The controversy began when the group published a systematic review and meta-analysis in 2024 examining evidence that Ctenocephalides felis, the cat flea, carries or transmits hemotropic Mycoplasma species, including Mycoplasma haemofelis and the smaller Candidatus Mycoplasma haemominutum. Their analysis reached a provocative conclusion: when researchers used species-specific primers targeting hemoplasma genes, hemoplasma DNA turned up in fleas only rarely, and the widely used Jensen primers—developed in 2001 to detect what was then called Haemobartonella felis—may actually be amplifying DNA from Spiroplasma, a related bacterium that naturally inhabits fleas. A subsequent author responded with a critique alleging methodological and interpretive flaws, and the new letter is the team’s detailed rebuttal.

The reply opens with an unusually candid acknowledgment. The authors thank their critic for identifying mistakes in the data collection underlying the meta-analysis and have issued corrected figures in their supplementary tables for three studies—those by Zarea and colleagues, Abdullah and colleagues, and Shaw and colleagues. They also accept a specific miscategorization: the Shaw study did, in fact, wash fleas before DNA extraction, and it followed polymerase chain reaction testing with sequencing in 16 of 44 flea DNA extracts from cats, confirming a match to M. haemominutum over a 193-base-pair fragment of the 16S rRNA gene. An updated table now reflects that correction. Yet the team argues that none of these fixes overturns its central conclusion.

The reasoning is technical but consequential. Even though the Shaw study washed its fleas—something that should, in principle, remove surface contaminants—roughly 40 percent of the fleas, 44 of 90, still tested positive with the Jensen primers. To the authors, that pattern strengthens rather than weakens their hypothesis: if washing does not reduce positivity, the amplified signal may come from bacteria residing within the flea itself, such as Spiroplasma species, rather than from hemoplasmas acquired in a blood meal. The Jensen and Manvell primer sets, which were grouped in the meta-analysis, share a reverse primer and differ by only a single base pair in the forward primer—a shift that is homologous among feline hemoplasmas and the Spiroplasma species of the cat flea. In the original Manvell study, numerous initial PCR positives were reclassified after sequencing revealed them to be Spiroplasma rather than hemoplasma, a cautionary tale about what 16S rRNA primers with insufficient specificity can pick up.

For the studies that used non-Jensen, species-specific primers, the corrected numbers barely move the needle. In the Zarea study, the proportion is ambiguous because two Ctenocephalides canis fleas removed from cats fell outside the inclusion criteria, and the original authors did not specify whether all seven hemoplasma-positive fleas were cat fleas; a conservative estimate is 7 of 91, a prevalence under 8 percent. In the Abdullah study, applying the inclusion criteria strictly means counting only flea pools collected from cats—210 pools—and, within those, only the two positive pools with a confirmed feline host, rather than the originally reported 3 of 467. Recalculating with 2 of 210, about 1 percent, makes no statistically meaningful difference. Across all studies using species-specific primers, the updated overall proportion of hemoplasma DNA amplified from cat fleas stands at 14 of 949, or 1.5 percent—a figure the authors describe as evidence that such detection remains exceedingly rare.

A second flashpoint concerns pooling. Fleas are often processed in groups for DNA extraction, and pool size directly affects the probability of detecting low-abundance bacterial DNA, yet few studies report how many fleas each pool contained. The authors concede that inconsistent pool sizes complicate meta-analysis and urge future researchers to report pool composition comprehensively. In the Willi study, 73 fleas from 17 cats were processed in pools of one to five fleas, and although the results section stated that all positive samples came from individually extracted arthropods, the discussion suggested that pooling might have lowered the apparent prevalence—an internal tension that makes the study impossible to classify cleanly as pooled or unpooled. In the Abdullah study, at least half of the supposed pools were single fleas, with no further pool-size information available. Even so, the authors note, the bottom line survives: with species-specific primers, only two individual fleas in the Willi study and two pools—likely but not definitively single-flea pools—out of more than 200 in the Abdullah study were positive.

The experimental heart of the original paper involved retesting samples from a 2012 study by Assarasakorn and colleagues in Bangkok, which had reported a high prevalence of hemoplasma DNA in fleas using Jensen primers. The reanalysis found a dramatically lower prevalence, and the critic suggested sample degradation as a possible explanation. The authors counter that a substantial body of research shows DNA remains stable when stored frozen for years, and they address the pooling critique with new detail. According to Lappin, a co-author of the original Bangkok study, fleas there were pooled by cat—many cats contributing a single flea, with a maximum of five per pool—and when cats carried more than five fleas, multiple pools were created but only one was tested. That practice explains the numerical discrepancy between the 50 pools tested originally and the 67 pools from the same 50 cats retested in the new work. All 67 original DNA extracts were retested at Colorado State University with Jensen primers and at North Carolina State University with Manvell primers. Of the 17 previously untested pools, 7—41 percent—were positive with Jensen primers, compared with 18 of 50, or 36 percent, of the originally tested pools, results now laid out explicitly in a new table.

On the washing experiments, the authors acknowledge a genuine limitation: because the hemoplasma status of the fleas was unknown, the effect of washing on detection could not be determined—a caveat they admit could have been emphasized more forcefully. Fleas with known hemoplasma infection were simply not available, and those experiments remain to be done. This concession, however, is paired with a firm defense of the broader evidence base. The experimental infection studies cited in the original review involved small numbers of animals, but the authors argue those numbers are appropriate given how labor-intensive it is to work with cat fleas and cats in controlled settings, and at the time of publication the results represented the only published experimental transmission study using fleas.

Fresh evidence from outside the dispute has since tilted the picture further. A new experimental study published after the original manuscript found less support for flea transmission and more for direct transmission of hemoplasmas. In that work, hemoplasma transmission occurred between flea-infested cats sharing an enclosure—one of six cats infected with M. haemofelis and three of six with M. haemominutum—but no transmission was observed between cats kept in separate enclosures that allowed flea movement but no direct contact. By contrast, Bartonella clarridgeiae, a bacterium from a genus known for arthropod-borne spread, transmitted among all flea-infested cats in the same enclosure and to two of eight cats in adjacent enclosures permitting only flea movement, demonstrating a far clearer vector role for the cat flea. Geography adds another layer of skepticism toward a flea-driven model: in the Swiss survey, higher hemoplasma prevalence in cats occurred in the south and west of the country, yet the alpine south has a subarctic to tundra climate, not the warm, humid conditions that favor flea abundance. Surveys in Japan, spanning subarctic to humid subtropical climates, and Italy, from tundra to hot-summer Mediterranean zones, likewise fail to tie hemoplasma prevalence to flea-friendly conditions, and both included flea-borne Bartonella surveillance that allows direct comparison.

The authors close by clarifying their intent: they never sought to dismiss the high hemoplasma prevalences reported with Jensen primers, but to caution that those primers may not be as hemoplasma-specific as originally believed—a concern reinforced by recent reports of incidental amplification of Wolbachia and other flea microbiome DNA with inadequately specific 16S rRNA primers. They accept that fleas could yet prove to be competent vectors, but argue that geographic surveys and the newest experimental evidence more strongly support direct transmission among cats, through biting, grooming, or fighting. The path forward, all parties seem to agree, runs through better experiments: standardized washing protocols, transparent pool-size reporting, sequencing confirmation with rigorous BLAST analysis, and head-to-head comparisons of conventional PCR, quantitative PCR, and other molecular methods. Until then, the true route by which these elusive bacteria colonize feline blood remains an open and actively contested question.

Subject of Research: Vector competence of the cat flea for feline hemotropic Mycoplasma species and the specificity of molecular detection methods

Article Title: A case of misleading conclusion: a critical reassessment of methodological and interpretive flaws of a recent meta-analysis on the vector role of cat fleas in feline hemotropic Mycoplasma species

Article References: Moore, C. O., Lashnits, E., Lappin, M., & Breitschwerdt, E. B. (2026). A case of misleading conclusion: a critical reassessment of methodological and interpretive flaws of a recent meta-analysis on the vector role of cat fleas in feline hemotropic Mycoplasma species. Parasites & Vectors, 19(1), Article 372. https://doi.org/10.1186/s13071-026-07315-2

Image Credits: AI Generated

DOI: 10.1186/s13071-026-07315-2

Keywords: cat flea, hemotropic Mycoplasma, Ctenocephalides felis, meta-analysis, PCR primer specificity, Spiroplasma, Mycoplasma haemofelis, vector-borne disease, feline hemoplasmosis, Ctenocephalides canis, 16S rRNA, direct transmission

The pathogens in question are a select group of bacteria that have made the plant vascular system their exclusive home. Xylella fastidiosa colonizes the xylem, the water-transport network of the plant, while Spiroplasmas, Phytoplasmas and Liberibacters confine themselves to the phloem, the sugar-rich tissue that distributes photosynthetic products. These bacteria cannot move between plants on their own. They depend on piercing-sucking insects, mostly hemipterans such as leafhoppers, planthoppers, spittlebugs and psyllids, whose needle-like stylets penetrate directly into the vascular tissues. In doing so, the insects ingest sap along with any microbes it contains and later deposit the bacteria into new hosts. Intriguingly, within the insect suborder Sternorrhyncha, only psyllids are known to transmit bacterial pathogens, while aphids and whiteflies, their close relatives, are primarily virus carriers.

The economic stakes are enormous. Citrus huanglongbing, or HLB, caused by the psyllid-transmitted Liberibacter species, has cut citrus yields in Florida by 74 percent since its detection there in 2005. In Italy, Xylella fastidiosa triggers olive quick decline syndrome, a disease that has killed millions of olive trees in the southern region of Puglia. Genetic analysis traced that outbreak to a Central American introduction of the pathogen around 2008, and phylogenetic work suggests the bacterium actually arrived in Europe as early as 1993. Xylella infects more than 700 plant species, including grape, in which it causes Pierce’s disease, and almond and citrus, where it produces leaf scorch and variegated chlorosis. The review also highlights the recent recognition of Xylella taiwanensis as a distinct species rather than a subspecies of X. fastidiosa.

Each pathogen has evolved a distinctive lifestyle suited to its vascular niche. Xylella, a Gram-negative bacterium related to Xanthomonas albilineans, uses Type IV pili to twitch its way through the nutrient-poor xylem, secreting cell wall-degrading enzymes such as polygalacturonase and endoglucanases to dissolve the pit membranes that separate adjacent vessels. It then forms sticky biofilms, anchored by extracellular polysaccharides and adhesins, that physically obstruct water flow. The resulting drought stress, stomatal closure and reduced photosynthesis eventually kill the plant. Spiroplasmas, by contrast, are helical, wall-less bacteria of the class Mollicutes that swim by propagating kinks along their bodies. Spiroplasma citri, discovered in 1970 and the first vector-borne bacterium ever cultured, causes citrus stubborn disease, while S. kunkelii produces maize stunt and S. phoeniceum infects periwinkle.

Phytoplasmas are stranger still. First observed in 1967 as mycoplasma-like organisms in infected phloem, they remain unculturable in the laboratory, forcing researchers to rely on DNA extracted from infected plants and on 16S rRNA gene sequencing for classification. Lacking both cell walls and flagella, they cannot produce the classic molecular patterns that plant immune receptors normally detect, and they appear to elicit immune responses through internal patterns instead. Their most dramatic effects are developmental: infected plants produce witches’ brooms, dense clusters of stunted twigs, or phyllody, in which flowers transform into leafy structures. These deformities prevent normal reproduction and turn the plant into a breeding ground for both the pathogen and its vectors. Phytoplasmas compensate for their lack of motility genes by using their immunodominant membrane protein to bind plant actin, apparently hitching rides through the host’s own cytoskeletal transport system.

The Liberibacters tell an evolutionary story of their own. Six species share a common ancestor, and genome analysis suggests the non-pathogenic Liberibacter crescens gave rise to the pathogenic Candidatus Liberibacter lineages. The pathogenic species are fastidious and unculturable, likely because their highly reduced genomes lack genes for thiamine and essential amino acids that L. crescens retains. Candidatus Liberibacter asiaticus is the primary HLB agent, transmitted by the Asian citrus psyllid Diaphorina citri, while Ca. L. solanacearum exists as at least six haplotypes: A and B, spread by the potato psyllid Bactericera cockerelli, cause zebra chip disease of potato, named for the dark streaks that appear in tubers after frying, and haplotypes C, D and E, carried by the carrot psyllid B. trigonica, attack umbelliferous crops.

Central to the review is the molecular arms race between these bacteria and plant immunity. Plants deploy pattern recognition receptors that detect bacterial flagellin and other pathogen-associated molecular patterns, triggering a first line of defense. Bacteria strike back with effector proteins. Phytoplasma effectors are among the best characterized: SAP05 hijacks the host 26S proteasome to degrade SPL and GATA transcription factors, promoting lateral shoots and sterile flowers; SAP11 and related proteins degrade TCP transcription factors to stimulate bud growth; SAP54 converts flowers into leaf-like structures; and SRP1 from rice orange leaf phytoplasma impairs chlorophyll precursor biosynthesis, producing yellow leaves that attract leafhoppers. Liberibacter effectors are equally sophisticated. CLas secretes SDE15 to target the programmed cell death regulator CsACD2, SDE1 to suppress papain-like cysteine proteases, and SDE3 and SDE4405 to manipulate autophagy, while the effector CLas4425 undermines salicylic acid signaling. For CLso, the effector Lso-HPE1 disrupts the tomato ubiquitin-proteasome system, and CKC_05701 suppresses programmed cell death and reactive oxygen production.

The relationship between pathogen and vector is just as intricate. Xylella is unique among these bacteria in forming biofilms in the foregut of its sharpshooter and spittlebug vectors, a semi-persistent, non-circulative association. It degrades chitin and uses adhesins such as XadA1 and XadA2, which bind strongly to chitin and cellulose, to anchor itself in the insect’s feeding apparatus. The phloem-limited bacteria, by contrast, establish persistent, circulative relationships: they cross the insect gut epithelium, survive in the hemolymph and eventually reach the salivary glands for reinoculation. Spiroplasma citri uses adhesion proteins including P89 and ScARPs for receptor-mediated endocytosis, and its phosphoglycerate kinase interacts with insect actin to promote colonization. Phytoplasmas enter insect cells through clathrin-mediated endocytosis, and their antigenic membrane proteins form complexes with insect microfilaments that determine vector specificity.

Liberibacters manipulate their psyllid hosts in remarkable ways. CLas localizes along the actin cytoskeleton of D. citri gut cells, and roughly 95 percent of its genes are active during gut colonization. In adult psyllids, CLas triggers apoptosis in the gut, which may reduce their transmission capacity, whereas nymphs show no such response and transmit more efficiently. CLso takes a preemptive approach: it induces expression of the anti-apoptotic gene IAPP5.2 early in infection, suppressing gut cell death to facilitate its own acquisition. The two zebra chip haplotypes provoke distinct immune responses in the potato psyllid, with CLsoB repressing apoptosis and achieving higher titers and more efficient transmission. Even insect symbionts matter: Wolbachia correlates positively with CLas titer in D. citri, and psyllids lacking Wolbachia acquire and transmit CLso less efficiently, suggesting microbiome-based control strategies.

The three-way interplay culminates in pathogen-driven manipulation of plant chemistry to recruit vectors. Phytoplasma effectors dampen jasmonic acid signaling and increase leafhopper fecundity, while SAP54 makes infected plants more attractive to leafhopper females. CLas induces citrus to release specific volatiles that deceptively attract D. citri and alters metabolite emissions to shift vector preference. Current management leans on insecticides and antibiotics, including oxytetracycline and streptomycin, approved by the U.S. EPA for Florida citrus, with trunk injection proving more efficient than foliar sprays. Promising alternatives include antimicrobial peptides such as nisin, which disrupts the Xylella lipid bilayer, and MaSAMP, a heat-stable peptide from HLB-resistant Microcitrus that both kills CLas and primes plant immunity. The authors point to CRISPR/Cas9 gene editing, plant immunomodulators such as salicylic acid analogues and GABA supplementation, and even light-engineering technologies as future directions. But with many of these pathogens still unculturable and genetically intractable, the review’s central message stands: cracking the bacterium-insect-plant triangle is the essential next step toward protecting global food security.

Subject of Research: Interactions among insect vectors, bacterial plant pathogens and host plants

Article Title: Bacterial vector-borne plant diseases: global issues caused by three-way interactions

Article References: Huang, Y., Hao, J., & Tang, X. (2025). Bacterial vector-borne plant diseases: global issues caused by three-way interactions. Crop Health, 3(1), Article 10. https://doi.org/10.1007/s44297-025-00049-0

Image Credits: AI Generated

DOI: 10.1007/s44297-025-00049-0

Keywords: Xylella fastidiosa, huanglongbing, Liberibacter, phytoplasma, Spiroplasma, insect vectors, psyllids, leafhoppers, plant immunity, effector proteins, phloem, xylem

The study focused on Niğde province, a region of central Türkiye where tortoises roam freely and their parasites follow. The research team, led by Bilge Karatepe and Mustafa Karatepe of the Department of Biotechnology, together with Fatma Mutlu and Zeynep Ergüleş, examined 88 spur-thighed tortoises, Testudo graeca, a species whose conservation status makes every detail of its parasite burden relevant. From these animals they collected 184 adult ticks, 167 males and just 17 females, preserving each specimen in sealed bottles of 70 percent alcohol before carrying them to the laboratory for identification under a stereo microscope.

Every single tick turned out to be Hyalomma aegyptium, confirming the reputation of this species as the quintessential tortoise tick. The infestation pattern was itself informative. Ticks were found on 62 of the 88 tortoises examined, an infestation rate of 70.4 percent. When the researchers broke the data down by month, they discovered that infestation rates varied significantly across the season, a statistically robust pattern with a p value below 0.05. The sex distribution of ticks on infested hosts, however, showed no significant variation, suggesting that male and female ticks exploit their reptile hosts in broadly similar ways even though males vastly outnumbered females in the sample.

With the ticks identified and counted, the team turned to the molecular heart of the study. Rather than testing each tick individually, they organized the 184 specimens into 68 pools, with separate pools for male and female ticks collected from each tortoise. This pooling strategy is a standard and efficient approach in tick microbiology, allowing researchers to screen large numbers of arthropods for rare infections without running hundreds of separate reactions. DNA was extracted from each pool and subjected to polymerase chain reaction, or PCR, using primers specifically designed to amplify marker genes from two bacterial genera of great interest to tick biologists.

The first target was Wolbachia, arguably the most widespread endosymbiotic bacterium on Earth. Wolbachia infects an enormous range of arthropods, manipulating the reproduction of its hosts in ways that can skew sex ratios, kill males, or enable reproductive parasitism. Because of these dramatic effects, Wolbachia has become a darling of both evolutionary biology and pest control, engineered into mosquito populations to block the transmission of dengue and other viruses. The researchers targeted the wsp gene, a surface protein gene of about 610 base pairs that serves as a reliable molecular fingerprint for the bacterium. The outcome was unambiguous: not one of the 68 tick pools produced a Wolbachia amplification product. Only the positive control, supplied by Serap Aksoy of the Yale School of Public Health, yielded the expected 610 base pair band, confirming that the assay worked perfectly and that the negative results were real biology rather than technical failure.

The second target told a different story. The team used primers directed at the 16S ribosomal RNA gene of Spiroplasma, a roughly 1000 base pair region that serves as the gold standard barcode for identifying bacteria. Spiroplasma is a fascinating genus of spiral-shaped, wall-less bacteria in the class Mollicutes, relatives of Mycoplasma. These microbes are best known from insects and plants, where they can act as pathogens, mutualists, or reproductive manipulators. In some insect species, Spiroplasma strains are male killers, selectively destroying male embryos to favor their own transmission through infected females, a phenomenon documented in organisms such as the small brown planthopper. In ticks, Spiroplasma has been reported sporadically, and its ecological role remains poorly understood.

When the researchers ran their Spiroplasma assays, four of the 68 pools, or 5.8 percent, came back positive for Spiroplasma DNA. Every one of the positive pools originated from male ticks, an intriguing observation given that the sample was overwhelmingly male to begin with. The amplified 16S rDNA fragments were analyzed with molecular evolutionary software, allowing the team to characterize the endosymbionts genetically and confirm their identity as Spiroplasma species. This detection represents the first molecular evidence of Spiroplasma in Hyalomma aegyptium ticks in Central Anatolia, a region where tick microbiology has been studied far less intensively than in coastal or western parts of the country.

The absence of Wolbachia is arguably as interesting as the presence of Spiroplasma. The authors note that their null result is consistent with several recent studies reporting low or absent Wolbachia prevalence in ixodid, or hard, ticks. This pattern contrasts sharply with the situation in soft ticks and in many insects, where Wolbachia is common. The reasons remain debated. Some researchers suggest that the physiology of hard ticks, including their long feeding intervals and unique reproductive biology, may simply be inhospitable to Wolbachia colonization. Others point to the possibility that competitive exclusion by other endosymbionts, such as Coxiella-like or Francisella-like bacteria that frequently dominate tick microbiomes, keeps Wolbachia out. Whatever the mechanism, the growing consensus is that hard ticks are not the Wolbachia reservoirs that earlier assumptions based on insect data might have predicted.

The tortoise tick itself deserves attention as a microbial vehicle. Hyalomma aegyptium has been implicated as a long-term carrier of Coxiella burnetii, the agent of Q fever, and previous work in Türkiye and neighboring countries has detected Borrelia turcica, Rickettsia species, Ehrlichia, and the endosymbiont Candidatus Midichloria mitochondrii in these ticks. Because tortoise ticks feed intensively on reptiles and occasionally attach to other animals, understanding their endosymbiont community helps distinguish true pathogens from harmless or even beneficial residents. Endosymbionts can also shape vector competence, the ability of a tick to acquire, maintain, and transmit disease agents, so mapping which microbes are present is a prerequisite for assessing public health risk. The Niğde survey contributes to a broader effort, spanning studies from Algeria and Qatar to Israel and the Balkans, to catalog the microorganisms associated with this single tick species across its vast range.

For the tortoises themselves, the findings carry conservation implications. Testudo graeca is under pressure across its range from habitat loss and collection, and heavy tick burdens can impose physiological costs on already vulnerable populations. Epidemiological studies of tick infestation in spur-thighed tortoises have begun to unravel the demographic consequences of parasitism, and knowing which microbes circulate in the ticks adds a layer of understanding about disease dynamics in these reptile communities. The Niğde study, supported by the Scientific Research Projects Coordination Unit of Niğde Ömer Halisdemir University, also demonstrates the value of regional molecular surveillance. A single season of fieldwork in one Anatolian province produced a novel regional record for Spiroplasma and a robust negative result for Wolbachia, both of which refine the emerging global picture of tick microbiomes. As climate trends continue to alter the distribution and activity of ticks worldwide, baseline data like these will become ever more valuable for predicting where tick-borne agents may emerge next, and for separating the genuine threats from the hidden symbionts that simply ride along.

Subject of Research: Molecular detection and characterization of Spiroplasma and Wolbachia endosymbionts in Hyalomma aegyptium ticks infesting spur-thighed tortoises in Central Anatolia

Article Title: Molecular Characterization of Spiroplasma Endosymbionts in Ticks on Tortoises in Niğde Province, Türkiye

Article References: Karatepe, B., Karatepe, M., Mutlu, F., & Ergüleş, Z. (2026). Molecular Characterization of Spiroplasma Endosymbionts in Ticks on Tortoises in Niğde Province, Türkiye. Acta Parasitologica, 71(5), Article 224. https://doi.org/10.1007/s11686-026-01410-1

Image Credits: AI Generated

DOI: 10.1007/s11686-026-01410-1

Keywords: Hyalomma aegyptium, Spiroplasma, Wolbachia, tick endosymbionts, Testudo graeca, tortoise ticks, Niğde, Türkiye, PCR, 16S rDNA, Central Anatolia, tick microbiome