Spotted fever Rickettsia and relapsing fever Borrelia in rodents from southern India

This study reports the detection of pathogenic spotted fever group *Rickettsia* and relapsing fever group *Borrelia* in rodent populations from southern India, revealing distinct tissue-specific detection patterns and highlighting a potential zoonotic transmission risk to humans and livestock.

The Gist

Imagine a bustling, invisible city living inside the bodies of the rats and mice that scurry through the forests and villages of southern India. This city is populated by tiny, microscopic invaders—bacteria that can sometimes jump from animals to humans, causing illness.

This paper is like a detective report sent by a team of scientists (the "detectives") who went into this region to see which of these microscopic invaders were hiding in the local rodent population. They weren't just looking for one criminal; they were hunting for five different families of bacteria: Rickettsia, Borrelia, Orientia, Leptospira, and Coxiella.

Here is the story of their investigation, broken down into simple concepts:

1. The Setting: A Mix of Forests and Farms

The scientists set up their "traps" (which were actually humane cages) in a unique landscape in Karnataka, India. This area is a mosaic—a patchwork quilt of dense forests, coffee plantations, and human villages.

• The Suspects: They caught four main types of rodents: some that live deep in the wild forests, and some that are "synanthropic" (a fancy word for animals that love hanging out near humans, like house rats).
• The Goal: They wanted to know: Who is carrying what? Are the forest rats different from the village rats? And are any of these rats carrying dangerous bacteria?

2. The Big Discovery: A "Low-Key" Epidemic

The team found that while these bacteria were present, they weren't running a massive, city-wide takeover. The infection rates were low, like finding a few stray cats in a neighborhood rather than a whole colony.

• The Winners: They found Rickettsia (about 7% of rats) and Borrelia (about 6% of rats).
• The Losers: They found almost no Leptospira (less than 1%), and they found zero Orientia and Coxiella.
• The Twist: The bacteria didn't care much about whether the rat was a "forest rat" or a "village rat." Both types of neighborhoods had the same low-level presence of these germs.

3. The "Whereabouts" Clue: Blood vs. Organs

This is the most fascinating part of the story. The scientists discovered that the two main bacterial families played by very different rules, like two different sports teams with different playing fields.

• The Rickettsia Team (The Organ Hiders):
  Imagine Rickettsia as a shy ghost that refuses to show up in the bloodstream. The scientists looked at the rats' blood and found nothing. But when they looked at the rats' internal organs (liver, spleen, etc.), the ghosts were there!

  • Analogy: It's like trying to find a specific type of fish. If you only look at the surface of the lake (blood), you see nothing. But if you dive down to the bottom (organs), you find them hiding in the weeds.

• The Borrelia Team (The Blood Swimmers):
  Borrelia is the opposite. It loves the open water. The scientists found these bacteria only in the blood and never in the organs.

  • Analogy: These are like sharks swimming in the open ocean. They don't hide in the weeds; they cruise the main highway (the bloodstream) to get picked up by their "taxi drivers" (ticks and mites) to spread to new hosts.

Why does this matter? It teaches us that if you want to find these germs, you have to know where to look. If you only test blood, you might miss Rickettsia. If you only test organs, you might miss Borrelia.

4. The Identity Crisis: Who Are These Germs?

The scientists didn't just find the bacteria; they took their "fingerprints" (DNA) to see exactly who they were.

• The Rickettsia Identity: They found two distinct groups. One group looked exactly like Rickettsia massiliae (a known human pathogen), and the other looked like Rickettsia honei. Interestingly, one type seemed to prefer the forest rats, while the other preferred the village rats. It's like two different gangs claiming different neighborhoods.
• The Borrelia Identity: This group was a diverse party. They found five different lineages. Some were new to science, and some were related to bacteria that infect livestock. One specific group was found in both forest rats and village rats, suggesting that the "taxi drivers" (ticks) might be moving back and forth between the forest and the village, carrying the bacteria with them.

5. The "Party" (Coinfection)

The scientists wondered: Are these rats carrying multiple bacteria at once? Like a person having a cold and the flu simultaneously?

• The Result: Not really. It was rare to find a rat with two or three different bacteria at the same time.
• The Takeaway: This suggests that these bacteria don't really hang out together. They likely have their own separate "delivery systems" (different ticks or fleas) and don't rely on each other to survive.

6. The Bottom Line: Why Should We Care?

Even though the infection rates were low, this study is a warning light.

• The Bridge: These rodents live in forests, farms, and villages. They are the bridge between the wild and our homes.
• The Risk: The bacteria they carry (like Rickettsia and Borrelia) are known to cause serious diseases in humans (like fever, headaches, and even life-threatening infections).
• The Lesson: Just because we don't see a massive outbreak doesn't mean the threat is gone. The "invisible city" is still there, quietly circulating.

In a nutshell:
The scientists went on a treasure hunt in southern India and found that while the "treasure" (dangerous bacteria) isn't everywhere, it is definitely there. It's hiding in specific places (blood vs. organs), it's diverse, and it's moving between the wild forests and human villages. By understanding exactly where these germs live and how they move, we can better protect farmers, plantation workers, and families from getting sick. It's a reminder that nature is full of hidden connections, and keeping an eye on the small things (like mice) helps us stay safe from the big things (like pandemics).

Technical Summary

1. Problem Statement

Zoonotic bacterial pathogens maintained in wildlife reservoirs pose a significant threat to public health, particularly in tropical regions like India where human-wildlife interactions are frequent due to agrarian livelihoods and urbanization. Despite the high rodent diversity in India (110 species), there is a critical gap in systematic surveillance regarding the diversity, ecology, and transmission pathways of vector-borne bacterial pathogens. Previous studies have largely focused on human clinical cases or serological surveys, leaving the enzootic cycles of specific genera—Rickettsia, Borrelia, Orientia, Leptospira, and Coxiella—in Indian rodent communities poorly characterized. Furthermore, the extent of pathogen co-circulation (coinfection) and host specificity within these communities remains largely unexplored.

2. Methodology

The study employed a multidisciplinary approach combining field ecology, molecular diagnostics, and phylogenetic analysis.

• Study Site and Sampling:

  • Location: Kadamane, a forest-plantation mosaic in Karnataka, southern India.
  • Sampling Period: January to March 2018.
  • Design: A grid-based framework (10×10 Sherman traps per hectare) covering forest, grassland, and human habitation habitats.
  • Subjects: Seven rodent species were captured, but molecular analysis focused on four species with sufficient sample sizes ($n \ge 10$): Rattus rattus (synanthropic), Rattus satarae (forest-associated), Mus cf. fernandoni, and Mus cf. famulus. Total $N=124$ for the study.
  • Sample Types: Dried blood spots (DBS) on FTA cards and pooled organ tissues (liver, spleen, kidney, lung, intestine) preserved in RNAlater.

• Molecular Diagnostics:

  • Target Genera: Rickettsia, Borrelia, Orientia, Leptospira, and Coxiella.
  • PCR Assays:
    • Rickettsia: OmpB gene (811 bp) from DBS.
    • Borrelia: 16S rRNA (650 bp) from DBS (nested PCR).
    • Orientia: 56-kDa protein (371 bp) from DBS.
    • Leptospira: 16S rRNA (289 bp) from kidney/spleen (nested PCR).
    • Coxiella: IS1111A (146 bp) from kidney/spleen.
    • Pooled Tissue Screening: To increase detection sensitivity, homogenates of pooled organs were screened for all five genera.
  • Controls: Positive controls were sequence-confirmed samples or cultured DNA; negative controls were included in every batch.

• Statistical and Phylogenetic Analysis:

  • Prevalence: Calculated with 95% confidence intervals (CI) using the Hmisc package in R.
  • GLMs: Generalized Linear Models assessed the effects of host species, sex, and their interaction on infection probability.
  • Coinfection: Pairwise and triple coinfection patterns (including previously collected Bartonella data) were analyzed.
  • Phylogenetics: Maximum-likelihood trees were constructed using MUSCLE alignment, Gblocks cleaning, and PhyML (10,000 bootstrap iterations). Genetic similarity (p-distance) was calculated against GenBank references.

3. Key Results

• Infection Prevalence:

  • Rickettsia: Detected in 7.26% (9/124) of rodents. Exclusively found in pooled organ tissues; no blood samples were positive.
    • R. rattus: 10% prevalence.
    • R. satarae: 12.12% prevalence.
    • No association found with host species or sex.
  • Borrelia: Detected in 6.45% (8/124) of rodents. Exclusively found in blood samples; no pooled tissues were positive.
    • Detected in R. rattus (10%), R. satarae (6.06%), and M. cf. fernandoni (13.63%).
  • Leptospira: Low prevalence (0.8%); only one R. satarae individual tested positive (found in kidney and pooled tissue).
  • Orientia & Coxiella: Not detected in any samples.

• Coinfection Patterns:

  • Coinfections were rare.
  • Rickettsia-Borrelia: 1.61% (2 individuals).
  • Rickettsia-Bartonella: 4.03% (5 individuals).
  • Borrelia-Bartonella: 2.41% (3 individuals).
  • Triple coinfection (Rickettsia-Borrelia-Bartonella) occurred in only one R. satarae individual (0.81%).
  • No significant associations were found between coinfection status and host species, sex, or the presence of other bacteria.

• Phylogenetic Diversity:

  • Rickettsia (Spotted Fever Group - SFG): Two distinct lineages identified.
    • Lineage R1 (R. rattus): 100% similarity to Rickettsia massiliae.
    • Lineage R2 (R. satarae): 99.7% similarity to Rickettsia honei.
    • Suggests host-specific structuring (synanthropic vs. forest species).
  • Borrelia (Relapsing Fever Group - RFG): Five distinct lineages identified.
    • Lineages B1–B3: Novel monophyletic cluster basal to known RFG/LDG groups; 92–94% similarity to Borrelia puertoricensis.
    • Lineage B4 (R. satarae): 99.6% similarity to Borrelia theileri (known to infect wildlife/livestock).
    • Lineage B5 (R. satarae): Sister taxon to Borrelia turcica.
    • Notably, Lineage B3 was shared between R. rattus and R. satarae, indicating broad host susceptibility for this lineage.
  • Leptospira: The single sequence was nested within the pathogenic clade, basal to L. interrogans and L. kirschneri, with 96.4% similarity to L. borgpetersenii.

4. Key Contributions

1. First Molecular Characterization: This is one of the first studies to use PCR-based molecular screening to characterize Rickettsia and Borrelia diversity in Indian rodents, moving beyond serology.
2. Tissue-Specific Detection Dynamics: The study highlights a critical methodological insight: Rickettsia (obligate intracellular) was only detectable in tissues, while Borrelia (extracellular) was only detectable in blood. This suggests that relying on a single sample type (e.g., blood only) may lead to significant underestimation of prevalence for specific pathogens.
3. Discovery of Novel Lineages: The identification of five distinct Borrelia lineages, including novel clusters basal to known groups, and two SFG Rickettsia lineages (R. massiliae and R. honei), expands the known bacterial diversity in the region.
4. Host Specificity vs. Generalism: The study reveals contrasting ecological patterns: Rickettsia lineages showed host specificity (segregated by R. rattus vs. R. satarae), whereas a specific Borrelia lineage (B3) crossed habitat and species barriers, suggesting different transmission dynamics.

5. Significance

• Public Health Implications: The detection of R. massiliae, R. honei, and B. theileri-like strains in rodents living in both forest and human habitation areas indicates a tangible spillover risk. These pathogens are known to cause severe human diseases (Spotted Fever, Relapsing Fever), and their presence in synanthropic rats (R. rattus) near human settlements is a direct occupational hazard for plantation workers and forest users.
• Surveillance Strategy: The findings underscore the necessity of systematic wildlife surveillance that includes diverse sample types (blood and tissues) and multiple genetic loci. Relying solely on human clinical data or single-tissue sampling misses critical reservoir data.
• Ecological Understanding: The low rate of coinfection suggests that these pathogens likely operate via independent transmission pathways (different vectors or transmission windows) rather than synergistic interactions within the host.
• Policy: The study calls for enhanced One Health approaches in India to monitor vector-borne diseases, particularly in the context of changing land use and human-wildlife interfaces.