Rodents are reservoirs for over 60 zoonotic pathogens, contributing to the global emergence and re-emergence of infectious diseases (Woolhouse and Gowtage-Sequeria, 2005; Luis et al., 2013). Representing 43% of mammalian diversity, rodents are integral to the animal-human interface in Bushbuckridge-East, South Africa, where their prevalence in homes reaches 76% (Huchon et al., 2002; Berrian et al., 2016). Studies in this area have connected rodent-borne zoonoses with acute febrile illnesses in humans, finding 9.5% of such patients positive for Bartonella spp. and showing exposure to Coxiella burnetii and Leptospira spp. in others (Simpson et al., 2018). Given the association of certain Bartonella species with rodents, monitoring these hosts is vital, because their role in transmitting pathogens in Bushbuckridge-East remains unclear. Prior research in the area identified Anaplasma phagocytophilum in Mastomys natalensis and Rattus tanezumi captured from urban and periurban areas using 16S rRNA and gltA gene sequencing, highlighting the need for continued surveillance (Kolo et al., 2020).

Mastomys natalensis and R. tanezumi, closely associated with humans, may transmit infections (Skinner and Chimimba, 2005; Bonwitt et al., 2017). Next-generation sequencing (NGS) has unveiled diverse bacterial communities across hosts (Greay et al., 2018), yet rodent microbiomes remain underexplored (Cohen et al., 2015; Razzauti et al., 2015; Rynkiewicz et al., 2015; Ge et al., 2018). Research in China and France focused on bacteria detected from rodent spleens (Razzauti et al., 2015; Ge et al., 2018), while studies in Israel and the US examined the microbiome of rodent blood and their ectoparasites (Cohen et al., 2015; Rynkiewicz et al., 2015). These studies emphasized the importance of understanding host and vector bacterial communities as tools for managing vector-borne diseases (Cohen et al., 2015; Rynkiewicz et al., 2015). Despite rodents’ role as reservoirs for tick-borne pathogens (Telfer et al., 2007), South Africa’s wild murid rodents’ pathogenic diversity is poorly documented, often limited to single-genus studies (Hatyoka et al., 2019a, 2019; Kolo et al., 2020).

Mastomys natalensis and M. coucha, morphologically similar multimammate mice, are prevalent in southern Africa’s natural and human environments (Skinner and Chimimba, 2005), inhabiting grain stores and homes (De Graaff, 1981). Mastomys coucha is the primary host for flea-borne Yersinia pestis the cause of bubonic plague (Davis, 1964), while M. natalensis is associated with the Lassa fever virus in West Africa (Skinner and Chimimba, 2005).

The aim of this project was to elucidate the bacterial pathogens present in the blood of Mastomys species captured from different habitats at the animal-human interface in the Bushbuckridge-East community by sequencing of near full-length 16S rRNA gene amplicons.

This study identified a variety of bacterial species in the blood of Mastomys and Steatomys spp., predominantly Bartonella spp., at 64% of sequences. All rodents were infected, aligning with prior research that showed Bartonella’s prevalence in rodent blood and fleas in Israel and the US (Cohen et al., 2015; Rynkiewicz et al., 2015), although Rynkiewicz et al. (2015) reported a higher infection rate (97.8%). In this study, B. grahamii represented 33.9% and 23.9% of bacterial sequences in M. natalensis and M. coucha. Previously detected in Gerbilliscus leucogaster in South Africa and Rattus norvegicus in Nigeria (Pretorius et al., 2004; Kamani et al., 2013), B. grahamii is associated with retinal occlusions, cat scratch disease and neuroretinitis in humans (Kerkhoff et al., 1999; Serratrice et al., 2003; Oksi et al., 2013). The co-infection with other Bartonella spp. highlights the need for further studies on Bartonella’s pathogenicity and transmission dynamics, given its public health implications. The presence of zoonotic B. grahamii in local rodents underscores the potential health risks to the community.

Rodents in peri-urban and communal rangelands had higher Bartonella burdens compared to those in protected areas, indicating a correlation between Bartonella spp. prevalence and human proximity.

Bartonella mastomydis constituted a significant portion of bacterial sequences in M. coucha (35.4%) and M. natalensis (14.9%). The organism has previously been detected in R. tanezumi flavipectus in China (Li et al., 2004). Its presence in South African rodents coupled with the molecular confirmation of invasive R. tanezumi in South Africa (Bastos et al., 2011) suggests spillover of infection between invasive and indigenous species (Hatyoka et al., 2019a).

Analysis of Bartonella gltA gene sequences confirmed B. mastomydis in M. natalensis and M. coucha across habitats. This strain has been found in other South African murids including Aethomys ineptus, Micaelamys natalensis and R. rattus (Mostert, 2010; Brettschneider et al., 2012; Hatyoka et al., 2019a). These studies pre-date the formal description of this species, all noting the close relationship of these strains to Bartonella elizabethae, a zoonotic species, thus 16S rRNA sequencing may misclassify B. mastomydis as B. elizabethae (Kosoy et al., 2018). These results represent the first confirmation of this organism in Mastomys in South Africa. Phylogeny of the gltA gene revealed a new Bartonella sp. in two M. natalensis specimens from Gottenburg and Manyeleti. Another taxon closely related to a Bartonella sp. AN-nh2 previously detected in Micaelamys namaquensis from the Free state, South Africa (Pretorius et al., 2004) was detected in R31 (M. natalensis) from Gottenburg.

Bartonella henselae, known for causing endocarditis and occult infections in immunocompromised humans (Hadfield et al., 1993; Breitschwerdt et al., 2007), was detected in R30 (Gottenburg). Bartonella henselae has been previously detected in cats and humans in South Africa (Kelly, 1996; Frean et al., 2002; Trataris et al., 2012). Its prior detection in two AFI patients in Bushbuckridge-East (Simpson et al., 2018) and now in Mastomys spp. from the same area indicates its potential role in human infections.

Our finding that Mastomys spp. across habitat areas were concurrently infected with multiple Bartonella spp. aligns with prior research showing a variety of Bartonella spp. in Chinese and Spanish rodents (Ying et al., 2002) (Márquez et al., 2008). Similarly, three distinct Bartonella lineages were detected in Rhabdomys pumilio (Western Cape) and A. ineptus (Gauteng) (Hatyoka et al., 2019a, 2019).

As arthropod vectors transmit Bartonella spp. (Breitschwerdt and Kordick, 2000), we examined the ectoparasites from the rodents, recovering 439 ectoparasites. Regulations of the South African Department of Agriculture, Land Reform and Rural Development (DALRRD) [which stated that whole body rodent carcasses must first be frozen at -80°C and stored at Hans Hoheisen Research Centre, Mpumalanga South Africa before transport on dry ice to a BSL3 provincial laboratory at Stellenbosch, South Africa where ectoparasites could be removed] prevented immediate screening of the ectoparasites for Bartonella spp. or any other pathogens at the time of the rodent capture. Nevertheless, the findings of fleas, ticks and mites is consistent with vectored transmission. Results of ectoparasite screening studies are currently ongoing and will be reported at a later time.

Overall, 17% of bacterial sequences in rodent blood corresponded to Pseudomonas spp., primarily from R21 and R159, from Manyeleti, with the organism making 91.6% and 81% of the total sequences obtained from the two samples. Pseudomonas spp. are opportunistic pathogens causing infection in immunocompromised rodents (Baker, 1998), yet their presence could stem from contamination, as they are known contaminants in 16S rRNA gene sequencing (Salter et al., 2014). DNA extraction kits and other laboratory reagents have also been implicated as sources of bacterial DNA contamination in microbiome studies (Salter et al., 2014). However, physical signs like tail growths and foot deformities associated with bumblefoot (ulcerative pododermatitis) observed in some of rodents in this study suggest valid Pseudomonas infections (Blair, 2013) ( Supplementary Figure S5 ).

Ochrobactrum spp. made up 7.2% of sequences from rodent blood, in M. natalensis (R29, R30, R31) from Gottenburg and Steatomys (R53) from Manyeleti. In R53, Ochrobactrum dominated comprising 85% of the total sequences. Recognized as opportunistic nosocomial pathogens (Hagiya et al., 2013), Ochrobactrum can present clinical symptoms that mimic the more virulent Brucella spp. Their close phylogenetic relationship has led to discussions on unifying Ochrobactrum and Brucella, with dissenting views (Hördt et al., 2020; Oren and Garrity, 2020). The CDC has recently unified these taxa (CDC, 2022) and the American Society for Microbiology Clinical and Public Health Microbiology Committee suggested that clinical laboratories report the affected organisms as Brucella (Ochrobactrum) species to differentiate them from the archetypal Brucella agent (She et al., 2023). Recently, 78 scientists highlighted significant flaws in the proposed nomenclature, citing insufficient phylogenetic analysis and exclusion of expert opinion, which could lead to serious risks for personnel handling zoonotic Brucella, particularly in poorer nations (Moreno et al., 2023). Therefore, based on the current knowledge on both organisms, we agree that Ochrobactrum and Brucella spp. be maintained as separate genera and have treated them as such.

Anaplasma centrale and A. marginale were detected in R20 (M. coucha) from Gottenburg and A. phagocytophilum in R98 (M. natalensis) from Hlalakahle. Typically, A. marginale and A. centrale infect cattle and other wild ruminant species (Aubry and Geale, 2011; Hosseini-Vasoukolaei et al., 2014; Wu et al., 2015; Khumalo et al., 2016, 2018). The detection of A. marginale and A. centrale in wild rodents was surprising as these species are thought to only infect ruminants. Because the number of sequences obtained was low, and sequence coverage was high (Rhoads and Au, 2015), their detection could be due to salivary secretions from infected ticks rather than successful establishment of infection. However, rodents in this study had a low tick burden and the Anaplasma positive rodents had no ticks on them at the time of capture. Anaplasma phagocytophilum detection in rodents, dogs and cattle from the study area has been previously reported (Kolo et al., 2020).

An Ehrlichia sp. was identified in R75 from Hlalakahle and R172 from Gottenburg (M. natalensis) with 99% identity to Ehrlichia sp. Ehf669 (AY309969) detected from Haemaphysalis ticks collected from dogs in Japan (Inokuma et al., 2004) and 98% identity to E. chaffeensis (NR_074500). Ehrlichia chaffeensis causes human monocytic ehrlichiosis (HME), a tick-borne zoonosis in the US transmitted by Amblyomma americanum ticks (Paddock and Childs, 2003). Previously, antibodies against E. chaffeensis have been detected in dogs in the Free State (Pretorius and Kelly, 1998). This study reports the first finding of an E. chaffeensis-like sequence in South African rodents.

Coxiella burnetii, the causative agent of Q fever, was detected in R12 (M. coucha) from Tlhavekisa marking its first detection from a wild rodent in South Africa. Q fever, manifesting as an acute febrile illness and chronic endocarditis in humans, and linked to livestock abortion (Vanderburg et al., 2014), is transmitted by ticks, aerosols or consumption of contaminated animal products (Maurin and Raoult, 1999). In South Africa, C. burnetii antibodies have been detected in cattle (Gummow et al., 1987), and wild dogs in the Kruger Park (Van Heerden et al., 1995).

We did not detect any Rickettsia spp. in rodent blood in this study contrasting with Essbauer et al. (2018) who detected pathogenic R. conorii, R. massiliae, R. felis and R. helvetica, in ear tissue of rodents sampled across South Africa and Namibia. Rickettsial pathogens are usually found in the dermis, vascular endothelium and spleen (Hawley et al., 2007; Bayliss et al., 2009) which may explain the absence of detection in blood samples. Essbauer et al. (2018) did not report any Rickettsia in M. natalensis, but found a 9% infection rate in M. coucha, which were identified as “Candidatus Rickettsia africaustralis”.

The detection of Anaplasma, Ehrlichia, B. henselae, and Coxiella in Mastomys spp. in this study was minimal, possibly lacking statistical significance. However, their zoonotic and veterinary significance warranted emphasis.

PCA analysis showed that rodents from Tlhavekisa and Hlalakahle shared similar blood microbiome profiles as opposed to rodents from Tlhavekisa and Gottenburg rodents which had distinct profiles. This is despite Gottenburg being closer to Tlhavekisa than Hlalakahle (7.7 km vs 8.1 km). The blood microbiome of Manyeleti rodents was also distinct, with Pseudomonas and Ochrobactrum spp. dominance. This finding supports studies by Gavish et al. (2014) and Gall et al. (2017) that suggested factors like geography, host diversity, and human interaction might influence bacterial diversity in hosts and vectors. PCA also revealed an association of positive correlations between Bartonella spp. and unclassified Rhizobiales and between Ochrobactrum spp., unclassified Brucellaceae, and B. henselae. Overall, the small sample size meant individual variations significantly influenced the results.

Our results suggest that the rodent blood microbiome is relatively species-sparse. Although the mean sequencing depth was low, the rarefaction curves suggest that the species richness in all samples was adequately captured, and further sequencing would not significantly increase the number of observed species. It is possible that the observed pattern reflects some degree of bias towards dominant species as PCR-based amplification and sequencing methods often favor highly abundant sequences, which may overshadow less abundant or rare species. Under-sampling rare species is a known limitation of low sequencing depths, and future studies could address this by increasing the sequencing depth to capture rare OTUs.

The study identified 17 OTUs across ten species and six genera, with Bartonella grahamii and B. mastomydis dominating the dataset. Approximately 90.0% of the total reads could be assigned to valid taxa, while reads classified as “Rare” accounted for 4.7% of the total reads. This taxonomic distribution indicates that the majority of the reads were assigned to non-rare taxa, suggesting a skew toward dominant species in the microbiome. These findings correspond with previous studies that show rodent blood microbiomes are often dominated by a few bacterial species due to selective pressures and niche specificity in the bloodstream environment (Gavish et al., 2014; Rynkiewicz et al., 2015). Another study found that the rodent blood microbiome is influenced by factors such as host immunity and interspecific bacterial interactions that favors the dominance of specific bacterial species (Cohen et al., 2015). This species-sparse nature of the rodent blood microbiome may explain why a mean sequencing depth of 2,602 reads was sufficient to capture the majority of OTUs present.

In conclusion, this study provides foundational data on bacterial diversity in the blood of indigenous murid rodents and highlights Mastomys spp. as key reservoirs of bartonellae. It reports the first confirmation of B. mastomydis detection in two cryptic Mastomys species in South Africa and documents the detection of important zoonotic pathogens including Ehrlichia spp., and C. burnetii.