Onchocerca lupi is a filarial nematode, a threadlike roundworm related to the parasites that cause river blindness in humans. Unlike many of its relatives, however, O. lupi targets the eyes of its hosts. In dogs and cats, infection produces a spectrum of signs ranging from ocular discharge and conjunctivitis to chronic nodular lesions involving the conjunctiva, the sclera, and the retrobulbar space behind the eyeball. The parasite has been documented in southwestern North America as well as in the Old World, and because it can also infect people, it carries genuine public health significance. Perhaps most troubling from a diagnostic standpoint is that many infections remain entirely subclinical, silently persisting in animals that never show obvious signs of disease and therefore never get tested.

The diagnostic toolbox for O. lupi has historically been limited. Confirmation of infection has relied primarily on microscopy and conventional PCR, or cPCR, applied either to adult worms recovered from nodules or to microfilariae, the larval stage, detected in skin snips. Real-time PCR, known as qPCR, has been applied mainly in large epidemiological surveys rather than routine clinical work. Each of these approaches has a critical weakness: amplification of O. lupi DNA can be inhibited by components of the sample matrix, producing false-negative results in animals that are actually infected. In a disease where a missed diagnosis can mean prolonged discomfort for a pet and continued zoonotic risk for its owners, that limitation matters.

Droplet digital PCR, or ddPCR, takes a fundamentally different approach to nucleic acid detection. Instead of relying on amplification curves and threshold comparisons, the technique partitions a sample into thousands of tiny water-in-oil droplets, each of which serves as an individual microreaction vessel. After amplification, each droplet is counted as either positive or negative, and the fraction of positive droplets is converted directly into an absolute number of target DNA copies in the original sample, without the need for a standard curve. This partitioning also dilutes out inhibitors that can derail conventional and real-time PCR reactions, making ddPCR particularly attractive for challenging sample types such as tissue biopsies and archived pathology specimens.

The research team set out with two goals: to validate a novel ddPCR assay capable of detecting O. lupi, and to assess how that assay performed compared with cPCR and qPCR in real clinical cases. The study population consisted of 202 suspected clinical cases submitted from 11 US states, the largest series of suspected O. lupi cases in companion animals assembled in the country to date. Dogs overwhelmingly dominated the caseload, accounting for 97 percent of the samples, or 196 of the 202 animals, while cats made up the remaining 3 percent, or six animals. The samples themselves were diverse: 34 were fragments of adult O. lupi specimens, 103 were subconjunctival nodule biopsies, 23 were interscapular skin snips, three were skin snips from the lower ear, and 39 were formalin-fixed paraffin-embedded tissue samples, the archival material that pathologists routinely generate. Critically, every one of these samples had already been assessed using both cPCR and qPCR, providing a direct head-to-head comparison.

The results were striking. Overall, ddPCR detected O. lupi DNA in 71.3 percent of the clinical cases, identifying 144 of 202 animals as positive. Among the dogs, ddPCR achieved a detection rate of 71.9 percent, or 141 of 196 animals, comfortably outperforming qPCR, which detected 64.8 percent, and conventional PCR, which managed only 54.1 percent. In other words, the digital method found positive results in dozens of animals that the older techniques had called negative. Among the six cats, all three diagnostic tests agreed, confirming infection in half of them. The pattern held across sample types, suggesting that the ddPCR assay’s advantage was not confined to a single kind of specimen but reflected a genuine improvement in analytical sensitivity.

Statistical analysis added nuance to the picture. The researchers used Cohen’s kappa, a measure of agreement between diagnostic methods that corrects for chance agreement, and found moderate agreement between cPCR and qPCR, with a kappa value of 0.74. Agreement between cPCR and ddPCR was fair, at 0.60, while qPCR and ddPCR showed almost perfect agreement, with a kappa of 0.81. A Cochran’s Q test was used to compare the pattern of positive and negative results across the three techniques. These statistics tell a coherent story: ddPCR agrees closely with qPCR but flags additional positives that both older methods miss, consistent with its ability to amplify DNA in samples where inhibitors or very low parasite burdens defeat conventional approaches.

The geographic scope of the study also yielded epidemiological insight. Positive detections were reported in 10 of the 11 states from which samples were submitted, although the authors note that travel history was not always available for the animals, making it difficult to draw firm conclusions about where infections were acquired. The finding nonetheless underscores that suspected O. lupi cases are being seen by veterinary practitioners across a wide swath of the United States, not merely in the southwestern states where the parasite was first documented in North America. For a zoonotic parasite, that broad footprint is a signal that clinicians well beyond the historical endemic zone should keep ocular onchocercosis on their differential lists when dogs and cats present with nodular or inflammatory eye lesions.

One of the assay’s most practical attributes is that it is host-agnostic, meaning it does not depend on the species of the infected animal and can therefore be applied across dogs, cats, and potentially other hosts. Combined with its demonstrated performance across multiple sample types, including the notoriously difficult FFPE archival tissues, this flexibility positions ddPCR as a tool not only for individual clinical diagnosis but also for the kind of large-scale surveillance studies needed to map the parasite’s true distribution. The authors argue that the validated assay supports improved epidemiological understanding and, ultimately, One Health-based prevention strategies that recognize the interconnected health of animals and people.

The study, which was partially funded by the Thomas B. and Jeannette E. Laws McCabe Fund at the University of Pennsylvania and sponsored in part by Elanco Animal Health through the CVBD World Forum Symposium, arrives at a moment when digital PCR is moving from specialized research laboratories into mainstream diagnostic use. For veterinarians confronting a dog with a mysterious conjunctival nodule, the message of this research is concrete: a negative conventional PCR result no longer means the parasite is absent. With a validated ddPCR assay now available, the invisible fraction of O. lupi infections, the subclinical and previously undiagnosed animals that quietly sustain transmission, may finally come into focus, and with it, a clearer picture of how this eye-dwelling worm spreads among the pets and people who share their homes.

Subject of Research: Validation of droplet digital PCR for enhanced molecular diagnosis of zoonotic Onchocerca lupi infection in companion animals

Article Title: Enhanced molecular diagnosis of Onchocerca lupi using droplet digital PCR in clinically suspected companion animals

Article References: Kelly, M. A., McLean, N. J., Peterson, S., Ferguson, S., Young, T., Foote, B., West, J., Campbell, J., Brown, K., Hakimi, H., Sobotyk, C., Weerarathane, P., & Verocai, G. G. (2026). Enhanced molecular diagnosis of Onchocerca lupi using droplet digital PCR in clinically suspected companion animals. Parasites & Vectors. https://doi.org/10.1186/s13071-026-07605-9

Image Credits: AI Generated

DOI: 10.1186/s13071-026-07605-9

Keywords: Onchocerca lupi, droplet digital PCR, molecular diagnostics, ocular onchocercosis, zoonotic parasites, companion animals, veterinary parasitology, filarial nematode, conventional PCR, real-time PCR, One Health, Parasites & Vectors

Blastocystis is one of the most commonly reported intestinal parasites on Earth. It is an anaerobic unicellular eukaryote that colonises the gastrointestinal tract of humans and an enormous range of animals, from rodents and livestock to birds and wildlife. Its genetic diversity is striking: more than forty subtypes have been defined on the basis of sequence analysis of the small subunit ribosomal RNA gene, and these subtypes differ markedly in their host distributions. Subtypes ST1 through ST4, along with ST6 to ST9 and ST12, are the ones most often recovered from people, with ST3 frequently dominating human populations. Higher-numbered subtypes, such as ST10 through ST17 and beyond, are overwhelmingly animal-associated, although the overlap between some human and animal subtypes is precisely what fuels concern about zoonotic transmission.

The clinical significance of Blastocystis remains one of the more debated questions in parasitology. Many carriers harbour the organism without any symptoms at all, while others experience acute or chronic diarrhoea, abdominal pain, bloating and irritable bowel syndrome-like complaints. Transmission is thought to occur mainly through water and food contaminated with the parasite’s resistant cysts, which makes exposure closely tied to sanitation, water quality and contact with infected animals or their faeces. Meta-analyses have estimated pooled prevalences of roughly eighteen to twenty-one percent in rodents worldwide and around twenty-nine percent in birds, figures that underscore how efficiently the parasite circulates through animal reservoirs and why wildlife surveillance matters for public health.

Iran offers a particularly interesting setting for such surveillance. The parasite is well documented in the country’s human population, with pooled prevalence estimates from systematic reviews ranging from about three to ten percent depending on region and detection method. Domestic animals carry their own burden: ruminants in some southwestern provinces show infection rates of up to fourteen percent, and camels have also been found positive. Iranian studies have identified zoonotic subtypes ST1 to ST3 in humans and livestock, alongside animal-associated subtypes such as ST5, ST10 and ST14, with evidence of shared genotypes between hosts reinforcing the possibility of transmission across species lines. Synanthropic wildlife adds to the picture, with crows and pigeons in Tehran carrying ST13 and ST14, and cattle in western provinces dominated by ST5.

Bats, meanwhile, are an exceptionally diverse group in Iran, with at least fifty-one species documented across habitats that range from arid lowlands to mountainous forest. As the second most speciose mammalian order after rodents, they are known reservoirs of numerous zoonotic viruses, bacteria and protozoa, which makes the near-total absence of Blastocystis data on them a conspicuous gap. Previous parasitological work on Iranian bats has concentrated on ecology, taxonomy and molecular phylogeny rather than enteric protozoa. International studies offered only fragmentary clues: in Mexico, a single positive bat sample turned up among 124 wild animal faecal specimens examined, and in China, none of ten fruit bat faecal samples from Hainan yielded Blastocystis even though Cryptosporidium was highly prevalent in the same animals.

To close this gap, a team led by researchers affiliated with Shiraz University of Medical Sciences captured 115 bats from five caves across three provinces between 2021 and 2022. Three of the caves lay in Fars province, at Jahrom, Marvdasht and Ghader-Abad, while the other two were in Kurdistan province near Bijar and in Kermanshah province near Kerend. The bats were caught with hand nets, held individually in cloth bags for about an hour while faeces were collected, and then released. Species were identified morphologically by an experienced biologist using standard field guides, with characters such as fur colour, ear shape and forearm length guiding the determinations. Samples destined for molecular analysis were preserved in ninety-five percent ethanol and stored at minus twenty degrees Celsius.

The sampled animals represented four species across four genera. The long-winged bat Miniopterus pallidus and the mouse-eared bat Myotis blythii were the most frequently captured, together accounting for the great majority of individuals, followed by the mouse-tailed bat Rhinopoma microphyllum and the Mediterranean horseshoe bat Rhinolophus euryale. In the laboratory, roughly one hundred to one hundred and fifty milligrams of each faecal sample were processed for DNA extraction using a stool kit after an initial washing and centrifugation protocol to remove ethanol residues. Extracted DNA was quantified on a spectrophotometer, and only samples meeting strict concentration and purity thresholds were carried forward to amplification.

Detection relied on polymerase chain reaction targeting a roughly six-hundred-base-pair fragment of the Blastocystis small subunit ribosomal RNA gene, using the primer pair RD5 and BhRDr. The reaction mixture and cycling conditions followed established protocols, and any weak bands triggered a second round of amplification with the same primers to enhance specificity. A human-derived positive control, previously sequenced and deposited in GenBank, confirmed that the assay could detect the parasite, and gel electrophoresis with ethidium bromide staining allowed visualisation of amplified fragments. Despite this validated pipeline, not one of the 115 bat faecal samples produced a positive signal. The result was a clean sweep of negatives across every species, every cave and every province included in the survey.

The authors argue that several biological and ecological factors may help explain why bats appear resistant to Blastocystis colonisation. Roosting high on cave ceilings in large colonies minimises direct contact with soil and contaminated ground, the primary environmental source of infective cysts. Most bat species are insectivorous, and even the frugivorous ones rarely forage on the ground, which sharply reduces exposure to faecal-oral transmission routes that plague ground-foraging rodents and omnivorous livestock. The physiological demands of flight may also play a role: rapid intestinal transit times and distinctive gut physiology in flying mammals could simply deny the slow-growing anaerobic parasite the conditions it needs to establish itself. Whatever the precise mechanism, the pattern is consistent with surveys from Europe, Brazil and elsewhere that have rarely or never detected Blastocystis in chiropterans even when screening diverse mammalian groups side by side.

The researchers are careful to acknowledge the limitations of their approach. Detection rested entirely on a single molecular target, without complementary microscopy, culture or a secondary genetic marker, so very low-burden or atypical infections could in theory have been missed. They note, however, that SSU rRNA-targeted PCR is among the most sensitive and specific methods available, with reported sensitivities often exceeding ninety percent compared with less than fifty percent for microscopy, and that the uniformly negative results mirror the low-to-absent prevalence reported for bats elsewhere. Future work combining molecular screening with morphological examination and additional gene targets would help cement the conclusion. For now, the study delivers valuable negative data: in the regions examined, bats seem to play at most a marginal role in the epidemiology of Blastocystis, and the zoonotic risk posed by these much-maligned mammals appears, on this front at least, to be negligible. Continued surveillance of wildlife reservoirs remains essential, but the Iranian caves have, for once, returned reassuringly empty results.

Subject of Research: Molecular survey of Blastocystis prevalence in wild bat populations in western and southwestern Iran

Article Title: No Trace of Blastocystis Parasite in Bats Caught in the Western and Southwestern Regions of Iran

Article References: Mosayebi, M. A., Pouryousef, A., Omidian, M., Shahabi, S., & Sarkari, B. (2026). No Trace of Blastocystis Parasite in Bats Caught in the Western and Southwestern Regions of Iran. Veterinary Medicine and Science, 12(6), Article e71242. https://doi.org/10.1002/vms3.71242

Image Credits: AI Generated

DOI: 10.1002/vms3.71242

Keywords: Blastocystis, bats, Iran, zoonotic parasites, PCR, intestinal protozoa, wildlife surveillance, Chiroptera, SSU rRNA, public health, cave ecology, parasitology

Entamoeba species are intestinal protozoa with a worldwide distribution, infecting humans and a broad range of animals. While some members of the genus, most famously Entamoeba histolytica, are responsible for amoebic dysentery and liver abscesses in people, others colonize the gut without causing obvious harm. Because these organisms are transmitted through fecally contaminated food and water, environments where humans and animals share space create ideal conditions for cross-species transmission. Non-human primates are of particular concern in this regard, since their physiological and genetic similarity to humans means that many of their parasites face relatively few barriers when jumping between hosts.

The research team, led by scientists at Anhui Science and Technology University, collected a total of 504 fecal samples from crab-eating macaques, also known as long-tailed macaques (Macaca fascicularis), housed at commercial breeding farms in two geographically distinct Chinese cities. Rather than relying on microscopy, which can confuse morphologically identical Entamoeba species, the researchers used polymerase chain reaction amplification and sequencing of the small subunit ribosomal RNA gene, a genetic marker that allows precise differentiation among six distinct Entamoeba species. This molecular approach is essential because species such as E. histolytica, E. dispar, and E. moshkovskii look identical under the microscope yet differ dramatically in their clinical significance.

The results revealed a layered picture of infection. Entamoeba coli was by far the most prevalent species, detected in 49.01 percent of samples, or 247 of the 504 specimens tested, with a 95 percent confidence interval spanning 44.6 to 53.4 percent. E. dispar followed at 36.51 percent, while E. histolytica, the most medically consequential species, was found in 8.13 percent of samples. The remaining species appeared at lower frequencies: E. chattoni at 5.95 percent, E. moshkovskii at 0.79 percent, and E. nuttalli at just 0.6 percent. The detection of E. histolytica in more than one in twelve animals is noteworthy, as this species remains a leading cause of parasitic death in humans globally.

Statistical analysis of the infection data uncovered a significant relationship between host age and the risk of carrying E. coli, with the association reaching a p-value below 0.0001. This strong age dependence suggests that older macaques accumulate infections over time, possibly through prolonged exposure to contaminated enclosures, changes in immune competence, or altered grooming and foraging behaviors. Understanding such risk factors is critical for breeding facilities, where managing parasite burden can affect animal welfare, the validity of biomedical research using these animals, and the safety of staff who work in close contact with the colonies.

The most striking findings emerged from the phylogenetic analysis. When the researchers reconstructed evolutionary relationships among the 247 E. coli isolates recovered from the macaques, they identified two distinct genotypes, designated SZ E. coli CEMs1 and SZ E. coli CEMs2. Both genotypes clustered firmly within the major E. coli clade and showed close genetic affinity with strains previously recovered from humans and other primate species. This pattern of shared lineages across host species is precisely what would be expected if the parasites are moving between humans and macaques, rather than evolving in isolated host-specific populations.

The presence of E. nuttalli, although rare in this cohort, adds another dimension to the zoonotic picture. This species, once confused with E. histolytica, is known to infect rhesus macaques and can cause liver abscesses in experimental animals, and previous studies have documented its genetic differentiation across macaque populations in Nepal, Myanmar, and China. Similarly, the detection of E. moshkovskii, an organism long considered a free-living amoeba but increasingly reported in human patients with diarrhea, underscores how fluid the boundaries between environmental, animal, and human Entamoeba lineages can be. The low prevalence of these species in the sampled colonies does not diminish their significance; rather, it highlights the value of sensitive molecular surveillance in detecting rare but potentially important infections.

From a practical standpoint, the findings carry implications for multiple stakeholders. For breeding facilities that supply macaques for biomedical research, high parasite prevalence complicates efforts to produce animals of defined health status, since subclinical infections can confound immunological and gastrointestinal studies. For public health authorities, the phylogenetic proximity of macaque-derived E. coli strains to human isolates argues for treating captive macaque colonies as potential zoonotic reservoirs warranting routine monitoring. And for the growing field of One Health, which examines the interconnected health of humans, animals, and environments, the study provides a concrete example of how intensively managed animal populations can harbor parasites with demonstrated potential for cross-host transmission.

The authors emphasize that enhanced surveillance is needed, both within breeding facilities and in contexts where macaques and humans interact more loosely, such as zoological gardens and areas where free-ranging macaques contact tourists. Prior studies of captive primates and their handlers in European zoos have already documented shared intestinal protists, suggesting that occupational exposure is a realistic concern. The Chinese breeding farms surveyed here represent a controlled setting, yet even under such conditions, nearly half the animals carried E. coli and a meaningful fraction harbored potentially pathogenic species, indicating that standard husbandry practices may not be sufficient to interrupt transmission cycles.

Looking forward, the genetic characterization of the two macaque E. coli genotypes opens avenues for finer-scale epidemiological work. Comparing these lineages with sequence data from human patients, livestock, and wild primates across Asia could clarify the directionality of transmission events and identify which host populations act as sources and which as sinks. Such work would build on a growing body of molecular epidemiology that has transformed Entamoeba from a morphologically defined genus into a genetically structured assemblage of species with distinct host associations and virulence potentials. As the new study demonstrates, even a parasite long regarded as a harmless commensal can reveal important truths about the porous boundaries between animal and human health, and about the responsibility that comes with keeping our closest relatives in close quarters.

Subject of Research: Zoonotic Entamoeba parasite prevalence and genetic diversity in captive crab-eating macaques in China

Article Title: Distribution of Zoonotic Entamoeba spp. in Crab-Eating Macaques Across China

Article References: Guo, Q., Gong, R., Ge, X., Li, M., Ma, J., Liu, X., & Li, W. (2026). Distribution of Zoonotic Entamoeba spp. in Crab-Eating Macaques Across China. Acta Parasitologica, 71(5), Article 216. https://doi.org/10.1007/s11686-026-01399-7

Image Credits: AI Generated

DOI: 10.1007/s11686-026-01399-7

Keywords: Entamoeba, crab-eating macaques, zoonotic parasites, E. histolytica, E. coli, E. dispar, SSU rRNA, phylogenetics, China, non-human primates, One Health, molecular epidemiology