Prevalence of Mycoplasmopsis agassizii across wild and captive Mediterranean tortoises

This study reveals that while *Mycoplasmopsis agassizii* is highly prevalent in captive Mediterranean tortoises in Spain, its occurrence in wild populations is species-specific, with *Testudo graeca* showing very low or absent infection rates compared to the comparatively higher prevalence found in wild *Testudo hermanni*.

The Gist

Imagine the world of tortoises as a giant, slow-moving neighborhood. In this neighborhood, there are two main families of residents: the Spur-thighed Tortoises (Testudo graeca) and Hermann's Tortoises (Testudo hermanni).

Recently, scientists discovered a tiny, invisible "neighborhood bully" called Mycoplasmopsis agassizii. This isn't a monster you can see with your eyes; it's a microscopic germ that causes a nasty cold in tortoises, giving them runny noses, swollen eyes, and making them feel very sick. This germ is famous for causing trouble in the United States, but nobody knew much about how it was behaving in the tortoise neighborhoods of Spain.

Here is the story of what the scientists found, explained simply:

The Two Worlds: The Zoo vs. The Wild

The researchers decided to play detective. They went to two different types of places to check for this germ:

1. The "Zoo" (Captive): These are tortoises living in recovery centers, shelters, or private collections. Think of these as crowded dorm rooms where everyone sleeps in the same small space.
2. The "Wild" (Free-range): These are tortoises living freely in nature, roaming hills and forests. Think of these as people living in spacious houses with lots of personal space.

They checked 259 tortoises in total. Here is what they found, and it's a bit like a mystery story with two very different endings.

The Mystery of the Two Families

1. The Hermann's Tortoise: The "Seasoned Veteran"
Imagine the Hermann's tortoise as an old-timer who has lived through many storms.

• In the Wild: When scientists checked the wild Hermann's tortoises in the northeast of Spain, they found the germ in about 40% of them. It was everywhere!
• In Captivity: In the shelters, it was also very common (about 50%).
• The Takeaway: This family seems to have lived with this germ for a long time. They've built up a sort of "callous" or immunity. The germ is there, but the tortoises seem to handle it okay, even in the wild. It's like a flu that everyone in a specific town has had before; it's annoying, but it's not wiping out the population.

2. The Spur-thighed Tortoise: The "Naive Newcomer"
Now, imagine the Spur-thighed tortoise as a new resident who has never seen this germ before.

• In the Wild: When scientists checked the wild Spur-thighed tortoises in the south of Spain, the result was shocking: Zero. Not a single wild tortoise had the germ. They were completely clean.
• In Captivity: However, when they checked the ones in shelters and zoos, the germ was rampant. In some places, two out of every three tortoises were infected.
• The Takeaway: This family is "naive." They have never met this germ in the wild. But in the crowded shelters, the germ spreads like wildfire because they are packed together. The scary part? When these wild tortoises do get sick, they get very sick. The study noted that in shelters, these tortoises die much more often than the Hermann's family.

The Big Danger: The "Pet Trade" Bridge

Think of the pet trade and animal shelters as a bridge connecting the "Zoo" world to the "Wild" world.

The scientists are worried because:

• The germ is hiding in the "Zoo" (shelters) in huge numbers.
• The wild Spur-thighed tortoises have no defense against it.
• If a sick tortoise from a shelter is accidentally released (or escapes) into the wild, or if a wild tortoise wanders into a shelter, the germ could jump across the bridge.

It would be like bringing a brand-new, super-virulent flu to a village of people who have never been exposed to it. The result could be a disaster for the wild population.

The "Mallorca" Twist

There was one tiny clue in the middle of the island of Mallorca. Scientists found a Hermann's tortoise living in a spot where only Spur-thighed tortoises were supposed to be. This tortoise was sick and had the germ. This suggests that humans have been moving tortoises around, mixing up the neighborhoods, which helps the germ jump between different groups.

The Bottom Line

This study is like a health warning label for conservationists.

• Good News: The wild Spur-thighed tortoises in Spain are currently safe and germ-free.
• Bad News: They are sitting ducks. If the germ jumps from the crowded shelters to the wild, it could be catastrophic.
• The Solution: We need to be very careful. We must screen tortoises in shelters before letting them go back to the wild, stop releasing pets into nature, and keep the "Zoo" and "Wild" worlds as separate as possible to protect the vulnerable wild families.

In short: The germ is a bully that thrives in crowded rooms. The Hermann's tortoises have learned to ignore the bully, but the Spur-thighed tortoises have never seen it before. If the bully escapes the crowded room and finds the wild tortoises, the consequences could be severe.

Technical Summary

1. Problem Statement

Mycoplasmopsis agassizii (formerly Mycoplasma agassizii) is a primary etiological agent of Upper Respiratory Tract Disease (URTD) in tortoises, linked to morbidity, mortality, and population declines. While the pathogen is well-documented in North American gopher tortoises and European captive collections, its epidemiology in wild European chelonian populations remains poorly understood.

• The Gap: There is a lack of baseline data regarding the prevalence of M. agassizii in wild Mediterranean tortoises in Spain.
• The Risk: The pet trade, along with the release or escape of captive individuals, creates pathways for pathogen spillover from high-density captive environments to wild populations. This is particularly concerning given the fragmented distribution of native species (Testudo graeca and Testudo hermanni) and the presence of introduced populations.
• Hypothesis: The authors hypothesized that infection prevalence would be significantly higher in captive individuals than in wild populations due to the role of captivity in facilitating pathogen transmission and maintenance.

2. Methodology

Study Design & Sampling:

• Species: The study focused on two native species: the Spur-thighed tortoise (Testudo graeca) and Hermann's tortoise (Testudo hermanni).
• Sample Size: A total of 259 tortoises were sampled between 2020 and 2025.
  • T. graeca: 127 wild (Almería, Doñana, Mallorca) and 63 captive (Recovery Centers in Mallorca, Alicante, Murcia).
  • T. hermanni: 46 wild (Catalonia, Mallorca) and 23 captive (Recovery Centers in Girona, Barcelona).
• Sampling Strategy: Random selection of individuals from both wild and captive populations. Clinical assessments were conducted; most were asymptomatic, though some captive individuals showed poor body condition.

Laboratory Analysis:

• Sample Collection: Oronasal epithelial and mucosal swabs (throat, tongue, palate) were collected using disposable gloves to prevent cross-contamination.
• Processing: Samples were preserved in ethanol, frozen, and processed at Laboklin GmbH & Co. KG (Germany).
• Detection Method: DNA/RNA extraction followed by PCR targeting the V3 variable region of the 16S rRNA gene (Brown et al., 1995).
  • Controls: Negative, positive, and extraction controls were included in every run.
  • Verification: Capillary electrophoresis (QIAxcel) confirmed the expected 576 bp product size.

Statistical Analysis:

• Modeling: Generalized Linear Mixed Models (GLMMs) with a binomial error distribution and logit link were used.
• Variables: Infection status (PCR +/-) was modeled as a function of Captivity Status (Wild vs. Captive), Species, and their Interaction.
• Random Effects: Sampling region was included as a random intercept to account for spatial clustering.
• Model Selection: Models were compared using AICc (Akaike Information Criterion corrected).

3. Key Results

Overall Prevalence Patterns:

• Captivity vs. Wild: Captive populations of both species showed high prevalence, whereas wild populations exhibited species-specific divergence.
• Species Differences:
  • Testudo graeca:
    • Captive: High prevalence (43% overall; 67% in Alicante/Murcia centers, 21% in Mallorca).
    • Wild: Extremely low prevalence. 0% in Almería and Doñana; only 1 positive case (13%) in Mallorca (out of 8 sampled).
  • Testudo hermanni:
    • Captive: High prevalence (52% overall; 67% in Barcelona, 43% in Girona).
    • Wild: High prevalence in mainland Catalonia (43% in Girona, 44% in Tarragona). Lower prevalence in Mallorca (14%), though the single positive case was a released individual in a T. graeca zone.

Statistical Findings:

• The best-supported model (AICc weight = 0.84) included Captivity Status, Species, and their Interaction.
• Predicted Probabilities:
  • T. graeca: Predicted prevalence dropped sharply from 0.48 (captive) to 0.009 (wild).
  • T. hermanni: Showed consistently high prevalence in both settings (0.49 captive vs. 0.36 wild).
• Significance: Wild T. graeca had significantly lower prevalence than any other group ($p < 0.005$).

4. Key Contributions

1. First Baseline Data: This study provides the first comprehensive assessment of M. agassizii prevalence in wild Mediterranean tortoises in Spain.
2. Species-Specific Epidemiology: It reveals a stark contrast between the two species:
   • T. graeca appears to be immunologically naive in mainland Spain, with wild populations showing near-zero infection.
   • T. hermanni is endemic to the pathogen in its wild populations in northeastern Spain, with infection rates comparable to captive settings.
3. Confirmation of Captivity as a Reservoir: The study confirms that captive environments act as major reservoirs for M. agassizii, with infection rates significantly higher than in most wild populations (except for T. hermanni in Catalonia).
4. Insight into Mortality: The authors note that while T. hermanni rarely dies in captivity despite high infection rates, T. graeca shows high mortality in recovery centers, suggesting M. agassizii may have more severe clinical impacts on T. graeca.

5. Significance and Conservation Implications

• Spillover Risk: The high prevalence in captive tortoises poses a severe threat to wild T. graeca populations, which appear to lack prior exposure. The introduction of the pathogen via the release of captive individuals could trigger catastrophic outbreaks in these naive populations.
• Management Recommendations:
  • Strict Biosecurity: Wildlife recovery centers must implement rigorous health screening and pathogen surveillance before any translocation or release.
  • Prevent Releases: Strict enforcement against the release of captive tortoises into the wild is critical to prevent pathogen spillover.
  • Species-Specific Strategies: Conservation strategies must differ by species. While T. hermanni in Catalonia is already exposed, T. graeca requires protection from introduction.
• Broader Context: The findings highlight how anthropogenic pressures (pet trade, habitat fragmentation, and captive breeding) interact with infectious diseases to threaten chelonian biodiversity. The study underscores the need for integrated disease ecology approaches in conservation planning.