Abstract
Coxiella burnetii, the etiological agent of human Q fever, can infect mammals, birds, and arthropods. The Canary Islands (Spain) are considered an endemic territory, with a high prevalence in both humans and livestock. Nonetheless, there is no epidemiological information about the wild and peridomestic cycles of C. burnetii. Tissue samples from rodents on farms (100) and wild rabbits (129) were collected and assessed by PCR to detect C. burnetii DNA. In parallel, ticks were also collected from vegetation (1169), livestock (335), domestic dogs (169), and wild animals (65). Globally, eight rodents (8%) and two rabbits (1.5%) were found to be positive, with the spleen being the most affected organ. Tick species identified were Hyalomma lusitanicum, Rhipicephalus turanicus, Rhipicephalus sanguineus, and Rhipicephalus pusillus. Hyalomma lusitanicum (80%) was the main species identified in vegetation, livestock, and wild animals, whereas Rhipicephalus sanguineus was the most prevalent in domestic dogs. Overall, C. burnetii DNA was detected in 6.1% of the processed ticks, distributed between those removed from livestock (11.3%), domestic dogs (6.9%), and from wild animals (6%). Ticks from vegetation were all negative. Results suggest that, in the Canary Islands, C. burnetii develops in a peridomestic rather than a wild cycle.
Introduction
C
Ticks play an important epidemiological role in the wild and peridomestic cycles of C. burnetii worldwide, and the bacterium has been isolated in more than 40 hard tick species and at least 14 soft tick species. Although ticks are not commonly involved in human and livestock outbreaks, they appear to play a major role in the transmission of the bacterium among other vertebrates, such as rodents, wild birds, and lagomorphs (Eldin et al. 2017). Different studies have isolated identical genotypes in humans, goats, and sheep, but not in cattle (Jado et al. 2012, Tilburg et al. 2012).
Geographically, Q fever is widely distributed throughout the world with the exception of New Zealand (OIE 2015). A significant number of human patients affected by Q fever have been reported in the Canary Islands, an archipelago of islands belonging to Spain off the northwestern coast of Africa (Bolaños et al. 2003b). Indeed, epidemiological surveys have reported an overall seroprevalence of 21.5% among the people of the Canaries, with differences between the islands (Bolaños et al. 2003a). To estimate the link between human and animal infection, other studies involving goats have shown an overall seroprevalence of 42% on the Canary Archipelago (Tejedor-Junco et al. 2016), with 60.4% detected on the island of Gran Canaria (Rodríguez et al. 2010). As a result, the Archipelago has been considered a Q fever-endemic region and several control programs have been proposed to contain the disease.
Nevertheless, the natural cycle of C. burnetii in domestic ruminant flocks and on the outside is currently unknown and its epidemiological importance should, therefore, be evaluated.
The aim of this study was to investigate the peridomestic and wild cycle of C. burnetii in the Canary Islands, where it is endemic. If significant, this information will be taken into consideration in future control measures to be implemented in the region.
Materials and Methods
Geographical area
Gran Canaria was selected as the most representative of the islands in the Canary Archipelago because most of the people affected by Q fever live on this island and also because it harbors the largest livestock population. The wild fauna on the island is mainly made up of small mammals, rabbits, birds, and reptiles. It should be pointed out that apart from small wild mammals, such as hedgehogs, shrews, and bats, rabbits are the only other wild mammals present in the Canary Archipelago. Ticks and animals were collected at 190 different sites, located mainly in the central and southeastern areas of Gran Canaria.
Peridomestic and wild animals under study
Animal and tick samples were collected at different sites across Gran Canaria between February 2010 and December 2011. After first obtaining permission from the relevant authorities, traps (Sherman and INRA traps) for small and large rodents were set on ruminant farms previously identified as positive for Q fever (Rodriguez et al. 2010), whereas larger cages, specifically designed to catch lagomorphs were placed and set at different sites in the countryside. Trapped animals were immediately transported to the laboratory and examined for ticks. Blood samples were taken under isoflurane anesthesia (only in rodents) and the animals were finally euthanized with an anesthesia overdose. A complete necropsy was performed and tissue samples (spleen and/or liver) were stored at −20°C until they were ready for PCR processing.
Tick collection and identification
To identify the main tick species present on the island, 1738 ticks were collected from different scenarios. Sampling sites were selected to take into account geographical, climatic, and ecosystem factors: (1) 1169 questing ticks were collected from vegetation at 124 points across the island using the blanket-dragging method (Sonenshine, 1993); (2) 335 feeding ticks from ruminants on 10 farms previously identified as positive for Q fever (Rodriguez et al. 2010); (3) 65 feeding ticks were collected from wild animals (13 rabbits, 24 hedgehogs, 28 birds). The animals were obtained either from specific traps or through the Wildlife Rehabilitation Center of Gran Canaria; and (4) 169 feeding ticks were obtained from domestic dogs attending several small veterinary clinics.
All ticks were identified in the laboratory using taxonomic keys (Gil-Collado et al. 1979, Manilla 1998) and were stored at −80°C until required for DNA purification.
DNA extraction and polymerase chain reaction assays
Briefly, genomic DNA was individually extracted from tissue samples (spleen and/or liver) of small rodents and rabbits and adult ticks, using the QIAamp DNA Mini Kit for extraction (Qiagen, Hilden, Germany), following the manufacturer's instructions. About 100–300 ng of DNA was amplified by PCR for the detection of C. burnetii (containing the transposase for insertion element IS1111) (Denison et al. 2007), using previously described PCR protocols (Berri et al. 2000). To prevent false-positives due to cross-contamination, sterile filter tips were used, PCR reactions and post-PCR analysis were performed in separate rooms, and a negative (water control) was used in each run.
Ethical isssues
The animals were managed in accordance with the International Guiding Principles for Biomedical Research Involving Animals, issued by the Council for the International Organizations of Medical Sciences.
Results
C. burnetii DNA in rodent and rabbit tissues
A total of 100 rodents (24 Mus musculus, 61 Rattus norvegicus, and 15 Rattus rattus) and 129 rabbits (Oryctolagus cuniculus) were captured during the study. Blood and tissue samples (spleen and/or liver) were tested by specific PCR assay. C. burnetii DNA was detected in eight (8%) rodents (all belonging to the species Rattus norvegicus); seven were from the spleen and just one from the liver. In the rabbits, two (1.5%) samples were positive by PCR, both from spleen tissue. Blood samples from the rodents and liver tissue samples from the rabbits were negative for C. burnetii.
Tick identification
Table 1 gives a detailed description of the number of ticks according to the various scenarios in which they were collected. Four species were identified: Hyalomma lusitanicum (80.4%), Rhipicephalus turanicus (8%), Rhipicephalus sanguineus (6%), and Rhipicephalus pusillus (5.6%). Globally, Hyalomma lusitanicum was the main species identified in vegetation, livestock, and wild animals, whereas Rhipicephalus sanguineus was the main tick species in domestic animals.
C. burnetii DNA in ticks
Results of C. burnetii genetic material detected in ticks are shown in Table 2. The overall prevalence of infected ticks was 6.1% (23/377), but ranged from 0% (0/133) for ticks collected from vegetation to 11.3% (17/151) for those taken from livestock. The prevalence in ticks obtained from domestic dogs was 6.9% (3/43) and in ticks from wild animals, 6% (3/50; 2 hedgehogs, 1 bird [Burhinus oedicnemus]). With respect to tick species, C. burnetii DNA was detected in 13 R. turanicus (11 from livestock, 2 from dogs), 9 H. lusitanicum (6 from livestock, 3 from wild animals), and 1 R. sanguineus (a dog). All specimens of R. pusillus were found to be negative.
Sixteen sheep, one pig.
Two Atelerix algirus, one Burhinus oedicnemus.
Discussion
In Spain, there is scant information about the role of small mammals and questing and feeding ticks in the C. burnetii wild cycle, and in the Canary Islands in particular, there has been none at all. The aim of our study was to clarify and add new information about the role of ticks in peridomestic and wild animals, and to improve our understanding of the natural cycle of C. burnetii in the Canary Islands.
The first observation of interest in our study was the difference between tick species observed on Gran Canaria and those identified in different areas of Spain, and even other European countries. The most frequently identified tick species in our study, of the four different species collected, was Hyalomma lusitanicum, representing 80% of all the specimens collected, followed by Rhipicephallus turanicus (8%). These results, however, vary according to the geographical area studied (Merino et al. 2005, Barandika et al. 2011)
Peridomestic rodents and wild rabbit species have commonly been involved in the natural cycle of C. burnetii. In a previous study, rodents carrying antibodies against C. burnetii were found on all the Canary Islands, except Tenerife, with a mean prevalence of 10.2% (Foronda et al. 2015). In our study involving peridomestic rodents captured on affected ruminant farms, C. burnetii DNA was detected in 8% (8/100), all Rattus norvegicus, and mainly in spleen tissue (in seven of eight cases), but not in blood samples taken from small mammals. The most likely transmission route, therefore, would be from infected ruminants to rodents.
In a study of a Q fever outbreak in humans and small ruminants in The Netherlands at the end of the last decade, 15.8% of brown rats (Rattus norvegicus), tested positive after serologic testing, and C. burnetii DNA was found in the internal organs (particularly the spleen) of 4.9% and 3% of brown (Rattus norvegicus) and black rats (Rattus rattus), respectively. Infected rodents were found all year round, leading to the conclusion that these species were not only spillover hosts, but could also serve as a reservoir capable of spreading and transmitting the pathogen (Reusken et al. 2011). These findings mean that peridomestic rodents must be taken into account to control the disease on farms. In support of this hypothesis, a study carried out in Germany that evaluated 119 rodents in several regions, where Q fever was endemic, although not infected farms, found no C. burnetii DNA in any of them (Pluta et al. 2010).
The presence of C. burnetii in rabbit tissue was lower than in peridomestic rodents (1.5% vs. 8%, respectively). In Europe, the C. burnetii genotypes detected in wild animals, wild rabbits, and Rattus rattus are related to those that infect human beings and livestock, according to multiple-locus variable number tandem repeat analysis (MLVA-6-marker) (González-Barrio et al. 2016). In the United States, high seroprevalence has been reported in wild rabbits (Enright et al. 1971), which would be of epidemiological importance since human Q fever infection has been reported following contact with rabbits (Marrie et al. 1986, González-Barrio et al. 2015). Rural areas contaminated with feces of C. burnetii-positive livestock as they graze in the fields could become a source of the infection of rabbits on the Island. C. burnetii DNA was detected in two spleen samples, confirming that, although limited, they can act as reservoirs for the bacterium.
With respect to ticks and their potential role in the natural cycle of C. burnetii, a significant percentage of collected ticks (23) were infected with this bacterium (6.1%); specifically, it was detected in 13 Rhipicephalus turanicus (56.5%), 9 H. lusitanicum (39.2%), and 1 R. sanguineus, which is not surprising because more than 40 tick species are naturally infected with C. burnetii and are able to transmit it by both vertical and horizontal routes (Lang 1990). All the positive ticks were obtained from animals, and all ticks collected from vegetation were negative. Seventeen were removed from livestock, three from domestic dogs, and three from wild animals, specifically two hedgehogs and one bird (Burhinus oedicnemus).
Livestock and, to a lesser extent, domestic dogs are commonly involved in the Q fever cycle and fed ticks are expected to yield positive results, although birds and hedgehogs are rarely reported. These latter species commonly live and feed in close proximity to livestock and may constitute a transmission route. In a study carried out in Japan, 167 (19%) of 863 wild birds were found positive, showing a tendency for high prevalence among birds living and/or feeding close to infected herds (To et al. 1998).
These results suggest that, in the Canary Islands, C. burnetii develops in a peridomestic rather than a wild cycle and is associated with infected flocks and ticks. Nevertheless, the limitations of this study mean that it it necessary to develop further studies to compare all sequences obtained and to characterize the genotypes of the strains found (Jado et al. 2012).
In conclusion, several animals and tick species collected close to livestock are potential sources for infection in the Canary Islands. These species are abundant in the region and could represent a risk to human health as well as to other domestic and wild animals. The peridomestic cycle of C. burnetii should be taken into account when a control program is implemented on ruminant farms in the Canary Islands.
Footnotes
Acknowledgments
This work was supported by funding from INIA, FAU2006-00002-C04-01. The authors would like to thank Pino Sosa for his laboratory assistance, Rubén Hernández and Antonio Cilleros for their contribution to this study, and Ms. Janet Dawson for her help in revising the English version of the article.
Author Disclosure Statement
No competing financial interests exist.