• View in gallery

    Mastomys natalensis breeding pair in Bamako, Mali (A), and Borrelia crocidurae in the feathered edge of a thin blood smear from an experimentally infected M. natalensis (B). Red blood cells 5–6 µm in diameter.

  • View in gallery

    Spirochetemia curves for Mastomys natalensis infected with Borrelia crocidurae showing the number of spirochetes observed on days 4–28 postinoculation. Animals in rows AD correspond to age, gender, and cage mates identified in Table 1. y axes vary in numbers counted to better show changes in patterns in animals with lower counts.

  • 1.

    Trape JF et al. 2013. The epidemiology and geographic distribution of relapsing fever borreliosis in West and North Africa, with a review of the Ornithodoros erraticus complex (Acari: Ixodida). PLoS One 11: e78473.

    • Search Google Scholar
    • Export Citation
  • 2.

    Leger A, 1917. Spirochète de las musaraigne (Crocidura stampflii Jentink). Bull Soc Path Exot 10: 280281.

  • 3.

    Boiron H, 1949. Considérations sur la fièvre récurrente a tiques au Sénégal. L’importance du rat comme réservoir de virus. Bull Soc Path Exot 42: 6270.

    • Search Google Scholar
    • Export Citation
  • 4.

    Mathis MM, Durieux C, 1934. Fréquence a Dakar de la spirochétose récurrente a tiques. Bull Acad Med 111: 37.

  • 5.

    Trape JF, Duplantier JM, Bouganali H, Godeluck B, Legros F, Cornet JP, Camicas JL, 1991. Tick-borne borreliosis in west Africa. Lancet 337: 473475.

  • 6.

    Godeluck B, Duplantier JM, Ba K, Trape JF, 1994. A longitudinal survey of Borrelia crocidurae prevalence in rodents and insectivores in Senegal. Am J Trop Med Hyg 50: 165168.

    • Search Google Scholar
    • Export Citation
  • 7.

    Diatta G, Trape JF, Legros F, Rogier C, Duplantier JM, 1994. A comparative study of three methods of detection of Borrelia crocidurae in wild rodents in Senegal. Trans R Soc Trop Med Hyg 88: 423424.

    • Search Google Scholar
    • Export Citation
  • 8.

    Trape JF, Godeluck B, Diatta G, Rogier C, Legros F, Albergel J, Pepin Y, Duplantier JM, 1996. The spread of tick-borne borreliosis in west Africa and its relationship to sub-saharan drought. Am J Trop Med Hyg 54: 289293.

    • Search Google Scholar
    • Export Citation
  • 9.

    Vial L, Diatta G, Tall A, Ba EH, Bouganali H, Durand P, Sokhna C, Rogier C, Renaud F, Trape JF, 2006. Incidence of tick-borne relapsing fever in west Africa: longitudinal study. Lancet 368: 3743.

    • Search Google Scholar
    • Export Citation
  • 10.

    Elbir H, FotsoFotso A, Diatta G, Trape JF, Arnathau C, Renaud F, Durand P, 2015. Ubiquitous bacteria Borrelia crocidurae in western African ticks Ornithodoros sonrai. Parasit Vectors 8: 477.

    • Search Google Scholar
    • Export Citation
  • 11.

    Schwan TG, Anderson JM, Lopez JE, Fischer RJ, Raffel SJ, McCoy BN, Safronetz D, Sogoba N, Maïga O, Traore SF, 2012. Endemic foci of the tick-borne relapsing fever spirochete Borrelia crocidurae in Mali, West Africa, and the potential for human infection. PLoS Negl Trop Dis 6: e1924.

    • Search Google Scholar
    • Export Citation
  • 12.

    Diatta G, Duplantier JM, Granjon L, Ba K, Chauvancy G, Ndiaye M, Trape JF, 2015. Borrelia infection in small mammals in West Africa and its relationship with tick occurrence inside burrows. Acta Trop 152: 131140.

    • Search Google Scholar
    • Export Citation
  • 13.

    Musser GG, Carleton MD, 2005. Superfamily Muroidea. Wilson DE, Reeder DM, eds. Mammal Species of the World. Baltimore, MD: The Johns Hopkins University Press, 8941531.

    • Search Google Scholar
    • Export Citation
  • 14.

    Barbour AG, 1984. Isolation and cultivation of Lyme disease spirochetes. Yale J Biol Med 57: 521525.

  • 15.

    Raffel SJ, Battisti JM, Fischer RJ, Schwan TG, 2014. Inactivation of genes for antigenic variation in the relapsing fever spirochete Borrelia hermsii reduces infectivity in mice and transmission by ticks. PLoS Pathog 10: e1004056.

    • Search Google Scholar
    • Export Citation
  • 16.

    Mooser H, 1958. Erythozyten-adhäsion und hämagglomeration durch rückfallfiefer-spirochäten. Z Tropenmed Parasitol 9: 93111.

  • 17.

    Burman N, Shamaei-Tousi A, Bergström S, 1998. The spirochete Borrelia crocidurae causes erythrocyte rosetting during relapsing fever. Infect Immun 66: 815819.

    • Search Google Scholar
    • Export Citation
  • 18.

    Shamaei-Tousi A, Martin P, Bergh A, Burman N, Brännstrom T, Bergström S, 1999. Erythrocyte-aggregating relapsing fever spirochete Borrelia crocidurae induces formation of miroemboli. J Infect Dis 180: 19291938.

    • Search Google Scholar
    • Export Citation
  • 19.

    Zumpt F, 1959. Is the multimammate rat a reservoir for Borrelia duttoni? Nature 184: 793794.

 
 
 

 

 
 
 

 

 

 

 

 

 

Host Competency of the Multimammate Rat Mastomys natalensis Demonstrated by Prolonged Spirochetemias with the African Relapsing Fever Spirochete Borrelia crocidurae

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  • 1 Laboratory of Virology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, Hamilton, Montana;
  • | 2 Zoonotic Diseases and Special Pathogens, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Canada;
  • | 3 Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, Hamilton, Montana

African multimammate rats, Mastomys natalensis, are widely distributed in sub-Saharan Africa and live in close association with humans. In West Africa, numerous field studies have shown these animals may be naturally infected with the relapsing fever spirochete Borrelia crocidurae, the primary cause of tick-borne relapsing fever in this region of the continent. However, naturally infected individual rats have never been examined over time; therefore, the true host competency of these rats for this spirochete is unknown. Therefore, using animals from an established laboratory colony of M. natalensis, rats were experimentally infected with B. crocidurae and their blood examined to 28 days postinoculation. These animals were highly susceptible to infection and displayed prolonged and cyclic spirochetemias. Our results demonstrate these peridomestic rodents are likely competent hosts for infecting argasid tick vectors and play a primary role in the enzootic cycle for B. crocidurae in West Africa.

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.312 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.

Figure 1.
Figure 1.

Mastomys natalensis breeding pair in Bamako, Mali (A), and Borrelia crocidurae in the feathered edge of a thin blood smear from an experimentally infected M. natalensis (B). Red blood cells 5–6 µm in diameter.

Citation: The American Journal of Tropical Medicine and Hygiene 101, 6; 10.4269/ajtmh.19-0590

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.

Table 1

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 genderCage*Age (weeks)Weight† (day 0)Weight† (day 28)Weight change† (%)Total spirochetes counted‡
1 ♀A103036+6 (20)0 (not infected)
2 ♀A1025250 (0)515
3 ♀A102529+4 (16)1,393
4 ♀A102629+3 (11.5)2,159
5 ♀B1239390 (0)676
6 ♀B124044+4 (10)327
7 ♀B1250500 (0)726
9 ♂C105255+3 (5.7)1,711
10 ♂C105662+6 (10.7)210
11 ♂C1060600 (0)693
12 ♂C106967−2 (2.9)721
13 ♂D127680+4 (5.3)357
14 ♂D129598+3 (3.2)611
15 ♂D127074+4 (5.7)633
16 ♂D128988−1 (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.

Figure 2.
Figure 2.

Spirochetemia curves for Mastomys natalensis infected with Borrelia crocidurae showing the number of spirochetes observed on days 4–28 postinoculation. Animals in rows AD correspond to age, gender, and cage mates identified in Table 1. y axes vary in numbers counted to better show changes in patterns in animals with lower counts.

Citation: The American Journal of Tropical Medicine and Hygiene 101, 6; 10.4269/ajtmh.19-0590

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

Acknowledgments:

We thank Marissa Woods and Greg Saturday for animal care and veterinary assistance.

REFERENCES

  • 1.

    Trape JF et al. 2013. The epidemiology and geographic distribution of relapsing fever borreliosis in West and North Africa, with a review of the Ornithodoros erraticus complex (Acari: Ixodida). PLoS One 11: e78473.

    • Search Google Scholar
    • Export Citation
  • 2.

    Leger A, 1917. Spirochète de las musaraigne (Crocidura stampflii Jentink). Bull Soc Path Exot 10: 280281.

  • 3.

    Boiron H, 1949. Considérations sur la fièvre récurrente a tiques au Sénégal. L’importance du rat comme réservoir de virus. Bull Soc Path Exot 42: 6270.

    • Search Google Scholar
    • Export Citation
  • 4.

    Mathis MM, Durieux C, 1934. Fréquence a Dakar de la spirochétose récurrente a tiques. Bull Acad Med 111: 37.

  • 5.

    Trape JF, Duplantier JM, Bouganali H, Godeluck B, Legros F, Cornet JP, Camicas JL, 1991. Tick-borne borreliosis in west Africa. Lancet 337: 473475.

  • 6.

    Godeluck B, Duplantier JM, Ba K, Trape JF, 1994. A longitudinal survey of Borrelia crocidurae prevalence in rodents and insectivores in Senegal. Am J Trop Med Hyg 50: 165168.

    • Search Google Scholar
    • Export Citation
  • 7.

    Diatta G, Trape JF, Legros F, Rogier C, Duplantier JM, 1994. A comparative study of three methods of detection of Borrelia crocidurae in wild rodents in Senegal. Trans R Soc Trop Med Hyg 88: 423424.

    • Search Google Scholar
    • Export Citation
  • 8.

    Trape JF, Godeluck B, Diatta G, Rogier C, Legros F, Albergel J, Pepin Y, Duplantier JM, 1996. The spread of tick-borne borreliosis in west Africa and its relationship to sub-saharan drought. Am J Trop Med Hyg 54: 289293.

    • Search Google Scholar
    • Export Citation
  • 9.

    Vial L, Diatta G, Tall A, Ba EH, Bouganali H, Durand P, Sokhna C, Rogier C, Renaud F, Trape JF, 2006. Incidence of tick-borne relapsing fever in west Africa: longitudinal study. Lancet 368: 3743.

    • Search Google Scholar
    • Export Citation
  • 10.

    Elbir H, FotsoFotso A, Diatta G, Trape JF, Arnathau C, Renaud F, Durand P, 2015. Ubiquitous bacteria Borrelia crocidurae in western African ticks Ornithodoros sonrai. Parasit Vectors 8: 477.

    • Search Google Scholar
    • Export Citation
  • 11.

    Schwan TG, Anderson JM, Lopez JE, Fischer RJ, Raffel SJ, McCoy BN, Safronetz D, Sogoba N, Maïga O, Traore SF, 2012. Endemic foci of the tick-borne relapsing fever spirochete Borrelia crocidurae in Mali, West Africa, and the potential for human infection. PLoS Negl Trop Dis 6: e1924.

    • Search Google Scholar
    • Export Citation
  • 12.

    Diatta G, Duplantier JM, Granjon L, Ba K, Chauvancy G, Ndiaye M, Trape JF, 2015. Borrelia infection in small mammals in West Africa and its relationship with tick occurrence inside burrows. Acta Trop 152: 131140.

    • Search Google Scholar
    • Export Citation
  • 13.

    Musser GG, Carleton MD, 2005. Superfamily Muroidea. Wilson DE, Reeder DM, eds. Mammal Species of the World. Baltimore, MD: The Johns Hopkins University Press, 8941531.

    • Search Google Scholar
    • Export Citation
  • 14.

    Barbour AG, 1984. Isolation and cultivation of Lyme disease spirochetes. Yale J Biol Med 57: 521525.

  • 15.

    Raffel SJ, Battisti JM, Fischer RJ, Schwan TG, 2014. Inactivation of genes for antigenic variation in the relapsing fever spirochete Borrelia hermsii reduces infectivity in mice and transmission by ticks. PLoS Pathog 10: e1004056.

    • Search Google Scholar
    • Export Citation
  • 16.

    Mooser H, 1958. Erythozyten-adhäsion und hämagglomeration durch rückfallfiefer-spirochäten. Z Tropenmed Parasitol 9: 93111.

  • 17.

    Burman N, Shamaei-Tousi A, Bergström S, 1998. The spirochete Borrelia crocidurae causes erythrocyte rosetting during relapsing fever. Infect Immun 66: 815819.

    • Search Google Scholar
    • Export Citation
  • 18.

    Shamaei-Tousi A, Martin P, Bergh A, Burman N, Brännstrom T, Bergström S, 1999. Erythrocyte-aggregating relapsing fever spirochete Borrelia crocidurae induces formation of miroemboli. J Infect Dis 180: 19291938.

    • Search Google Scholar
    • Export Citation
  • 19.

    Zumpt F, 1959. Is the multimammate rat a reservoir for Borrelia duttoni? Nature 184: 793794.

Author Notes

Address correspondence to Tom G. Schwan, Laboratory of Bacteriology, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 903 South 4th St., Hamilton, MT 59840. E-mail: tschwan@nih.gov

Financial support: This work was funded by the International Centers for Excellence in Research (ICER) and the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institute of Health.

Authors’ addresses: Kristin Boardman, Cary Institute of Ecosystem Studies, Millbrook, NY, E-mail: kristinboardman94@yahoo.com. Kyle Rosenke, Heinz Feldmann, and Tom G. Schwan, Rocky Mountain Laboratories, National Institute of Allergy and Infectious Diseases, Hamilton, MT, E-mails: kyle.rosenke@nih.gov, feldmannh@niaid.nih.gov, and tschwan@nih.gov. David Safronetz, National Microbiology Laboratory, Public Health Agency of Canada, Winnipeg, Canada, E-mail: david.safronetz@canada.ca.

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