One particular Anaplasma phagocytophilum ecotype infects cattle in the Camargue, France
Parasites & Vectors volume 10, Article number: 371 (2017)
Anaplasma phagocytophilum is a zoonotic tick-borne pathogen responsible for granulocytic anaplasmosis, a mild to a severe febrile disease that affects man and several animal species, including cows and horses. In Europe, I. ricinus is the only proven vector for this pathogen, but studies suggest that other tick genera and species could be involved in its transmission. Our objective was to assess the presence and genetic diversity of A. phagocytophilum in domestic animals and different tick species from the Camargue region, located in the south of France.
A total of 140 ticks and blood samples from 998 cattle and 337 horses were collected in Camargue and tested for the presence of A. phagocytophilum DNA by msp2 quantitative real-time PCR. Molecular typing with four markers was performed on positive samples.
Anaplasma phagocytophilum DNA was detected in 6/993 (0.6%) cows, 1/20 (5%) Haemaphysalis punctata, 1/57 (1.75%) Rhipicephalus pusillus, and was absent in horses (0%). All cattle A. phagocytophilum presented a profile identical to an A. phagocytophilum variant previously detected in Dermacentor marginatus, Hyalomma marginatum, and Rhipicephalus spp. in Camargue.
Our results demonstrate that one particular A. phagocytophilum variant infects cattle in Camargue, where I. ricinus is supposed to be rare or even absent. Dermacentor marginatus, Rhipicephalus spp. and Hyalomma spp., and possibly other tick species could be involved in the transmission of this variant in this region.
Anaplasma phagocytophilum is a tick-borne intragranulocytic alpha-proteobacterium that can infect many mammalian species. It is known to be the causative agent of tick-borne fever (TBF) in domestic ruminants, a disease with significant economic impact in Europe, and of equine granulocytic anaplasmosis (EGA) in horses in both the USA and Europe . Anaplasma phagocytophilum also infects cats, dogs and humans and causes human granulocytic anaplasmosis (HGA), an emerging zoonotic disease in Asia, the USA, and Europe.
Anaplasma phagocytophilum epidemiological cycles are complex and involve different ecotypes, vectors, and mammalian host species. To date, these epidemiological cycles are poorly understood, especially in Europe. Known arthropod vectors of A. phagocytophilum are ticks belonging to the genus Ixodes: I. ricinus in Europe, I. scapularis in eastern USA, I. pacificus and I. spinipalpis in the western USA, and I. persulcatus in Asia and Russia . However, several studies suggest that other tick genera and species could be involved in A. phagocytophilum transmission, but their vector competence is yet to be proven . For example, I. trianguliceps could play the role of an A. phagocytophilum vector in an independent epidemiological cycle in the UK and central Europe, involving different rodent species as reservoir hosts [3, 4]. In addition, it has been suggested that Rhipicephalus spp. could be involved in A. phagocytophilum transmission in the French Camargue region, a 1500 km2 marshland area in the south of France, and from where I. ricinus is supposed to be absent [5, 6]. Here, livestock is almost exclusively constituted of a local fighting bull breed, the Camargue breed, raised in extensive systems, which exposes them to arthropod bites. It is the same for the Camargue horses, which are also bred extensively. Even though up to 26% of horses have been found seropositive for A. phagocytophilum , live bacteria or bacterial DNA have never been detected in the animals. In addition, Camargue cattle have never been investigated for A. phagocytophilum infection. For these reasons, the presence/absence and the epidemiological cycle(s) of A. phagocytophilum in Camargue should be investigated.
Our objective was to assess the presence and genetic diversity of A. phagocytophilum in domestic animals and different tick species in Camargue. To achieve this, 140 ticks, and blood samples from 998 cattle (Bos taurus) and 337 horses (Equus caballus) were collected in Camargue and tested for the presence of A. phagocytophilum DNA by quantitative real-time PCR targeting the msp2 gene. Positive samples were then molecularly typed via four sequences located within the genes ankA, pleD, msp4 and typA, among those genes used for MLST by Chastagner et al. [5, 8].
Camargue is a 1500 km2 area in the south of France, from where I. ricinus has never been collected to date. Camargue is a region of marshlands where herds are almost exclusively constituted of a local fighting bull breed and a local horse breed raised in extensive conditions. Three French administrative departments extend into this area: Bouches-du-Rhône, Gard, and Hérault.
In 2015, blood samples from 337 horses (269 located in Bouches-du-Rhône, 47 in Gard, and 21 in Hérault) were collected. Blood from horses was preferentially collected near areas where ticks positive for A. phagocytophilum were detected in previous studies . Ticks (engorged or not) feeding on horses was also collected during the same time. One-hundred and forty ticks were found on examined horses, 108 in Bouches-du-Rhône, 19 in Gard, and 13 in Hérault (Additional file 1: Table S1). Between 2013 and 2015, blood samples from 998 cattle (i.e. 10% of the 10,000 French Camargue cattle; 235 in Gard and 765 in Hérault), older than 24 months and belonging to the Camargue or Brave breeds, were collected (Fig. 1). These blood samples from cattle had been collected in a previous study unrelated to A. phagocytophilum, which explains why no tick sample was associated with these cattle samples. Moreover, collecting ticks on bovines in this region could be very hazardous as they are bred for fighting.
Ticks were morphologically identified to the species level following Pérez-Eid’s recommendations .
For DNA extraction, the NucleoSpin® Blood QuickPure kit (Macherey-Nagel, Bethlehem, USA) (blood samples and engorged ticks), or the NucleoSpin® Tissue kit (Macherey-Nagel) (non-engorged ticks) were used according to manufacturer’s instructions. DNA extracts were then stored at -20 °C prior to testing.
Quantitative real-time PCR
Anaplasma phagocytophilum DNA was detected by qPCR, targeting a 77 bp fragment of the major surface protein 2 (msp2) gene as previously described by Courtney et al. . To confirm the presence of A. phagocytophilum DNA, each msp2 qPCR-positive sample was also tested for another gene specific to A. phagocytophilum, with qPCR targeting a fragment of the ankA and groEL genes, with the primers designed by Dugat et al. .
For each sample, qPCR targeting the bovine, equine and ticks glyceraldehyde-3-phosphate dehydrogenase (gapdh) gene was also performed to demonstrate the efficiency of DNA extraction and the absence of PCR inhibitors in the sample, using the primers designed [12, 13]. All samples had to be gapdh PCR-positive to be included in subsequent analyses.
For ankA, groEL and gapdh, qPCR assays were performed using the Maxima SYBR Green qPCR Master Mix (2×) Kit (Thermo Fisher Scientific, Villebon-sur-Yvette, France) in a 25 μl total reaction volume, with Master Mix at a 1× final concentration, 0.3 μM of each primer and 5 μl of purified DNA. Negative controls were included in each run. qPCR cycling was performed on the LightCycler480 Multiwell Plate 96 system (Roche, Basel, Switzerland) as follows: 95 °C for 10 min, then 40 cycles of 10 s at 95 °C, 30 s at 60 °C and 30 s at 72 °C. The signal emitted was detected at the end of each annealing-extension step. A threshold was automatically set, and the threshold cycle value (Ct) was consequently determined. All real-time PCR reactions were performed in duplicate.
Samples positive for A. phagocytophilum were initially typed using the eight loci selected from a recently developed - MLST adapted for A. phagocytophilum [5, 8]. These loci were the following: ankA, groESL, gyrA, msp4, pleD, polA, recG, typA.
Sequencing results were analysed using Bioedit software version 7.2.5 (Ibis Biosciences, Carlsbad, USA). Each sequence was deposited in GenBank (KU859923–KU859946). Nucleotide sequences were then aligned to all the sequences available in GenBank, including those obtained by Chastagner et al.  (GenBank accession numbers: JX197073–JX197368) using the programme MEGA7 (Molecular Evolutionary Genetics Analysis Version 7.0.18) . Gene sequences were aligned using ClustalW while applying the IUB matrix.
A total of 140 ticks were collected from horses: 5 Dermacentor reticulatus (3.5%), 20 Haemaphysalis punctata (14.2%), 4 Haemaphysalis sp. (2.8%), 57 Rhipicephalus pusillus (40.4%), 19 Rhipicephalus sanguineus (13.5%), 1 Rhipicephalus turanicus (0.7%), and 34 Rhipicephalus sp. (24.2%). None of the 140 collected ticks belonged to the genus Ixodes.
Detection of A. phagocytophilum DNA
For domestic animals, 993/998 (99.5%) cow samples and 269/337 (79.8%) horse samples gave positive gapdh PCR results and were included in subsequent analyses. 6/993 (0.6%, 95% CI: 0.2–1.3%) cow samples were msp2 PCR-positive. These six positive cows belonged to the same herd, located in Hérault, and all cows had been born in this herd. No msp2 amplification was observed in any of the 269 horses tested (0%, 95% CI: 0–1.4%) (Table 1, Additional file 1: Table S1). Statistically, cattle were not more frequently infected by A. phagocytophilum than horses (Fisher’s exact test, P = 0.35).
For the tick samples, 137/140 (97%) were gapdh PCR-positive and were included in subsequent analyses. Only 2/137 (1.5%, 95% CI: 0.2–5.2%) were msp2 PCR-positive: one R. pusillus male and one H. punctata male (Table 1, Additional file 1: Table S1). These two ticks were collected from two different horse farms next to the town of Arles (Bouches-du-Rhône).
The eight msp2 PCR-positive samples were also positive for ankA and groEL qPCRs.
In the positive H. punctata and R. pusillus, DNA quantity was too low to enable complete genotyping. However, genotyping could be performed on the six positive cow samples. All eight genes included in this technique gave positive PCR results, but positive sequencing results were generated only for four loci: ankA, pleD, msp4, and typA. The sequence quality of groESL, gyrA, recG and polA loci was too poor to be analysed properly. Subsequently, too little sample DNA remained to perform a second round of genotyping. All six cow samples presented 100% identity at these four loci. The sequences of these genes were then aligned to all the sequences available on GenBank, including those obtained by Chastagner et al. . For all four loci, the six positive cow samples shared 100% identity with the single A. phagocytophilum genotype identified in 40 Rhipicephalus spp., D. marginatus and H. marginatum . However, when comparing the msp4 sequences, the Camargue genotype was located in a different cluster than A. phagocytophilum variants from cattle living in several other French areas . Moreover, a significant part of the available sequences of A. phagocytophilum from humans in different areas in the USA (HGE1, HZ, NY18 and Webster) were also located in this cluster, as well as one sequence from a horse living in the USA (Additional file 2: Table S2). Apart one sequence from a red deer in Slovenia and one sequence from a donkey in Italy, they were the only non-Camargue samples to belong to this cluster.
Anaplasma phagocytophilum can infect many mammalian species worldwide and is known to be the causative agent of TBF and EGA, two diseases with high economic impact in Europe . On this continent, I. ricinus is the main vector, and to date the only proven vector, of A. phagocytophilum. In the present study, we investigated for the first time the presence and genetic diversity of A. phagocytophilum both in ticks and domestic animals in Camargue, a 1500 km2 area in the south of France from where I. ricinus is supposed to be absent, due to unfavourable ecosystem conditions for this species.
To our knowledge, this study is the first large-scale screening of A. phagocytophilum in cattle from a particular French region and is the first to report A. phagocytophilum DNA in cows from Camargue. Six on 998 cows, which all belonged to the same herd, were found positive for A. phagocytophilum. Our results are consistent with those obtained by Torina et al.  in Sicily (5/374 cows, 1.3%, 95% CI: 0.4–3.1%), a region in which I. ricinus is rare, whereas Dermacentor marginatus, H. marginatum and Rhipicephalus spp. are commonly collected. Our results demonstrate that A. phagocytophilum infects cattle in Camargue.
However, this region is considered a I. ricinus-free area: indeed, no I. ricinus were collected in our study or during several studies conducted between 2007 and 2010 . Moreover, A. phagocytophilum DNA has already been detected in R. bursa, R. sanguineus, R. turanicus, R. pusillus, D. marginatus and H. marginatum in Camargue . Taken together, these results indicate A. phagocytophilum is most likely transmitted by the vector(s) other than I. ricinus in this region. Thus we investigated the presence of A. phagocytophilum in different tick genera and species collected in Camargue. At the level of our sampling, only two ticks were qPCR-positive: one R. pusillus male and one H. punctata male. This is the first report of A. phagocytophilum in R. pusillus, whereas H. punctata has recently been suspected to be a vector of A. phagocytophilum in Spain . This species could then also potentially transmit A. phagocytophilum to cows in Camargue. However, the high number of A. phagocytophilum-infected tick species found in Camargue from previous studies raises questions about their vector competence. Many of these ticks could have acquired A. phagocytophilum (or only its DNA) by passively feeding on an infected animal, without then being able to transmit the pathogen. Unfortunately, due to the low quantity of tick sample DNA, we were not able to determine the genotype of A. phagocytophilum present in these ticks and compare it to the genotype present in the cows typed in our study. However, it is noteworthy that the profile of the six A. phagocytophilum cow samples for the four tested genes (ankA, pleD, msp4 and typA) was identical to that of A. phagocytophilum from 40 Rhipicephalus spp., D. marginatus and H. marginatum sampled in Camargue during a previous study . This profile was shared by ticks that had been collected throughout the three French administrative departments of Camargue, which covers 1500 km2. In addition, this genotype has never been detected in ticks and animals from other regions in France. These observations strongly suggest that only one variant, which is transmitted by one or several tick species, infects cows in Camargue in an epidemiological cycle independent from I. ricinus. This “specialisation” could have led to decreased A. phagocytophilum diversity in Camargue, resulting in the circulation of this single variant.
Interestingly, the msp4 cluster to which this variant belongs also includes sequences originating from humans in the USA. It is particularly remarkable to notice that this a priori result is in complete accordance with that previously observed using MLVA, with identical profiles between the samples from Camargue and the human Webster profile, the only American human sample that could be tested by MLVA . The sequence identity, for both markers, of samples originating from regions as far apart from one another (Camargue and the USA) could be due to homoplasy.
None of the 269 studied horses was infected by A. phagocytophilum, whereas in prior studies occurring between 2001 and 2010, 8.6 to 26% of examined horses were seropositive [7, 16, 18]. In this context, the absence of any positive PCR result in horses in our study was a priori surprising as 42 out of the 337 horses tested in the present study displayed clinical signs compatible with equine granulocytic anaplasmosis. This could be explained by the fact that most of the diseased horses received imidocarb. This treatment was administered because Theileria equi infection is the main cause of fever in horses in this area. Imidocarb is also commonly used for the treatment of cattle anaplasmosis. Thus, the use of this babesicid molecule could most likely explain the negative PCR results.
Finally, the reservoir host(s) of the Camargue variant must be identified. This unique and stable variant suggests that it could be adapted to a restricted number of reservoir host(s) and/or vector(s). This variant belongs to the ankA cluster I, which mostly contains variants obtained from humans, dogs, cats, and horses, and several variants obtained from domestic and wild ruminants [5, 8]. Haemaphysalis punctata females are known to have a trophic preference for both wild and domestic ruminants, and R. pusillus for rabbits . For these reasons, wild ruminants and rabbits should be investigated as potential A. phagocytophilum reservoirs in Camargue, even if there is some doubt about the vector competence of H. punctate and R. pusillus. Furthermore, it also remains to be demonstrated that the same unique variant is also present in these ticks.
Due to these observations, prevalence studies should be continued in both ticks and domestic animals throughout the coming years in order to determinethe A. phagocytophilum vector competence of the tick species present in Camargue, the reservoir host of A. phagocytophilum in Camargue, the level of infection in cattle and horses and the clinical impact of disease in these species, and the zoonotic potential of this variant.
In conclusion, our results strongly suggest that one particular A. phagocytophilum variant infects cows in Camargue, an area where I. ricinus is supposed to be rare or even absent. A variant that presented the same profile based on our four markers was already identified in Rhipicephalus spp., D. marginatus, H. marginatum, H. punctata and R. pusillus by Chastagner et al. . These ticks could be involved in A. phagocytophilum transmission in this particular region, but additional studies are needed before confirming this theory. The vertebrate and invertebrate actors of this epidemiological cycle must now be confirmed or identified to develop appropriate surveillance measures. Finally, the zoonotic potential of this variant should also be investigated.
Equine granulocytic anaplasmosis
- gapdh :
Human granulocytic anaplasmosis
Multilocus sequence typing
Major surface protein 2
Quantitative real-time PCR
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The authors thank Valérie Poux for tick identification, Severine Barry for tick DNA extraction, Elsa Jourdain for scientific support and Christelle Gandouin, Corinne Bouillin, and Joanne Befort for their technical support. This work was performed within the Laboratory of Excellence (Labex) of Integrative Biology of Emerging Infectious Diseases (IBEID).
This work was supported by the Alfort National Veterinary School (ENVA), by the French Agency for Food, Environmental and Occupational Health and Safety (Anses) and by the French National Institute for Agricultural Research (INRA). TD was funded by Anses.
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The data supporting the conclusions of this article are included within the article and Additional file 1.
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The domestic animals used in this study met the definition of “farm animals”, which are not currently covered by French regulations (Décret 2013–118 dated the 1st February 2013, from the French Ministry of Agriculture). The owners of the animals provided permission for studies to be performed on animal samples collected during sanitary investigations. Samples were de-identified so that breeding farms remained anonymous.
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Dugat, T., Leblond, A., Keck, N. et al. One particular Anaplasma phagocytophilum ecotype infects cattle in the Camargue, France. Parasites Vectors 10, 371 (2017). https://doi.org/10.1186/s13071-017-2305-3