Ecological niche partitioning and urbanization-driven restructuring of flea-borne pathogen communities in stray carnivores of Peninsular Malaysia: discovery of uncharacterised Bartonella lineages

Flea-borne bacteria are emerging zoonotic pathogens of increasing public health importance, yet their ecological distribution, community structure, and genetic diversity across contrasting landscapes in Southeast Asia remain poorly understood. This study investigated flea infestation, flea-borne bacterial pathogens, and their ecological associations in stray cats and dogs across urban, peri-urban, and rural landscapes of Peninsular Malaysia. A total of 296 stray cats and dogs were examined for flea infestation, and all recovered fleas (n = 636) were identified morphologically and screened individually for Bartonella spp., Rickettsia spp., and Orientia tsutsugamushi using polymerase chain reaction (PCR) assays followed by Sanger sequencing and phylogenetic analysis. Risk factors associated with flea infestation, Bartonella spp. and Rickettsia spp. occurrence were evaluated using generalized linear models (GLMs), generalized linear mixed-effects models (GLMMs), and Firth's penalized logistic regression, respectively. Pathogen community structure was assessed using alpha diversity indices, non-metric multidimensional scaling (NMDS), permutational multivariate analysis of variance (PERMANOVA), and tripartite ecological network analysis. Overall flea infestation prevalence was 32.09% (95/296), with infestation significantly higher in cats (39.44%) than dogs (13.25%) (χ 2 = 10.672, df = 1, p = 0.001). All 636 fleas exhibited complete host-associated segregation, with Ctenocephalides felis (n = 485) occurring exclusively on cats and C. orientis (n = 151) exclusively on dogs. Flea-borne pathogens were detected in 71.86% (457/636) of fleas, comprising Bartonella henselae , B. clarridgeiae , B. koehlerae , several uncharacterized Bartonella lineages, Rickettsia felis , and R. asembonensis . Urban landscapes were associated with significantly higher Bartonella prevalence in C. felis (70.05%), whereas C. orientis was overwhelmingly dominated by R. asembonensis (94.04%). Ecological analyses demonstrated significant landscape-associated variation in pathogen community composition among C. felis populations but not C. orientis . Phylogenetic analysis identified a distinct, well-supported clade of previously uncharacterized Bartonella lineages (bootstrap = 100%). No O. tsutsugamushi was detected in any flea sample. Flea-borne pathogen communities in Peninsular Malaysia are structured by strong host–vector associations and ecological landscape, with peri-urban environments supporting greater pathogen diversity and urban landscapes demonstrated higher detection of Bartonella in C. felis . The discovery of previously uncharacterized Bartonella lineages expands the known diversity of flea-associated Bartonella in Malaysia and highlights the importance of integrating molecular epidemiology with community ecology to improve surveillance of emerging flea-borne zoonoses.