Staphylococcus saprophyticus is a pathogen of the cattle tick Rhipicephalus (Boophilus) microplus

The southern cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae) is a persistent vector for infective tick-borne diseases in cattle such as babesiosis and anaplasmosis (Cossio-Bayugar, Miranda, and Holman 2005). The endemic nature of this bovine blood-sucking ectoparasite is considered an obstacle for development of the cattle industry in tropical and subtropical cattle grazing areas world-wide (Nu ez, Mu oz-Cobe as, and Molted 1985). The basic strategy in preventing monetary losses due to tick infestation of livestock and achieving relative control of cattle tick populations and tick transmitted diseases depends on the chemical treatment of tick-infested animals with different acaricides (de Castro 1997). Pesticide resistance against virtually all commercially available acaricide formulas is common among cattle tick populations, (Cossio-Bayugar, Miranda-Miranda, Portilla-Salgado, and Osorio-Miranda, 2009). The search for alternative tick control methods, necessitated the search for unknown bacteria in different developmental stages of Rhipicephalus spp. ticks (Amoo, Dipeolu, Akinboade, and Adeyemi 1987; Noda, Munderloh, and Kurtti 1997). These studies included the description of bacterial endosymbionts of the brown dog tick Rhipicephalus sanguineus not related to known tick-borne bacteria transmitted to humans and animals (Noda, Munderloh, and Kurtti 1997). Most notably, among the bacteria found on Rhipicephalus spp. ticks are several species of staphylococci bacteria isolated from eggs, larvae and engorged female cattle ticks Rhipicephalus (Boophilus) decoloratus and R. (Boophilus) geigy (Amoo, Dipeolu, Akinboade, and Adeyemi 1987). Further studies, reported the isolation of S. aureus and S. epidermidis from haemolymph obtained from engorged females of the brown dog tick R. sanguineus. Furthermore, both species of bacteria were isolated from eggs and larval progeny of the analysed adult ticks (Adejinmi and Ayinmode 2008). In addition to the bacteria already reported in ticks of different developmental stages, the presence of abundant staphylococci bacteria has been well-documented on the skin and mucosa of R. microplus bovine host and other farm animals (Devriese, Schleifer, and Adegoke 1985; Devriese, Laevens, Haesebrouck, and Hommez 1994; Ieven, Verhoeven, Pattyn, and Goossens 1995; Hajek, Meugnier, Bes, Brun, Fiedler, Chmela, Lasne, Fleurette, and Freney 1996; Zadoks and Watts 2009). The abundance of bacteria on the bovine host and the process of blood feeding by Rhipicephalus ticks may explain the mechanism by which ticks in parasitic stages acquire commensal staphylococci bacteria commonly found on animal hosts, as tick larvae wander freely over the host in search of patches of skin rich in vascularisation (Nu ez, Mu oz-Cobe as, and Molted 1985), possibly acquiring the host's bacteria in the process. Host staphylococci bacterial contamination of R. microplus has been suggested by previous observations of infected R. microplus ticks detected during experimental infestation of bovines (Miranda-Miranda, Cossio-Bayugar, Quezada-Delgado, Sachman-Ruiz, and Reynaud-Garza 2009). Unfortunately, very little information has been published regarding the pathogenic effect of ruminant skin staphylococci bacteria on R. microplus ticks. The objective of this study was to identify the bacteria species found in a disease constantly observed on Rhipicephalus microplus ticks.

A R. microplus acaricide susceptible reference strain (Su) was used for all experiments. Ticks were cultured and maintained at the Centro Nacional de Servicios de Constatación en Salud Animal (CENAPA-SAGARPA), Mexico. The Su tick strain was raised by infesting bovines with 2 X 10⁴ 10 to 15-day-old larvae according to procedures previously reported (Cossio-Bayugar, Miranda, and Holman 2005). Engorged female adult ticks were collected 21 days after infestation and thoroughly inspected to identify ticks with characteristic symptoms of infection or exudate at the genital orifice area. Adult ticks without obvious signs of infection were placed in sterile petri dishes for oviposition in groups of 10, and incubated at 28 °C in 80% relative moisture until complete oviposition was achieved. The egg masses were collected, weighed and aliquots of 200 mg were placed in clean cotton-plugged vials at 28 °C until larval eclosion.

Bacterial isolates were obtained from adult ticks showing symptoms of infection and/or genital orifice exudate by taking an exudate sample using a sterile bacteriological loop, inoculating the exudate on sterile petri dishes containing soy trypticase agar (STA) prepared according to the manufacturer's instructions (Difco-BD) and incubating overnight at 37°C. Larvae obtained as the progeny of uninfected ticks were killed by freezing for 2 h at -20 °C. Afterwards, batches of 5 to 10 larvae were selected and placed on sterile Petri dishes containing STA and incubated overnight at 37°C. Bacterial colonies growing on agar and samples of tick exudates were analysed and compared by Gram staining under an optical microscope according to previous reports (Blazevic and Ederer 1975; Freney, Kloos, Hajek, Webster, Bes, Brun, and Vernozy-Rozand 1999; Zadoks and Watts 2009). The bacterial colonies isolated on agar were maintained by continuous propagation on STA and as frozen suspensions in glycerol broth at -25°C according to previous report (Hajek, Meugnier, Bes, Brun, Fiedler, Chmela, Lasne, Fleurette, and Freney 1996).

The following properties were determined by previously described methods (Blazevic and Ederer 1975; Devriese, Schleifer, and Adegoke 1985; Devriese, Laevens, Haesebrouck, and Hommez 1994; Hajek, Ludwig, Schleifer, Springer, Zitzelsberger, Kroppenstedt, and Kocur, 1992; Hajek, Meugnier, Bes, Brun, Fiedler, Chmela, Lasne, Fleurette, and Freney 1996; Ieven, Verhoeven, Pattyn, and Goossens 1995; Freney, Kloos, Hajek, Webster, Bes, Brun, and Vernozy-Rozand 1999; Goyal, Singh, Kumar, Kaur, Singh, Sunita, and Mathur 2006; Vickers, Chopra, and O'Neill 2007): Gram staining, cell and colony morphology, colony pigmentation and production of the enzymes catalase, plasma coagulase, gelatinase, caseinase, -galactosidase, alkaline phosphatase, lecitinase and lactase. In addition, determination of production of acid under aerobic conditions from the carbohydrates: L (+) arabinose, D (+) galactose, glycerol, lactose, D (+) mannose, sucrose and turanose was performed. Intrinsic novobiocin resistance was determined on STA containing 10 µg/ml novobiocin.

Ten millilitres of liquid trypticase-soy bacteriological medium were inoculated with a single colony from a representative bacterial culture from isolate V, and then incubated overnight with agitation at 37 °C. The bacterial culture was divided into two 5 ml samples. One sample was used on engorged ticks without further processing, and the other sample was filtered through a 0.22 µm membrane. The resulting filtrate was used and the cell pellet was discarded. A total of 288 recently detached, fully engorged female ticks were divided into five groups: Group A was the normal control and contained 50 ticks picked up at random without any further treatment. The rest of the groups were comprised of ticks that were carefully inspected for the absence of any sign of previous bacterial infection and divided into five additional groups: Group B contained 48 ticks and was left untreated as the uninfected tick control. Group C contained 48 ticks, and was sprayed using a sterile glass sprayer containing 4 ml of bacterial culture. Group D contained 47 ticks and was sprayed with 4 ml of filtered bacterial culture. Group E contained 48 ticks that were each intracuticularly injected with 20 µl of bacterial culture. Group F contained 47 ticks that were each injected with 20 µl of filtered bacterial culture (Table 2). All groups were incubated at 28°C in 80% relative moisture for 20 days. After incubation, all ticks were inspected for symptoms of bacterial infection and presence of exudates. If present, exudates samples were taken for Gram staining analysis and catalase assays.

Larval progeny were obtained after collecting several egg masses from apparently healthy engorged ticks. The egg masses were incubated in batches of approximately 200 mg in clean cotton-plugged glass vials kept at 28°C and 80% relative moisture until hatching. Ten day old larvae were killed by freezing at -20°C for 2 h and submitted for SEM imaging at Carl Zeiss de México S.A. de C.V. using a Zeiss EVO model 40 microscope.

A 2 ml bacterial culture from isolate V was prepared in soy trypticase broth according to the manufacturer's instructions (Difco-BD) containing 10 mg/ml of novobiocin. A single colony of a representative bacterial culture from isolate V was used for inoculating the tube, and the bacterial culture was incubated at 37°C with agitation for eight hours. A commercial kit (Wizard genomic, Promega) suitable for genomic DNA isolation was used for obtaining the bacterial DNA according to the manufacturer's instructions. The tube was centrifuged at 13000 X g for two minutes, the supernatant was discarded and the cell pellet was resuspended in 480 µl of 50 mM EDTA. 60 µl of 10 mg/ml lysozyme was added and the mixture was gently agitated and incubated for 60 min at 37°C. The sample was centrifuged at 13000 X g for 2 min, the supernatant removed and the cells were resuspended in 600 µl nuclei lysis solution and incubated at 80°C for 5 min, 3 µl of RNase solution was added inverting the tube five times to mix and the tube was incubated at 37°C for 60 minutes, 200 µl of protein precipitation solution was added to the cell lysate and vortex vigorously for 20 seconds. The cell lysate was finally incubated on ice for 5 min and centrifuged at 13000 X g for 3 min. The supernatant was transferred to a 1.5 ml tube containing 600 µl of isopropanol gently inverting the tube to mix the sample. The bacterial genomic DNA was concentrated by centrifugation at 13000 X g for 2 min, the supernatant was discarded and 600 µl of 70% ethanol was added inverting the tube several times to wash the DNA. The tube was centrifuged at 13000 X g and once again and the supernatant discarded. The DNA pellet was air dried for 15 min and resuspended in rehydration solution.

DNA concentrations were determined by optical density estimation at 260 nanometers according to a previously described procedure (Sambrook and Russel 2001). As previously described (Weisburg, Barns, Pelletier, and Lane 1991; Ghebremedhin, Layer, Konig, and Konig 2008) 16S ribosomal DNA amplification was achieved by PCR reactions containing 1X PCR buffer, 100 pg of bacterial DNA, 10 pmol of forward primer 5'-AGAGTTTGATCMTGGCTCAG-3', 10 pmol of backward primer 5'-GGTTACCTTGTTACGACTT-3', dNTP's to a concentration of 2mM, 1.5 mM of MgCl₂, and two units of Taq polymerase in a total volume of 20 µl. The cycling conditions were 94°C for 4 min followed by 25 cycles of 95° C for 1 min, 54° C for 45 sec, 72°C for 1 min, and a 4°C hold in a Sprint thermal cycler (Thermo Scientific USA). The PCR amplicons were verified by 1% agarose electrophoresis in 1X TBE buffer (Sambrook and Russel 2001). Amplicons were cloned directly into the pGEM-T-easy vector T/A cloning system (Promega) according to the manufacturer's instructions. One µl of PCR product was placed into 5 µl 2X T4 ligation buffer and 5 ng of pGEM-T-easy vector, incubating the mixture for 1 h at room temperature.

Plasmids containing the amplicons were used to transform high efficiency competent E. coli Jm 109 bacteria Promega USA) by placing 50 µl recently thawed cells in a 200 µl tube containing 2 µl of plasmid-amplicon ligation reaction, gently mixing the tube contents by flicking and incubating on ice for 20 min, the mixture was heat shocked for 45 seconds at 42°C and immediately returned to the ice for 2 min. Transformed cells were added to 950 µl of SOC medium and incubated for 90 min at 37°C with vigorous agitation; 50, 100 and 200 µl of transformed cell mixture were plated on Luria agar containing 60 µg/ml of ampicilin, 30 µg/ml IPTG and 50 µg/ml of Xgal. The plates were incubated overnight at 37°C according to Sambrook and Russel (2001). Six individual white colonies were used to seed 5 ml Luria broth containing 60 µg/ml of ampicilin, and bacterial cultures were grown at 37°C with agitation for 12 h. E. coli cell pellets were collected by centrifugation and subjected to a plasmid isolation protocol using a commercial kit (Wizard Miniprep Promega USA). Isolated plasmid DNA concentration was determined by Optical Density estimation at 260 nanometers and screened for cloned amplicons by Eco R1 restriction enzyme digestion of the plasmid,

DNA insertions were verified by 1% agarose electrophoresis (Sambrook and Russell 2001). All six purified plasmids containing the PCR amplicons, were sequenced using the primers m13/pUC forward 5'-GTT TTC CCA GTC ACG TTG TA-3' or m13/pUC reverse 5'-TTG TGA GCG GAT AAC AAT TTC-3 for a total of 12 sequence reactions by the Institute of Biotechnology of the National Autonomous University of Mexico using Terminator Cycle Sequencing Ready Reaction BigDye® terminators v3.1 (PE Applied Biosystems) and automated sequencing using an ABI PRISM Model 3730 Xl genetic analyser automated sequencer with data collection software version 3.0. The sequences were analysed using the online DNA sequence software analysis at the National Center for Biotechnology Information of the National Institute of Health NCBI/NIH (www.ncbi.nih.gov). The search for similar reported sequences by genomic BLAST algorithm, considered only the microbial genomes that included 1047 bacterial, 60 archeal and 183 eukaryotic genomes and used the BLASTn mode algorithm similarity search (Altschul, Madden, Schaffer, Zhang, Zhang, Miller, and Lipman 1997). Eighty-one 16S rRNA sequences in the Genbank database were reported for the Staphylococcus genus, and were used to reconstruct a phylogenetic tree using the MEGA 4 algorithm, which included the 16S rRNA gene sequence obtained from bacterial isolate V, for the purpose of comparison and estimation of phylogenetic accuracy. Four 16S rRNA sequences belonging to different species of the genus Macrocuccus spp. were used as an outgroup or unrelated bacteria, and the unrooted phylogenetic tree was constructed by the neighbour-joining method (Saitou and Nei 1987) using the Jukes-Cantor one parameter (Jukes and Cantor 1969). A bootstrap resampling analysis of 1000 replicates was performed to estimate the confidence of the phylogenetic tree topologies (Felsenstein 1985).

A total of 764 adult engorged females were raised on bovines and 38 of them (4.98%), exhibited characteristic symptoms of infection or exudate at the genital orifice area (Figure 1a). Microscope preparations of tick exudate showed abundant clusters of Gram-positive cocci bacteria (Figure 1c).

Figure 1. Infected tick symptoms and exudate microscopic examination. Engorged females were selected showing evident symptoms of a disease identified by a yellow exudate apparently secreted through the genital orifice (a). Compared to an asymptomatic engorged female (b). Optical microscopic analysis of the tick's exudate exhibit abundant clusters of Gram-positive cocci bacteria (c). Objective immersion 100 X.

Seven bacterial isolates, identified by Roman numerals I to VII, were obtained from different adult ticks exhibiting symptoms of bacterial infection. One bacterial isolate was obtained from larvae placed on sterile Petri dishes containing STA and was identified by Roman numeral VIII. All bacterial colonies grew larger than 5 mm in diameter and showed intense yellow pigmentation and entire edges. Microscopic analysis revealed that the bacterial cells were Gram-positive cocci bacteria. All isolated bacteria showed positive catalase reaction and resistance to 10 µg/ml novobiocin, but no coagulase activity in the presence of bovine plasma nor proteolytic activity in the presence of gelatin or casein. All bacteria were able to produce acid by fermenting glycerol, lactose, D(+) mannose, sucrose and turanose, however, they were unable to use L(+) arabinose or L(+) lactose.

Cultured bacteria sprayed over healthy ticks produced 21% infection prevalence, whereas control ticks sprayed with filtered culture media showed no sign of infection. On the other hand, ticks injected with bacteria showed 25% infection prevalence, whereas filtrate injected control ticks showed only 2% infection prevalence. Control groups corroborated that tick infection was associated with cultured S. saprophyticus bacterial cells during the Koch-Henle postulates experiment (Table 1).

Table 1. Koch-Henle postulates experiment data

PCR assays using DNA purified from bacterial isolate V produced a DNA amplicon of approximately 1450 base pairs (bp) and was visualised by agarose gel electrophoresis (Data not shown). The PCR amplicon cloning procedure utilised several clones of the sequencing vector containing the 1450 bp PCR amplicon. One of these clones was subjected to multiple sequencing reactions on plus and minus DNA chains, produced a consensus sequence of 1432 bp. This DNA sequence was electronically submitted to the Genbank sequence database (www.ncbi.nih.gov) and was matched to the bacterial Isolate V 16S rRNA gene with the Genbank accession number GQ422825.

The BLAST DNA sequence similarity analysis on Isolate V 16S rRNA gene, demonstrated an identity of 99.65% in 1427 bases out of 1432 to Staphylococcus saprophyticus subsp. saprophyticus ATCC 15305 Genbank accession number NC_007350.1 (Kuroda, Yamashita, Hirakawa, Kumano, Morikawa, Higashide, Maruyama, Inose, Matoba, Toh, Kuhara, and Hattori Mohta 2005). The BLAST algorithm identified three closely related staphylococci bacteria species other than S. saprophyticus including S. haemolyticus, S. warneri and S. aureus that shared between 98.32 to 97.41% identity. The phylogenetic tree reconstructed by the MEGA 4 algorithm showed that bacterial Isolate V 16S rRNA phylogenetic identity was located within the S. saprophyticus clade grouped with all previously reported 16S rRNA sequences for S. saprophyticus subsp. saprophyticus and S. saprophyticus subsp. bovis (Figure 2). Larvae placed on STA media yielded one bacterial isolate that exhibited identical biochemical properties as those of bacterial isolates obtained from the sick adult ticks. SEM imaging showed that the analysed larvae carried multiple cocci bacteria covering the larval cuticles (Figure 3).

Figure 2. Jukes-Cantor species unrooted phylogenetic tree. The Neighbor-joining model was used based on 81 sequences of 16S rRNA genes of Staphylococcus species and Macrococcus spp. as outgroup. The support values on the bipartitions correspond to the percent occurrence of 1000 replicates boostrapping. The ISOLATE V grouped in the S. saprophyticus clade is located within the gray box.

Figure 3. Scanning electron microscope imaging larvae analysis. S. saprophyticus was isolated from healthy larvae that show abundant cocci bacteria on the tick's cuticle around the palps and chelicerae (a) and around the tarsus of an anterior leg (b). Clear arrows indicate examples of the bacteria.

This study was motivated by the long-held observations that bovine tick infestation under controlled conditions consistently yielded a small percentage (approximately 5%) of engorged females, with obvious symptoms of an infectious disease identifiable by the production of a yellow exudate accumulated around the tick's capitulum. This exudate, which is apparently secreted through the genital orifice (figure 1a), invariably affected oviposition and prevented completion of the infected tick's life cycle (Miranda-Miranda, Cossio-Bayugar, Quezada-Delgado, Sachman-Ruiz, and Reynaud-Garza, 2009). This phenomenon was considered worthy of a thorough observation of tick exudate by optical microscopy, which led to detection of significant levels of clusters of Gram-positive cocci bacteria (Figure 1c) with morphology resembling that of the genus Staphylococcus spp. (Freney, Kloos, Hajek, Webster, Bes, Brun, and Vernozy-Rozand 1999; Zadoks and Watts 2009). This microscopic observation was the starting point for further bacteriological analysis involving basic techniques specific to staphylococci bacteria (Blazevic and Ederer 1975; Freney, Kloos, Hajek, Webster, Bes, Brun, and Vernozy-Rozand 1999; Zadoks and Watts 2009).

Exudate samples yielded highly pigmented bacterial colonies on STA plates and exhibited conserved Gram-positive cocci bacteria microscopic morphology that was identical to that of the bacteria previously observed in the sick ticks' exudates (Figure 1c). Biochemical characteristics demonstrated that the isolated bacteria were catalase-positive, a property only observed in Gram-positive cocci bacteria belonging to Staphylococcus spp. (Freney, Kloos, Hajek, Webster, Bes, Brun, and Vernozy-Rozand 1999; Zadoks and Watts 2009) which confirmed the genus identity. All bacterial isolates showed intrinsic novobiocin-resistance which is a distinctive property of very few staphylococci species including S. lentus, S. sciuri, S. xylosus, S. cohnii, S. gallinarum and S. saprophyticus (Devriese, Schleifer, and Adegoke 1985; Devriese, Laevens, Haesebrouck, and Hommez 1994; Hajek, Meugnier, Bes, Brun, Fiedler, Chmela, Lasne, Fleurette, and Freney 1996; Zadoks and Watts 2009). This narrowed the species identification to only six potential candidates. Further biochemical comparison of proteases and acid production demonstrated a close match with S. saprophyticus, as this species is the only one among the six novobiocin-resistant staphylococci species candidates incapable of producing proteolytic enzymes such as gelatinase and caseinase (Devriese, Schleifer, and Adegoke 1985; Devriese, Laevens, Haesebrouck, and Hommez 1994; Hajek, Meugnier, Bes, Brun, Fiedler, Chmela, Lasne, Fleurette, and Freney 1996; Zadoks and Watts 2009). S. saprophyticus species identification was supported by the 16S rRNA genotyping that showed 99.65% sequence identity with S. saprophyticus subsp. saprophyticus by BLAST algorithm (Altschul, Madden, Schaffer, Zhang, Zhang, Miller, and Lipman 1997). BLAST algorithm analysis of the ten closest matches identified other species of staphylococci bacteria with high identity to Isolate V such as S. haemolitycus (98.39%), S. warneri (97.97%) and seven different strains of S. aureus subsp. aureus (97.48-97.41%). However, according to the literature, none of these staphylococci species exhibited intrinsic novobiocin resistance (Devriese, Schleifer, and Adegoke 1985; Devriese, Laevens, Haesebrouck, and Hommez 1994; Hajek, Meugnier, Bes, Brun, Fiedler, Chmela, Lasne, Fleurette, and Freney 1996; Zadoks and Watts 2009), which leaves S. saprophyticus supported both by biochemical phenotyping and 16S rRNA genotyping as the only possible species for the bacteria isolated from infected R. microplus. There are two S. saprophyticus subspecies reported in the literature: S. saprophyticus subsp. saprophyticus, described as a human pathogenic bacteria associated with urinary infection in young women (Kuroda, Yamashita, Hirakawa, Kumano, Morikawa, Higashide, Maruyama, Inose, Matoba, Toh, Kuhara, and Hattori Mohta 2005; Vickers, Chopra, and O'Neill 2007), and S. saprophyticus subsp. bovis which has been isolated from skin and nostrils of goats, sheep and cows, where this bacterium subspecies is described as part of the flora commonly identified on farm animals without any association to a specific disease (Devriese, Schleifer, and Adegoke 1985; Devriese, Laevens, Haesebrouck, and Hommez 1994; Hajek, Meugnier, Bes, Brun, Fiedler, Chmela, Lasne, Fleurette, and Freney 1996; Zadoks and Watts 2009).

Ticks used during this study were obtained from controlled infestations of cattle. For this reason, we would expect that S. saprophyticus isolated from ticks would be related to the strain previously isolated from cow skin and nostrils (Devriese, Schleifer, and Adegoke 1985; Devriese, Laevens, Haesebrouck, and Hommez 1994; Hajek, Meugnier, Bes, Brun, Fiedler, Chmela, Lasne, Fleurette, and Freney 1996; Zadoks and Watts 2009). However, when the 16S rRNA gene sequence of bacterial isolate V was used by the MEGA 4 algorithm, a phylogenetic tree showed that isolate V fully integrated within the S. saprophyticus clade and exhibited a closer phylogenetic proximity to S. saprophyticus subsp. saprophyticus (Figure 2).

The BLAST algorithm confirmed a 99.65% match to S. saprophyticus subsp. saprophyticus, and excluded S. saprophyticus subsp. bovis from the ten closest matches, even though the 16S rRNA gene sequence reported for S. saprophyticus subsp. bovis was available among the bacterial sequences selected for the BLAST sequence similarity analysis. Further genotyping of other gene sequences such as hsp60, rpoB, sodA, dnaJ and tuf genes should be undertaken to make a conclusive report regarding the similarity of the isolated bacteria to previously reported S. saprophyticus subspecies (Shah, Iihara, Noda, Song, Nhung, Ohkusu, Kawamura, and Ezaki 2007; Ghebremedhin, Layer, Konig, and Konig, 2008; Zadoks and Watts 2009).

Cultured S. saprophyticus initially isolated from naturally infected ticks was able to induce the same type of infection on healthy engorged female ticks experimentally infected under controlled conditions with an infection prevalence five times higher than the observed natural prevalence (Table 1). Healthy ticks experimentally infected with cultured S. saprophyticus, also produced identical symptoms to those originally observed in the natural tick infection, which fulfilled Koch-Henle postulates and demonstrated that S. saprophyticus is the causative agent of the lethal bacteriological infection observed on R. microplus. During this study, S. saprophyticus was not only isolated on bacteriological medium from sick engorged females, but also from larvae that hatched from eggs oviposited under laboratory controlled conditions by apparently healthy females. These eggs were collected from Petri dishes and incubated in cotton-plugged clean glass vials, so the only way for the larvae to acquire S. saprophyticus was from their apparently healthy progenitor tick. Therefore, it is reasonable to infer that the bacteria are present on healthy engorged female ticks showing no signs of infection.

SEM imaging analysis of larvae obtained from the same batch used for isolating staphylococci bacteria on STA plates showed cocci bacteria thriving on the larval cuticles (Figure 3), which explains why S. saprophyticus isolation from larvae was possible in the first place. Although SEM imaging does not indicate the cocci bacteria species, it is evident that the cocci bacteria on the larval cuticles are constant in their spherical shape and size, and exhibit clusters of bacterial cells (Figure 3b), which is a distinctive morphology of Staphylococcus sp. (Freney, Kloos, Hajek, Webster, Bes, Brun, and Vernozy-Rozand 1999). The bacteria visualised by SEM grew on bacteriological medium from the same batch of tick larvae used for the SEM imaging analysis. These larval isolated bacteria, identified as isolate VIII, showed identical biochemical properties as those bacterial isolates from sick adult ticks, suggesting that S. saprophyticus is commonly found in at least two developmental stages of R. microplus, although it triggers a conspicuous disease only in a few adult ticks. SEM images provide a possible explanation for why only a small number of engorged females acquire the bacterial infection. It is evident that the sole presence of the bacteria on the cuticle is not enough to cause disease, the bacteria have to find their way to the inside of the tick or at least into the oviduct were the infection appears to occur. SEM images showed a small number of larvae with cocci bacteria on the chelicerae (Figure 3a), a situation that facilitates transmission during the tick's blood feeding process; these bacterial cells are in the optimal position to contaminate the host's blood that finally enters the engorged female. During the SEM imaging of the larvae; most of the images showed cocci bacteria on the larval cuticles, but very few of the analysed larvae exhibited cocci bacteria in the mouth parts (Figure 3). This could explain the low prevalence of the natural infection and this may be the hypothesis that motivates further studies on this subject. Another hypothesis supported by the experimental data may be that cattle ticks acquire the bacteria during a natural infestation of the bovine host where previous reports have identified S. saprophyticus (Devriese, Schleifer, and Adegoke 1985; Hajek, Meugnier, Bes, Brun, Fiedler, Chmela, Lasne, Fleurette, and Freney 1996). This hypothesis is partially supported by previous reports that animal hosts may contaminate Rhipicephalus spp. ticks with commensal staphylococci bacteria (Amoo, Dipeolu, Akinboade, and Adeyemi 1987; Adejinmi and Ayinmode 2008).

As demonstrated by this study and previous observations (Miranda-Miranda, Cossio-Bayugar, Quezada-Delgado, Sachman-Ruiz, and Reynaud-Garza, 2009), under certain circumstances, staphylococci bacteria are able to produce a lethal infection in fully engorged ticks. The precise mechanism of bacterial infection of the engorged females must be clarified to make a comprehensive assessment for the potential use of S. saprophyticus as an alternative control method of the cattle tick and tick-transmitted diseases. Further studies on this subject should be considered carefully if alternative control methods are implemented, especially because of ever-increasing R. microplus acaricide resistance spreading worldwide.

We would like to thank to Karl Zeiss de México S.A. de C.V. and Dr. Marco A. Ramirez O for their invaluable assistance on the scanning electron microscope imaging analysis and M. en C. Rene Hernández Vargas at the Institute of Biotechnology of the National Autonomous University of Mexico, for his assistance during the DNA sequence and preparation of this manuscript.

This research was partially funded by: SEP-CONACYT; Grant number: 58609. CONACYT; Grant number: 49498. and PAPIIT-UNAM; Grant number: IN211707.

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