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.     

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.     

Spatial distribution of Dermacentor reticulatus and Ixodes ricinus in Hungary: evidence for change?

A survey was conducted to investigate the spatial distribution of Ixodes ricinus and Dermacentor reticulatus in Hungary and to compare these data with the results of a previous large-scale survey. In the survey conducted in the 1950s, D. reticulatus adults were detected in two isolated areas of two counties, and the presence of these ticks in the collection was explained by accidental introduction. In the present survey, D. reticulatus became the second most common species occurring in all 16 counties involved in the monitoring and showed high prevalence. The change in the spatial distribution of this tick species, the increase of incidence of Babesia canis infection in Hungary, and the increasing number of canine babesiosis case reports from other Central and Central Eastern European countries since the 1970s suggest an expansion of the geographic range of D. reticulatus and intensification of the transmission rate of B. canis and probably other D. reticulatus-borne diseases (e.g. tularemia and tick-borne lymphadenopathy) in the region. The spatial distribution of I. ricinus was roughly in line with the results of the earlier survey. I. ricinus was the most common tick species being present in all 16 counties with the highest prevalence. Nevertheless, the comparison of the data of the previous and current survey cannot be used for fine-scale analysis; thus, it cannot be dismissed that the spatial distribution of I. ricinus also changed during the past decades. The spatial distribution patterns of tick-borne encephalitis in Hungary and other Central Eastern European countries may indicate such a change.     

Seroprevalence of Borrelia burgdorferi, Anaplasma phagocytophilum, Ehrlichia canis, and Dirofilaria immitis among dogs in Canada

The seropositivity of dogs to Borrelia burgdorferi, Anaplasma phagocytophilum, and Ehrlichia canis antibodies, and Dirofilaria immitis antigen was assessed in Canada. Borrelia burgdorferi had the highest seroprevalence, while that of Dirofilaria immitis has not changed significantly in the past 20 y. The risk for these vector-borne infectious agents in Canadian dogs is low but widespread with foci of higher prevalence.     

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.     

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.     

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.     

Typical and atypical manifestations of Anaplasma phagocytophilum infection in dogs

Eighteen clinically ill dogs, naturally infected with Anaplasma phagocytophilum, were examined at a veterinary practice in Baxter, Minnesota. A clinical examination, complete blood cell count, enzyme- linked immunosorbent assay (ELISA) for A phagocytophilum, Borrelia burgdorferi, and Ehrlichia canis antibodies and Dirofilaria immitis antigen, and a polymerase chain reaction test for A phagocytophilum DNA were obtained for all dogs. Physical examination findings included fever, arthropathy, lymphadenopathy, epistaxis, acute gastritis, cervical hyperpathia, and central nervous system dysfunction. Complete blood cell count abnormalities included thrombocytopenia, morulae in neutrophils, anemia, leukopenia, eosinopenia, lymphopenia, and monocytosis. Seroreactivity to A phagocytophilum was found in 61%, B burgdorferi antibodies in 17%, and D immitis antigen in 5% of the dogs. Fever, arthropathy, neurologic dysfunction, and epistaxis are clinical syndromes that can be associated with A phagocytophilum infection. Treatment with doxycycline resulted in rapid resolution of clinical signs in all dogs.     

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.     

Prevalence of selected zoonotic and vector-borne agents in dogs and cats in Costa Rica

To estimate the prevalence of enteric parasites and selected vector-borne agents of dogs and cats in San Isidro de El General, Costa Rica, fecal and serum samples were collected from animals voluntarily undergoing sterilization. Each fecal sample was examined for parasites by microscopic examination after fecal flotation and for Giardia and Cryptosporidium using an immunofluorescence assay (IFA). Giardia and Cryptosporidium IFA positive samples were genotyped after PCR amplification of specific DNA if possible. The seroprevalence rates for the vector-borne agents (Dirofilaria immitis, Borrelia burgdorferi, Ehrlichia canis, and Anaplasma phagocytophilum) were estimated based on results from a commercially available ELISA. Enteric parasites were detected in samples from 75% of the dogs; Ancylostoma caninum, Trichuris vulpis, Giardia, and Toxocara canis were detected. Of the cats, 67.5% harbored Giardia spp., Cryptosporidium spp., Ancylostoma tubaeforme, or Toxocara cati. Both Cryptosporidium spp. isolates that could be sequenced were Cryptosporidium parvum (one dog isolate and one cat isolate). Of the Giardia spp. isolates that were successfully sequenced, the 2 cat isolates were assemblage A and the 2 dog isolates were assemblage D. D. immitis antigen and E. canis antibodies were identified in 2.3% and 3.5% of the serum samples, respectively. The prevalence of enteric zoonotic parasites in San Isidro de El General in Costa Rica is high in companion animals and this information should be used to mitigate public health risks.     

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.     

Diagnosis of Lyme disease in two cows by the detection of Borrelia burgdorferi DNA

Two cows from different herds in a district of Switzerland known to harbour ixodid ticks had erythematous lesions on the hairless skin of the udder, were in poor general condition with a poor appetite and decreased milk production, and had a stiff gait and swollen joints. Borrelia burgdorferi sensu strictu DNA was detected in samples of synovial fluid and milk from one of the cows and Borrelia afzelii DNA was detected in synovial fluid from the other by means of a real-time PCR.     

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.     

Evidence of Bartonella sp. in questing adult and nymphal Ixodes ricinus ticks from France and co-infection with Borrelia burgdorferi sensu lato and Babesia sp

Ticks are known vectors for a wide range of pathogenic microorganisms. Their role in the transmission of some others is so far only suspected. Ticks can transmit multiple pathogens, however, little is known about the co-existence of these pathogens within questing ticks. We looked for the presence of DNA from three micro-organisms, Bartonella sp., Borrelia burgdorferi sensu lato and Babesia sp. which are known or suspected tick-borne pathogens, using a cohort of 92 questing Ixodes ricinus ticks collected from pastures in northern France. DNA was extracted from each individual tick and the presence of the three pathogens was investigated using Polymerase Chain Reaction (PCR) amplification. Nine among 92 samples (9.8%) demonstrated PCR products using Bartonella specific primers, 3 among 92 (3.3%) using Borrelia burgdorferi sensu lato specific primers and 19 among 92 (20.6%) using Babesia specific primers. Seven among 92 samples (7.6%) were PCR positive for at least two of the pathogens and one sample was positive for all three. Adult ticks (12/18; 67%) showed significantly higher infection rates compared to nymphs (11/74; 15%) for all three pathogens (P < 0.001). This study is the demonstration of the simultaneous presence of Bartonella sp., Borrelia burgdorferi sensu lato and Babesia sp. in questing Ixodes ricinus ticks.     

GIS-facilitated spatial epidemiology of tick-borne diseases in coyotes (Canis latrans) in northern and coastal California

Ixodes pacificus is the main tick vector for transmission of Anaplasma phagocytophilum and Borrelia burgdorferi to large vertebrates in California. The present study was undertaken in I. pacificus-infested counties in California to examine spatial and temporal relationships among A. phagocytophilum and B. burgdorferi-exposed coyotes with vegetation type and climate. The overall A. phagocytophilum and B. burgdorferi seroprevalences were 39.5% (N=215) and 18.9% (N=148), respectively, with no association with sex. PCR for A. phagocytophilum and B. burgdorferi was negative in all blood and kidney samples. Increased seroprevalence was a positive function of rainfall. Ehrlichial seropositivity was increased in blue-oak foothill pine, montane hardwood, and redwood vegetation regions, and decreased in coastal sagebrush and cropland. Increased exposure to B. burgdorferi occurred in blue oak woodland.     

West-to-east differences of Babesia canis canis prevalence in Dermacentor reticulatus ticks in Slovakia

Babesia canis canis is the most frequent causative agent of canine babesiosis in Central Europe, frequently causing severe disease. Recently, many new endemic foci of this disease have been reported from European countries. Growing incidence of canine babesiosis was recorded also in Slovakia during the last decade, from first cases in eastern Slovakia ten years ago to recent cases all over the south of the country. We have used nested PCR-RFLP method to study prevalence of B. c. canis in its natural tick vector Dermacentor reticulatus, collected at three geographically isolated lowland areas of southern Slovakia situated in the southeast, southwest, and west of Slovakia, respectively. The highest prevalence of B. c. canis was observed in D. reticulatus from eastern Slovakia (14.7%; n=327), whereas the prevalence in southwest was significantly lower (2.3%; n=1205). Notably, all 874 D. reticulatus ticks collected at Záhorská ní?ina lowland (W Slovakia) were B. c. canis-negative. Recorded differences in Babesia prevalence concurs well with the shift in incidence of clinical cases of canine babesiosis as observed by vet practitioners. Presented results revealed that eastern Slovakia represents an area of high risk of B. c. canis infection, whereas western areas of the country still remain Babesia canis-free.     

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.     

Babesia canis rossi infection in a Texas dog

A 5-month-old intact male Boerboel dog, imported from South Africa 1 week previously, was presented to a Texas veterinarian for lethargy, anorexia, and labored breathing. The dog was febrile, anemic, leukopenic, thrombocytopenic, and slightly azotemic. Results of the IDEXX SNAP-4Dx enzyme immunoassay were negative for Dirofilaria immitis antigen and antibodies against Ehrlichia canis, Borrelia burgdorferi, and Anaplasma phagocytophilum. An EDTA blood sample analyzed at Oklahoma State University Center for Veterinary Health Sciences revealed nonregenerative anemia, neutropenia, and large protozoal piroplasms in 0.7% of the RBCs. Piroplasms were 2-5μm long and varied in shape from round to oval to piriform; extracellular merozoites were also observed. Nested PCR was performed on DNA extracted from blood using primers that amplify the 18s rRNA gene from all known Babesia species, and the product was sequenced. Basic Local Alignment Search Tool analysis of the 437 base sequence revealed 99-100% similarity to Babesia canis rossi, 92-93% similarity to Babesia canis canis, and 92% similarity to Babesia canis vogeli. The dog responded well to treatment with imidocarb. PCR analysis of a second blood sample 2 weeks later was negative for Babesia spp. DNA. This case represents the first diagnosis of B. canis rossi infection in the United States.