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.     

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.     

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.     

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.     

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.     

Prevalence of antibodies against Borrelia burgdorferi sensu lato and Anaplasma phagocytophilum and their clinical relevance in dogs in Munich, Germany

Although prevalences of antibodies against Borrelia (B.) burgdorferi sensu lato (sl) and Anaplasma (A.) phagocytophilum have been reported to be high in the German dog population, the importance of the diseases caused by both agents is still not well characterized in a field situation.The aim of this study was (1) to determine the prevalence of antibodies to B. burgdorferi sl and A. phagocytophilum in dogs in Munich, Germany, and (2) to assess the clinical presentation and laboratory values of antibody-positive dogs and compare them to a negative control group. In total, 448 randomly selected dogs were screened for antibodies to B. burgdorferi sl and A. phagocytophilum with the SNAP 4Dx assay (IDEXX, Laboratories, Inc., USA). Dogs carrying antibodies against B. burgdorferi sl and/or A. phagocytophilum were classified as "positive"(n=100), the following 100 negative dogs served as control group. In both groups, physical examination and laboratory parameters were compared. 22 (4.9%) dogs had antibodies to B. burgdorferi sl, 78 (19.4%) to A. phagocytophilum, nine (2.0%) to both agents. Bernese Mountain Dogs had significantly more often antibodies against B. burgdorferi sl. Negative dogs were more often diagnosed as "healthy" compared to A. phagocytophilum antibody-positives that showed more often elevated body temperature and poor general condition; beyond that, there were no differences in clinical and laboratory abnormalities between both groups. Although dogs tested negative were more often considered healthy, there were no differences in parameters considered "specific" for both infections between dogs with and without antibodies. Hence, tests detecting antibodies against both agents are not able to detect animals with the clinical disease.     

Tick parasitism and antibodies to Borrelia burgdorferi in cats

Ticks were removed from naturally infested cats, and serum samples from these cats were tested for antibodies to Borrelia burgdorferi. Twenty-two of 93 cats (23.7%) had one or more motile stages of Ixodes dammini attached. Of 2 larvae and 20 nymphs removed from cats, 1 larva and 2 nymphs were infected with B burgdorferi. Spirochetes were not found in tissues of 13 female and 4 male ticks. Ten of 71 serum samples analyzed by indirect fluorescent antibody staining or ELISA contained antibodies to this spirochete. Maximal antibody titers were 1:256 and 1:2,560, respectively. At titers greater than or equal to 1:160 in ELISA, seropositivity ranged from 8.8% (n = 34 sera tested from 34 cats) in May through July to 33.3% (n = 12 cats tested) during February through April. In clinical studies of 30 cats, there were nearly equal percentages of seropositive cats with limb or joint disorders not accompanied by fever, anorexia, or fatigue (5 of 21 cats) and cats with these signs of illness but lacking lameness (2 of 9 cats.)     

Survey of ixodid tick species on domestic cats in Japan

Various species of ixodid ticks, attached to domestic cats in Japan, were identified in spring (April to June) and autumn (September to November). In the spring, a total of 282 ticks, including 61 larvae, 70 nymphs, 127 females and 24 males were collected from 126 cats. Of these, 264 were identified up to the species level. In the spring, Haemaphysalis longicornis was the most frequently (39.7%, 50/126) found tick species on feline hosts, followed by Ixodes ovatus (35.0%, 44/126), Ixodes nipponensis (15.9%, 20/126) and Haemaphysalis flava (9.5%, 12/126). Small numbers of Haemaphysalis megaspinosa, Haemaphysalis japonica, Ixodes persulcatus, Ixodes granulatus and Amblyomma testudinarium were also recovered. H. longicornis was the most frequently found tick species on cats around riversides or river basins, while I. ovatus and I. nipponensis were more frequently found on cats kept near woodland or related areas. I. nipponensis was more frequently found on castrated males. No major statistical differences in the frequency of tick attachment among sex, age or hair length for the three major tick species were found. Of 205 ticks including 173 (84.4%) larvae, 27 (13.2%) nymphs, 4 (2.0%) females and 1 (0.5%) male recovered from 62 cats in autumn, only 32 (15.6%) were identified. Most of the larvae were fully- or partly-engorged Haemaphysalis spp., and it was difficult to identify them further by morphological characterization.     

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.     

Detection of Invasive Borrelia burgdorferi Strains in North‐Eastern Piedmont,Italy

Following reports of human cases of Lyme borreliosis from the Ossola Valley, a mountainous area of Piemonte, north‐western Italy, the abundance and altitudinal distribution of ticks, and infection of these vectors with Borrelia burgdorferi sensu lato were evaluated. A total of 1662 host‐seeking Ixodes ricinus were collected by dragging from April to September 2011 at locations between 400 and 1450 m above sea level. Additional 104 I. ricinus were collected from 35 hunted wild animals (4 chamois, 8 roe deer, 23 red deer). Tick density, expressed as the number of ticks per 100 m², resulted highly variable among different areas, ranging from 0 to 105 larvae and from 0 to 22 nymphs. A sample of 352 ticks (327 from dragging and 25 from wild animals) was screened by a PCR assay targeting a fragment of the 16S rRNA gene of B. burgdorferi s.l. Positive samples were confirmed with a PCR assay specific for the 5S‐23S rRNA intergenic spacer region and sequenced. Four genospecies were found: B. afzelii (prevalence 4.0%), B. lusitaniae (4.0%), B. garinii (1.5%) and B. valaisiana (0.3%). Phylogenetic analysis based on the ospC gene showed that most of the Borrelia strains from pathogenic genospecies had the potential for human infection and for invasion of secondary body sites.     

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.     

Studies on the critical water mass, rehydration capability and potential, acute chill tolerance and supercooling point of Argas (Persicargas) walkerae (Acari: Argasidae)

The critical water mass, defined as the water mass remaining in a dehydrated tick in the non-ambulatory state, differed only slightly between light and heavy mass groups of Argas walkerae and averaged 23.6% and 23.2%, respectively, in males and 28.4% and 28.0%, respectively, in females. All ticks survived dehydration to 50%, 75% or 100% of their critical water mass, and 95% of them rehydrated during their subsequent incubation at 95% relative humidity (RH) and 28 degrees C for 14 days and regained their ambulatory status. Unfed adults were able to balance water loss frequently over a period of several months. When ticks were repeatedly dehydrated at 0% RH for 14 days, females and males suffered 50% mortality after 16 and 19 cycles of de- and rehydration, respectively, over a period of 278 days and 337 days, respectively. Water itself was not attractive to either dehydrated or non-dehydrated ticks and drinking was not observed. After submergence in water for 3 days, most of the dehydrated adult ticks gained mass. Judged by 50% mortality, larvae tolerated short-term extreme chilling to -24 degrees C, nymphs 1 to -22 degrees C, nymphs II to -20 degrees C, females and males to -19 degrees C. None survived tissue freezing. At a chilling rate of 0.3 degrees C/min, mean supercooling points (SCP) ranged from -25.9 degrees C in eggs to -16.5 degrees C in unfed females. The SCP of all other stages was significantly higher than that of eggs. Mean SCPs of unfed adult ticks dehydrated to 50% or 75% of their critical water mass were significantly lower than that of fully hydrated ticks. The SCPs of ticks acclimated by several weeks exposure to 0 degrees C or to 38 degrees C were significantly lower than those of adult ticks kept constantly at 28 degrees C.     

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.     

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.     

Infection rates with Cowdria ruminantium of nymphs and adults of the bont tick Amblyomma hebraeum collected in the field in Zimbabwe

Cowdria ruminantium (heatwater) infection rates of field populations of the bont tick, Amblyomma hebraeum, were determined at two locations in the southern lowveld of Zimbabwe. At Mbizi Quarantine Station, unfed adult males and females, and nymphs were collected at intervals over a 2-year period using traps. At Lemco Ranch, engorged nymphs were collected on three occasions from weaner calves and allowed to moult to adults. The unfed ticks were fed in small pools on heartwater-susceptible sheep, some of which became infected. The infection rates of the ticks were then estimated statistically. Depending on the date of collection and locality, these rates were in the range 0.0-44.9% for males, 20.0-36.1% for females and 0.0-13.4% for nymphs. Most of these rates are considerably higher than those previously believed to occur.     

Ixodes (Afrixodes) drakensbergensis n. sp. from domestic and wild animals in Natal, Republic of South Africa.

Ixodes (Afrixodes) drakensbergensis n. sp., is described from females, males, nymphs and larvae collected on a drag at Giant's Castle Nature Reserve, Natal, Republic of South Africa; it was also taken from an eland in the same area and from goats and a cow in the adjacent Tank Area 118. The occurrence of I. (A.) drakensbergensis on domestic animals suggests that it may be of economic importance in this area. Information is provided to separate the new species from other closely related Ixodes species that occur or may occur in this region.     

全沟硬蜱围食膜初步观察研究

本工作用光镜和电镜技术对全沟硬蜱的围食膜进行了初步观察研究。结果表明，饥饿幼虫、若虫、成虫以及吸血期雄性成虫不具围食膜。吸血期幼虫、若虫及雌性成虫最晚分别于吸血开始后20、20及18小时出现了单层结构的围食膜；缓慢吸血期围食膜的厚度持续增加，而在快速吸血期其厚度却会显著减小。饱血后发育期围食膜不断增厚，愈来愈拱曲，与肠壁的距离不断扩大，并形成多层结构；至少分别于饱血后12、25及11天，仍能在幼虫、若虫及雌性成虫肠腔观察到完好状态的围食膜。全沟硬蜱是我国北方地区多种血液传播病原体的重要媒介，其围食膜的发现及围食膜形态变化的初步描述，对进一步认识这些病原体在全沟硬蜱体内迁移扩散规律、发育动态以及向宿主的传播途径，可提供有益的启示。     

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.     

Population structure of Argas arboreus (Acari: Argasidae) ticks associated with seasonally abandoned mixed heronries, dominated by cattle egrets (Bubulcus ibis), in South Africa

During winter populations of Argas arboreus from heronries of the cattle egret, Bubulcus ibis, in South Africa are composed of adults, with some predominance of males, and II-IV instar nymphs, in a state of diapause. The period of tick activity, including reproduction and development of eggs, larvae and N1 nymphs, is synchronized with the nesting and breeding season of their avian hosts. It begins during spring with the return of birds to the heronry, and ceases in autumn through induction of reproductive diapause in engorged females, and behavioural diapause in unfed nymphs and adult ticks. Many ticks showed morphological anomalies and malformations, the study of which could possibly be used for monitoring of environmental pollution.