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Table of Contents
LETTER TO EDITOR
Year : 2020  |  Volume : 13  |  Issue : 2  |  Page : 94-96

First evidence of Bartonella phoceensis and Candidatus Mycoplasma haemomuris subsp. ratti in synanthropic rodents in Malaysia


1 Tropical Infectious Diseases Research and Education Centre (TIDREC), University of Malaya, Malaysia
2 Department of Parasitology, Faculty of Medicine, University of Malaya, Malaysia
3 Faculty of Medicine and Defence Health, National Defence University of Malaysia, Malaysia
4 Tropical Infectious Diseases Research and Education Centre (TIDREC); Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Malaysia
5 Department of Parasitology, Faculty of Medicine; Centre of Excellence for Research in AIDS (CERIA), University of Malaya, Malaysia

Date of Submission12-Aug-2019
Date of Decision14-Dec-2019
Date of Acceptance16-Dec-2019
Date of Web Publication14-Jan-2020

Correspondence Address:
Tiong Kai Tan
Department of Parasitology, Faculty of Medicine, University of Malaya
Malaysia
Van Lun Low
Tropical Infectious Diseases Research and Education Centre (TIDREC), University of Malaya, Malaysia
Malaysia
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1995-7645.275418

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How to cite this article:
Low VL, Tan TK, Ibrahim J, AbuBakar S, Lim YA. First evidence of Bartonella phoceensis and Candidatus Mycoplasma haemomuris subsp. ratti in synanthropic rodents in Malaysia. Asian Pac J Trop Med 2020;13:94-6

How to cite this URL:
Low VL, Tan TK, Ibrahim J, AbuBakar S, Lim YA. First evidence of Bartonella phoceensis and Candidatus Mycoplasma haemomuris subsp. ratti in synanthropic rodents in Malaysia. Asian Pac J Trop Med [serial online] 2020 [cited 2020 Sep 20];13:94-6. Available from: http://www.apjtm.org/text.asp?2020/13/2/94/275418



Rodent-borne leptospirosis is by far the most common bacterial zoonosis and it is an important emerging global public health concern in Southeast Asia. Bacterial pathogens associated with rodents, especially those that live in close association with humans have been underreported. To fill this knowledge gap, the present study was undertaken to explore other neglected disease agents that can naturally infect synanthropic rodents. Both Bartonella and Mycoplasma pose major health threats to various animal hosts and they are also emerging zoonoses and pathogens of public health concern. With these in mind, we aimed to detect the presence of Bartonella and Mycoplasma bacteria in synanthropic rodents from a densely populated capital city of Malaysia, Kuala Lumpur.

Rodent collection has been described elsewhere in detail[1]. Briefly, a total of 134 synanthropic rodents comprising Rattus (R.) rattus diardii, R. norvegicus, R. argentiventer, R. tiomanicus, and R. exulans were trapped from two human populated areas (Sentul and Chow Kit) in Kuala Lumpur using steel wire traps. Blood was collected from the heart using a needle and syringe and placed into EDTA tube. Genomic DNA was extracted from whole blood using the QIAamp DNA Blood Mini Kit (QIAGEN Inc.,Valencia, CA).

Bartonella DNA detection was performed using polymerase chain reaction (PCR) with primers (325-5’CTT CAG ATG ATG ATC CCA AGC CTT TTG GCG-3’), and (1100-5’ GAA CCG ACG ACC CCC TGC TTG CAA AGC-3’) which amplify a portion of the 16S-23S rRNA intergenic spacer region[2]. PCR amplification was performed in a final volume of 25 μL containing 25-50 ng genomic DNA, 12.5 μL of MyTaq Red Mix (Bioline Reagents Ltd., London, UK) and 10 pmol of each forward and reverse primer. PCR was performed using the Applied Biosystems Veriti 96-Well Thermal Cycler (Applied Biosystems, Inc., Foster City, CA) with the following thermal protocol: initial denaturation at 95 °C for 2 min followed by 55 cycles of denaturing at 94 °C for 15 s, annealing at 66 °C for 15 s, and extension at 72 °C for 15 s, and final extension at 72 °C for 1 min.

For Mycoplasma DNA detection, samples were subjected to 16S rRNA gene amplification using a universal Mycoplasma primer pair (HBT-F-5’ ATA CGG CCC ATA TTC CTA CG-3’ and HBT-R-5’ TGC TCC ACC ACT TGT TCA-3’)[3], 25-50 ng genomic DNA in a total volume of 25 μL, with the following thermal protocol: 95 °C for 15 min and 50 cycles of 95 °C for 10 s, 55 °C for 15 s and 72 °C for 30 s, and 72 °C for 1 min.

Purified PCR amplicons were sequenced using an ABI PRISM 377 Genetic Analyzer (Applied Biosystems, Inc.). Representative sequences of Bartonella (MK953939-MK953940) and Mycoplasma (MK959182) generated from this study were deposited in the National Center for Biotechnology Information GenBank. A neighbour-joining (NJ) phylogenetic tree was plotted using MEGA6 (https://megasoftware.net/). The NJ bootstrap values were estimated using 1 000 replicates with Kimura’s two-parameter model of substitution (K2P distance). Brucella abortus (X95889) and Brucella melitensis (AY513568) were used as outgroups for the construction of Bartonella and Mycoplasma phylogenetic trees, respectively. Based on the 16S-23S fragment of Bartonella, successful PCR amplification was found in 5 out of 134 samples (3.73%). NJ phylogenetic analysis revealed that all five sequences clustered with Bartonella (B.) phoceensis sequence (AY515123) retrieved from the National Center for Biotechnology Information GenBank. B. phoceensis showed a sister relationship with B. rattimassiliensis, and distantly separated from the other Bartonella species [Figure 1]. For Mycoplasma, three samples (2.24%) were positive to 16S rRNA fragment and successfully sequenced. The three sequences formed a monophyletic clade with the sequences of Candidatus Mycoplasma (M.) haemomuris subsp. ratti (AB758434, AB758435 and AB758439) and showed a close relationship with Candidatus M. haemomuris subsp. musculi [Figure 2].
Figure 1: Neighbour-joining phylogenetic tree of Bartonella spp. based on the 16S-23S intergenic spacer region. Bootstrap values (NJ) are shown on the branches. Newly generated sequences are in bold.

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Figure 2: Neighbour-joining phylogenetic tree of Mycoplasma spp. based on the 16S rRNA sequences. Bootstrap values (NJ) are shown on the branches. Newly generated sequences are in bold.

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This study provides the first evidence for the presence of B. phoceensis and Candidatus M. haemomuris subsp. ratti in synanthropic rodents in Malaysia. Their prevalence rates (3.73% for Bartonella and 2.24% for Mycoplasma), however, were considered low in comparison to that previously reported in Malaysia. In the earlier study, five different Bartonella species (i.e., B. tribocorum, B. rattimassiliensis, B. coopersplainsensis, B. elizabethae, and B. queenslandensis) (13.7%) were isolated from kidney and spleen homogenates of rats[4]. Nevertheless, the detection protocols adopted (16S-23S versus gltA and rpoB; and blood versus kidney and spleen) may have contributed to the discrepancies in the recovered species in both studies. Among the detected species, B. rattimassiliensis, B. tribocorum, and B. elizabethae were implicated as causing human infections in Thailand[5]. The pathogenicity and zoonotic potential of B. phoceensis are unknown.

Mycoplasma spp. from various animals in Malaysia were reported at varying frequencies such as M. haemofelis (11.7%) in cats[6], M. wenyonii and Candidatus M. haemobos (69.0%) in cattle[7], and several species in various animal samples received in a veterinary diagnostic laboratory[8] .

Investigation of Mycoplasma in rodents in Malaysia is underappreciated, though M. arthritidis was reported in three rats (out of 10 rats) in a previous study[9]. In contrast, in the present study, we detected Candidatus M. haemomuris subsp. ratti, a subspecies of M. haemomuris which was recently incriminated as the anaemic pathogen of rats in Japan, and differentiated from Candidatus M. haemomuris subsp. musculi which mainly infects mice[10]. Animal Mycoplasma spp. rarely infect humans, but their zoonotic potential could not be disregarded since cases of human haemoplasma infection have been documented.

In conclusion, we report here the occurrence of B. phoceensis and Candidatus M. haemomuris subsp. ratti in synanthropic rodents for the first time in Malaysia. Our results suggest that synanthropic rodents can serve as the reservoir for these vector-borne pathogens. Nevertheless, their routes of transmission and zoonotic potential require further investigation.

Ethics statement

The study protocol [PAR/20/09/2011/J (R)] was reviewed and approved by the Institutional Animal Care and Use Committee, University of Malaya, Malaysia.

Conflict of interest statement

The authors declare that there are no competing interests.

Acknowledgement

This study was financially supported by the University of Malaya research grant (RP021C-16SUS) and Higher Institution Centre of Excellence (HICoE) program (MO002-2019).

Authors’ contributions

V.L.L. and T.K.T. wrote the manuscript and performed the experiments, J.I., S.A. and Y.A.L.L. contributed to the final version of the manuscript.



 
  References Top

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Tan TK, Low VL, Ng WH, Ibrahim J, Wang D, Tan CH, et al. Occurrence of zoonotic Cryptosporidium and Giardia duodenalis species/genotypes in urban rodents. Parasitol Int 2019; 69: 110-113.  Back to cited text no. 1
    
2.
Diniz PP, Maggi RG, Schwartz DS, Cadenas MB, Bradley JM, Hegarty B, et al. Canine bartonellosis: Serological and molecular prevalence in Brazil and evidence of co-infection with Bartonella henselae and Bartonella vinsonii subsp. berkhoffi. Vet Res 2007; 38: 697-710.  Back to cited text no. 2
    
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Criado-Fornelio A, Martinez-Marcos A, Buling-Saraña A, Barba-Carretero JC. Presence of Mycoplasma haemofelis, Mycoplasma haemominutum and piroplasmids in cats from southern Europe: A molecular study. Vet Microbiol 2003; 93(4): 307-317.  Back to cited text no. 3
    
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Tay ST, Mokhtar AS, Zain SN, Low KC. Isolation and molecular identification of Bartonellae from wild rats (Rattus species) in Malaysia. Am J Trop Med Hyg 2014; 90(6): 1039-1042.  Back to cited text no. 4
    
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Kosoy M, Bai Y. Bartonella bacteria in urban rats: A movement from the jungles of Southeast Asia to metropoles around the globe. Front Ecol Evol 2019; 7: 88.  Back to cited text no. 5
    
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Aklilu E, Shaharulnizim N, Francis JJ, Anurrdin SH. Molecular investigation of Mycoplasma haemofelis in stray cats in Kota Bharu, Kelantan. Trop Biomed 2016; 33(4): 608-612.  Back to cited text no. 6
    
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Mohd Hasan LI, Kho KL, Koh FX, Hassan Nizam QN, Tay ST. Molecular evidence of hemoplasmas in Malaysian cattle and ticks. Trop Biomed 2017; 34(3): 668-674.  Back to cited text no. 7
    
8.
Dahlia H, Tan LJ, Zarrahimah Z, Chandrawathani P, Ramlan M. Isolation of Mycoplasma species from various animal hosts in Malaysia. Malaysian J Vet Res 2011; 2(1): 53-57.  Back to cited text no. 8
    
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Premaalatha B, Nurulaini R, Zawida Z, Norakmar I, Imelda LV, Adnan M, et al. A survey of bacterial and parasitic infections of rats caught in the Veterinary Research Institute (VRI), Ipoh. Malaysian J Vet Res 2010; 1(1): 45-50.  Back to cited text no. 9
    
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Harasawa R, Fujita H, Kadosaka T, Ando S, Rikihisa Y. Proposal for ‘Candidatus Mycoplasma haemomuris subsp. musculi’ in mice, and ‘Candidatus Mycoplasma haemomuris subsp. ratti’ in rats. Int J Syst Evol Microbiol 2015; 65: 734-737.  Back to cited text no. 10
    


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  [Figure 1], [Figure 2]



 

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