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

Van Lun Low, Tiong Kai Tan, Jamaiah Ibrahim, Sazaly AbuBakar, Yvonne Ai Lian Lim

1Tropical Infectious Diseases Research and Education Centre (TIDREC), University of Malaya, Malaysia

2Department of Parasitology, Faculty of Medicine, University of Malaya, Malaysia

3Faculty of Medicine and Defence Health, National Defence University of Malaysia, Malaysia

4Department of Medical Microbiology, Faculty of Medicine, University of Malaya, Malaysia

5Centre of Excellence for Research in AIDS (CERIA), University of Malaya, Malaysia

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 ℃ for 2 min followed by 55 cycles of denaturing at 94 ℃ for 15 s, annealing at 66 ℃ for 15 s, and extension at 72 ℃ for 15 s, and final extension at 72 ℃ 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 ℃for 15 min and 50 cycles of 95 ℃ for 10 s, 55 ℃ for 15 s and 72 ℃for 30 s, and 72 ℃ 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).

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].

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.

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.