In West Africa, Borrelia crocidurae is the primary cause of relapsing fever of humans and this spirochete is maintained in enzootic cycles involving multiple species of small mammals and its argasid tick vector Ornithodoros sonrai.1 This spirochete was first found in the shrew Crocidura stampflii (now Crocidura poensis) over a hundred years ago in Dakar, Senegal.2 Since this seminal discovery, numerous species of small mammals have been found to be naturally infected with B. crocidurae when captured during field investigations, including the multimammate rat Mastomys natalensis.3–12 These rats often live in peridomestic settings in close association with humans and comprise the most widespread species of rodents in sub-Saharan Africa.13 This behavior and wide geographic distribution make them ideally suited as potential sources of zoonotic pathogens. In Mali, M. natalensis was the most frequently captured animal indoors, and in the two nearby villages of Kalibombo and Doucombo, 11 individuals (∼10% of those examined) were infected with B. crocidurae.11 However, these animals were sampled for infection only once after being euthanized and blood samples collected,11 which is the case for all prior studies investigating wild small mammals for infection with B. crocidurae in West Africa. We are unaware of any studies that tested any species of African rodent for levels and duration of spirochetemia by B. crocidurae in the same individuals over time. Therefore, to better understand these rats’ potential significance as a natural host for B. crocidurae, experimental spirochete infections of M. natalensis were performed in the laboratory and the animals were followed out to 28 days postinoculation.
A M. natalensis breeding colony was first established at the Malaria Research and Training Center, University of Sciences, Techniques and Technologies of Bamako, Mali, with animals captured in Doneguebougou, Mali (12°48′18″N and 7°58′49″W) (Figure 1A). Voucher specimens of skulls of M. natalensis collected from this village are in the Smithsonian Institution, National Museum of Natural History, Collection of African Mammals, Washington, DC (USNM numbers 599351–599353, 599355, 599356, and 599361–599363) and mitochondrial cytochrome b DNA sequences are in GenBank (accession numbers JX292861 and JX292862).11 In November 2013, 20 adult pairs of rats were shipped to the Rocky Mountain Laboratories (RML), Hamilton, MT, where a breeding colony was established.
On November 2, 2017, 16 animals comprising eight males and eight females from two litters were marked with ear tags, caged by gender with litter mates in groups of four individuals (Table 1), and allowed to adjust to their new caging for 1 week before inoculation. On day 0, animals (10 or 12 weeks old) were weighed to the nearest gram and inoculated with B. crocidurae Doucombo, a strain from a pool of 11 O. sonrai ticks (five nymphs, five males, and one female) collected in September 2011 in Doucombo, Mali.11 Spirochetes in a −80°C frozen culture were thawed, grown, passaged twice in BSK-II medium,14 counted with a Petroff-Hausser counting chamber and Nikon Eclipse E600 dark-field microscope (Nikon Instruments Inc., Melville, NY), and diluted to 1,000 bacteria in 200 µL of medium, and the inoculum administered by intraperitoneal injection to each animal. All procedures performed on the animals were carried out while anesthetized by inhalation of isoflurane (Fluriso, VetOne; MWI Veterinary Supply, Boise, ID). This study was approved by the RML Animal Care and Use Committee (protocol #2017-042) and performed in a BSL-2+ laboratory as approved by the Institutional Biosafety Committee.
Mastomys natalensis inoculated with Borrelia crocidurae, gender, age, body weights and weight change, and total number of spirochetes counted in each animal
|Rat no. and gender||Cage*||Age (weeks)||Weight† (day 0)||Weight† (day 28)||Weight change† (%)||Total spirochetes counted‡|
|1 ♀||A||10||30||36||+6 (20)||0 (not infected)|
|2 ♀||A||10||25||25||0 (0)||515|
|3 ♀||A||10||25||29||+4 (16)||1,393|
|4 ♀||A||10||26||29||+3 (11.5)||2,159|
|5 ♀||B||12||39||39||0 (0)||676|
|6 ♀||B||12||40||44||+4 (10)||327|
|7 ♀||B||12||50||50||0 (0)||726|
|9 ♂||C||10||52||55||+3 (5.7)||1,711|
|10 ♂||C||10||56||62||+6 (10.7)||210|
|11 ♂||C||10||60||60||0 (0)||693|
|12 ♂||C||10||69||67||−2 (2.9)||721|
|13 ♂||D||12||76||80||+4 (5.3)||357|
|14 ♂||D||12||95||98||+3 (3.2)||611|
|15 ♂||D||12||70||74||+4 (5.7)||633|
|16 ♂||D||12||89||88||−1 (1.1)||435|
* Litter mates from 2 litters, 10 and 12 weeks old, caged separately in cages A, B, C, and D.
† Body weight in grams.
‡ Cummulative total of spirochetes counted in 25 blood smears taken days 4–28 postinoculation.
On day 0, rat #8 succumbed to anesthesia during inoculation, leaving 15 animals for the remainder of the study. On days 4–28 postinoculation, each animal was observed for overt signs of clinical disease (lethargy, ruffed hair, and humped back), anesthetized, weighed, and a drop of blood collected from the tail vein by puncture with a sterile lancet. We chose not to measure body temperature at the time of blood sampling because of the excitability of the animals and the possible influence our handling might have on raising their temperature. Thin blood smears were made on glass microscope slides, fixed and stained with the 3-Stain Diff Quick Dip Kit (Cancer Diagnostics, Durham, NC), and examined with a Nikon Eclipse E800 microscope (Nikon Instruments Inc.) at ×1,000 magnification with a ×100 oil immersion objective lens. Fifty microscopic fields of the feathered edge of each smear were examined, spirochetes were counted each day, and the numbers were totaled for each animal at the end of the study.
Spirochetes became detectible in the blood on days 4–6 postinoculation in 14 of 15 animals (Figure 1B). Rat #1, the first animal inoculated, remained negative throughout the study. During the 22 days that followed, the 14 infected rats all displayed at least one prolonged detectable spirochetemia lasting for 10–20 continuous days (X = 12.8 days), with the number of spirochetes observed daily varying considerably (Figure 2). The most B. crocidurae seen in the highest peak of spirochetemia on a single day for each animal ranged from a low of 47 spirochetes in rat #13 (day 15) to a high of 510 spirochetes in rat #9 (day 13) (Figure 2). The cyclic fluctuations in the number of spirochetes observed in the blood were typical for a relapsing fever spirochete. Initial spirochetemias were followed during subsequent days by a series of lower and then higher bacterial cell counts, typical of relapses during the course of infection, until spirochetes were no longer detectable (spirochetes last seen on days 16–27 postinoculation). The four older males (Figure 2, #13, 14, 15, and 16) had spirochetemias with two or three distinct relapses following the first peak, similar to what one sees with the North American relapsing fever spirochete, Borrelia hermsii, when infecting laboratory mice (Mus musculus).15 Yet, whereas B. hermsii typically achieves its highest cell density in the first spirochetemia, this is not what we observed for the B. crocidurae infections in M. natalensis. In all but one animal (Figure 2, rat #6), the highest spirochetemias were achieved during one of the relapses rather than during the first bacteremia.
The total number of spirochetes counted in each animal suggested that the younger animals were more susceptible to higher spirochetemias, which was true. When we compared the total number of spirochetes observed in the 10-week-old versus 12-week-old animals, the difference was highly significant (X2 = 608.40; P < 0.01). However, when the initial body weights of the 14 infected animals (25–95 g) were compared with the total numbers of B. crocidurae counted in each of them (210–2,159 spirochetes) (Table 1), the inverse relationship between body weight and number of spirochetes was not significant (slope = −11.15; R2 = 0.2057; P = 0.1034).
The animals showed no overt signs of clinical illness and were clean and well-groomed throughout the study. They remained alert, moved quickly, and required anesthesia in their holding cages before their removal and transfer to the procedural room to prevent their escape. Whereas a few animals either lost weight or remained the same, eight of the 14 rats gained weight over the 4 weeks when the study was terminated (Table 1). The animal gaining the most weight (20%) was rat #1, for which no spirochetemia was detected.
Our results demonstrate that M. natalensis is highly susceptible to infection with B. crocidurae. These animals displayed prolonged spirochetemias while remaining healthy, which makes them ideally suited as hosts for infecting ticks with spirochetes. The pattern of prolonged bacteremia with delayed peaks of increasing spirochetemias may reflect a form of immune evasion due to the spirochetes being cloaked in rosettes with aggregated red blood cells, as has been observed with B. crocidurae infections in laboratory mice (M. musculus).16,17 Experimentally infected laboratory mice (M. musculus, BALB/c) also showed prolonged and relapsing spirochetemias when infected with an unidentified strain of B. crocidurae,18 which demonstrates a useful animal model. Also, an early study showed that M. natalensis was a competent experimental host for Borrelia duttoni.19 Our results provide the foundation for future studies examining the relapsing phenomenon of B. crocidurae in M. natalensis, their immunological and temperature responses to infection, and to explore other aspects of host competency such as latent infections of the central nervous system, which has been demonstrated primarily in other species of West African rodents.7,9,12
We thank Marissa Woods and Greg Saturday for animal care and veterinary assistance.
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