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Identification and genetic characterization of Piroplasmida and Anaplasmataceae agents in feeding Amblyomma variegatum ticks from Benin
Veterinary Parasitology: Regional Studies and Reports 14 (2018) 137–143
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Original article
Identification and genetic characterization of Piroplasmida and Anaplasmataceae agents in feeding Amblyomma variegatum ticks from Benin
T
Paul Franck Adjou Moumounia,b, Huanping Guoa, Yang Gaoa, Mingming Liua, Aaron Edmond Ringoa, Eloiza May Galona, Patrick Vudrikoa,b, Rika Umemiya-Shirafujia,b, ⁎ Noboru Inouec, Hiroshi Suzukia,b, Xuenan Xuana,b, a
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-855, Japan Research Center for Global Agromedicine, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-855, Japan c Obihiro University of Agriculture and Veterinary Medicine, Hokkaido 080-855, Obihiro, Japan b
A R T I C LE I N FO
A B S T R A C T
Keywords: Tick-borne pathogens Amblyomma variegatum Cattle Benin
In many African countries including Benin, the reluctance of some livestock owners to blood collection from their cattle makes epidemiological surveys cumbersome and prevents regular monitoring of tick-borne diseases. In the present study, Amblyomma variegatum ticks were used to find out more about bovine tick-borne pathogens. DNA extracts from 910 adult ticks collected off cattle in North East Benin were examined for Babesia bigemina, B. bovis, Theileria taurotragi, T. annulata, T. orientalis, T. parva, T. mutans, Anaplasma marginale and Ehrlichia ruminantium using pathogen-specific PCR assays and sequence analyses. Altogether, 21.6% of the ticks carried at least one pathogen. A. marginale (142/910) was the most frequent pathogen, followed by E. ruminantium (57/910), B. bovis (10/910), T. mutans (3/910) and B. bigemina (1/910). Theileria taurotragi, T. annulata, T. orientalis, T. parva were not detected in the samples. Babesia bigemina, B. bovis and T. mutans were present in only one location whereas A. marginale and E. ruminantium were found in ticks from 7/8 locations surveyed. Coinfections occurred in 7.1% of all positive ticks. The analyses of partial sequences of B. bovis spherical body protein 4, B. bigemina rhoptry-associated protein-1a, T. mutans 18S rRNA and A. marginale major surface protein 5 showed high sequence conservation and homologies between Benin isolates and those from other African countries. However, E. ruminantium pCS20 partial sequences were different from published West African isolates and presented similar genetic variation with South and East African isolates. These results provide information on the pathogens circulating in North East Benin and suggest that Am. variegatum, one of the most abundant ticks in Africa, may play a role in the transmission of A. marginale.
1. Introduction Babesia bovis and B. bigemina, Theileria spp. (protozoa, order Piroplasmida), Anaplasma marginale and Ehrlichia ruminantium (bacteria, Anaplasmataceae family) are the causative agents of babesiosis, theileriosis, anaplasmosis, and heartwater, respectively. These tickborne diseases (TBD) are frequent in Africa and cause great losses in the cattle industry (Bock et al., 2004; Allsopp, 2010; Kocan et al., 2010; Kalume et al., 2011). Presently, safe and fully effective vaccines against these diseases are not available (Kocan et al., 2010; Kalume et al., 2011; Suarez and Noh, 2011; Gohil et al., 2013). Efforts to mitigate disease impact mainly consist of tick control and treatments which require regular monitoring of disease epidemiology. Although sensitive and
specific methods for detection and characterization are available, the epidemiology of tick-borne pathogens in Africa is still not well understood. In many African countries including Benin, epidemiological studies are hindered by the reluctance of livestock owners to blood collection from their cattle. Ticks are obligate hematophagous arthropods, which means that all life stages ingest blood meals. They may carry vectored pathogens as well as those collected during feeding and therefore are useful material for the identification of circulating pathogens. Following the advent of molecular techniques, several methods for DNA extraction have been developed, and a wide range of sensitive PCR assays can now identify pathogens in ticks (Sparagano et al., 1999). Consequently, ticks are increasingly used to elucidate the epidemiology of tick-borne pathogens
⁎ Corresponding author at: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan. E-mail address: [email protected] (X. Xuan).
https://doi.org/10.1016/j.vprsr.2018.10.006 Received 6 February 2018; Received in revised form 15 October 2018; Accepted 26 October 2018 Available online 29 October 2018 2405-9390/ © 2018 Elsevier B.V. All rights reserved.
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district, North East Benin (Fig. 1). Prior to DNA extraction, tick species identification and morphological classification were carried out using a binocular microscope (Olympus SZX16, Japan) with standard taxonomic keys established by Walker et al. (2003). After DNA extraction (Adjou Moumouni et al., 2016), tick species were confirmed by molecular techniques (PCR, sequencing and BLASTn analysis). Briefly, the primer pair NS1 (5’-GTAGTCATATGCTTGTCTC-3′) and NS2 (5’-GGCT GCTGGCACCAGACTTGC -3′) were used to amplify a 550 bp fragment of tick 18S nuclear rDNA gene (Black et al., 1997) in 10 randomly selected DNA extracts. The thermocycling conditions consisted of an initial denaturation (5 min, 94 °C) followed by 30 cycles of denaturation (30 s, 94 °C) annealing (45 s, 52 °C) and extension (1 min, 72 °C) then a final extension step (7 min, 72 °C). Both microscopic examination and BLASTn analysis of amplicon sequences (GenBank accession number: MH706981) identified the tick samples as Am. variegatum.
in various parts of the world including African countries (Adham et al., 2009; Ogo et al., 2012; Reye et al., 2012; Nakayima et al., 2014). Ticks, compared to animal blood, are easy to obtain and represent an interesting alternative to overcome the difficulties of blood sampling. In Benin, however, despite a diverse tick fauna including around 17 cattletick species (Vercruysse et al., 1982; De Clercq et al., 2012), ticks have rarely been exploited to collect information on bovine pathogens. Therefore, this study was carried out with the aim of using ticks to know more about bovine tick-borne pathogens and their transmission cycles in Benin. Tick vectors of bovine pathogens that are reported in the country include Rhipicephalus microplus (B. bovis, B. bigemina and A. marginale), R. annulatus (B. bovis, B. bigemina and A. marginale), R. decoloratus (B. bigemina and A. marginale), R. geigyi (B. bovis and B. bigemina), Hyalomma rufipes (A. marginale) and Amblyomma variegatum (T. mutans and E. ruminantium) (Biguezoton et al., 2016; De Clercq et al., 2012; Vercruysse et al., 1982). Here, Am. variegatum (Fabricius, 1794), a three-host tick and one of the most abundant and widely distributed cattle-tick in the country (Farougou et al., 2007a; Farougou et al., 2007b; Biguezoton et al., 2016) was used as a template. Nine hundred and ten (910) ticks collected from cattle in North East Benin were analyzed for B. bovis, B. bigemina, Theileria spp., A. marginale and E. ruminantium using species-specific PCR assays and sequencing.
2.2. Detection of microbial DNAs The samples were screened for the presence of piroplasmid and Anaplasmataceae DNA using pathogen-specific nested PCR assays. The following genetic markers were employed: B. bovis spherical body protein 4 (SBP-4), B. bigemina rhoptry-associated protein-1a (RAP-1a) (Terkawi et al., 2011), Theileria mutans 18S rRNA, T. taurotragi 18S rRNA, T. parva 18S rRNA (Simuunza et al., 2011), T. annulata major merozoite surface antigen (D'Oliveira et al., 1995; Martín-Sánchez et al., 1999), T. orientalis major piroplasm surface protein (Ota et al., 2009), A. marginale major surface protein 5 (Msp5) (Ybañez et al., 2013) and E. ruminantium pCS20 (Molia et al., 2008). PCR screening of each tick DNA sample was carried out in a final total volume of 13 μl which was composed of 2 μl of DNA template, 1 μl (10 μM) of each primers, 5 μl of 2× Ampidirect plus (Shimadzu, Japan), 0.075 μl of Ex
2. Materials and methods 2.1. Tick DNA samples The study samples consisted of DNA extracts of 910 adult Am. variegatum (789 males and 121 females). These ticks were collected from cattle in traditional farms in 8 different locations (Beterou, Suya, Tasso, Nikki centre, Sekere, Sikki, Dunkassa and Bouka) of Borgou
Fig. 1. Map of Borgou district showing tick collection sites. The ticks were collected in 8 locations covering 4 different divisions (Tchaourou, Nikki, Sinende, Kalale) of Borgou district in North East Benin. The black stars indicate tick collection sites and the numbers between brackets represent the number of ticks collected in each area. This map was adapted from Adjou Moumouni et al., 2016. 138
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Table 1 Results of Anaplasma marginale and Ehrlichia ruminantium detection in Amblyomma variegatum ticks collected in Borgou district, Benin. Samples
A. marginale positive ticks a
Location
No. of ticks
Beterou Suya Tasso Nikki centre Sekere Sikki Dunkassa Bouka Total
28 (13/15) 16 (13/3) 200 (169/31) 82 (71/11) 213 (195/18) 87 (71/16) 127 (106/21) 157 (151/6) 910 (789/121)
a
E. ruminantium positive ticks
Male (%)
Female (%)
Total (%)
Male (%)
Female (%)
Total (%)
1 (7.7) 0 64 (37.9) 9 (12.7) 21 (10.8) 7 (9.9) 6 (5.7) 17 (11.3) 125 (15.8)
1 (6.7) 0 9 (29) 1 (9.1) 1 (5.6) 3 (18.8) 1 (4.8) 1 (16.7) 17 (14)
2 (7.1) 0 73 (36.5) 10 (12.2) 22 (10.3) 10 (11.5) 7 (5.5) 18 (11.5) 142 (15.6)
1 (7.7) 0 8 (4.7) 5 (7) 15 (7.7) 8 (11.3) 5 (4.7) 10 (6.6) 52 (6.6)
1 0 2 0 0 1 0 1 5
2 (7.1) 0 10 (5) 5 (6.1) 15 (7) 9 (10.3) 5 (3.9) 11 (7) 57 (6.3)
(6.7) (6.5)
(6.3) (16.7) (4.1)
Total number of ticks collected (Male/Female).
Taq polymerase (Takara, Japan) and 3.925 μl of distilled water. DNA of B. bigemina (Argentina strain), B. bovis (Texas strain), T. parva (Muguga G6, ILRI), T. annulata (Ankara C9, Edinburgh University), E. ruminantium (Welgevonden strain) and cattle DNA samples positive for T. orientalis, T. mutans, T. taurotragi and A. marginale (Adjou Moumouni et al., 2015) were used as positive controls and double-distilled water as negative control. Amplicons were visualized under UV transilluminator using 2% FastGene agarose gel (Nippon Genetics, Japan) after electrophoresis.
INFO™ software (CDC, USA, version 7.1.1). Statistically significant differences were determined at p < 0.05. 3. Results 3.1. Piroplasmid infections Among the 910 ticks that were analyzed, 197 (21.6%) were found to carry at least one bovine pathogen. Piroplasms were rarely found in the ticks (11/910). All the samples were negative for T. parva, T. taurotragi, T. annulata and T. orientalis. Meanwhile, B. bigemina, B. bovis and T. mutans were only detected in samples from 1/8 locations. Babesia bigemina was identified in a single tick collected in Nikki centre. Analysis of RAP-1a partial sequence showed that the B. bigemina in this study (KU042084; 411 bp) is 100% identical to the pathogen detected in cattle in the same area (KX685379, KX685381), Indonesia (KY484520), Syria (AB617643) and Thailand (AB594817). Babesia bovis was found in 10 (5 males and 5 females) ticks from Beterou. The two B. bovis SBP-4 sequences obtained (KU042085, KU042086; 521 bp) shared 99.8% identity with each other and were 100% and 99% identical to an isolate previously recovered from cattle in the same area (KX685398) and in Kenya (KP347555). Meanwhile, T. mutans was present in 3 (2 males and 1 female) ticks from Beterou and its 18S rRNA partial sequence (MF159031; 220 bp) was 100% identical to isolates from Benin (KX685406), Uganda (KU206320), Mozambique (FJ869899) and Kenya (AF078815).
2.3. Cloning and sequencing Amplicons were purified from agarose gel using the QIAquick Gel Extraction Kit (QIAGEN GmbH, Germany) and sequenced as previously described (Adjou Moumouni et al., 2015). Cloning in pGEM-T Easy Vector (Promega, USA) and sequencing with vector primers were done to amplicon sequences that were not long enough for genetic characterization. Dye Terminator Cycle Sequencing Kit (Applied Biosystems, USA) and ABI PRISM 3100 genetic analyzer (Applied Biosystems, USA) were used in all sequencing assays. All sequences obtained in this study are available in GenBank of the NCBI database under the accession numbers: KU042079, KU042084 to KU042086, and MF159030 to MF159037. 2.4. Sequence analysis Recovered sequences were analyzed using the BLASTn tool of NCBI and sequence comparison was performed using the pairwise alignment of EMBOSS NEEDLE software (http://www.bioinformatics.nl/cgi-bin/ emboss/needle). Phylogenetic analyses were done in MEGA version 7 (Kumar et al., 2016) using Muscle algorithm and the maximum likelihood method.
3.2. Anaplasmataceae infections Infection with bacteria of the Anaplasmataceae family was frequent in the samples (189/910) and was found in 7/8 study areas (Table 1). A. marginale was detected in 142 ticks (15.6%) whereas E. ruminantium was detected in 57 ticks (6.3%). No statistical difference was observed in the infection rates between male and female ticks. Genetic characterization revealed that A. marginale Msp5 sequences were similar across the study areas and shared 100% identity with
2.5. Statistical analysis The distribution of detected pathogens was analyzed using EPI
Table 2 BLASTn results for Anaplasmataceae DNA sequences recovered from Amblyomma variegatum ticks collected in Borgou district, Benin. DNA sequences
Highest blastn match
Pathogen
Target gene
GenBank ID
Length (bp)
GenBank ID (origin)
% Identity
A. marginale
Msp5
E. ruminantium
pCS20
KU042079 MF159030 MF159032 MF159033 MF159034 MF159035 MF159036 MF159037
469 555 278 278 278 278 278 278
KX685369 (Benin); KU042083/KU042081(Egypt) CP006847 (Australia) KP347554 (Kenya); KU042080 (Egypt) AY236070 (South Africa) AY236070 (South Africa) KX373601 (Mozambique); AB218277 (Sudan) AY236061 (Guadeloup, French west indies) AY236059/ AY236058 (South Africa) AY236065 (Sankat strain, Ghana) AY236065 (Sankat strain, Ghana)
100 100 98 99 99 99 99 99
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Fig. 2. Phylogenetic tree showing the relationship between Benin Ehrlichia ruminantium and reference stocks based on partial pCS20 region nucleic acid sequences. The tree was constructed in MEGA ver.7 with the maximum likelihood method and using Tamura 3-parameter with Invariant sites (T92+ I) model. Ehrlichia chaffeensis was used as an outgroup. Sequences determined in this study are shown in bold font. Numbers on internodes indicate percentages of 1000 bootstrap replicates. The scale bars indicate the estimated number of substitutions per site.
GenBank sequences (Table 2). E. ruminantium pCS20, however, showed high genetic diversity. Six nucleotide sequences with pairwise identities ranging from 90.3% to 99.3% were obtained. Single nucleotide polymorphisms (SNPs) were observed in all the sequences. Some of the SNPs were synonymous mutations while others were non-synonymous. Similarity among deduced amino acid sequences showed that the six genotypes form three groups, hereafter referred to as A (MF159032 and MF159033), B (MF159034 and MF159035) and C (MF159036 and MF159037). B sequences were found in six areas (Beterou, Nikki centre, Tasso, Sekere, Bouka and Dunkassa) whereas A and C sequences were retrieved from two (Tasso and Dunkassa) and four areas (Tasso, Sekere, Sikki, Bouka), respectively. None of the E. ruminantium sequences of this study was 100% identical to any registered sequences in the GenBank data (Table 2) and therefore a phylogenetic tree was constructed. The pCS20 phylogenetic tree separated the E. ruminantium isolates into three major clades (Fig. 2). Benin E. ruminantium sequences were found in all the clades. B sequences were together with isolates from the Caribbean, Sudan, and Southern Africa (Clade 1) while C sequences formed a clade with West African (Senegal, Ghana, Mali) and South Africa Kumm1 isolates (Clade 2). Meanwhile, A sequences were found related to South Africa Kumm2 isolate (Clade 3).
Table 3 Frequency of single and co-infections with bovine tick-borne pathogens in Amblyomma variegatum ticks collected in Borgou district, Benin. No. of positive ticksa Nature of infection Single infections B. bigemina B. bovis A. marginale E. ruminantium Co-infections B. bovis + T. mutans B. bovis + E. ruminantium A. marginale + E. ruminantium B. bovis + T. mutans + A. marginale B. bovis + T. mutans + E. ruminantium Total a
Male (%)
Female (%)
Total (%)
1 (0.1) 2 (0.3) 115 (14.6) 42 (5.3)
0 4 (3.3) 16 (13.2) 3 (2.5)
1 (0.1) 6 (0.7) 131 (14.4) 45 (4.9)
1 (0.1) 1 (0.1) 9 (1.1) 1 (0.1) 0 172 (21.8)
0 0 1 (0.8) 0 1 (0.8) 25 (20.7)
1 (0.1) 1 (0.1) 10 (1.1) 1 (0.1) 1 (0.1) 197 (21.6)
The total number of ticks tested is 910 (789 Male/121Female).
4. Discussion In the present study, adult Am. variegatum obtained from cattle in North East Benin were molecularly screened in order to identify circulating bovine pathogens and contribute to a better understanding of the impact of the tick on TBD transmission. Although all life stages of Am. variegatum parasitize cattle (Barré and Uilenberg, 2010), adult ticks were targeted because they have had more blood meals and therefore are more likely to be carrying pathogens transstadially maintained or acquired from their hosts. Our current findings provide insight into the tick-borne pathogens circulating in 4 different divisions of Borgou district (Tchaourou, Nikki,
3.3. Coinfections Coinfection was present in 7.1% (14/197) of positive ticks and in 4/ 8 study areas. A. marginale + E. ruminantium was the most frequent (10/14) combination and was observed in 2, 5, and 3 ticks from Tasso, Sekere and Bouka, respectively. The other coinfections were B. bovis + T. mutans, B. bovis + E. ruminantium, B. bovis + T. mutans + A. marginale, and B. bovis + T. mutans + E. ruminantium and were found in ticks from Beterou (Table 3). 140
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Fig. 3. Map of Borgou district with the distribution of bovine tick-borne pathogens in Tchaourou, Nikki, Kalale and Sinende divisions. Pathogens identified in ticks (this study) and in cattle (Adjou Moumouni et al., 2018) are presented. The black stars indicate tick collection sites. Colours indicate pathogen species, and the circle and triangle indicate whether pathogens were molecularly detected in ticks or in cattle blood samples.
isolates previously obtained in cattle from the same area are another proof of the relatedness of pathogens found in Am. variegatum ticks and in cattle. Although our results proved that feeding Am. variegatum was a valid alternative for investigating the presence of Babesia spp. in 2 study areas, the sampling strategy prevented us from ascertaining the occurrence of the protozoa in other areas. The fact that A. marginale-positive ticks were in all but one study area indicates that A. marginale infection is common among Benin cattle. Accordingly, high rates of infection with A. marginale have been reported in cattle in Borgou district (Adjou Moumouni et al., 2018; Pangui and Salifou, 1992; Farougou et al., 2007b). Tick species that are biological vectors of A. marginale might be responsible for the maintenance of the bacteria and its subsequent presence in blood meals. However, this study is not the first to report the presence of A. marginale in Amblyomma ticks naturally feeding on livestock. In Madagascar (Pothmann et al., 2016), Gambia (Faburay et al., 2007) and Ethiopia (Teshale et al., 2015), A. marginale was found in feeding Am. variegatum. Adult Am. gemma (Fyumagwa et al., 2009) in Tanzania and several life stages of Am. cajennense and Am. maculatum (da Silva et al., 2015) in Brazil were also reported to harbour A. marginale. In reports where several ticks species were investigated, Amblyomma ticks generally showed lower infection rates than biological vectors (Fyumagwa et al., 2009; Ogo et al., 2012; Reye et al., 2012; da Silva et al., 2015; Ehounoud et al., 2016). Amblyomma spp. may not have the vector competence of already described tick vectors but the premise that they may play a role in the transmission of A. marginale cannot be discarded. Studies evaluating the potential of laboratory-reared Amblyomma ticks in transmitting A. marginale may be needed to clarify the epidemiology of anaplasmosis in Africa. The detection of T. mutans and E. ruminantium in the samples relates
Kalale and Sinende; Fig.1). The occurrence of B. bovis and T. mutans in Beterou, B. bigemina in Nikki centre, and A. marginale in Beterou, Nikki centre and Tasso corroborates a previous report of these pathogens in cattle from Tchaourou and Nikki divisions (Adjou Moumouni et al., 2018). To the best of our knowledge, the presence of A. marginale in Kalale and Sinende divisions has never been investigated. Hence, the A. marginale-positive ticks observed in Dunkassa, Bouka, Sekere and Sikki are the first confirmation of the circulation of this important rickettsia in the aforementioned divisions. Likewise, E. ruminantium presence in Tchaourou, Nikki, Kalale and Sinende divisions is described for the first time. The data reported here extend the known distribution of tickborne pathogens in Borgou district (Fig.3). This study found that Am. variegatum infection rates varied depending on the pathogen and the sampling site. Several factors may explain the observed pathogen distribution. Am. variegatum has not been described as a vector or reservoir for B. bigemina, B. bovis and A. marginale (Bock et al., 2004; Jongejan and Uilenberg, 2004; Kocan et al., 2010); therefore, these pathogens may have originated from the blood meal ingested prior to collection. Factors such as the prevalence of Babesia spp. and A. marginale infections in cattle in the sampling areas, parasitemia in cattle from which the ticks were collected and the state of engorgement of the ticks are determinant of tick infection rates. The overall Babesia spp. infection rate is in agreement with the report of Ogo et al. (2012) which indicated low infection rates for B. bigemina among feeding Am. variegatum in Nigeria. The previous study in Borgou district showed that B. bovis infection was frequent among cattle from Tchaourou while B. bigemina had a high prevalence in cattle from Nikki (Adjou Moumouni et al., 2018). This may explain why B. bovis and B. bigemina positive ticks were found in samples from these two areas. The identities of the B. bovis SBP-4 and B. bigemina RAP-1a sequences to 141
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different E. ruminantium genotypes, a phenomenon previously described (Allsopp and Allsopp, 2007). The fact that two Benin genotypes shared similarity with Kumm2, an isolate known to grow only on sheep cells (Allsopp, 2010), suggests that they may have originated from sheep, which in Benin are always herded along with cattle. Although current results shed some light on E. ruminantium genetic diversity in Benin, the study of core-function genes such as 16S rRNA, gltA, groEL, ftsZ, sodB, nuoB is required for complete characterization and pathogenicity evaluation.
to the role of the tick in the life cycle of these pathogens. Two factors may explain the low T. mutans infection rate: the parasitemia level of the cattle which the ticks were feeding on, and interactions between T. mutans and Am. variegatum tissues. Ticks do not transovarially transmit Theileria species (Bishop et al., 2004). For adult ticks to be infected, they should feed on an infected host during their nymphal stages or acquire the pathogen during their blood meal. However, not all parasites in the blood meal will survive and only a few will stay in the midgut (Bishop et al., 2004). Previous studies showed high prevalence of T. mutans infection in cattle from Bourgou district (Adjou Moumouni et al., 2018; Pangui and Salifou, 1992; Farougou et al., 2007b). The data in this study, therefore indicate that most of the ticks were feeding on uninfected or low parasitemia animals. The T. mutans prevalence here is similar to reports in Ethiopia (Hornok et al., 2014) and Nigeria (Ogo et al., 2012; Reye et al., 2012). The identification of T. mutans in ticks from Beterou but not in the other areas may indicate that at the sampling time, cattle from that area had higher infection rates. Am. variegatum ticks transstadially maintain E. ruminantium, making their infection rates highly dependent on pathogen prevalence among the hosts they feed on (Allsopp, 2010). In addition, ticks that fed on infected hosts during larval or nymphal stage remain infected for life (Camus and Barré, 1992). Hence, the overall infection rate in this study (57/910) indicates that many of the ticks fed on non-susceptible or noninfected hosts during their life cycle. The low prevalence of E. ruminantium infection in cattle from Borgou district (Adjou Moumouni et al., 2018) may also explain our results. The range of infection rates obtained (0% -10.3%) is similar to values recorded in North East and Central Benin (Farougou et al., 1998; Farougou et al., 2012), Nigeria (Ogo et al., 2012), Senegal (Gueye et al., 1993), Ivory Coast (Ehounoud et al., 2016), Burkina Faso (Adakal et al., 2010), Gambia (Faburay et al., 2007), Sudan (Muramatsu et al., 2005) and Ethiopia (Teshale et al., 2015). However, higher infection rates were reported in North West Benin (Farougou et al., 2012) and Cameroon (Esemu et al., 2013). Sampling areas and season-based variations may explain the difference between our results and the other studies. Here, ticks were collected in May–June (Adjou Moumouni et al., 2016) which is the beginning of the rainy season and the peak of adult Am. variegatum abundance in North East Benin (Farougou et al., 2007a) whereas the samples analyzed in North West Benin were obtained in the middle of rainy season and dry season (Farougou et al., 2012). The sampling design may have also affected our results. Areas with a higher number of tick samples presented more positive and it is therefore likely that the low tick number (16) may have reduced the likelihood of detecting the pathogen in Suya (Table 1). The present findings indicate that Benin cattle are at risk of contracting heartwater. Studies relating seasonal variations in Am. variegatum abundance, activity and infection rates will help in better understanding the epidemiology of E. ruminantium in Benin. The polymorphism of E. ruminantium pCS20 sequences indicates the presence of several strains in the country. The presence of SNPs was previously reported among South and East African isolates (Allsopp and Allsopp, 2007) but this is the first report of SNPs in West Africa. A previous study reported that the pCS20 sequences from West Africa isolates were all identical to one isolate from South Africa (Allsopp et al., 2003). However, in this study, none of the isolates was 100% identical to the previously registered West African isolates. This is confirmed by the phylogenetic tree (Fig. 2) which showed that sequences from Benin isolates were found in all three clades previously described for E. ruminantium stocks (Van Heerden et al., 2004). The pCS20 is a genomic region well known to provide sensitive and specific detection of E. ruminantium (Allsopp et al., 2003; Allsopp, 2010) and the use of degenerate primers (Molia et al., 2008) has certainly contributed to unravelling the genetic diversity of the bacteria. The presence of multiple genotypes in Benin may be a consequence of uncontrolled animal movement and trade. Benin frequently hosts transhumant herds and serves as transit point for cattle from West and Central African countries. This may have led to genetic recombination between
5. Conclusion The present study reports the identification and genetic characterization of tick-borne protozoa and bacteria in feeding Am. variegatum ticks from Benin. Several pathogens that are threats to animal health were found prevalent and their sequence homology with registered isolates from Benin and other countries was elucidated. These findings show that feeding Am. variegatum are useful for identification of circulating pathogens. However, extending tick sampling to other species will certainly increase the efficiency of ticks as templates for pathogen detection. Hence, animal health workers and farmers should be trained to identify and collect ticks. The results of this study affirm the need to raise the awareness of Benin veterinarians on the potential of tick-borne pathogens to affect animal health and productivity. Conflict of interest None of the authors of this work has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. Ethical statement Tick sampling as well as sample processing were carried out according to ethical guidelines permitted by Obihiro University of Agriculture and Veterinary Medicine (Permit for DNA experiment: 1219-3; Pathogen: 201210-5). Acknowledgements The authors would like to thank Dr. Nakao Ryo from Hokkaido University, Japan for supplying E. ruminantium (Welgevonden strain) DNA sample. We also acknowledge Dr. MAMA SAMBO Adamou, coordinator of APIDev-ONG, Benin, Mr. Dadidje Y. Francois and cattle owners for facilitating sample collection. This study was supported by a Grant-in-Aid for Scientific Research from Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan, and a Grant from Japan Society for the Promotion of Science (JSPS) Core-to-Core Program, Japan. The first author was supported by a research grant fellowship from JSPS for Young Scientists, Japan. References Adakal, H., Gavotte, L., Stachurski, F., Konkobo, M., Henri, H., Zoungrana, S., Huber, K., Vachiery, N., Martinez, D., Morand, S., Frutos, R., 2010. Clonal origin of emerging populations of Ehrlichia ruminantium in Burkina Faso. Infect. Genet. Evol. 10, 903–912. Adham, F.K., Abd-El-Samie, E.M., Gabre, R.M., El Hussein, H., 2009. Detection of tick blood parasites in Egypt using PCR assay I-Babesia bovis and Babesia bigemina. Parasitol. Res. 105, 721–730. Adjou Moumouni, P.F., Aboge, G.O., Terkawi, M.A., Masatani, T., Cao, S., Kamyingkird, K., Jirapattharasate, C., Zhou, M., Wang, G., Liu, M., Iguchi, A., Vudriko, P., Ybanez, A.P., Inokuma, H., Shirafuji-Umemiya, R., Suzuki, H., Xuan, X., 2015. Molecular detection and characterization of Babesia bovis, Babesia bigemina, Theileria species and Anaplasma marginale isolated from cattle in Kenya. Parasit. Vectors 8, 496. Adjou Moumouni, P.F., Terkawi, M.A., Jirapattharasate, C., Cao, S., Liu, M., Nakao, R., Umemiya-Shirafuji, R., Yokoyama, N., Sugimoto, C., Fujisaki, K., Suzuki, H., Xuan, X., 2016. Molecular detection of spotted fever group rickettsiae in Amblyomma variegatum ticks from Benin. Ticks Tick. Borne. Dis. 7, 828–833.
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Adjou Moumouni, P.F., Aplogan, G.L., Katahira, H., Gao, Y., Guo, H., Efstratiou, A., Jirapattharasate, C., Wang, G., Liu, M., Ringo, E.A., Umemiya-Shirafuji, R., Suzuki, H., Xuan, X., 2018. Prevalence, risk factors, and genetic diversity of veterinary important tick-borne pathogens in cattle from Rhipicephalus microplus -invaded and noninvaded areas of Benin. Ticks Tick. Borne. Dis. 9, 450–464. Allsopp, B.A., 2010. Natural history of Ehrlichia ruminantium. Vet. Parasitol. 167, 123–135. Allsopp, M.T.E.P., Allsopp, B.A., 2007. Extensive genetic recombination occurs in the field between different genotypes of Ehrlichia ruminantium. Vet. Microbiol. 124, 58–65. Allsopp, M.T.E.P., Van Heerden, H., Steyn, H.C., Allsopp, B.A., 2003. Phylogenetic relationships among Ehrlichia ruminantium isolates. Ann. N. Y. Acad. Sci. 990, 685–691. Barré, N., Uilenberg, G., 2010. Propagation de parasites transportés avec leurs hôtes: cas exemplaires de deux espèces de tiques du bétail. Rev. Sci. Tech. l'OIE 29, 135–147. Biguezoton, A., Adehan, S., Adakal, H., Zoungrana, S., Farougou, S., Chevillon, C., 2016. Community structure, seasonal variations and interactions between native and invasive cattle tick species in Benin and Burkina Faso. Parasit. Vectors 9, 43. Bishop, R., Musoke, A., Morzaria, S., Gardner, M., Nene, V., 2004. Theileria: intracellular protozoan parasites of wild and domestic ruminants transmitted by ixodid ticks. Parasitology 129 (Suppl), S271–S283. Black, W.C., Klompen, J.S., Keirans, J.E., 1997. Phylogenetic relationships among tick subfamilies (Ixodida: Ixodidae: Argasidae) based on the 18S nuclear rDNA gene. Mol. Phylogenet. Evol. 7, 129–144. Bock, R., Jackson, L., de Vos, A., Jorgensen, W., 2004. Babesiosis of cattle. Parasitology 129 (Suppl), S247–S269. Camus, E., Barré, N., 1992. The role of Amblyomma variegatum in the transmission of heartwater with special reference to Guadeloupe. Ann. N. Y. Acad. Sci. 653, 33–41. da Silva, J.B., da Fonseca, A.H., Barbosa, J.D., 2015. Molecular characterization of Anaplasma marginale in ticks naturally feeding on buffaloes. Infect. Genet. Evol. 35, 38–41. De Clercq, E.M., Vanwambeke, S.O., Sungirai, M., Adehan, S., Lokossou, R., Madder, M., 2012. Geographic distribution of the invasive cattle tick Rhipicephalus microplus, a country-wide survey in Benin. Exp. Appl. Acarol. 58, 441–452. D'Oliveira, C., Weide, M., van der Habela, M.A., Jacquiet, P., Jongejan, F., 1995. Detection of Theileria annulata in blood samples of carrier cattle by PCR. J. Clin. Microbiol. 33, 2665–2669. Ehounoud, C.B., Yao, K.P., Dahmani, M., Achi, Y.L., Amanzougaghene, N., Kacou N'Douba, A., N'Guessan, J.D., Raoult, D., Fenollar, F., Mediannikov, O., 2016. Multiple Pathogens Including potential New Species in Tick Vectors in Côte d'Ivoire. PLoS Negl. Trop. Dis. 10, 1–18. Esemu, S.N., Besong, W.O., Ndip, R.N., Ndip, L.M., 2013. Prevalence of Ehrlichia ruminantium in adult Amblyomma variegatum collected from cattle in Cameroon. Exp. Appl. Acarol. 59, 377–387. Faburay, B., Geysen, D., Munstermann, S., Taoufik, A., Postigo, M., Jongejan, F., 2007. Molecular detection of Ehrlichia ruminantium infection in Amblyomma variegatum ticks in the Gambia. Exp. Appl. Acarol. 42, 61–74. Farougou, S., Toguebaye, B.S., Tounkara, K., Sy, D., Akakpo, A.J., 1998. Epidemiology of heartwater in Bénin. 1 - preliminary study of the germ presence in tick vectors. Rev. Med. Vet. 149, 953–958. Farougou, S., Kpodekon, M., Tassou, A.W., 2007a. Seasonal abundance of ticks (Acari: Ixodidae) infesting cattle in the Sudan area of Benin: case of the departments of Borgou and Alibori. Rev. Afr. Santé Prod. Anim. 5, 61–67. Farougou, S., Tassou, A.W., Tchabode, D.M., Kpodekon, M., Boko, C., Youssao, A.K.I., 2007b. Tiques et hémoparasites du bétail dans le Nord-Bénin. Rev. Med. Vet. 158, 463–467. Farougou, S., Adakal, H., Biguezoton, A.S., Boko, C., 2012. Prévalence de l’ infection d’ Amblyomma variegatum par Ehrlichia ruminantium dans les élevages extensifs du Bénin. Rev. Med. Vet. 163, 261–266. Fyumagwa, R.D., Simmler, P., Meli, M.L., Hoare, R., Hofmann-Lehmann, R., Lutz, H., 2009. Prevalence of Anaplasma marginale in different tick species from Ngorongoro Crater, Tanzania. Vet. Parasitol. 161, 154–157. Gohil, S., Herrmann, S., Günther, S., Cooke, B.M., 2013. Bovine babesiosis in the 21st century: advances in biology and functional genomics. Int. J. Parasitol. 43, 125–132. Gueye, A., Mbengue, M., Diouf, A., 1993. Epidemiologie de la cowdriose au Senegal. 1. Etude de la transmission et du taux d'infection d'Amblyomma variegatum (Fabricius, 1794) dans la region des Niayes. Rev. Elev. Med. Vet. Pays Trop. 46, 441–447. Hornok, S., Abichu, G., Meli, M.L., Tánczos, B., Sulyok, K.M., Gyuranecz, M., Gönczi, E.,
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Original article
Identification and genetic characterization of Piroplasmida and Anaplasmataceae agents in feeding Amblyomma variegatum ticks from Benin
T
Paul Franck Adjou Moumounia,b, Huanping Guoa, Yang Gaoa, Mingming Liua, Aaron Edmond Ringoa, Eloiza May Galona, Patrick Vudrikoa,b, Rika Umemiya-Shirafujia,b, ⁎ Noboru Inouec, Hiroshi Suzukia,b, Xuenan Xuana,b, a
National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-855, Japan Research Center for Global Agromedicine, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080-855, Japan c Obihiro University of Agriculture and Veterinary Medicine, Hokkaido 080-855, Obihiro, Japan b
A R T I C LE I N FO
A B S T R A C T
Keywords: Tick-borne pathogens Amblyomma variegatum Cattle Benin
In many African countries including Benin, the reluctance of some livestock owners to blood collection from their cattle makes epidemiological surveys cumbersome and prevents regular monitoring of tick-borne diseases. In the present study, Amblyomma variegatum ticks were used to find out more about bovine tick-borne pathogens. DNA extracts from 910 adult ticks collected off cattle in North East Benin were examined for Babesia bigemina, B. bovis, Theileria taurotragi, T. annulata, T. orientalis, T. parva, T. mutans, Anaplasma marginale and Ehrlichia ruminantium using pathogen-specific PCR assays and sequence analyses. Altogether, 21.6% of the ticks carried at least one pathogen. A. marginale (142/910) was the most frequent pathogen, followed by E. ruminantium (57/910), B. bovis (10/910), T. mutans (3/910) and B. bigemina (1/910). Theileria taurotragi, T. annulata, T. orientalis, T. parva were not detected in the samples. Babesia bigemina, B. bovis and T. mutans were present in only one location whereas A. marginale and E. ruminantium were found in ticks from 7/8 locations surveyed. Coinfections occurred in 7.1% of all positive ticks. The analyses of partial sequences of B. bovis spherical body protein 4, B. bigemina rhoptry-associated protein-1a, T. mutans 18S rRNA and A. marginale major surface protein 5 showed high sequence conservation and homologies between Benin isolates and those from other African countries. However, E. ruminantium pCS20 partial sequences were different from published West African isolates and presented similar genetic variation with South and East African isolates. These results provide information on the pathogens circulating in North East Benin and suggest that Am. variegatum, one of the most abundant ticks in Africa, may play a role in the transmission of A. marginale.
1. Introduction Babesia bovis and B. bigemina, Theileria spp. (protozoa, order Piroplasmida), Anaplasma marginale and Ehrlichia ruminantium (bacteria, Anaplasmataceae family) are the causative agents of babesiosis, theileriosis, anaplasmosis, and heartwater, respectively. These tickborne diseases (TBD) are frequent in Africa and cause great losses in the cattle industry (Bock et al., 2004; Allsopp, 2010; Kocan et al., 2010; Kalume et al., 2011). Presently, safe and fully effective vaccines against these diseases are not available (Kocan et al., 2010; Kalume et al., 2011; Suarez and Noh, 2011; Gohil et al., 2013). Efforts to mitigate disease impact mainly consist of tick control and treatments which require regular monitoring of disease epidemiology. Although sensitive and
specific methods for detection and characterization are available, the epidemiology of tick-borne pathogens in Africa is still not well understood. In many African countries including Benin, epidemiological studies are hindered by the reluctance of livestock owners to blood collection from their cattle. Ticks are obligate hematophagous arthropods, which means that all life stages ingest blood meals. They may carry vectored pathogens as well as those collected during feeding and therefore are useful material for the identification of circulating pathogens. Following the advent of molecular techniques, several methods for DNA extraction have been developed, and a wide range of sensitive PCR assays can now identify pathogens in ticks (Sparagano et al., 1999). Consequently, ticks are increasingly used to elucidate the epidemiology of tick-borne pathogens
⁎ Corresponding author at: National Research Center for Protozoan Diseases, Obihiro University of Agriculture and Veterinary Medicine, Inada-cho, Obihiro, Hokkaido 080-8555, Japan. E-mail address: [email protected] (X. Xuan).
https://doi.org/10.1016/j.vprsr.2018.10.006 Received 6 February 2018; Received in revised form 15 October 2018; Accepted 26 October 2018 Available online 29 October 2018 2405-9390/ © 2018 Elsevier B.V. All rights reserved.
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district, North East Benin (Fig. 1). Prior to DNA extraction, tick species identification and morphological classification were carried out using a binocular microscope (Olympus SZX16, Japan) with standard taxonomic keys established by Walker et al. (2003). After DNA extraction (Adjou Moumouni et al., 2016), tick species were confirmed by molecular techniques (PCR, sequencing and BLASTn analysis). Briefly, the primer pair NS1 (5’-GTAGTCATATGCTTGTCTC-3′) and NS2 (5’-GGCT GCTGGCACCAGACTTGC -3′) were used to amplify a 550 bp fragment of tick 18S nuclear rDNA gene (Black et al., 1997) in 10 randomly selected DNA extracts. The thermocycling conditions consisted of an initial denaturation (5 min, 94 °C) followed by 30 cycles of denaturation (30 s, 94 °C) annealing (45 s, 52 °C) and extension (1 min, 72 °C) then a final extension step (7 min, 72 °C). Both microscopic examination and BLASTn analysis of amplicon sequences (GenBank accession number: MH706981) identified the tick samples as Am. variegatum.
in various parts of the world including African countries (Adham et al., 2009; Ogo et al., 2012; Reye et al., 2012; Nakayima et al., 2014). Ticks, compared to animal blood, are easy to obtain and represent an interesting alternative to overcome the difficulties of blood sampling. In Benin, however, despite a diverse tick fauna including around 17 cattletick species (Vercruysse et al., 1982; De Clercq et al., 2012), ticks have rarely been exploited to collect information on bovine pathogens. Therefore, this study was carried out with the aim of using ticks to know more about bovine tick-borne pathogens and their transmission cycles in Benin. Tick vectors of bovine pathogens that are reported in the country include Rhipicephalus microplus (B. bovis, B. bigemina and A. marginale), R. annulatus (B. bovis, B. bigemina and A. marginale), R. decoloratus (B. bigemina and A. marginale), R. geigyi (B. bovis and B. bigemina), Hyalomma rufipes (A. marginale) and Amblyomma variegatum (T. mutans and E. ruminantium) (Biguezoton et al., 2016; De Clercq et al., 2012; Vercruysse et al., 1982). Here, Am. variegatum (Fabricius, 1794), a three-host tick and one of the most abundant and widely distributed cattle-tick in the country (Farougou et al., 2007a; Farougou et al., 2007b; Biguezoton et al., 2016) was used as a template. Nine hundred and ten (910) ticks collected from cattle in North East Benin were analyzed for B. bovis, B. bigemina, Theileria spp., A. marginale and E. ruminantium using species-specific PCR assays and sequencing.
2.2. Detection of microbial DNAs The samples were screened for the presence of piroplasmid and Anaplasmataceae DNA using pathogen-specific nested PCR assays. The following genetic markers were employed: B. bovis spherical body protein 4 (SBP-4), B. bigemina rhoptry-associated protein-1a (RAP-1a) (Terkawi et al., 2011), Theileria mutans 18S rRNA, T. taurotragi 18S rRNA, T. parva 18S rRNA (Simuunza et al., 2011), T. annulata major merozoite surface antigen (D'Oliveira et al., 1995; Martín-Sánchez et al., 1999), T. orientalis major piroplasm surface protein (Ota et al., 2009), A. marginale major surface protein 5 (Msp5) (Ybañez et al., 2013) and E. ruminantium pCS20 (Molia et al., 2008). PCR screening of each tick DNA sample was carried out in a final total volume of 13 μl which was composed of 2 μl of DNA template, 1 μl (10 μM) of each primers, 5 μl of 2× Ampidirect plus (Shimadzu, Japan), 0.075 μl of Ex
2. Materials and methods 2.1. Tick DNA samples The study samples consisted of DNA extracts of 910 adult Am. variegatum (789 males and 121 females). These ticks were collected from cattle in traditional farms in 8 different locations (Beterou, Suya, Tasso, Nikki centre, Sekere, Sikki, Dunkassa and Bouka) of Borgou
Fig. 1. Map of Borgou district showing tick collection sites. The ticks were collected in 8 locations covering 4 different divisions (Tchaourou, Nikki, Sinende, Kalale) of Borgou district in North East Benin. The black stars indicate tick collection sites and the numbers between brackets represent the number of ticks collected in each area. This map was adapted from Adjou Moumouni et al., 2016. 138
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Table 1 Results of Anaplasma marginale and Ehrlichia ruminantium detection in Amblyomma variegatum ticks collected in Borgou district, Benin. Samples
A. marginale positive ticks a
Location
No. of ticks
Beterou Suya Tasso Nikki centre Sekere Sikki Dunkassa Bouka Total
28 (13/15) 16 (13/3) 200 (169/31) 82 (71/11) 213 (195/18) 87 (71/16) 127 (106/21) 157 (151/6) 910 (789/121)
a
E. ruminantium positive ticks
Male (%)
Female (%)
Total (%)
Male (%)
Female (%)
Total (%)
1 (7.7) 0 64 (37.9) 9 (12.7) 21 (10.8) 7 (9.9) 6 (5.7) 17 (11.3) 125 (15.8)
1 (6.7) 0 9 (29) 1 (9.1) 1 (5.6) 3 (18.8) 1 (4.8) 1 (16.7) 17 (14)
2 (7.1) 0 73 (36.5) 10 (12.2) 22 (10.3) 10 (11.5) 7 (5.5) 18 (11.5) 142 (15.6)
1 (7.7) 0 8 (4.7) 5 (7) 15 (7.7) 8 (11.3) 5 (4.7) 10 (6.6) 52 (6.6)
1 0 2 0 0 1 0 1 5
2 (7.1) 0 10 (5) 5 (6.1) 15 (7) 9 (10.3) 5 (3.9) 11 (7) 57 (6.3)
(6.7) (6.5)
(6.3) (16.7) (4.1)
Total number of ticks collected (Male/Female).
Taq polymerase (Takara, Japan) and 3.925 μl of distilled water. DNA of B. bigemina (Argentina strain), B. bovis (Texas strain), T. parva (Muguga G6, ILRI), T. annulata (Ankara C9, Edinburgh University), E. ruminantium (Welgevonden strain) and cattle DNA samples positive for T. orientalis, T. mutans, T. taurotragi and A. marginale (Adjou Moumouni et al., 2015) were used as positive controls and double-distilled water as negative control. Amplicons were visualized under UV transilluminator using 2% FastGene agarose gel (Nippon Genetics, Japan) after electrophoresis.
INFO™ software (CDC, USA, version 7.1.1). Statistically significant differences were determined at p < 0.05. 3. Results 3.1. Piroplasmid infections Among the 910 ticks that were analyzed, 197 (21.6%) were found to carry at least one bovine pathogen. Piroplasms were rarely found in the ticks (11/910). All the samples were negative for T. parva, T. taurotragi, T. annulata and T. orientalis. Meanwhile, B. bigemina, B. bovis and T. mutans were only detected in samples from 1/8 locations. Babesia bigemina was identified in a single tick collected in Nikki centre. Analysis of RAP-1a partial sequence showed that the B. bigemina in this study (KU042084; 411 bp) is 100% identical to the pathogen detected in cattle in the same area (KX685379, KX685381), Indonesia (KY484520), Syria (AB617643) and Thailand (AB594817). Babesia bovis was found in 10 (5 males and 5 females) ticks from Beterou. The two B. bovis SBP-4 sequences obtained (KU042085, KU042086; 521 bp) shared 99.8% identity with each other and were 100% and 99% identical to an isolate previously recovered from cattle in the same area (KX685398) and in Kenya (KP347555). Meanwhile, T. mutans was present in 3 (2 males and 1 female) ticks from Beterou and its 18S rRNA partial sequence (MF159031; 220 bp) was 100% identical to isolates from Benin (KX685406), Uganda (KU206320), Mozambique (FJ869899) and Kenya (AF078815).
2.3. Cloning and sequencing Amplicons were purified from agarose gel using the QIAquick Gel Extraction Kit (QIAGEN GmbH, Germany) and sequenced as previously described (Adjou Moumouni et al., 2015). Cloning in pGEM-T Easy Vector (Promega, USA) and sequencing with vector primers were done to amplicon sequences that were not long enough for genetic characterization. Dye Terminator Cycle Sequencing Kit (Applied Biosystems, USA) and ABI PRISM 3100 genetic analyzer (Applied Biosystems, USA) were used in all sequencing assays. All sequences obtained in this study are available in GenBank of the NCBI database under the accession numbers: KU042079, KU042084 to KU042086, and MF159030 to MF159037. 2.4. Sequence analysis Recovered sequences were analyzed using the BLASTn tool of NCBI and sequence comparison was performed using the pairwise alignment of EMBOSS NEEDLE software (http://www.bioinformatics.nl/cgi-bin/ emboss/needle). Phylogenetic analyses were done in MEGA version 7 (Kumar et al., 2016) using Muscle algorithm and the maximum likelihood method.
3.2. Anaplasmataceae infections Infection with bacteria of the Anaplasmataceae family was frequent in the samples (189/910) and was found in 7/8 study areas (Table 1). A. marginale was detected in 142 ticks (15.6%) whereas E. ruminantium was detected in 57 ticks (6.3%). No statistical difference was observed in the infection rates between male and female ticks. Genetic characterization revealed that A. marginale Msp5 sequences were similar across the study areas and shared 100% identity with
2.5. Statistical analysis The distribution of detected pathogens was analyzed using EPI
Table 2 BLASTn results for Anaplasmataceae DNA sequences recovered from Amblyomma variegatum ticks collected in Borgou district, Benin. DNA sequences
Highest blastn match
Pathogen
Target gene
GenBank ID
Length (bp)
GenBank ID (origin)
% Identity
A. marginale
Msp5
E. ruminantium
pCS20
KU042079 MF159030 MF159032 MF159033 MF159034 MF159035 MF159036 MF159037
469 555 278 278 278 278 278 278
KX685369 (Benin); KU042083/KU042081(Egypt) CP006847 (Australia) KP347554 (Kenya); KU042080 (Egypt) AY236070 (South Africa) AY236070 (South Africa) KX373601 (Mozambique); AB218277 (Sudan) AY236061 (Guadeloup, French west indies) AY236059/ AY236058 (South Africa) AY236065 (Sankat strain, Ghana) AY236065 (Sankat strain, Ghana)
100 100 98 99 99 99 99 99
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Fig. 2. Phylogenetic tree showing the relationship between Benin Ehrlichia ruminantium and reference stocks based on partial pCS20 region nucleic acid sequences. The tree was constructed in MEGA ver.7 with the maximum likelihood method and using Tamura 3-parameter with Invariant sites (T92+ I) model. Ehrlichia chaffeensis was used as an outgroup. Sequences determined in this study are shown in bold font. Numbers on internodes indicate percentages of 1000 bootstrap replicates. The scale bars indicate the estimated number of substitutions per site.
GenBank sequences (Table 2). E. ruminantium pCS20, however, showed high genetic diversity. Six nucleotide sequences with pairwise identities ranging from 90.3% to 99.3% were obtained. Single nucleotide polymorphisms (SNPs) were observed in all the sequences. Some of the SNPs were synonymous mutations while others were non-synonymous. Similarity among deduced amino acid sequences showed that the six genotypes form three groups, hereafter referred to as A (MF159032 and MF159033), B (MF159034 and MF159035) and C (MF159036 and MF159037). B sequences were found in six areas (Beterou, Nikki centre, Tasso, Sekere, Bouka and Dunkassa) whereas A and C sequences were retrieved from two (Tasso and Dunkassa) and four areas (Tasso, Sekere, Sikki, Bouka), respectively. None of the E. ruminantium sequences of this study was 100% identical to any registered sequences in the GenBank data (Table 2) and therefore a phylogenetic tree was constructed. The pCS20 phylogenetic tree separated the E. ruminantium isolates into three major clades (Fig. 2). Benin E. ruminantium sequences were found in all the clades. B sequences were together with isolates from the Caribbean, Sudan, and Southern Africa (Clade 1) while C sequences formed a clade with West African (Senegal, Ghana, Mali) and South Africa Kumm1 isolates (Clade 2). Meanwhile, A sequences were found related to South Africa Kumm2 isolate (Clade 3).
Table 3 Frequency of single and co-infections with bovine tick-borne pathogens in Amblyomma variegatum ticks collected in Borgou district, Benin. No. of positive ticksa Nature of infection Single infections B. bigemina B. bovis A. marginale E. ruminantium Co-infections B. bovis + T. mutans B. bovis + E. ruminantium A. marginale + E. ruminantium B. bovis + T. mutans + A. marginale B. bovis + T. mutans + E. ruminantium Total a
Male (%)
Female (%)
Total (%)
1 (0.1) 2 (0.3) 115 (14.6) 42 (5.3)
0 4 (3.3) 16 (13.2) 3 (2.5)
1 (0.1) 6 (0.7) 131 (14.4) 45 (4.9)
1 (0.1) 1 (0.1) 9 (1.1) 1 (0.1) 0 172 (21.8)
0 0 1 (0.8) 0 1 (0.8) 25 (20.7)
1 (0.1) 1 (0.1) 10 (1.1) 1 (0.1) 1 (0.1) 197 (21.6)
The total number of ticks tested is 910 (789 Male/121Female).
4. Discussion In the present study, adult Am. variegatum obtained from cattle in North East Benin were molecularly screened in order to identify circulating bovine pathogens and contribute to a better understanding of the impact of the tick on TBD transmission. Although all life stages of Am. variegatum parasitize cattle (Barré and Uilenberg, 2010), adult ticks were targeted because they have had more blood meals and therefore are more likely to be carrying pathogens transstadially maintained or acquired from their hosts. Our current findings provide insight into the tick-borne pathogens circulating in 4 different divisions of Borgou district (Tchaourou, Nikki,
3.3. Coinfections Coinfection was present in 7.1% (14/197) of positive ticks and in 4/ 8 study areas. A. marginale + E. ruminantium was the most frequent (10/14) combination and was observed in 2, 5, and 3 ticks from Tasso, Sekere and Bouka, respectively. The other coinfections were B. bovis + T. mutans, B. bovis + E. ruminantium, B. bovis + T. mutans + A. marginale, and B. bovis + T. mutans + E. ruminantium and were found in ticks from Beterou (Table 3). 140
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Fig. 3. Map of Borgou district with the distribution of bovine tick-borne pathogens in Tchaourou, Nikki, Kalale and Sinende divisions. Pathogens identified in ticks (this study) and in cattle (Adjou Moumouni et al., 2018) are presented. The black stars indicate tick collection sites. Colours indicate pathogen species, and the circle and triangle indicate whether pathogens were molecularly detected in ticks or in cattle blood samples.
isolates previously obtained in cattle from the same area are another proof of the relatedness of pathogens found in Am. variegatum ticks and in cattle. Although our results proved that feeding Am. variegatum was a valid alternative for investigating the presence of Babesia spp. in 2 study areas, the sampling strategy prevented us from ascertaining the occurrence of the protozoa in other areas. The fact that A. marginale-positive ticks were in all but one study area indicates that A. marginale infection is common among Benin cattle. Accordingly, high rates of infection with A. marginale have been reported in cattle in Borgou district (Adjou Moumouni et al., 2018; Pangui and Salifou, 1992; Farougou et al., 2007b). Tick species that are biological vectors of A. marginale might be responsible for the maintenance of the bacteria and its subsequent presence in blood meals. However, this study is not the first to report the presence of A. marginale in Amblyomma ticks naturally feeding on livestock. In Madagascar (Pothmann et al., 2016), Gambia (Faburay et al., 2007) and Ethiopia (Teshale et al., 2015), A. marginale was found in feeding Am. variegatum. Adult Am. gemma (Fyumagwa et al., 2009) in Tanzania and several life stages of Am. cajennense and Am. maculatum (da Silva et al., 2015) in Brazil were also reported to harbour A. marginale. In reports where several ticks species were investigated, Amblyomma ticks generally showed lower infection rates than biological vectors (Fyumagwa et al., 2009; Ogo et al., 2012; Reye et al., 2012; da Silva et al., 2015; Ehounoud et al., 2016). Amblyomma spp. may not have the vector competence of already described tick vectors but the premise that they may play a role in the transmission of A. marginale cannot be discarded. Studies evaluating the potential of laboratory-reared Amblyomma ticks in transmitting A. marginale may be needed to clarify the epidemiology of anaplasmosis in Africa. The detection of T. mutans and E. ruminantium in the samples relates
Kalale and Sinende; Fig.1). The occurrence of B. bovis and T. mutans in Beterou, B. bigemina in Nikki centre, and A. marginale in Beterou, Nikki centre and Tasso corroborates a previous report of these pathogens in cattle from Tchaourou and Nikki divisions (Adjou Moumouni et al., 2018). To the best of our knowledge, the presence of A. marginale in Kalale and Sinende divisions has never been investigated. Hence, the A. marginale-positive ticks observed in Dunkassa, Bouka, Sekere and Sikki are the first confirmation of the circulation of this important rickettsia in the aforementioned divisions. Likewise, E. ruminantium presence in Tchaourou, Nikki, Kalale and Sinende divisions is described for the first time. The data reported here extend the known distribution of tickborne pathogens in Borgou district (Fig.3). This study found that Am. variegatum infection rates varied depending on the pathogen and the sampling site. Several factors may explain the observed pathogen distribution. Am. variegatum has not been described as a vector or reservoir for B. bigemina, B. bovis and A. marginale (Bock et al., 2004; Jongejan and Uilenberg, 2004; Kocan et al., 2010); therefore, these pathogens may have originated from the blood meal ingested prior to collection. Factors such as the prevalence of Babesia spp. and A. marginale infections in cattle in the sampling areas, parasitemia in cattle from which the ticks were collected and the state of engorgement of the ticks are determinant of tick infection rates. The overall Babesia spp. infection rate is in agreement with the report of Ogo et al. (2012) which indicated low infection rates for B. bigemina among feeding Am. variegatum in Nigeria. The previous study in Borgou district showed that B. bovis infection was frequent among cattle from Tchaourou while B. bigemina had a high prevalence in cattle from Nikki (Adjou Moumouni et al., 2018). This may explain why B. bovis and B. bigemina positive ticks were found in samples from these two areas. The identities of the B. bovis SBP-4 and B. bigemina RAP-1a sequences to 141
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different E. ruminantium genotypes, a phenomenon previously described (Allsopp and Allsopp, 2007). The fact that two Benin genotypes shared similarity with Kumm2, an isolate known to grow only on sheep cells (Allsopp, 2010), suggests that they may have originated from sheep, which in Benin are always herded along with cattle. Although current results shed some light on E. ruminantium genetic diversity in Benin, the study of core-function genes such as 16S rRNA, gltA, groEL, ftsZ, sodB, nuoB is required for complete characterization and pathogenicity evaluation.
to the role of the tick in the life cycle of these pathogens. Two factors may explain the low T. mutans infection rate: the parasitemia level of the cattle which the ticks were feeding on, and interactions between T. mutans and Am. variegatum tissues. Ticks do not transovarially transmit Theileria species (Bishop et al., 2004). For adult ticks to be infected, they should feed on an infected host during their nymphal stages or acquire the pathogen during their blood meal. However, not all parasites in the blood meal will survive and only a few will stay in the midgut (Bishop et al., 2004). Previous studies showed high prevalence of T. mutans infection in cattle from Bourgou district (Adjou Moumouni et al., 2018; Pangui and Salifou, 1992; Farougou et al., 2007b). The data in this study, therefore indicate that most of the ticks were feeding on uninfected or low parasitemia animals. The T. mutans prevalence here is similar to reports in Ethiopia (Hornok et al., 2014) and Nigeria (Ogo et al., 2012; Reye et al., 2012). The identification of T. mutans in ticks from Beterou but not in the other areas may indicate that at the sampling time, cattle from that area had higher infection rates. Am. variegatum ticks transstadially maintain E. ruminantium, making their infection rates highly dependent on pathogen prevalence among the hosts they feed on (Allsopp, 2010). In addition, ticks that fed on infected hosts during larval or nymphal stage remain infected for life (Camus and Barré, 1992). Hence, the overall infection rate in this study (57/910) indicates that many of the ticks fed on non-susceptible or noninfected hosts during their life cycle. The low prevalence of E. ruminantium infection in cattle from Borgou district (Adjou Moumouni et al., 2018) may also explain our results. The range of infection rates obtained (0% -10.3%) is similar to values recorded in North East and Central Benin (Farougou et al., 1998; Farougou et al., 2012), Nigeria (Ogo et al., 2012), Senegal (Gueye et al., 1993), Ivory Coast (Ehounoud et al., 2016), Burkina Faso (Adakal et al., 2010), Gambia (Faburay et al., 2007), Sudan (Muramatsu et al., 2005) and Ethiopia (Teshale et al., 2015). However, higher infection rates were reported in North West Benin (Farougou et al., 2012) and Cameroon (Esemu et al., 2013). Sampling areas and season-based variations may explain the difference between our results and the other studies. Here, ticks were collected in May–June (Adjou Moumouni et al., 2016) which is the beginning of the rainy season and the peak of adult Am. variegatum abundance in North East Benin (Farougou et al., 2007a) whereas the samples analyzed in North West Benin were obtained in the middle of rainy season and dry season (Farougou et al., 2012). The sampling design may have also affected our results. Areas with a higher number of tick samples presented more positive and it is therefore likely that the low tick number (16) may have reduced the likelihood of detecting the pathogen in Suya (Table 1). The present findings indicate that Benin cattle are at risk of contracting heartwater. Studies relating seasonal variations in Am. variegatum abundance, activity and infection rates will help in better understanding the epidemiology of E. ruminantium in Benin. The polymorphism of E. ruminantium pCS20 sequences indicates the presence of several strains in the country. The presence of SNPs was previously reported among South and East African isolates (Allsopp and Allsopp, 2007) but this is the first report of SNPs in West Africa. A previous study reported that the pCS20 sequences from West Africa isolates were all identical to one isolate from South Africa (Allsopp et al., 2003). However, in this study, none of the isolates was 100% identical to the previously registered West African isolates. This is confirmed by the phylogenetic tree (Fig. 2) which showed that sequences from Benin isolates were found in all three clades previously described for E. ruminantium stocks (Van Heerden et al., 2004). The pCS20 is a genomic region well known to provide sensitive and specific detection of E. ruminantium (Allsopp et al., 2003; Allsopp, 2010) and the use of degenerate primers (Molia et al., 2008) has certainly contributed to unravelling the genetic diversity of the bacteria. The presence of multiple genotypes in Benin may be a consequence of uncontrolled animal movement and trade. Benin frequently hosts transhumant herds and serves as transit point for cattle from West and Central African countries. This may have led to genetic recombination between
5. Conclusion The present study reports the identification and genetic characterization of tick-borne protozoa and bacteria in feeding Am. variegatum ticks from Benin. Several pathogens that are threats to animal health were found prevalent and their sequence homology with registered isolates from Benin and other countries was elucidated. These findings show that feeding Am. variegatum are useful for identification of circulating pathogens. However, extending tick sampling to other species will certainly increase the efficiency of ticks as templates for pathogen detection. Hence, animal health workers and farmers should be trained to identify and collect ticks. The results of this study affirm the need to raise the awareness of Benin veterinarians on the potential of tick-borne pathogens to affect animal health and productivity. Conflict of interest None of the authors of this work has a financial or personal relationship with other people or organizations that could inappropriately influence or bias the content of the paper. Ethical statement Tick sampling as well as sample processing were carried out according to ethical guidelines permitted by Obihiro University of Agriculture and Veterinary Medicine (Permit for DNA experiment: 1219-3; Pathogen: 201210-5). Acknowledgements The authors would like to thank Dr. Nakao Ryo from Hokkaido University, Japan for supplying E. ruminantium (Welgevonden strain) DNA sample. We also acknowledge Dr. MAMA SAMBO Adamou, coordinator of APIDev-ONG, Benin, Mr. Dadidje Y. Francois and cattle owners for facilitating sample collection. This study was supported by a Grant-in-Aid for Scientific Research from Ministry of Education, Culture, Sports, Science, and Technology (MEXT), Japan, and a Grant from Japan Society for the Promotion of Science (JSPS) Core-to-Core Program, Japan. The first author was supported by a research grant fellowship from JSPS for Young Scientists, Japan. References Adakal, H., Gavotte, L., Stachurski, F., Konkobo, M., Henri, H., Zoungrana, S., Huber, K., Vachiery, N., Martinez, D., Morand, S., Frutos, R., 2010. Clonal origin of emerging populations of Ehrlichia ruminantium in Burkina Faso. Infect. Genet. Evol. 10, 903–912. Adham, F.K., Abd-El-Samie, E.M., Gabre, R.M., El Hussein, H., 2009. Detection of tick blood parasites in Egypt using PCR assay I-Babesia bovis and Babesia bigemina. Parasitol. Res. 105, 721–730. Adjou Moumouni, P.F., Aboge, G.O., Terkawi, M.A., Masatani, T., Cao, S., Kamyingkird, K., Jirapattharasate, C., Zhou, M., Wang, G., Liu, M., Iguchi, A., Vudriko, P., Ybanez, A.P., Inokuma, H., Shirafuji-Umemiya, R., Suzuki, H., Xuan, X., 2015. Molecular detection and characterization of Babesia bovis, Babesia bigemina, Theileria species and Anaplasma marginale isolated from cattle in Kenya. Parasit. Vectors 8, 496. Adjou Moumouni, P.F., Terkawi, M.A., Jirapattharasate, C., Cao, S., Liu, M., Nakao, R., Umemiya-Shirafuji, R., Yokoyama, N., Sugimoto, C., Fujisaki, K., Suzuki, H., Xuan, X., 2016. Molecular detection of spotted fever group rickettsiae in Amblyomma variegatum ticks from Benin. Ticks Tick. Borne. Dis. 7, 828–833.
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