. In 66 ticks (8.5%) from 34 mice (10.2%) borreliae could be demonstrated. These discoveries came from 9 of 14 investigated forest regions in Lower Saxony.     

Evaluation of different media and a BGM cell culture assay for isolation of Borrelia burgdorferi sensu lato from ticks and dogs

A co-culture assay for isolation of Borrelia burgdorferi sensu lato (sl.) from naturally infected ticks and dogs suspected of Lyme borreliosis (LB) was evaluated using buffalo-green-monkey (BGM) cells as the mammalian component. Four different media were tested for their ability to provide sufficient growth conditions for spirochetes and BGM cells. A total of 176 Ixodes ricinus ticks and 268 specimens from 98 dogs were used to compare cell-free culture with the BGM co-culture. A 1:1 mixture of Barbour-Stoenner-Kelly medium (BSK) and Eagle's minimum essential medium (EMEM) supported the growth of the two test strains, B. burgdorferi sensu stricto B31 and B. valaisiana VS116 to the same extent as BSK medium and the growth as well as the viability of BGM cells in this medium were the same as in EMEM. Using the 1:1 mixture of BSK and EMEM, borrelial growth measured in co-culture with BGM cells did not differ significantly from corresponding values obtained in cell-free cultures. In cell-free culture the isolation rate of B. burgdorferi sl. from ticks was significantly higher in BSK/EMEM 1:1 than in BSK medium (P < 0.01). Co-culture with BGM cells had no significant influence on the isolation rate of borreliae from ticks. However, a significant amount of isolates were obtained by one of the procedures only. Analysing canine specimens accordingly, spirochetes were grown from the blood of one dog after four weeks in BGM cell co-culture. The isolate was classified as B. afzelii by PCR-coupled restriction fragment length polymorphism analysis.     

Estimating Lyme disease risk using pet dogs as sentinels

The reported number of cases of Lyme disease, Borrelia burgdorferi sensu lato, is thought to have increased in the UK over the past decade, but consistent surveillance data are lacking. Here the prevalence of B. burgdorferi in ticks attached to pet dogs was examined - using them as sentinels for human disease risk. Dogs give a good indication of the exposure of their human owners to infected ticks, since they largely share the same environment and visit the same outdoor areas. PCR was used to test 739 tick samples collected from 3534 dogs selected at random as they visited veterinary practices over a period of six months. Overall, the prevalence of infected ticks on all dogs was 0.5% giving an estimated 481 infected ticks per 100,000 dogs. The data suggest that the prevalence of Borrelia in the UK tick population is considerably higher than most recent estimates indicate.     

Presence and distribution of Borrelia burgdorferi sensu lato species in internal organs and skin of naturally infected symptomatic and asymptomatic dogs, as detected by polymerase chain reaction.

Tissues from Dutch family dogs symptomatic for borreliosis according to established criteria and from infected but asymptomatic dogs were tested for Borrelia burgdorferi sensu lato DNA using a polymerase chain reaction. Subsequently, B. burgdorferi sensu stricto, B. garinii, B. afzelii, and B. valaisiana were identified by hybridization. Symptomatic dogs showed a higher prevalence of Borrelia in liver samples (9 of 15) than asymptomatic dogs (9 of 43) p = 0.0049. Overall, B. garinii was the most prevalent species and occurred together with up to three other species in on liver sample. B. burgdorferi sensu stricto however, was predominantly detected in samples of synovial membranes, skin, cerebrospinal fluid, bladder, heart, and bone marrow. Nine out of 10 symptomatic dogs with a very high antibody titre were positive for Borrelia DNA by PCR in one or more of these tissues. We conclude that dissemination in naturally infected European dogs occurs and that the two most prevalent species, B. burgdorferi sensu stricto and B. garinii, differ in their tropism.     

Investigation of Bartonella infection in ixodid ticks from California

A total of 1253 ixodid ticks (254 tick pools) collected between the end of 1995 and the spring of 1997 from six California counties (El Dorado, Los Angeles, Orange, Santa Cruz, Shasta and Sonoma) were examined for the presence of Bartonella DNA by PCR of the citrate synthase gene. Of 1,119 adult Ixodes pacificus ticks tested, 26 (11.6%) of 224 pools, each containing five ticks, were positive (minimum percentage of ticks harboring detectable Bartonella DNA, 2.3%). Bartonella PCR-positive ticks were identified in five counties but none of the ticks from Los Angeles County was positive. Among 47 nymphal I. pacificus ticks collected in Sonoma County, one (10%) positive pool out of 10 pools was identified (minimum percentage of ticks harboring detectable Bartonella DNA, 2.1%). Among the 54 Dermacentor occidentalis grouped in 12 pools from Orange County, one pool (8.3%) was PCR positive for Bartonella and similarly one pool (14.3%) was positive among the 30 Dermacentor variabilis ticks grouped in seven pools. None of the three D. occidentalis from El Dorado County were positive. None of the nine tick pools positive for Ehrlichia phagocytophila were positive for Bartonella. Following our previous findings of Bartonella PCR-positive adult I. pacificus ticks in central coastal California, this is the first preliminary report of the presence of Bartonella DNA in I. pacificus nymphs and in Dermacentor sp. ticks. Distribution of Bartonella among ixodid ticks appears widespread in California.     

Molecular evidence of tick-transmitted infections in dogs and cats in the United Kingdom

PCR analysis was used to determine the prevalence of tick-transmitted infections in 120 systemically ill dogs and 60 cats recruited over a period of three months from 52 veterinary practices in the UK. The animals had not travelled outside the UK and had one or more of the following clinical criteria: acute or recurrent pyrexia, anaemia and/or thrombocytopenia, polyarthritis/muscle pain, splenomegaly/lymphadenopathy, and intraocular inflammation with systemic signs. Blood samples from the animals were tested for the presence of DNA from Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum by using simple PCR targeting. B. burgdorferi sensu lato was detected in five dogs and two cats, and A. phagocytophilum was detected in one dog and one cat. These results provide the first molecular evidence of naturally occurring B. burgdorferi sensu lato infection in cats in the UK and confirm that A. phagocytophilum infection is present in cats. There were no statistically significant associations between the infections and the clinical signs shown by the dogs and cats.     

A survey of ixodid ticks feeding on cattle and prevalence of tick-borne pathogens in the Black Sea region of Turkey

The study reports the frequency of infestation and the prevalence of tick-borne pathogens in feeding adult ticks detached from cattle in two climatic zones of the Black Sea region of Turkey. A total of 2160 adult ticks were collected during 2007-2008. Of these, 1062 were randomly selected, divided into 224 pools, and tested for the presence of bovine Theileria, Babesia, and Anaplasma species. Eleven tick species were recognized on cattle in the study. Hyalomma marginatum was widely disrubuted in the semi-arid bioclimatic zone, but few specimens were collected in the humid bioclimatic zone. The most prevalent tick species in the humid climatic zone was Ixodes ricinus. Infection rates were calculated as the maximum likelihood estimation with 95% confidence intervals (CI). Overall, 4% (CI 2.87-5.44) of 224 tick pools were found to be positive for the pathoges by Reverse line blot. Maximum likelihood estimation of the infection rate varied among tick species, ranging from 2.68% (CI 0.16-12.68) in Haemaphysalis sulcata to 10.49% (CI 4.07-23.66) in Rhipicephalus bursa. The most prevalent tick-borne pathogen was Anaplasma phagocytophilum at 6.78% (CI 3.41-12.18) followed by A. centrale (6.56%, CI 0.42-31.47), Anaplasma/Ehrlichia spp. (3.61%, CI 1.99-6.06), Babesia spp. (3.33%, CI 1.65-6.03), and T. buffeli/orientalis (2.71%, CI 0.73-7.18). Sequencing results indicated that Babesia spp. shared 99% to 100% similarity with the unnamed Babesia sp. Kashi 1 and 2, Babesia sp. Kayseri 1 and Babesia sp.CS58. Anaplasma/Ehrlichia spp. were 98% and 100% identical to Ehrlichia canis and Ehrlichia sp. Omatjenne strain, respectively.     

The low seroprevalence of tick-transmitted agents of disease in dogs from southern Ontario and Quebec

Infectious diseases caused by pathogens transmitted by ticks and other insect vectors are an important cause of morbidity and mortality in both dogs and humans throughout North America. The purpose of this study was to determine the seroprevalence of selected vector-transmitted pathogens in southern Ontario and Quebec. Samples submitted to the Vector Borne Disease Diagnostic Laboratory (VBDDL) at the North Carolina State University College of Veterinary Medicine were evaluated for antibodies to Ehrlichia canis, Anaplasma phagocytophilum, Babesia canis, Bartonella henselae, Borrelia burgdorferi, Bartonella vinsonii subspecies berkhoffii, and Rickettsia rickettsii. Information regarding breed and the city or province from which the sample originated was recorded; however, travel history was unknown for the majority of dogs. Overall seroprevalence to these tick-borne pathogens in southern Ontario and Quebec is low compared with most regions of the United States, suggesting that veterinarians in this region of Canada should pursue diagnostic evidence of infection in dogs with a travel history or prior residence in areas endemic for exposure to tick-borne infections.     

The ability of a topical novel combination of fipronil, amitraz and (S)-methoprene to protect dogs from Borrelia burgdorferi and Anaplasma phagocytophilum infections transmitted by Ixodes scapularis

Healthy, purpose-bred laboratory beagle dogs that had not been exposed to ticks and were seronegative for Borrelia burgdorferi and Anaplasma phagocytophilum were randomly assigned to four groups of eight dogs each. Control group 1 was not treated. Groups 2, 3 and 4 were treated with a single topical application of a new formulation of fipronil, amitraz and (S)-methoprene (CERTIFECT?, Merial Limited, GA, USA) at 28, 21 or 14 days prior to tick infestation, respectively. Each dog was infested with 25 female and 25 male field-collected adult Ixodes scapularis ticks that had infection rates of 66% for B. burgdorferi sensu stricto and 23% for A. phagocytophilum, as determined by polymerase chain reaction. Two and five days after tick infestation, control dogs had an average of 9.5 and 13.9 attached adult female ticks, respectively, whilst the 24 treated dogs remained tick-free aside from a single tick on the 2nd day after infestation. Serial serological tests demonstrated that the ticks successfully infected 8/8 control dogs with B. burgdorferi and co-infected 6/8 with A. phagocytophilum. B. burgdorferi infection also was confirmed in most control dogs by culture (6/8) and PCR (7/8) of skin biopsies. In contrast, CERTIFECT protected all 24 treated dogs against infection by both B. burgdorferi and A. phagocytophilum, as demonstrated by their negative serological tests throughout the study and the absence of any positive skin biopsy culture or PCR in these dogs.