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The role of cats in the eco-epidemiology of spotted fever group diseases
research-article
Author(s):
Ferran Segura ,
Immaculada Pons ,
Jaime Miret ,
Júlia Pla ,
Anna Ortuño ,
María-Mercedes Nogueras
Publication date (Electronic):
1 August 2014
Journal:
Parasites & Vectors
Publisher:
BioMed Central
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Abstract
Background
Mediterranean Spotted Fever (MSF), whose etiological agent is R. conorii, is one of the oldest described vector-borne infectious diseases. Although it is endemic in the Mediterranean area, clinical cases have also been reported in other regions. R. massiliae-Bar29 is related to MSF cases. This strain is distributed worldwide. R. conorii and R. massiliae-Bar29 are transmitted by ticks. Dogs are considered the sentinel of R. conorii infection. Cats could also be involved in their transmission. Rickettsia felis, etiological agent of Flea-borne spotted fever , is mainly transmitted by the cat flea, Ctenocephalides felis. Up to now, the role of cats in its transmission is not entirely elucidated. The aim of the study is to analyze the infection in cats by these microorganisms.
Methods
The study was undertaken in Northeastern Spain. Twenty municipalities of seven regions participated in the study. 212 cats (pets and stray cats) were analyzed. Variables surveyed were: date of collection, age, sex, municipality, source, living place, outdoor activities, health status, type of disease, contact with other animals, and ectoparasite infestation. Sera were evaluated by indirect immunofluorescence antibody assay (IFA). Molecular detection (real-time PCR and sequencing) and cultures were performed on blood samples.
Results
There were 59 (27.8%) cats seroreactive to one or more microorganisms. Considering cross-reactions, the seroprevalences were 15.6%-19.5% ( R. massiliae-Bar29), 1.9%-6.2% ( R. conorii), and 5.2%-7.5% ( R. felis). A weak association was observed between SFG seropositivity and tick infestation. Ticks found on seropositive cats were Rhipicephalus pusillus, R. sanguineus and R. turanicus. DNA of Rickettsia was detected in 23 cats. 21 of them could be sequenced. Sequences obtained were identical to those sequences of SFG rickettsiae similar to R. conorii and R. massiliae. No amplification of R. felis was obtained.
Related collections
Most cited references46
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Development of a quantitative real-time polymerase chain reaction assay specific for Orientia tsutsugamushi.
Two specific and sensitive polymerase chain reaction (PCR) assays were developed to detect and quantitate Orientia tsutsugamushi, the agent of scrub typhus, using a portion of the 47-kD outer membrane protein antigen/ high temperature requirement A gene as the target. A selected 47-kD protein gene primer pair amplified a 118-basepair fragment from all 26 strains of O. tsutsugamushi evaluated, but it did not produce amplicons when 17 Rickettsia and 18 less-related bacterial nucleic acid extracts were tested. Similar agent specificity for the real-time PCR assay, which used the same primers and a 31-basepair fluorescent probe, was demonstrated. This sensitive and quantitative assay determination of the content of O. tsutsugamushi nucleic acid used a plasmid containing the entire 47-kD gene from the Kato strain as a standard. Enumeration of the copies of O. tsutsugamushi DNA extracted from infected tissues from mice and monkeys following experimental infection with Orientia showed 27-5552 copies/microL of mouse blood, 14448-86012 copies/microL of mouse liver/spleen homogenate, and 3-21 copies/microL of monkey blood.
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Warmer Weather Linked to Tick Attack and Emergence of Severe Rickettsioses
Philippe Parola, Cristina Socolovschi, Luc Jeanjean … (2008)
Introduction The recent outbreak of Chikungunya virus in the Indian Ocean Islands and India [1] that reached Europe in 2007 [2], has illustrated the current medical importance of globalising vector-transmitted infections [3]. The impact of ticks on human public health was recognised with the emergence of Lyme disease 25 years ago [4]. Since then, around 15 emerging tick-borne rickettsioses have emerged [5]. Ticks strive for the best conditions for their life cycle and to find and bite a host [6]. Climate and the availability of hosts are among the major factors influencing ticks [6],[7]. The impact of climate change on tick-borne diseases has been the topic of controversial debate in the scientific literature [7],[8]. Additionally, it has been suggested that global warming would result in a northward expansion of several tick species, including I. ricinus and I. scapularis the vectors of Lyme disease in Europe and North America, respectively [7],[9], and Rhipicephalus sanguineus group ticks [10]. Rh. sanguineus, the dog brown tick, is considered the first globalised tick as it has become the most widespread tick throughout the world due to its specialised feeding on the domestic dog [11],[12]. In addition to the dog pathogens Babesia canis and Ehrlichia canis [12], Rh. sanguineus is known to transmit two life threatening rickettsial diseases in humans, Mediterranean spotted fever (MSF) caused by R. conorii in the old world [13], and Rocky Mountain spotted fever (RMSF) caused by R. rickettsii in America [14]. Rh. sanguineus also transmits R. massiliae, a worldwide emerging pathogen [15] with a single documented case of infection [16]. Rh. sanguineus is well adapted to urban environments and lives in close contact with humans. However, Rh. sanguineus rarely feeds on humans, particularly in temperate countries [11]. Herein, we describe the investigation of a focus of rickettsioses in southern France during the exceptionally warm April and May months of 2007. Patients suffered from severe R. conorii and R. massiliae infections, and we found that this cluster of cases resulted from unexpected proliferation and aggressive behaviour of Rh. sanguineus ticks infected by these rickettsiae. We demonstrate experimentally that Rh. sanguineus readily bites humans when exposed to higher temperatures. Methods Case reports Patient 1 On May 24th, 2007, a 25-year-old man developed fever, night sweats, headache, two skin necrotic lesions on the buttocks and the thighs, respectively, and a maculopapular rash. On June 10th he complained of acute visual loss. A bilateral chorioretinitis was diagnosed ( Figure 1 ). A maculopapular rash was still present involving the palms and soles ( Figure 1 ). He was initially treated with intravenous ganciclovir as well as eye drops as a cytomegalovirus infection was suspected. On June 23rd (day 29), a late serum tested positive for spotted fever group rickettsiae. Oral doxycycline plus ofloxacin was started with an intravenous methylprednisolone regimen. The clinical course was favourable, but three months later the recovery of visual acuity was incomplete and fundus examination of the left eye showed a disorganisation of the macular neuroretina. When interviewed again, the patient reported contact with ticks when he visited a friend at the beginning of May. Written informed consent has been obtained from this patient to report case details and pictures. 10.1371/journal.pntd.0000338.g001 Figure 1 Chorioretinitis with macular involvement in patient 1 with Rickettsia massiliae infection. Fundus examination shows numerous intraretinal haemorrhages around three white medium-sized lesions including one located near the macula. An important macular oedema was observed in the left eye (Panel A), and a mild inflammation with a white medium-sized retinal lesion was present in the right eye (Panel B). Panel C shows a fluorescein angiography with retinal vascular leakage around hypofluorescent lesions in the right eye. Optical Coherence Tomography showed an important macular oedema on the left eye (panel D) compared to the right eye (panel E). Panel F shows a maculopapular rash still present three weeks after the onset of ophthalmic symptoms. Inoculation eschars were still visible on the trunk (Panel G and H) and on the feet (Panel I). Patient 2 A previously healthy 30-year-old man (a friend of patient 1) developed high fever, night sweats and headache in May 2007 followed by a maculopapular rash involving the palms 10 days later. A cicatricial inoculation eschar was found on the skin of the left axilla. He presented with acute bilateral visual loss associated with confusion, loss of hearing and tinnitus. A bilateral chorioretinis was diagnosed. An empiric doxycycline plus ofloxacin treatment was started and he recovered. He reported contact with ticks when he visited the same friend as patient 1. Written informed consent has been obtained from this patient to report case details. Laboratory testing Serum, spinal fluid, aqueous humor and tissue specimens were obtained from both patients. Sera were tested for IgG and IgM antibodies by immunofluorescence (IF) assay [17] using 10 rickettsial antigens [18]. When cross-reactivity was noted between several antigens, and the difference in titers between antigens was lower than two-fold, western blotting following cross-adsorption was performed [17]. DNA was extracted from ground eschar biopsies and aqueous humor specimens from patient 1 and from acute sera from both patients [19]. These extracts were used as templates in a nested PCR assay incorporating primers selected from specific regions of the pgsA gene present in both R. conorii and R. massiliae genomes (GenBank accession numbers NC 003103 and CP 000683, respectively). Primers specifically designed for this study included pgsAF1 (5′- AGATAATGTAGATGAGATACC-3′), pgsAR1 (5′-GTTAAAAAAGCGGCAATCCA-3′), pgsA F2 (5′-TTTTTAGTTAGCGGTCTTCGG-3′) and pgsAR2 (5′- TTGAGCCTAGTATCAATATCG-3′). The so-called “suicide PCR” procedure has been followed, that is a nested PCR using single-use primers targeting a gene never amplified previously in the laboratory. This procedure avoids “vertical” contamination by amplicons from previous assays, one of the limitations of extensive use of PCR [20]. Sequences of positive PCR products were compared to GenBank [21]. Detection of Rickettsia spp. in ticks by PCR and Multi-spacer typing (MST) genotyping Tick DNA was extracted and rickettsiae were detected in each sample through the PCR amplification of a 382-bp fragment of the gltA gene [22]. Sterile water and DNA extracted from uninfected ticks from our laboratory were used as negative controls. Sequences obtained from PCR products were identified by comparison with GenBank [22]. All gltA-positive DNA samples were tested using multi-spacer typing [23]. For each sample, three intergenic spacers, (dksA-xerC, mppA-purC and rpmE-tRNAfMet) were amplified and sequenced using the primer pairs dksAF-dksAR, mppAF-mppAR and rpmEF-rpmER, respectively. All amplicon sequences were compared to GenBank [22],[23]. Epidemiological, entomological and climatic investigations On July 5th 2007, the house where the patients reported to have been bitten by ticks was visited. The owners were interviewed. Ticks were collected from walls of the house and from the garden ( Figure 2 ) and were morphologically identified [24]. The climatic conditions in Nîmes in April and May 2007 were studied using the Météo-France's web site (http://www.meteo.fr/meteonet_en/index.htm). 10.1371/journal.pntd.0000338.g002 Figure 2 Entomological survey in the homesites where two patients where infected by Rickettsia conorii and R. massiliae. Panels A and B show the house and the garden where the patients were bitten by ticks. Panel C shows the collection of ticks by “flagging” or “dragging” a blanket over vegetation (Panel C). Ticks become attached to the blanket and can be removed periodically (Panel D). Panel E shows the garage where a dog used to sleep. Many Rhipicephalus sanguineus were found in crevices and cracks in the wall (Panel G and H) and on a blanket (F) and were collected (panel I) for molecular detection of rickettsias. Testing the affinity of Rh. sanguineus for biting humans We used larvae, nymphs and adults from a pathogen-free laboratory colony of Rh. sanguineus, that were colonized starting August 2006 when engorged Rh. sanguineus females were collected in Oran, Algeria, and maintained in environmental incubators at 25°C and 90% RH with a day/night photoperiod of 16∶8 (L∶D) h until they oviposited. Eggs and all life-cycle stages of subsequent generation were maintained under the same environmental conditions. For their blood meal, all stages were placed on a rabbit to feed until repletion [25]. The third generation of ticks was used for the experiment. For all stages, two batches were put on the arm of 3 human volunteers (3 of the authors including PP, IB, DR) who gave written consent to participate. This healthy volunteer study was approved by the Ethical Review Committee of the Faculty of Pharmacy, Algiers, Algeria. One batch was maintained the night before the test 25°C, and the other was maintained at 40°C. All ticks from the different groups were stored in environmental incubators with 90% relative humidity. All ticks were removed after 40 minutes and the number of attached ticks was compared between conditions (Mantel-Haenszel test). Three experiments were processed for larvae and nymphs, two for adults. An additional experiment was made that compared in the same experimental design, the affinity for biting of nymphs maintained at 32°C and 25°C. Results Diagnosis of rickettsioses in patients Using IF, antibodies against all spotted fever group rickettsial antigens were detected in patient 1 at the same level (IgG 2,048, IgM 16, on July 8th). In patient 2, the difference in titers between several antigens was lower than two-fold (IgG 1,024, IgM 256 for R. conorii ; IgG 1,024, IgM 128, for all other antigens). Western blot and cross-adsorption assays indicated that antibodies were specifically directed against R. massiliae in patient 1 and R. conorii in patient 2 ( Figure 3 ). “Suicide PCR” was positive from two samples (of seven tested) obtained from the eye acqueous humor and the eschar biopsy of patient 1. Amplicon sequencing confirmed that patient 1 was infected with R. massiliae, as the obtained pgsA sequence was 98.9% similar to R. massiliae. 10.1371/journal.pntd.0000338.g003 Figure 3 Western blot assay (WB) and cross adsorption studies of 2 patients with severe tick-borne rickettsioses in Nîmes, southern France, 2007. WB procedures were performed as described elsewhere [47] using 20 µl of a 1 mg/ml suspension of rickettsial antigen per lane. The cross-adsorption assay using R. massiliae and R. conorii antigens followed by WB on the resulting supernatant was performed as previously described [47]. Columns Rc and Rm depict western blots using R. conorii and R. massiliae antigens, respectively. Molecular weights (MW) are indicated on the left (arrow = 135 kDa). Untreated sera is acute sera tested by WB. For patient 1, when adsorption is performed with R. massiliae antigens (columns AdM), it results in the disappearance of homologous and heterologous antibodies. In contrast, when absorption is performed with R. conorii antigens (columns AdC), only homologous antibodies disappear indicating that antibodies are specific for R. massiliae. For patient 2, when adsorption is performed with R. conorii antigens (columns AdC), it results in the disappearance of homologous and heterologous antibodies. However, when it is performed with R. massiliae antigens (columns AdM), only homologous antibodies disappear indicating that antibodies are specific for R. conorii. Epidemiological and entomological investigation in patients' home sites The owner, a 50 year-old nurse of the house were patient 1 and patient 2 had been bitten by tick was interviewed. Her dog that used to roam freely and used to rest and sleep in the garage and many rooms of the house, died due to a gastric torsion in April 2007. She reported that the ticks on this dog were numerous and particularly aggressive to people in April 2007, including before its death. The ticks regularly bit her, and her son, a 30 year-old man (not tested here) who presented at that time with a febrile syndrome and a maculo-papular rash. He did not sick medical care. However, when a veterinary doctor was consulted about the dog, he suggested that the son could have a “boutonneuse fever”, the other name of Mediteranean spotted fever, and suggested a doxycycline treatment. Fever disappeared on day 2 of a 200 mg daily doxycycline treatment and the son remained well. The case-patients reported tick bites when they were outside, although they also noticed ticks in several walls inside the house. The entomological investigation was performed after the patients had cleared of brushwood and sprayed the garden with acaricides. A total of 218 nonengorged ticks, all identified as adult Rh. sanguineus, were collected in less than one hour. Ten ticks were collected with flannel flags dragged over vegetation. In the garage, 25 ticks were recovered from a blanket and 90 were collected from the walls inside ( Figure 2 ). Outside, 93 ticks were collected from the walls of the house. Identification and genotyping of rickettsiae detected in ticks Rickettsial DNA was detected in 37/133 ticks tested by PCR (28%). Twenty-four (18%) exhibited a 100% sequence similarity to R. conorii subsp. conorii strain Malish (AE008677). Thirteen specimens (10%) showed 100% sequence similarity with R. massiliae (U59720). Each of the dksA-xerC, mppA-purC, and rpmE-tRNAfMet intergenic spacers were amplified from gltA-positive ticks. For all 24 R. conorii-positive ticks, mppA-purC sequences were 100% similar to R. conorii genotype A (AY345089), and rpmE-tRNAfMet sequences were 100% similar to R. conorii genotype B (AY345092), but dksA-xerC sequences represented a new genotype named AX (EU081773). For all 13 R. massiliae-positive ticks, dksA-xerC sequences were 100% similar to R. massiliae genotype AE (CP000683), rpmE-tRNAfMet sequences were new (genotype AD, EU250277), and mppA-purC sequences were also new (genotype AH, EU250278). All together, the two rickettsia represented new genotypes. Climatic conditions In April 2007, the weather in southern France was associated with the highest temperatures noted since 1950 (+3 to +4°C compared to seasonal norms) [26], particularly in the Gard region (Nimes being the main town). After April 15th, in Nîmes, maximal temperatures were continuously between 25°C and 30°C ( Figure 4 ). A total of 15 “warm days” (>25°C) were recorded, in contrast to the seasonal norm of 0.6. The total duration of sunshine during the month was also increased when compared to seasonal norms (260h14min; that is+40 h). Few periods of rainfall were recorded between December 2006 and April 2007, with a total of 114.4 mm during this period, making this the 4th dryest since 1921. In May, new records were reached with temperatures higher than 33°C between the 22nd and 24th [26]. 10.1371/journal.pntd.0000338.g004 Figure 4 Monthly mean temperature (minimal and maximal) averaged from 1971 to 2000 in Nîmes southern France. The star symbolizes April 2007 which was the warmest April since 1950 when the focus of infection from an attack of Rh. sanguineus ticks was investigated (modified from [26]). Affinity of Rh. sanguineus for biting humans In the 3 experiments with larvae, 27–67% of the ticks previously maintained at 40°C attached to the skin, whereas 0–6% of those at room temperature attached (p<0.05 in all cases). Among the nymphs, 10–20% of the batches previously maintained at 40°C attached to the skin ( Figure 5 ), whereas none of those at room temperature attached. Overall, for larvae and nymphs, the number of ticks attached to the skin was dramatically higher for the group maintained at 40°C ( Table 1 ). No difference appeared in adults, as no specimen but one attached to the skin within 40 mn. In the fourth additional experiment the affinity for biting of nymphs maintained at 32°C was also significantly higher than at 25°C ( Table 1 ). 10.1371/journal.pntd.0000338.g005 Figure 5 Rhipicephalus sanguineus nymphs found attached to human skin 40 min after having been placed on the arm. These ticks had been maintained at 40°C for one night before being placed on the arm. 10.1371/journal.pntd.0000338.t001 Table 1 Pathogen-free laboratory Rhipicephalus sanguineus put on the arm of a human volunteer. Ticks stages/température Number of tick tested by experiment Number of tick found attached after 40 minutes (%) Larvae/25°C Exp. 1 30 2 (6.7%)a Exp. 2 60 0b Exp. 3 30 0c Larvae/40°C Exp. 1 30 8 (27%)a Exp. 2 60 37 (66%)b Exp. 3 30 17 (57%)c Nymphs/25°C Exp. 1 30 0d Exp. 2 10 0 Exp. 3 30 0e Exp. 4 30 1 (3.3%) Nymphs/40°C Exp. 1 30 6 (20%)d Exp. 2 10 1 Exp. 3 30 4 (13.3%)e Nymphs/32°C Exp. 4 30 9 (30%) Adults/25°C Exp. 1 20 0 Exp. 2 30 0 Adults/40°C Exp. 1 30 0 Exp. 2 20 1 One batch of each stage of 3 experiments was maintained the night before the test at room temperature (25°C), and the other was maintained at 40°C. All ticks were removed after 40 minutes. Experiments 1 to 3 were performed using larvae and nymphs, 2 weeks old ticks. Experiment 4 was performed using 2 months old nymphs. a, b, c, d, e : p<0.05 (Mantel-Haenszel test). Discussion When investigating these grouped cases of severe spotted fevers first presumed to be MSF caused by R. conorii, two rickettsial pathogens were in fact identified. This report describes the second human case of R. massiliae infection and was documented using the IF reference serology assays [5], completed by western blot and cross absorption studies and definitely confirmed with the use of molecular tools. R. massiliae is a worldwide rickettsia that was isolated in 1992 and thereafter detected in Rhipicephalus spp. in Europe and Africa [5], Argentina [27], and recently in Arizona, USA [15]. The recognition of the pathogenicity of R. massiliae occurred in 2005 when molecular tools were used to identify a rickettsial isolate obtained 20 years before from a man hospitalised in Italy with fever, an eschar, and a maculopapular rash [16]. In fact, R. massiliae is the sole pathogenic rickettsia known to be prevalent in America, Africa and Europe. In the present report, the predominant symptom was acute visual loss, and both diagnosis and treatment were delayed. Although eye involvement has been reported in spotted fever group rickettsioses, these manifestations are underdiagnosed or frequently misdiagnosed [28]–[30]. Clinicians should suspect rickettsioses in patients with febrile acute visual loss, particularly during the warmest and most common months for Rh. sanguineus-transmitted diseases. Indeed, we have identified here that the source of the focus was an unexpected attack of Rh. sanguineus. Additionally, we have shown for the first time that the population of rickettsias found in a focus of infection was clonal, as all R. massiliae- and R. conorii-positive samples had a unique MST genotype. The rate of infection of Rh. sanguineus was high, particularly for R. conorii (18%). In contrast, the rate of infection is usually lower that 1% [5],[31]. This, combined with an unusual rate of tick attack, was responsible for multiple inoculation escar in our patients. This is an unusual finding in most tick-borne rickettsial diseases, including MSF, because the probability of being bitten simultaneously by several infected ticks has been considered to be rare. This finding is characteristic of few other tick-borne rickettsioses, an example being African tick bite fever caused by R. africae, due to the aggressive behavior of the tick vectors and a high tick infection rate [5],[32]. Rh. sanguineus lives in peridomestic environments shared with dogs but is known to have a low affinity for humans. Hosts other than dogs are usually only infested when dogs are present to maintain a population of the tick [33]. In this setting, the dog allowed a large infestation of ticks, which had no place to go when the dog died. The risk of the people to be bitten was therefore greatly increased, very much like relapsing fever in the American West, where infected soft ticks accumulate in cabins when their squirrel hosts die during the winter [34]. This highlights the importance of the so-called “zooprophylaxis” – that risk is small when there are alternative hosts than people upon which a vector can focus. However, in the present report, ticks started to be particularly aggressive to people before the death of the dog. Also, the son of the owner presented with a tick-borne eruptive fever, before the dog died. Therefore, the death of the dog could not be considered as the cause of this unusual cluster of Rh. sanguineus transmitted rickettsioses.We provide some evidence that this cluster of cases was related to unusually warm temperatures. As shown herein, April 2007 was the warmest April since 1950, with summer-like temperatures [26]. Also, considerable evidence was accumulated by investigators in the 1940's about the role of Rh. sanguineus as a vector of rickettsioses in warm countries such as Mexico [5]. In Europe and North Africa, although Rh. sanguineus starts to be active in May and June [24], most cases of MSF are diagnosed during July and August. This is probably linked to an increased aggressiveness and propensity of Rh. sanguineus to bite hosts in warmer conditions, as demonstrated for other Rhipicephalus species biting cattle [35],[36]. During the 1970s, the increase in the number of MSF cases observed in southern Europe [37] was correlated with higher temperatures and lower rainfall in Spain, and with a decrease in the number of days of frost during the preceding year in France [38],[39]. The cases of MSF recognized in Oran, Algeria in 1993 peaked in 2005 together with the hottest summer of the past decades [18]. More recently, maximum temperature levels during the previous summer were associated with increases in MSF incidence in Sardinia [40]. Finally, during the French heat wave in 2003, with the hottest summer of the preceding 50 years, 22 Rh. sanguineus, including specimens infected by R. conorii and R. massiliae, were found attached to an homeless person who died of MSF in August [41]. This case was highly unusual in regard to the intensity of the parasitism by Rh. sanguineus which had never been reported before in patients or by entomologic investigators who spent their lives collecting ticks [38]. Herein, we have demonstrated by our experimental model that the aggressiveness of immature stages of Rh. sanguineus to bite human is modulated by external temperature. It is important to remind that ixodids do not cause pain while feeding and immature stages are frequently not detected on people because of their small size [6]. Furthermore, we have recently demonstrated the similar effects of higher temperature on the speed of attachment of all stages of Rh. sanguineus ticks, including adults using a rabbit model in a similar experimental design and a 48 hour observation period [42]. We conclude that the host seeking and feeding behaviors of Rh. sanguineus in the present focus were modified by the warmer climatic circumstances and became highly aggressive for the owners and visitors of the house. Rh. sanguineus, a tick of African origin, is now of global importance [11]. The public health importance of the globalisation of vector-borne diseases has been illustrated with West-Nile fever that emerged in 1999 in the USA and has become the dominant vector-borne viral disease [43]. More recently, a returned traveller served as a source of a local Chikungunya virus outbreak in Europe [2], where Aedes albopictus, the recently globalised Asian tiger mosquito is prevalent, as it also is in America [44]. Although ticks have long been considered vectors of geographic diseases because of their preferred environmental conditions and biotopes [6], some vectors have also been globalised. R. africae, the agent of African tick bite fever, has been found in the West Indies where it was introduced from Africa during the 18th century through Amblyomma variegatum ticks on cattle. Now, this rickettsia threatens the American mainland [45]. Rh. sanguineus has spread globally between 50°N and 35°S because of its ability to survive in human home sites [11]. Climate variability will change local weather in sites where brown dog tick infestations occur. If global trends in weather over the long term unfold as predicted, weather will be more variable and may comprise warmer temperatures, droughts and heat waves, as well as more monsoons, or more intense snowstorms, depending on the site [9],[46]. Based on the present investigation and previous epidemiological clinical and experimental data ( Table 2 ), and on a global perspective, we predict that increased temperature will lead to an increased period of activity of Rh. sanguineus and an increased aggressiveness and proclivity to bite humans, and that increased incidence of Rh. sanguineus-transmitted diseases will be observed ( Figure 6 ). On a flip side, cooler weather, if any, in sites where Rh. sanguineus are currently endemic would imply less human biting. However, in the context of warming [9], the public health burden of this tick will increase. After R. conorii, R. rickettsii and the worldwide emerging pathogen R. massiliae [5], other rickettsial agents such as R. rhipicephali [5], or yet undescribed microorganisms, could be found soon as emerging pathogens transmitted by the globalised and multipotent vector, Rh. sanguineus. 10.1371/journal.pntd.0000338.g006 Figure 6 Global distribution of spotted fever group rickettsias potentially transmitted by Rh. sanguineus, threatening humans all over the world, including 3 recognized pathogens R. conorii, R. massiliae, R. rickettsii (which is also transmitted by Dermacentor spp, and Amblyomma spp), and R. rhipicephali, a rickettsia of unknown pathogenicity. 10.1371/journal.pntd.0000338.t002 Table 2 Evidence of the influence of warmer weather and climate on Rh. sanguineus transmitted rickettsioses in humans. Epidemiological evidence • France, this report. Unsual cluster of cases in an atypical period of the year: April 2007 was the warmest April since 1950, with summer-like temperature • Southern USA, and Central America: role of Rh. sanguineus as a vector of R. rickettsii in warm states (Arizona) [14] or countries (Mexico) [5]. • Europe and North Africa: Rh. sanguineus starts to be active in May and June [24], but most cases of MSF are diagnosed during the warmest months, July and August. • Southern Europe, the 1970s: the increase in the number of MSF cases [37] was correlated with higher temperatures and lower rainfall in Spain, and with a decrease in the number of days of frost during the preceding year in France [39]. • Oran, Algeria: the cases of MSF peaked in 2005 together with the hottest summer of the past decades [18]. • Sardinia, Italy: maximum temperature levels associated with increases in MSF incidence in Sardinia [40]. Clinical evidence • France, French heat wave in 2003: hottest summer of the preceding 50 years, 22 Rh. sanguineus, including specimens infected by R. conorii and R. massiliae, were found attached to an homeless person, who died of MSF [41]. • This report: multiple escars unusual finding in MSF, because the probability of being bitten simultaneously by several infected Rh. sanguineus ticks is considered to be rare. • Multiple eschars in MSF reported in the warmest countries of southern Europe (Spain) [48]. Experimental models • Increased aggressiveness and propensity of Rh. sanguineus to bite unusual hosts (rabbit) in warmer conditions [42] • This study: Increased aggressiveness and propensity of Rh. sanguineus to bite hosts in warmer conditions
- Record: found
- Abstract: found
- Article: found
Molecular Detection and Characterization of Tick-borne Pathogens in Dogs and Ticks from Nigeria
Joshua KAMANI, Gad Baneth, Kosta Mumcuoglu … (2013)
Introduction Several tick-borne bacteria and parasites are important pathogens of humans and animals [1]. Being haematophagous, ticks are capable of transmitting disease agents such as viruses, bacteria and protozoa. Historically, they have been considered second only to mosquitoes in their ability to transmit disease agents [2]. Ticks attach to their hosts, facilitating transmission of infectious agents to the host and their spread to different geographical regions via traveling pets or other means of transportation [3]. Globalization and increased international trade, urbanization, climate change and increased travel and mobility of pets have resulted in rapid extension of the zoogeographical range for many tick species [1]. In areas where canine vector-borne diseases are endemic, dogs can be simultaneously or sequentially infected with more than one vector-borne agent [3], [4]. Because blood sucking vectors contain infected host blood and pathogens, they are reliable indicators for the existence of pathogens in a specific area [5]. Therefore, it is recommended to periodically screen animals and vectors for pathogen carriage. Several molecular surveys have evaluated the existence of multiple vector borne pathogens in specific regions including, Europe [4], Middle East [6], Asia [7] and Africa [8]. Epidemiological surveillance of disease occurrence and prevalence is required to map local risk, to acquaint physicians and veterinarians with the prevalence of pathogens and emergence of new infectious agents and forecast possible vector-borne infection outbreaks. This can be achieved by the use of molecular diagnostic techniques, data analysis and mathematical models as well as veterinary clinical surveillance. In Nigeria, the diagnosis of vector-borne pathogens (VBPs) is usually based on the microscopic detection of pathogens in peripheral blood smears, sometimes serology is employed and rarely molecular methods are used. Microscopic diagnosis may lack sensitivity and is time consuming while serology usually indicates exposure rather than active infection, and might mislead due to serological cross reactions with other closely related organisms. Conversely, molecular detection is more sensitive and specific. As data on canine vector-borne infections in Nigeria is scarce [9], [10], this study aimed at broadening the knowledge on these canine pathogens and their ectoparasites. The objective of this study was to molecularly detect and, characterize various vector-borne pathogens in dogs and ticks in four states of Nigeria. Materials and Methods Study area The study was conducted in the 4 Nigerian states; Plateau (9°10′N9°45′E), Kaduna (10°20′N7°45′E), Kwara (8°30′N 5°00′E) and Rivers (4°45′N 6°50′E) (Figure 1). 10.1371/journal.pntd.0002108.g001 Figure 1 Map of Nigeria, West Africa showing states (shaded) where samples were collected. Legends describe the location of veterinary clinics and hospitals where samples were collected. Ethics statement The study protocol was read and approved by The National Veterinary Research Institute Vom, Nigeria Ethical Committee on Animal Use and Care. Animals were treated in a humane manner and in accordance with authorizations and guidelines for Ethical Conduct in the Care and Use of Nonhuman Animals in Research of the American Psychological Association (APA) for use by scientists working with nonhuman animals (American Psychological Association Committee on Animal Research and Ethics in 2010). Sampling of dogs One hundred and eighty one dogs from 4 states of Nigeria presented to private or government veterinary hospitals between August and December 2011 were selected. The selection criteria included dogs infested with ticks or manifesting clinical signs of tick borne diseases, such as anemia, weakness, pyrexia, anorexia and haemoglobinuria. Demographic data, signalment (age, sex, and breed) and clinical signs were recorded for each dog. Five ml of blood were collected from the cephalic or jugular vein into sterile EDTA tubes, and kept at 4°C until arrival at the laboratory. Sampling of ticks Ticks were collected from dogs infested at the time of presentation into test tubes containing absolute ethanol and transported to the laboratory. Thereafter, samples were preserved at −20°C and transported in a cool box to Israel for identification and DNA analysis. A total of 258 ticks were collected from 42 domestic dogs. After identification, ticks from each dog were grouped according to their species. One to three ticks from each dog were pooled for analysis. Seventy six tick pools were processed for DNA extraction. Most of the ticks selected were partially or fully engorged adult females, nymphs and larvae. DNA extraction Blood samples DNA was extracted from EDTA-anticogulated blood using the Illustra blood genomic Prep Mini Spin kit (GE Healthcare UK Limited) according to the manufacturer's instructions. DNA concentrations were determined by measuring the absorbance at 260 nm (A 260) with a NanoVue Spectrophotometer (GE Healthcare, UK Limited). Ticks Ticks were minced using a sterile scalpel blade before homogenization in sterile Eppendorf tubes containing 50 µl phosphate buffered saline (PBS). Total DNA was extracted from each tick pool using the Illustra tissue and cell genomic Prep Mini Spin kit (GE Healthcare UK Limited), adjusted in 100 µl of TE buffer and stored at −20°C until further use. Real Time PCR for detection of Ehrlichia and Anaplasma spp A 97 base pair segment of the 16S ribosomal RNA (16S rRNA) gene of Ehrlichia canis and Anaplasma platys was targeted using primers 16S-F and 16S-R as previously described [6]. DNA from E. canis 611 tissue culture was obtained from The Koret School of Veterinary Medicine, The Hebrew University of Jerusalem, Rehovot Israel and was used as a positive control. DNA from blood of a specific pathogen free (SPF) dog was used as a negative control. The negative and non-template controls (NTC) as well as the positive control were included in each reaction in duplicates. Non template control reactions were done using the same procedures and reagents described above but without DNA added to the PCR reaction to rule out PCR contaminations. Real Time PCR for detection of Rickettsia spp Initial detection of Rickettsia sp. was performed by screening all DNA samples for the presence of a 133-bp citrate synthase gene (gltA) fragment using primers rico173F and rico173R as previously described [11]. Positive samples from this reaction were further analyzed for the presence of a 178–189-bp fragment of the outer membrane protein A gene (ompA), using primers 107F and 299R as previously described [12]. DNA extracted from cultured R. conorii israelensis was used as a positive control, and two negative control samples containing all the ingredients of the reaction except DNA were used for all trials. Conventional PCR assays for Babesia and Hepatozoon spp A 408 base-pair fragment of the 18S ribosomal RNA (18S rRNA) gene of Babesia spp. was targeted using primers PIRO-A and PIRO-B as previously described [13]. For the detection of Hepatozoon spp., the forward primer HEP-F and the reverse primer HEP-R were used as previously reported targeting a 666 bp fragment of the 18S rRNA gene [14], [15]. Positive controls of naturally infected dogs with Babesia vogeli (Bab 36799) and Hepatozoon canis (HEP 7423), as well as negative DNA controls from colony-bred dogs negative by PCR for vector-borne pathogens were run with each corresponding PCR reaction. Non-template control (NTC) reactions were done using the same procedures and reagents described above but without DNA added to the PCR reaction to rule out contaminations. PCR was performed using Syntezza PCR-Ready High Specificity (Syntezza Bioscience, Israel). Amplification was performed using a programmable conventional thermocycler (Biometra, Goettingen, Germany). PCR products were electrophoresed on 1.5% agarose gels stained with ethidium bromide and checked under UV light for the size of amplified fragments by comparison to a 50 bp DNA molecular weight marker. Sequencing DNA amplicons from positive samples were purified (EXOSAP-IT, USB, Cleveland, Ohio, USA) and sequenced using forward primers at the Center for Genomics Technologies, Hebrew University of Jerusalem, Israel. DNA sequences obtained were evaluated with Chromas Lite software version 2.01(Technelysium pty Ltd) and compared for similarity to sequences deposited in GenBank, using the BLAST program hosted by NCBI, National Institutes of Health, Bethesda, MD USA http://www.ncbi.nlm.nih.gov/BLAST. Selected sequences were deposited in GenBank. Statistics Data generated in the study were analyzed by a χ2 test using the Statistix 8 software. Association of pathogen DNA between dogs and ticks was computed using the Medcal statistical software [16]. P values 36 months, 5%, while no information on age was available for 15 (8.3%) dogs. Seventy eight (43.1%) of the dogs studied belonged to a local (Nigerian) breed, 61 (33.7%) belonged to pure foreign breeds, 27 (14.9%) were of cross- breeds while no information on breed was available for 15 (8.3%) dogs. The majority of the sampled dogs were from Plateau State (150; 82.9%), followed by Rivers State (17; 9.4%), Kaduna State (11; 6.1%) and Kwara State (3; 1.7%). 10.1371/journal.pntd.0002108.t001 Table 1 Distribution of vector-borne pathogens among dog population studied. Variables No. (%) examined No. (%) positive for Total E. canis A. platys B. rossi B. vogeli Theileria sp. H. canis Rickettsia spp. gltA OmpA Sex Male 66 (36.5) 4 (6.1) 5 (7.6) 3 (4.5) 0 (0) 0 (0) 27 (40.9) 5 (7.6) 1 (1.5) 44 (24.3) Female 102 (56.4) 11 (10.8) 7 (6.9) 9 (8.8) 0 (0) 1 (0.9) 38 (37.3) 9 (8.8) 0 (0) 75 (41.4) DNA 13 (7.2) 8 (61.5) 0 (0) 0 (0) 1 (1.7) 0 (0) 10 (76.9) 2 (15.3) 0 (0) 21 (11.6) Age group (months) 0–6 58 (32.0) 7 (12.1) 3 (5.2) 5 (8.6) 0 (0) 0 (0) 21 (36.2) 6 (10.3) 0 (0) 42 (22.7) 7–12 56 (30.9) 7 (12.5) 4 (7.1) 4 (7.1) 0 (0) 0 (0) 24 (42.9) 2 (3.6) 0 (0) 41 (22.1) 13–24 36 (19.9) 2 (5.6) 5 (13.9) 1 (2.8) 0 (0) 0 (0) 15 (41.7) 5 (13.9) 1 (2.8) 28 (15.5) 25–36 7 (3.9) 0 (0) 0 (0) 1 (14.3) 0 (0) 1 (14.3) 1 (14.3) 0 (0.0) 0 (0) 3 (1.7) >36 9 (5.0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 2 (22.2) 1 (11.1) 0 (0) 3 (1.7) DNA 15 (8.3) 7 (46.7) 0 (0) 1 (6.7) 1 (6.7) 0 (0) 12 (80.0) 2 (13.3) 0 (0) 23 (12.7) Breed Local 78 (43.1) 7 (9.0) 5 (6.4) 5 (5.1) 0 (0) 0 (0) 35 (44.9) 9 (11.5) 1 (1.3) 61 (33.1) Exotic 61 (33.7) 6 (9.8) 6 (9.8) 6 (9.8) 0 (0) 1 (1.6) 16 (26.2) 4 (6.6) 0 (0) 39 (215) Cross 27 (14.9) 3 (11.1) 1 (3.7) 1 (7.4) 0 (0) 0 (0) 12 (44.4) 1 (3.7) 0 (0) 18 (9.9) DNA 15 (8.3) 7 (46.7) 0 (0) 0 (0) 1 (6.7) 0 (0) 12 (80.0) 2 (13.3) 0 (0) 22 (12.2) Sampling Area Rivers state 17 (9.4) 2 (11.8) 2 (11.8) 2 (11.8) 0 (0) 0 (0) 2 (11.8) 0 (0) 0 (0) 8 (4.4) Kwara state 3 (1.7) 0 (0) 1 (33.3) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 1 (0.6) Kaduna state 11 (6.1) 7 (63.3) 0 (0) 0 (0) 1 (9.1) 0 (0) 9 (81.8) 2 (18.2) 0 (0) 19 (10.5) Plateau State a. Jos North 41 (22.7) 7 (17.1) 4 (9.8) 6 (14.6) 0 (0) 0 (0) 11 (26.8) 2 (4.9) 0 (0) 30 (16.0) b. Jos South 84 (46.4) 6 (7.1) 4 (4.8) 4 (4.8) 0 (0) 1 (1.2) 30 (35.7) 10 (11.9) 1 (1.2) 55 (30.4) c. Lantang North 25 (13.8) 1 (4.0) 1 (4.0) 0 (0) 0 (0) 0 (0) 23 (92.0) 2 (8.0) 0 (0) 27 (14.9) Total 181 (100) 23 (12.7) 12 (6.6) 12 (6.6) 1 (0.6) 1 (0.6) 75 (41.4) 16 (8.8) 1 (0.6) 140 (77.3) DNA = Data not available; E. canis = Ehrlichia canis; A. platys = Anaplasma platys; B. rossi = Babesia rossi; B. vogeli = Babesia vogeli; H. canis = Hepatozoon canis. Overall estimate of infection with VBPs was 77.3% (140/181) in sampled dogs. Single infections occurred in 73(40.3%) dogs while co- infection with more than one pathogen occurred in 67 (37%) of the dogs examined. Co- infections with H. canis were most prevalent (14.4%) followed by E. canis and Rickettsia spp. (6.6%) each (Table 2). Single infections occurred mostly in dogs within the age range of 9–12 months. Co-infections were mostly detected in dogs between 2–12 months of age. 10.1371/journal.pntd.0002108.t002 Table 2 Number of dogs infected with single or multiple vector- borne pathogens. Nature of infection Pathogen species detected in dogs E. canis A. platys B. rossi B. vogeli Theileria sp. H. canis Rickettsia sp. Total (%) Single infection 10 6 6 0 1 46 4 73 (40.3) Co infection with E. canis - 0 1 0 0 10 1 12 (6.6) A. platys 0 - 1 0 0 5 2 8 (4.4) B. rossi 1 1 - 0 0 4 1 7 (3.9) B. vogeli 1 0 0 - 0 1 - 2 (1.1) Theileria sp. 0 0 0 0 0 0 0 0(0) H. canis 10 4 3 1 0 - 8 26 (14.4) Rickettsia sp. 1 1 1 0 0 9 - 12 (6.6) Total 23 12 12 1 1 75 16 140 (77) E. canis = Ehrlichia canis; A. platys = Anaplasma platys; B. rossi = Babesia rossi; B. vogeli = Babesia vogeli; H. canis = Hepatozoon canis. Hepatozoon canis was the most frequently detected pathogen in dogs (41.4%), followed by E. canis, Rickettsia spp., Babesia rossi and A. platys (12.7%, 8.8%, 6.6% and 6.6% respectively). Babesia vogeli, Theileria sp. and R. conorii israelensis were detected in one dog each (Table 1). There was no significant difference (p>0.05) in prevalence of these pathogens between the various groups of dogs studied (Table 1). Sequences of pathogens derived from dog's blood in this study were deposited in GenBank under the following accession numbers; H. canis (JQ976620–JQ976629); B. rossi (JQ976603–JQ976616); B. vogeli (JQ976617); Theileria sp. (JQ976619); E. canis (JQ976630–JQ976641) and A. platys (JQ976642–JQ976653). Pathogen DNA in ticks A total of 258 ticks (128 adults, 124 nymphs and 6 larvae) partially or fully engorged belonging to two genera Rhipicephalus and Haemaphysalis, removed from 42 dogs were examined for various VBPs. Ehrlichia canis, H. canis and Rickettsia spp. DNA were detected in R. sanguineus, R. turanicus and H. leachi ticks. NA of various VBPs was detected in all the different tick species examined in this study. A total of 76 tick pools were tested out of which 18 (23.7%), 16(21.1%), 8(10.5%) and 4(5.3%) were positive for the DNA of E. canis, H. canis, Rickettsia spp. and Candidatus N. mikurensis respectively, while A. platys and R. conorii israelensis DNA were detected in one tick pool each (Table 3). Sequences from ticks were assigned the following accession numbers: H. canis (JX027010–JX027020), Candidatus N. mikurensis (JX027021–JX027024), E. canis (JQ976654–JQ976665), A. platys (JQ976666) and R. conorii israelensis (JX259321 and JX259322). 10.1371/journal.pntd.0002108.t003 Table 3 Comparison of DNA sequence similarities between pathogens detected in dogs and ticks in this study and GenBank deposited sequences. Pathogen sequences from dog blood Pathogen sequences from ticks Pathogen genotype (No. positive)-Accession No. First Genbank Match Accession No. (% sequence similarity) Pathogen genotype (No. positive)- Accession No. First GenBank Match Accession No. (% sequence similarity) Babesia spp. B. rossi (1) –JQ976615 Babesia rossi -AB303074.1 (100) - - B. rossi (9) - JQ976603 Babesia rossi -AB303074.1 (99) - - B. rossi (2) –JQ976612 Babesia rossi -AB303074.1 (98) - - B. vogeli (1) -JQ976617 Uncultured Babesia clone seqBCV79-JN717134.1 (99) - - Theileria sp. Theileria sp (1)-JQ976622 Theileria ovis -GU726904.1 (98) - - Ehrlichia spp. E. canis (17) - JQ976631 Uncultured Ehrlichia sp. clone -JQ260861 (100) E. canis (15) - JQ976654 Uncultured Ehrlichia spp -JQ260861 (100) E. canis (2)- JQ976630 Uncultured Ehrlichia sp. clone-JQ260861 (99) E. canis (2) –JQ976659 E. chaffensis-JQ085940.1 (99) E. canis (3) - JQ976639 Uncultured Ehrlichia sp. clone-JQ260861 (97) Candidatus Neoehrlichia mikurensis C. N mikurensis (4) - JX027021 C.N. mikurensis- JQ359051 (100) Anaplasma spp. A. platys (8) - JQ976650 Anaplasma platys isolate A.pl -JQ 396431(100) A. platys (1) - JQ976666 Anaplasma platys -JQ396431 (99) A. platys (1) –JQ976643 Anaplasma platys isolate A.pl-JQ 396431 (99) A.platys (1) - JQ976648 Anaplasma platys isolate A.pl-JQ 396431 (97) Anaplasma sp (1)-JQ976642 uncultured Anaplasmataceae bacterium-JN581373.1 (99) Hepatozoon canis H. canis (6) –JQ976617 Hepatozoon canis -DQ 111754 (99) H. canis (2)-JX027013 Hepatozoon canis -DQ111754 (100) H. canis (3) JQ976626 Hepatozoon canis -EU289222 (99) H. canis (5)-JX027011 Hepatozoon canis -DQ111754 (99) H. canis (1)-JQ976629 Hepatozoon canis -JF 459994 (99) H. canis (3) - JX027010 Hepatozoon canis-GU376457 (99) Rickettsia spp. Rickettsia sp (gltA) (16) JX259323 Uncultured Rickettsia sp JQ664729 (100) Rickettsia sp (gltA) (6) JX259324 Uncultured Rickettsia sp JQ664729 (100) R. c. israelensis (1) JX259321 R. c. israelensis (EU122392 (100) R. c. israelensis (1) JX259322 R. c. israelensis EU122392 (100) E. canis = Ehrlichia canis; A. platys = Anaplasma platys; B. rossi = Babesia rossi; B. vogeli = Babesia vogeli; H. canis = Hepatozoon canis; E. chaffeensis = Ehrlichia chaffeensis; C. N mikurensis = Candidatus N. mikurensis; R. c. israelensis = Rickettsia. conorii israelensis. Comparison between the presence of pathogen DNA in blood and ticks from the same dog Pathogen DNA as single or multiple infections were detected in 26/76 (34.2%) tick pools removed from dogs with tick infestation at the time of clinical presentation and sampling. Blood and ticks from 7 dogs (nos. 1, 8, 33, 34, 37, 38 and 39) were free from DNA of the various VBPs tested for in this study. DNA of pathogens was detected in ticks removed from 7 other dogs (nos. 3, 4, 12, 18, 25, 31 and 36) but none was detected in the blood of their dog host. Conversely, DNA of various pathogens was detected in 7 dogs (nos. 5,7,13, 16, 28, 29 and 49), but no pathogen DNA was detected in ticks removed from them. Ticks collected from 4 dog (nos. 2, 15, 22 and 23) as well as the dogs from which they were removed were both positive for E. canis DNA. Similarly, DNA of H. canis was detected in 3 dogs (nos. 20, 21 and 24) and ticks removed from each of them. However, DNA of H. canis only was detected in 3 dogs (nos. 11, 35 and 42) but DNA of both H. canis and E. canis was detected in ticks removed from them. Different pathogen's DNA was detected in 10 dogs (nos. 6, 9, 10 14, 17, 19, 26, 27, 35 and 40) as compared to the ticks removed from them. Nine (11.8%) of the tick pools were co-infected by two or more pathogens. There was a significant association between H. canis DNA in dogs and ticks removed from them (Relative risk = 2.69; 95% Confidence Interval = 1.2–5.8; Z = 2.51; p = 0.012). However, there was no significant association between the detection of pathogen DNA in dogs blood and ticks removed from them for E. canis, (RR = 1.56; 95% CI = 0.44–5.45; Z = 0.69; p = 0.49), A. platys (RR = 0.59; 95% CI = 0.025–14.3; Z = 0.32; p = 0.75) or B. rossi (RR = 0.07; 95% CI = 0.004–1.13; Z = 1.88; p = 0.061). Babesia spp. and Theileria spp. DNA were not detected in any of the tick pools tested. Identity of pathogen sequences amplified from dogs and ticks Babesia rossi and B. vogeli sequences from this study had 98–100% and 99% similarities, respectively, with the first matched BLAST result from GenBank (Table 3), while Theileria sp. had 98% sequence similarity with Theileria ovis. Ehrlichia canis sequences from blood of dogs and ticks in this study had 97–100% and 100% similarities, respectively, with the first matched BLAST result from GenBank. Two sequences had 99% similarity with Ehrlichia chaffeensis as the first GenBank match from BLAST. However, attempts to validate the identity of this species by PCR for secretory genes (SodB/VirB 3, VirB 4, and VirB 9) did not yield confirmatory results. Four sequences from ticks had 100% sequence identity to Candidatus N. mikurensis. Anaplasma platys sequences from dogs and ticks had 97–100% and 99% sequences similarities, respectively, with the first matched BLAST result from GenBank. Similarly, H. canis sequences from dogs and ticks had 99% and 99–100% similarities, respectively, with H. canis sequences deposited in GenBank (Table 3). The rickettsial gltA gene fragment was detected in 16 of 181 (8.8%) dog blood samples and in 8 of 76 (10.5%) tick pools examined. Rickettsial ompA DNA was found in one (0.6%) blood and one tick sample. Both sequences were identical and were 100% similar to ompA fragment from R. conorii israelensis. All the sequences detected in this study from dogs and the ticks removed from each of them were highly identical to each other (99–100%) for all the pathogens identified (Table 3). Discussion Ticks and other haematophagous arthropods play a major role in the epidemiology of diseases of humans and animals globally. Their distribution and abundance determines the epidemiology of vector borne infections. The results of this study provide molecular evidence for the presence of E. canis, H. canis, A. platys, B. rossi, B. vogeli, Theileria sp., closely related to T. ovis, Candidatus N. mikurensis and R. conorii israelensis in dogs and ticks from Nigeria. DNA of at least one vector borne pathogen was detected in 77% of the dogs and 45% of the tick pools examined. This is the first report documenting the identification of Candidatus N. mikurensis, R. conorii israeliensis, A. platys and Theileria sp. in dogs and ticks from Nigeria. In fact, it is the first detection of the zoonotic pathogen, Candidatus N. mikurensis, in Africa. Candidatus N. mikurensis is an emerging pathogen first described in 2004 affecting humans and animals with varying clinical manifestation [17]. This pathogen was reported in several hosts including Ixodes spp. ticks, Rattus norvegicus, humans and dogs from Japan, Switzerland and Germany [17]–[19]. Although Ixodes ticks of medical and veterinary importance are not found in Nigeria, R. norvegicus are common and serve as small mammal hosts to multi- host ticks during their life cycle. As engorged ticks were screened in this study, it is possible that R. sanguineus ticks acquired Candidatus N. mikurensis from infected R. norvegicus or dogs. Due to the fact that this agent is a potential threat to humans, physicians should consider this pathogen in their differential diagnosis list in complicated unexplained fever of unknown etiology cases in Nigeria. Rickettsia gltA DNA was detected in 8.8% and 10.5% of dogs and ticks respectively in this study. These estimates are almost similar to the 7.8% previously reported [9] but lower than the 20.6% infection rate reported for R. africae in ticks collected from cattle and vegetation in Nigeria [10]. Another report of prevalence of 0.4% R. conorii and 94–100% R. africae in Rhipicephalus evertsi was made in Guinea and Liberia, West Africa [20]. In the present study, 8.8% of blood samples and 10.5% of tick pools were positive for the rickettsial gltA but only one blood sample (0.6%) and one R. sanguineus tick pool (1.3%) were found positive for the rickettsial ompA gene, and their sequences were 100% identical to R. conorii israelensis. This is the first report indicating the presence of the agent of Mediterranean Spotted Fever in Nigeria. Rickettsiae with ompA gene are considered to be pathogenic, while those who exclude this gene are probably non-pathogenic endosymbionts [21]. Rickettsia africae, the etiologic agent of African tick fever in humans has been detected in ticks from Nigeria [9], [10] and other West African countries [20] but not in our study. It is possible R. sanguineus, R. turanicus and H. leachi found on dogs in this study are not competent vectors for this organism [20]. The detection of A. platys infection in Nigeria is also reported for the first time in this study. The estimate of 6.6% infection rate in dogs in this study is higher than the 4% reported for dogs in Italy [22], but lower than 16% reported in Venezuela [23]. Anaplasma platys is a thrombocytotropic bacteria of dogs that causes canine infectious cyclic thrombocytopaenia characterized by clinical abnormalities such as fever, anorexia, petechial haemorrhages, and uveitis [24]. Theileria sp. with 98% sequence similarity to T. ovis from a sheep in Iran [25] was detected in one dog in this study (Table 3). Theileria spp. have been reported in dogs from South Africa [26] and Spain [27]. The Theileria sp. in this study appears to be molecularly different from the previously described species. The high estimate of H. canis (41.4%) and E. canis (12.7%) infections reported in dog blood in this study are higher than the 22% and 5%, respectively reported earlier in Zaria-Nigeria using microscopic examination of blood smears [28]. However, the estimate of 6.6% for B. rossi in this study is lower than previous reports of 10.2% [29] and 11.0% [30], and higher than 2.0% for dogs in Nigeria [28], but close to the 9.0% reported in Sudan [8]. Similarly, the estimate of 41.4% H. canis infection in this study is higher than the 20.3% previously reported in dogs from Nigeria, but almost similar to the 42.3% reported in Sudan [8]. The higher estimate of H. canis DNA in this study can be attributed to the sensitivity of the techniques used, enabling the detection of E. canis and H. canis at low bacterial and parasite loads. Babesia canis and B. gibsoni were not detected in any of the samples tested. This finding is in agreement with earlier molecular surveys in dogs from Nigeria [30] and can be attributed to the absence of their tick vectors in Nigeria. Although a case of B. rossi and B. canis co-infection in a local Nigerian dog that never left the country has been reported [31], the source of that infection could not be elucidated. One recent study in Nigeria did not detect Ehrlichia spp. in R. sanguineus ticks [9], while another study reported a prevalence of 5.7% Ehrlichia spp. in ticks collected from cattle [10] which is much lower than the 23.7% detected in this study. The difference in prevalence may be attributed to variation in techniques used and source of samples. Dogs are competent reservoir- hosts of several zoonotic pathogens and are infested by many blood-feeding arthropods. The role of ticks as vectors of these pathogens can be asserted considering the high sequence similarities (99–100%) between the pathogens detected from the host and those detected in ticks removed from them (Table 3). Of the 18 tick pools positive for E. canis, and 16 positive for H. canis, 22% and 38% of the pools were from E. canis and H. canis positive hosts, respectively. There was a significant association between the detection of H. canis DNA in dogs and ticks removed from the same dog, but no association was found for E. canis, A. platys or B. rossi. As all ticks were removed from dogs while they were attached and most of them were partially or fully engorged, it is impossible to ascertain whether the ticks were fed with infected blood or if they served as vectors and transmitted the pathogen to their present host. Considering the fact that the tick species included in this study have multi- host life cycle, they could have acquired infection during feeding on a previous infected host and transmitted the infection during their next feeding. Dog breeding is a lucrative business in Nigeria, where dogs are used for trade and security. Dogs also serve as a food source and their meat is considered as a delicacy among some ethnic groups in Nigeria. These can expose humans directly or indirectly to zoonotic agents during handling of dogs and ticks carrying pathogens, or during processing and consumption of their meat. Further investigation is required to elucidate the role of ticks and the effect of these pathogens in causing diseases in humans in Nigeria. In conclusion, this study has confirmed that several vector borne pathogens of humans and animals are highly prevalent in Nigeria and West Africa where the incidence of tick-borne infections appears to be underestimated. Physicians and veterinarians should be aware of the existence of these pathogens in Nigeria and should include them in the differential diagnoses for clinical illnesses with compatible clinical signs. Screening targeted groups for VBPs as well as humans with fever of unknown origin or undiagnosed cases for infection with R. conorii israelensis and Candidatus N. mikurensis is recommended.
Author and article information
Contributors
Ferran Segura: fsegura@tauli.cat
Immaculada Pons: ipons@tauli.cat
Jaime Miret: jaimemiret@gmail.com
Júlia Pla: julia.pla@hotmail.com
Anna Ortuño: ana.ortuno@uab.es
María-Mercedes Nogueras: mnogueras@tauli.cat
Journal
Journal ID (nlm-ta): Parasit Vectors
Journal ID (iso-abbrev): Parasit Vectors
Title:
Parasites & Vectors
Publisher:
BioMed Central
(London
)
ISSN
(Electronic):
1756-3305
Publication date
(Electronic):
1
August
2014
Publication date PMC-release: 1
August
2014
Publication date Collection: 2014
Volume: 7
Issue: 1
Electronic Location Identifier: 353
Affiliations
Article
Publisher ID: 1549DOI: 10.1186/1756-3305-7-353
PMC ID: 4141105
PubMed ID: 25084969
SO-VID: 2ee496e8-9e53-4c48-a0da-03186fe637de
Copyright © © Segura et al.; licensee BioMed Central Ltd. 2014
License:
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
History
Date
received
: 16
April
2014
Date
accepted
: 15
July
2014
Categories
Subject:
Research
Custom metadata
issue-copyright-statement © The Author(s) 2014
ScienceOpen disciplines: Parasitology
Data availability:
ScienceOpen disciplines: Parasitology