Molecular Detection of Bartonella in Lions, Zimbabwe

Abstract

Domestic cats around the world are commonly infected with Bartonella and there is growing evidence that wild felids can also be infected. To provide further data on Bartonella infections in African lions (Panthera leo). We used PCR and sequencing of the 16S rRNA to detect Bartonella in whole blood DNA samples from a convenience sample of 84 lions. Four out of 84 (5%) lions were positive by PCR. The 305-bp sequence of the amplicons was identical in all four lions and had highest similarity to the recognized species Bartonella grahamii (304/305 matches) and Bartonella henselae (303/305 matches). It was identical to unidentified strains of Bartonella from mice in the US, and voles in Lapland and Alaska. Our Zimbabwe data adds to that available from South Africa to show infections with Bartonella are not uncommon in lions in Southern Africa and perhaps the continent. The range of Bartonella species that infect lions and their effects on the animals’ health remains to be determined.

Keywords: Lion; Bartonella; Zimbabwe

Introduction

Bartonella are Gram-negative bacteria that are found principally in erythrocytes in their reservoir hosts. The organisms grow slowly on conventional agar media and with the advent of molecular testing there has been a dramatic increase in our knowledge of the species with the five known species in 1993 [1] expanding to over 33 today [2]. One of the first new species to be described was Bartonella henselae, the agent of cat scratch disease, bacilliary angiomatosis and endocarditis in people. The organism is commonly found in domestic cats that can remain bacteremic for long periods, even years, although only very infrequently, if ever, showing clinical signs [3].

Five Bartonella spp. have now been identified in domestic cats: B. henselae, Bartonella clarridgeiae, Bartonella koehlerae, Bartonella bovis (formerly B. weissii) and Bartonella quintana [2]. Bartonella henselae has also been found in wild felid species in Africa, in lions (Panthera leo) [4,5] and a cheetah [6]. Further, a species intermediate between B. henselae and B. koehlerae (Namibian cheetah strain 1178) was isolated from a wild cheetah in Namibia [5].

Studies of wild cats in the US have shown exposure to Bartonella spp. is high with seroprevalences of up to 53% [7]. Bartonella henselae has been shown to occur in wild felids [8] as well as a new subspecies, B. koehlerae subsp. boulouisii and B. koehlerae subsp. bothieri, for which mountain lions and bobcats are the natural reservoirs, respectively [2].

To provide more data on Bartonella infections in lions in southern Africa we used a PCR to analyzed DNA remaining from a study on tick-borne diseases in lions [9].

Materials and Methods

The DNA used in the study had been extracted from convenience samples of whole blood from 84 adult captive lions from Zimbabwe (Gweru, N=67; Masvingo, N=6; Dollar Block farm N=8; Hwange, N=3) for a previous study on tick-borne pathogens in lions [9]. That study had been reviewed and approved by the Institutional Animal Care and Use Committee of the Ross University School of Veterinary Medicine, St Kitts. For the current study, the DNA was analyzed by PCR as described previously [10] for a 305-bp section of the 16S rRNA with forward (5’-AGCGCACTCTTTTAGAGTGAGCGG-3’) and reverse primers (5’-CATGGCTGGATCAGGGTTGCC-3’) that detect the recognized Bartonella in GenBank. Positive control DNA was from B. henselae and B. clarridgeiae while negative control DNA was from the related species Brucella melitensis, Br. chilestrain, Wolbachia, Coxiella burnetii, Rickettsia felis, R. rickettsiae, Anaplasma phagocytophilum and A. marginale. Amplicons of positive PCRs were sequenced (GenScript, Nanjing, Jiangsu, China) and compared with sequences available in GenBank.

Results and Discussion

Four (5%) of the lions were PCR positive with amplicons that were all identical. Comparing the sequences of the amplicons with those of recognized species in GenBank revealed the Bartonella in the lions was closest to a number of strains of Bartonella grahamii with which it had 1 mismatch (304/305 matches, 99%, 4e-155). The flea-borne B. grahamii has been widely described in wild rodents, including in Nigeria, and has been associated with eye conditions in people [11]. The next most closely related species was B. henselae with which the lion strain had 2 mismatches (303/305; 99%; 2e-153). Bartonella henselae has previously been identified by PCR and sequencing in both wild (4%; 2/58) [5] and semi-captive (2%; 1/65) lions [12]. In the latter study, anti-Bartonella antibodies were detected by ELISA in 29% of the lions. In a survey in Zambia, however, none of 48 wild lions had evidence of Bartonella infection by PCR [13] and, in a survey in Zimbabwe, none of 4 lions were culture-positive [6]. Bartonella henselae has also been isolated from a captive cheetah in Zimbabwe [6] and a Bartonella intermediate between B. henselae and B. koehlerae (Namibian cheetah strain 1178) was isolated from a wild cheetah in Namibia [5].

Our lion Bartonella had identical GenBank sequences with a number of isolates that have not been definitively identified. These included ones found in voles from Lapland (KT961186) [14] and Alaska (EU97535) [15], a cat flea (FJ981670), and a mouse (Peromyscus leucopus) in the Midwestern USA (U71322) [16]. It is important to note that, although our PCR detected a variable region of the 16S rRNA, it is recommended that a number of other genes, such as the gltA [10], are also sequenced to ensure the correct classification of newly identified strains. Shortcomings in using only one gene sequence for speciation are apparent from the data on the Midwestern mouse isolate referred to above. Although it had an identical 16S rRNA sequence (U71322) to our Bartonella and to B. grahamii, when sequencing was performed on PCR products amplified from the gltA it was found the isolate was most closely related to Bartonella vinsonii. Sequencing of further genes would have been required to more clearly define the isolate. Unfortunately we had only very limited DNA available to us and were, then, unable to confirm the identity of the lion Bartonella by sequencing other genes.

In summary, our study shows lions in Zimbabwe can also be infected with Bartonella which together with data from South Africa indicate infections are widespread in the region and perhaps the continent. Further, our data is consistent with other studies showing there are as yet uncharacterized Bartonella in Southern African, not only in lions [5] but also bats [17], mice [18], and small mammals [12]. Further studies are needed to characterize the clinical significance of infections with Bartonella in lions, especially in relation to reportedly common coinfections with other vector borne agents [9] and FIV [19]. There are also possible public health implications, in particular as HIV infections are common in Southern Africa and studies have shown high prevalences of HIV-positive patients (up to 23%) are positive for Bartonella in PCRs on blood samples [20].

Acknowledgments

This project was supported by the Ross University School of Veterinary Medicine, the Animal and Wildlife Area Research and Rehabilitation (AWARE) Trust of Zimbabwe.

Conflict of Interest

The authors declared no competing interests.

Author Contributions

Ke Huang, Patrick Kelly, and Chengming Wang designed the study and wrote the manuscript. Lisa Marabini and Keith Dutlow collected the samples and Ke Huang, Jilei Zhang, and Yi Yang performed the laboratory analyses. All authors participated in the review of the manuscript.

References

1. Brenner DJ, O'Connor SP, Winkler HH, Steigerwalt AG. Proposals to unify the genera Bartonella and Rochalimaea, with descriptions of Bartonella quintana comb. nov., Bartonella vinsonii comb. nov., Bartonella henselae comb. nov., and Bartonella elizabethae comb. nov., and to remove the family Bartonellaceae from the order Rickettsiales. Int. J. Syst. Bacteriol. 1993; 43: 777-786.
2. Chomel BB, Molia S, Kasten RW, Borgo GM, Stuckey MJ, Maruyama S, et al. Isolation of Bartonella henselae and two new Bartonella subspecies, Bartonella koehlerae subspecies boulouisii subsp. nov and Bartonella koehlerae subspecies bothieri subsp nov from free-ranging Californian mountain lions and bobcats. PLoS ONE. 2016; 11: e0148299.
3. Yamamoto K, Chomel BB, Kasten RW, Hew CM, Weber DK, Lee WI, et al. Experimental infection of domestic cats with Bartonella koehlerae and comparison of protein and DNA profiles with those of other Bartonella species infecting felines. J. Clin. Micro. 2002; 40: 466-474.
4. Pretorius AM, Beati L, Birtles RJ. Diversity of bartonellae associated with small mammals inhabiting Free State province, South Africa. Int. J. Syst. Evol. Microbiol. 2004; 54: 1959-1967.
5. Molia S, Kasten RW, Stuckey MJ, Boulouis HJ, Allen J, Borgo GM, et al. Isolation of Bartonella henselae, Bartonella koehlerae subsp. koehlerae, Bartonella koehlerae subsp. bothieri and a new subspecies of B. koehlerae from free-ranging lions (Panthera leo) from South Africa, cheetahs (Acinonyx jubatus) from Namibia and captive cheetahs from California. Epidemiol. Infect. 2016; 144: 3237-3243.
6. Kelly PJ, Rooney JJ, Marston EL, Jones DC, Regnery RL. Bartonella henselae isolated from cats in Zimbabwe. Lancet. 1998; 351: 1706.
7. Yamamoto K, Chomel BB, Lowenstine LJ, Kikuchi Y, Phillips LG, Barr BC, et al. Bartonella henselae antibody prevalence in free-ranging and captive wild felids from California. J. Wildl. Dis. 1998; 34: 56-63.
8. Girard YA, Swift P, Chomel BB, Kasten RW, Fleer K, Foley JE, et al. Zoonotic vector-borne bacterial pathogens in California mountain lions (Puma concolor), 1987-2010. Vect. Borne. Zoonotic. Dis. 2012; 12: 913-921.
9. Kelly P, Marabini L, Dutlow K, Zhang J, Loftis A, Wang C. Molecular detection of tick-borne pathogens in captive wild felids, Zimbabwe. Parasit. Vectors. 2014; 7: 514.
10. Huang K, Kelly PJ, Zhang J, Yang Y, Qi K, Wang C. Molecular detection of Bartonella spp. in China and St. Kitts. Can. J. Infect. Dis. Med. Microbiol. 2019.
11. Kamani J, Morick D, Mumcuoglu KY, Harrus S. Prevalence and diversity of Bartonella species in commensal rodents and ectoparasites from Nigeria, West Africa. PLoS Negl. Trop. Dis. 2013; 7: e2246.
12. Pretorius AM, Kuyl JM, Isherwood DR, Birtles RJ. Bartonella henselae in African lion, South Africa. Emerg. Infect. Dis. 2004; 10: 2257-2258.
13. Williams BM, Berentsen A, Shock BC, Teixiera M, Dunbar MR, Becker MS, et al. Prevalence and diversity of Babesia, Hepatozoon, Ehrlichia, and Bartonella in wild and domestic carnivores from Zambia, Africa. Parasitol. Res. 2014; 113: 911-918.
14. Koskela KA, Kalin-Mänttäri L, Hemmilä H, Smura T, Kinnunen PM, Niemimaa J, et al. Metagenomic Evaluation of Bacteria from Voles. Vector Borne Zoonotic Dis. 2017; 17: 123-133.
15. Matsumoto K, Cook JA, Goethert HK, Telford SR. Bartonella sp. Infection of voles trapped from an interior Alaskan site where ticks are absent. J. Wildl. Dis. 2010; 46: 173-178.
16. Hofmeister EK, Kolbert CP, Abdulkarim AS, Magera JM, Hopkins MK, Uhl JR, et al. Cosegregation of a novel Bartonella species with Borrelia burgdorferi and Babesia microti in Peromyscus leucopus. J. Infect. Dis. 1998; 177: 409-16.
17. Dietrich M, Tjale MA, Weyer J, Kearney T, Seamark E, Nel LH, et al. Diversity of Bartonella and Rickettsia spp. in Bats and Their Blood-Feeding Ectoparasites from South Africa and Swaziland. PLoS One. 2016; 11: e0152077.
18. Hatyoka L, Froeschke G, Kleynhans D, van der Mescht L, Heighton S, Matthee S, et al. Bartonellae of synanthropic four-striped mice (Rhabdomys pumilio) from the Western Cape Province, South Africa. Vect. Borne Zoonotic Dis. 2019; 19: 242-248.
19. McDermid KR, Snyman A, Verreynne F, Carroll JP, Penzhorn BL, Yabsley MJ. Surveillance for viral and parasitic pathogens in a vulnerable African lion (Panthera leo) population in the northern Tuli game reserve, Botswana. J. Wildl. Dis. 2017; 53: 54-61.
20. Trataris AN, Rossouw J, Arntzen L, Karstaedt A, Frean J. Bartonella spp. in human and animal populations in Gauteng, South Africa, from 2007 to 2009. Onderstepoort J. Vet. Res. 2012; 79: 452.