Denaturing Gradient Gel Electrophoresis Analysis of Bacteria in Italian Ticks and First Detection of Streptococcus equi in Rhipicephalus bursa from the Lazio Region

Abstract

Tick-borne diseases are an increasing problem for the community. Ticks harbor a complex microbial population acquired while feeding on a variety of animals. Profiling the bacterial population by 16S rDNA amplification and denaturing gradient gel electrophoresis enables detection of the broad spectrum of bacteria that settles in the ticks. This study identified known and unknown tick-infecting bacteria in samples from Italy. Seven adult ticks from different hosts and origins were analyzed: two Rhipicephalus sanguineus ticks from dogs (Lombardia), two Rhipicephalus bursa ticks from bovines (Lazio), and three Ixodes ricinus ticks from humans (Marche). The major result was the first report of the zoonotic agent Streptococcus equi in ticks. S. equi is a species complex of highly contagious pathogens. Subsequent to S. equi detection in a R. bursa tick removed from a bovine of Lazio in 2012, we studied 95 R. bursa samples collected from 3 bovines, 3 ponies, and 1 sheep grazing in the same area in 2012 and from 6 ponies grazing there in 2017. The results of a specific PCR assay indicated a not sporadic occurrence of S. equi in ticks. This finding provides a basis for assessing the potential of ticks to harbor and disperse S. equi.

Introduction

Ticks (Ixodidae) are vectors of bacteria, viruses, and protozoa that cause zoonoses. Some researchers have raised the question of whether these arthropods are implicated not only in the already known tick-borne diseases (TBDs) but also in the transmission of other pathogens. For instance, the identification of the sand-fly transmitted protozoon Leishmania infantum in the dog-tick Rhipicephalus sanguineus supports the hypothesis of ticks as potential vectors of this parasite (Dantas-Torres et al. 2011).

Regarding bacterial communities associated with arthropod vectors, several studies exploring ticks were carried out using 16S rDNA amplification and denaturing gradient gel electrophoresis (DGGE) (Rudolf et al. 2009, Tveten and Sjastad 2011, Xu et al. 2015). In the present work, we report a list of bacteria identified through DGGE: in Ixodes ricinus, Rhipicephalus bursa, and R. sanguineus ticks from Italy, several Proteobacteria (Class: Alpha, Beta and Gamma) and Firmicutes (Class: Clostridia and Bacilli) were detected: Rickettsia peacockii, Burkholderia sp., Coxiella-like endosymbiont, Xanthomonas sp., and Clostridium sp., Staphylococcus sp., Streptococcus equi., respectively.

S. equi is a serious pathogenic species complex transmissible by direct contact, which includes three subspecies: S. e. equi, S. e. zooepidemicus, and S. e. ruminatorum. S. e. equi causes strangles in equines (Neamat-Allah and Damaty 2016), whereas S. e. zooepidemicus and S. e. ruminatorum are zoonotic agents associated with horses and ruminants (Downar et al. 2001, Fernandez et al. 2004). Since to our knowledge S. equi has never been described in ticks, its identification in R. bursa was particularly interesting. Analysis of a 100 R. bursa removed from asymptomatic bovines and ponies in the Lazio Region showed a significant circulation of the bacterium in ticks. Our results provide the basis for additional studies to assess the association between ticks and S. equi, as demonstration of the tick's role in pathogen diffusion might be important for prevention of serious zoonoses.

Materials and Methods

Ticks

Seven adult ticks from different hosts and origins were analyzed through DGGE: two R. sanguineus ticks from dogs, Lombardia (Varese, 2012); two R. bursa ticks from bovines, Lazio (Roccassecca, Frosinone, 2012); and three I. ricinus ticks from humans, Marche (Macerata, 2016).

Ninety-five nymph/adult R. bursa ticks from grazing animals (13), in a rural area near the municipality of Roccasecca, were analyzed through a specific S. equi-PCR test: the 2012 collection had 1 tick from 1 sheep, 6 ticks from bovines (3), and 10 ticks from ponies (3), while the 2017 collection had 78 ticks from ponies (6). PCR screening included some adult ticks visibly engorged (fed), and nymphal or adult ticks that looked not-engorged or not-containing blood (likely, just acquired from environment/grazing).

Primary identification of the tick species was based on morphologic criteria and then confirmed through molecular analysis. All ticks analyzed in this study (102) were removed from asymptomatic hosts (dog, bovine, sheep, pony, or human). In particular, the ponies (nine) belonged to a fine local breed called “Pony of Esperia” that has its origin from mountains near Esperia (Frosinone, Lazio) and their health conditions were guaranteed by the breeder.

DNA extraction and identification of tick species

Adult ticks were removed from the hosts and stored in sterile tubes containing 70% v/v ethanol. Each tick was washed twice in 1× PBS and sterile water, and air-dried before DNA extraction. The arthropods were treated using an automatic tissue homogenizer (Bertin Technologies), and the DNAs were extracted using a JetFlex Genomic DNA Purification kit (Life Technologies) following the manufacturer's instructions and stored at −20°C. The tick species were confirmed using primers targeting a region of mitochondrial 12S rDNA and the PCR protocol described in Beati and Keirans (2001). Dream Taq (Fermentas) was used for amplifications, and reactions were performed in a Biometra ThermoCycler (Life Science). A 360 bp amplicon was purified and the sequence analyzed using BLASTN (NCBI database).

Denaturing gradient gel electrophoresis

The highly variable V3 region of the bacterial 16S rRNA gene was targeted through a nested PCR as described by Hu et al. (2013). Fifty nanogram of DNA was amplified in the first PCR step, then a dilution 1:50 was used as template in the second PCR step, using the DreamTaq (Fermentas) following the manufacturer's instructions. All the reactions were performed in a Biometra ThermoCycler (Life Science). The nested-PCR products were loaded onto a 10% (w/v) poly-acrylamide (40% acrylamide/bio-acrylamide) gel containing a linear denaturing gradient of 35% to 65%, where 100% denaturing acrylamide was defined as containing 7 M urea and 40% formamide.

DGGE were performed using The DCode™ Universal Mutation Detection System (Bio-Rad). The gel was run at 50 V for 17 h at 60°C in 1× TAE buffer (Bio-Rad), stained in 3× solution of gel Red (Life Technologies) and photographed under UV light. Dominant DGGE bands were excised from the gel and the DNA was eluted using the PCR clean-up Gel extraction Kit (Macherey Nagel). The bands were re-amplified and purified. Sequence analysis was carried out using BLASTN (NCBI database).

PCR test for the detection of S. equi subsp. in ticks

Ticks were analyzed using a multiplex PCR developed by Preziuso and Cuteri (2012). For the reaction, we used the sodA-F/sodA-R primer, which amplified a 235 bp region of the superoxide dismutase-A gene of S. equi species complex, and the seeI-F/seeI-R primer, which amplified a 520 bp region of the superantigen gene SeeI specific for S. equi subsp. equi (Alber et al. 2004). The discrimination between S. e. zooepidemicus and S. e. ruminatorum was not possible due to the high genetic identity of the subspecies.

Results

DGGE analysis

DGGE bacterial profiling of different tick species was obtained from seven samples from Italy: two R. sanguineus ticks (dogs, Varese, Lombardia) (lanes 1, 3); two R. bursa ticks (bovines, Frosinone, Lazio) (lanes 2, 4); and three I. ricunus ticks (humans, Macerata, Marche) (lanes 5, 6, 7) (Fig. 1). Fifteen amplicons were analyzed and identified as the following bacterial sequences (a–j): uncultured Staphylococcus sp. (a, i), Burkholderia sp. (b), Coxiella-like endosymbiont (c), uncultured bacterium (d), S. equi (e), uncultured Clostridiales (f, h), Xanthomonas sp. (g), and R. peacockii (j).

FIG. 1.

DGGE patterns of three tick species. Lanes 1, 3 = Rhipicephalus sanguineus; lanes 2, 4 = Rhipicephalus bursa; lanes 5, 6, 7 = Ixodes ricinus. Sequenced amplicons (dominant bands) are highlighted by black bars (a–j), the corresponding bacterial sequences: Unc. Staphylococcus sp. (a, i); Burkholderia sp. (b); Coxiella-like endosymbiont (c); Unc. bacterium (d); Streptococcus equi (e); Unc. Clostridium sp. (f); Xanthomonas sp. (g); Unc. Clostridiales (h); Rickettsia peacockii (j). DGGE, denaturing gradient gel electrophoresis.

Nine bacteria were classified as Firmicutes (5/10) of the orders Clostridiales, Bacillales and Lactobacillales, or Proteobacteria (4/10) of the classes Alpha, Beta, and Gamma; one sequence matched to an unc. bacterium (1/10) (Table 1). Members of both phyla were detected in the three tick species analyzed. All the listed bacteria were described as components of microbiota of ticks except S. equi.

Table 1.

Analysis of Bacterial Profile in Italian Ticks by Denaturing Gradient Gel Electrophoresis

Tick species	Ticks N°	Host, origin	Bacteria	Accession	% identity
Tick species	Ticks N°	Host, origin	Bacteria	number in NCBI	% identity
Rhipicephalus sanguineus	2	Dog, Varese (2012)	Burkholderia sp.^a	EU589420.1	96
			Coxiella-like endosymbiont^b	CP011126.1	98
			Unc. Staphylococcus sp.^c	KJ910364.1	99
Rhipicephalus Bursa	2	Bovine, Frosinone^d (2012)	Streptococcus equi ^e	CP001129.1	97
			Unc. Bacterium	KR514398.1	99
			Unc. Clostridium sp.^f	EF10535.1	98
			Xanthomonas sp.^b	KX518637.1	99
Ixodes ricinus	3	Human, Macerata (2016)	Rickettsia peacockii ^g	NR118837.1	99
			Unc. Clostridiales^f	HM076965.1	98
			Unc. Staphylococcus sp.^c	FJ384491.1	100

Beta-proteobacteria.

Gamma-proteobacteria.

Firmicutes, Bacillales.

The R. bursa ticks were collected from a bovine that grazed in a rural area near the municipality of Roccasecca (Frosinone, Lazio Region).

Firtmicutes, Lactobacillales.

Firmicutes, Clostridiales.

Alpha-proteobacteria.

The two R. sanguineus ticks showed similar DGGE profiles characterized by three dominant bands (a, b, and c), whose sequence analysis revealed homology to unc. Staphylococcus sp. (Bacillales), Burkholderia sp. (Beta-proteobact.), and Coxiella-like symbiont (Gamma-proteobact.).

Staphylococcus is a well-known pathogen that causes many types of diseases in humans and warm-blooded animals. It had already been reported in Rhipicephalus microplus, Ixodes sp., and Dermacentor sp. ticks (Egyed and Makrai 2014, Xu et al. 2015). Burkholderia sp. causes different diseases both in humans and animals (Boone et al. 2017), and Burkholderia cepacia had already been isolated in Ixodes holocyclus and I. ricinus (Murrell et al. 2003, Stojek and Dutkiewicz 2004). Coxiella-like symbionts were detected in different ticks including R. sanguineus (Machado-Ferreira et al. 2016), and their benefits to arthropod development and reproduction are documented (Koropatnick et al. 2004).

Similar profiles were also shown in the two I. ricinus ticks characterized by two dominant bands (h, i), whose sequence analysis revealed homology to Clostridiales and unc. Staphylococcus sp., whereas the third tick showed only one dominant band (j, profile 7, Fig. 1) identified as R. peacockii (Alpha-proteobact.). The order Clostridiales includes environmental saprophyte and human pathogens, and a recent study detected members of the order in Ixodes scapularis and Dermacentor variabilis (Clow et al. 2018). R. peacockii is an obligate intracellular bacterium replicating in arthropod cells, able to maintain its natural infection through trans-stadial and trans-ovarial transmission (Niebylski et al. 1997). It was described in R. microplus and Dermacentor andersoni ticks (Niebylski et al. 1997, Xu et al. 2015).

The two R. bursa ticks showed different bacterial profiles. In a sample, three dominant bands were identified (d–f) whose sequence analysis revealed homology to unc. bacterium, S. equi (Lactobacillales), and unc. Clostridium sp. (Clostridia). The second tick showed one dominant band (g, profile 4, Fig. 1) identified as Xanthomonas sp. (Gamma-proteobact.). The unc. bacterium showed an identity to microbes found in the reproductive tracts of bovine, thus the tick could have acquired the microbe from the host.

Remarkably, the pathogen S. equi had not been previously observed in ticks, a finding worthy of further investigation. Instead, Clostridium sp. includes pathogenic bacteria able to produce toxins that were already reported in Hyalomma marginatum and Hyalomma excavatum removed from humans (Keskin et al. 2017), and Xanthomonas sp., which is one of the most agriculturally important pathogens (Akimoto-Tomiyama et al. 2018), was already reported in I. ricinus and R. microplus (Rudolf et al. 2009, Xu et al. 2015).

Analysis of the presence of S. equi subspecies in R. bursa ticks by specific PCR

Subsequent to preliminary detection of S. equi in a R. bursa tick removed from a cow in a rural area in Frosinone (Lazio), 95 ticks collected from cattle, sheep, and ponies grazing the same area were analyzed (Table 2). The study was carried out using a specific PCR assay able to discriminate S. e. equi and S. e. zooepidemicus/ruminatorum (a high genetic identity does not allow further classification in the subspecies zooepidemicus or ruminatorum by molecular methods). Ticks positive for S. e. zooepidemicus/ruminatorum were 24.2% (41.2% and 20.5% in collections of 2012 and 2017, respectively).

Table 2.

Streptococcus equi-PCR Test in Rhipicephalus bursa from the Lazio Region (Italy)

Ticks positive ^a /total	Year of collection	Animals
3/6	2012	Bovines (three)
4/10	2012	Ponies (three)
0/1	2012	Sheep (one)
16/78	2017	Ponies (six)
23/95

Ticks positive for S. e. zooepidemicus/ruminatorum. All ticks were collected from asymptomatic hosts (13) that grazed in the same rural area near the municipality of Roccasecca (Frosinone, Lazio Region). The ponies (9) were of the breed “Pony of Esperia” and have been raised in the wild.

As expected, bacterial detection in analyzed samples was independent of host blood, in fact S. equi (zooepidemicus/ruminatorum) is considered a pathogen of reproductive and respiratory systems of horses and other animals, and it does not cause bacteremia in asymptomatic animals. Bacteremia due to S. e. zooepidemicus has been rarely reported and only in human patients with severe diseases (Poulin and Boivin 2009, Veldeman et al. 2018).

Discussion

The tick distribution in Europe has modified recently due to climatic changes, a situation that is favoring the spread of TBDs (Hai et al. 2014). Cases of emerging TB pathogens and the number of undiagnosed infectious diseases, especially in tropical regions, could suggest that there are still new unidentified pathogens associated with ticks (Mediannikov and Fenollar 2014).

Analyzing the bacterial population in ticks may provide a better understanding of the potential of ticks to spread pathogens. In this context, molecular investigations are very useful for revealing pathogens not yet identified in ticks. Several studies have been carried out through automated metagenomics methods to profile the complete bacterial communities associated with ticks. In particular, Carpi et al. (2011) provided extensive analysis of the bacterial population of I. ricinus from Italy. Despite the power of automated approaches, even recently some studies exploring the bacterial microbiota of ticks were accomplished through DGGE, demonstrating that this is a useful method for this kind of analysis (Cheng and Liu 2017).

Here, we report a first study of the bacterial community in three species of Italian ticks through DGGE. Most of the bacteria detected were already described as components of the microbiota of ticks, but in other species (Xu et al. 2015). Instead, we found for the first time Staphylococcus sp. and Burkholderia sp. in R. sanguineus; Clostridiales and R. peacockii in I. ricinus; Clostridium sp. and Xanthomonas sp. in R. bursa; and S. e. zooepidemicus/ruminatorum in ticks. Positivity for S. e. zooepidemicus/ruminatorum was detected in about 1/4 of the R. bursa ticks from Lazio (Central Italy). Interestingly, R. bursa was described as a prevalent species in areas of Lazio grazed by equine and cattle (Toma et al. 2015) and it was also reported infesting ruminants, dogs, and humans (Walker et al. 2000).

The screening revealed S. e. zooepidemicus/ruminatorum in ticks collected in 2012 and 2017, suggesting that the circulation of the pathogen in ticks was not sporadic. Likely ticks acquire this zoonotic agent by host mucosae, independently of bloodmeal. This study highlights the possibility that ticks might contribute to the diffusion of S. equi subspecies. We suggest that R. bursa ticks may act for mechanical spreading of these bacteria, whereas additional data are requested to consider ability of ticks to transmit the pathogenic agent also as competent vectors. Assessing this risk might improve efforts to control these important zoonoses, providing new insights for prophylaxis.

Footnotes

Acknowledgments

The authors would like to thank Dr. Alessandro Chiodera (Infectious Diseases Unit, Hospital of Macerata, Italy) and Dr. Edoardo Battista (veterinary and “Pony of Esperia” breeder) for providing ticks from Macerata and Roccasecca (Frosinone, Lazio) respectively. They would also like to thank Sheila Beatty for editing the English usage in the article. This research was funded by the European Union FP7/2007-2013_FP7/2007-2011 under grant agreement no. 281222 to Irene Ricci and UNICAM-FAR (Fondo Ateneo Ricerca, Università di Camerino) 2014–2015 to Guido Favia.

Author Disclosure Statement

No conflicting financial interests exist.

References

Akimoto-Tomiyama

, Tanabe

, Kajiwara

, Minami

, et al. Loss of chloroplast-localized protein phosphatase 2Cs in Arabidopsis thaliana leads to enhancement of plant immunity and resistance to Xanthomonas campestris pv. campestris infection. Mol Plant Pathol, 2018; 19:1184–1195.

Alber

, El-Sayed

, Lämmler

, Hassan

, et al. Multiplex polymerase chain reaction for identification and differentiation of Streptococcus equi subsp. zooepidemicus and Streptococcus equi subsp. equi . J Vet Med B Infect Dis Vet Public Health, 2004; 51:455–458.

Beati

, Keirans

. Analysis of the systematic relationships among ticks of the genera Rhipicephalus and Boophilus (Acari: Ixodidae) based on mitochondrial 12S ribosomal DNA gene sequences and morphological characters. J Parasitol, 2001; 87:32–48.

Boone

, Henning

, Hilbert

, Neubauer

, et al. Are brucellosis, Q fever and melioidosis potential causes of febrile illness in Madagascar?. Acta Trop, 2017; 172:255–262.

Carpi

, Cagnacci

, Wittekindt

, Zhao

, et al. Metagenomic profile of the bacterial communities associated with Ixodes ricinus ticks. PLoS One, 2011; 6:e25604.

Cheng

, Liu

. PCR denaturing gradient gel electrophoresis as a useful method to identify of intestinal bacteria flora in Haemaphysalis flava ticks. Acta Parasitol, 2017; l62:269–272.

Clow

, Weese

, Rousseau

, Jardine

. Microbiota of field-collected Ixodes scapularis and Dermacentor variabilis from eastern and southern Ontario, Canada. Ticks Tick Borne Dis, 2018; 9:235–244.

Dantas-Torres

, Latrofa

, Otranto

. Quantification of Leishmania infantum DNA in females, eggs and larvae of Rhipicephalus sanguineus . Parasit Vectors, 2011; 4:56.

Downar

, Willey

, Sutherland

, Mathew

, et al. Streptococcal meningitis resulting from contact with an infected horse. J Clin Microbiol, 2001; 39:2358–2359.

10.

Egyed

, Makrai

. Cultivable internal bacterial flora of ticks isolated in Hungary. Exp Appl Acarol, 2014; 63:107–122.

11.

Fernandez

, Blume

, Garrido

, Collins

, et al. Streptococcus equi subsp. ruminatorum subsp. nov., isolated from mastitis in small ruminants. Int J Syst Evol Microbiol, 2004; 54:2291–2296.

12.

Hai

, Almeras

, Socolovschi

, Raoult

, et al. Monitoring human tick-borne disease risk and tick bite exposure in Europe: Available tools and promising future methods. Ticks Tick Borne Dis, 2014; 5:607–619.

13.

, Wang

, Chen

, Ma

. Differences in the structure of the gut bacteria communities in development stages of the Chinese white pine beetle (Dendroctonus armandi). Int J Mol Sci, 2013; 14:21006–21020.

14.

Keskin

, Bursali

, Snow

, Dowd

, et al. Assessment of bacterial diversity in Hyalomma aegyptium, H. marginatum and H. excavatum ticks through tag-encoded pyrosequencing. Exp Appl Acarol, 2017; 73:461–475.

15.

Koropatnick

, Engle

, Apicella

, Stabb

, et al. Microbial factor-mediated development in a host-bacterial mutualism. Science, 2004; 306:1186–1188.

16.

Machado-Ferreira

, Vizzoni

, Balsemão-Pires

, Moerbeck

, et al. Coxiella symbionts are widespread into hard ticks. Parasitol Res, 2016; 115:4691–4699.

17.

Mediannikov

, Fenollar

. Looking in ticks for human bacterial pathogens. Microb Pathog, 2014; 77:142–148.

18.

Murrell

, Dobson

, Yang

, Lacey

, et al. A survey of bacterial diversity in ticks, lice and fleas from Australia. Parasitol Res, 2003; 89:326–334.

19.

Neamat-Allah

, Damaty

. Strangles in Arabian horses in Egypt: Clinical, epidemiological, hematological, and biochemical aspects. Vet World, 2016; 9:820–826.

20.

Niebylski

, Schrumpf

, Burgdorfer

, Fischer

, et al. Rickettsia peacockii sp. nov., a new species infecting wood ticks, Dermacentor andersoni, in western Montana. Int J Syst Bacteriol, 1997; 47:446–452.

21.

Poulin

, Boivin

. A case of disseminated infection caused by Streptococcus equi subspecies zooepidemicus . Can J Infect Dis Med Microbiol, 2009; 20:59–61.

22.

Preziuso

, Cuteri

. A multiplex polymerase chain reaction assay for direct detection and differentiation of β-hemolytic Streptococci in clinical samples from horses. J Equine Vet Sci, 2012; 32:292–296.

23.

Rudolf

, Mendel

, Sikutová

, Svec

, et al. 16S rRNA gene-based identification of cultured bacterial flora from host-seeking Ixodes ricinus, Dermacentor reticulatus and Haemaphysalis concinna ticks, vectors of vertebrate pathogens. Folia, 2009; 54:419–428.

24.

Stojek

, Dutkiewicz

. Studies on the occurrence of Gram-negative bacteria in ticks: Ixodes ricinus as a potential vector of Pasteurella . Ann Agric Environ Med, 2004; 11:319–322.

25.

Toma

, Khoury

, Bianchi

, Severini

, et al. Preliminary investigation on tick fauna in the neighborhood of Tarquinia, Lazio, Italy. Ann Ist Super Sanità, 2015; 51:67–70.

26.

Tveten

, Sjåstad

. Identification of bacteria infecting Ixodes ricinus ticks by 16S rDNA amplification and denaturing gradient gel electrophoresis. Vector Borne Zoonotic Dis, 2011; 11:1329–1334.

27.

Veldeman

, De Wilde

, Vogelaers

, Lerut

, et al. Acute renal failure with need for renal replacement therapy as a complication of zoonotic S. zooepidemicus infection: Case report and review of the literature. Acta Clin Belg, 2018; 73:144–150.

28.

Walker

, Keirans

, Horak

. The Genus Rhipicephalus (Acari: Ixodidae): A Guide to the Brown Ticks of the World. Cambridge: Cambridge University Press, 2000.

29.

, Cheng

, Yang

, Yan

. Identification of intestinal bacterial flora in Rhipicephalus microplus ticks by conventional methods and PCR-DGGE analysis. Exp Appl Acarol, 2015; 66:257–268.