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Abstract Background Thelaziosis is a neglected vector-borne disease caused by parasitic nematode worms of the genus Thelazia which affects various hosts. Limited attention has been given to ungulate-associated Thelazia species. Current diagnosis of thelaziosis and the identification/differentiation of species heavily relies on morphological features. Therefore, we conducted an epidemiological study in Romanian cattle, with the aim to obtain morphological and molecular data that can be used for species identification. Methods The eyes of 705 slaughtered cattle were sampled and subjected to morphological identification, morphometric analysis, and molecular characterization. PCR amplification and sequence analysis were performed based on the cytochromec oxidase subunit 1 (COI) gene. Statistical tests assessed the correlations between infection parameters and ecological or biogeographical factors. A novel PCR method was developed based on the consensus sequence from each species. Specific forward primers were designed for each of the three species, and a reverse primer (COIintR) was used for all reactions. A consensus thermal profile was established by gradient PCR amplification of each species separately. Results Of the sampled cattle, 19.3% were infected with Thelazia spp. Prevalence varied significantly with ecogeographical factors. A total of 585 Thelazia nematodes were recovered, with T. rhodesi being the most abundant, followed by T. skrjabini and T. gulosa. Morphometric and molecular analyses supported the morphological identification, yielding unique sequences for each species. From the 59 T. rhodesi specimens sequenced, 29 unique sequences were obtained, with a 99.1–99.85% nucleotide identity to the only other COI sequence present in GenBank®. All nine T. gulosa isolates were unique (99.37–100% nucleotide identity to other sequences), while T. skrjabini specimens displayed 98.47–100% nucleotide identity to the sole available sequence. Conclusions Bovine thelaziosis is prevalent in Romania, raising concerns for animal welfare and potential economic impacts. Infected cattle grazing alongside vulnerable wild ruminants, such as the European bison, may affect conservation efforts. Our newly developed multiplex PCR shows promise as a valuable surveillance tool, enabling the detection of occult infections in apparently healthy animals through lachrymal secretion testing. Graphical Abstract

Abstract Background Equine thelaziosis is a neglected vector-borne parasitic disease in modern veterinary medicine, lacking recent reports. It is transmitted by Musca autumnalis, and potentially other Muscidae species, by ingesting the lachrymal secretions of its equine host. The distribution of both Thelazia lacrymalis and its intermediate hosts remains largely unknown throughout Europe, with most studies dating back 20 years. The aim of this study was to assess the presence, prevalence and distribution of T. lacrymalis in horses from Romania. Methods The eyes of 273 horses, slaughtered at two abattoirs from the Northwestern and Western regions of Romania, were examined for the presence of T. lacrymalis between March and November 2021. Upon detection, the nematodes were collected and morphologically identified using the keys from literature. Following identification, one specimen from each animal was selected for molecular analysis while the rest underwent detailed morphometric measurements. Mapping and distribution, according to ecoregions, was done using the QGis 3.20 software, while sequences obtained were compared to those available in GenBank through BLAST analysis using the MEGA X software. Results Of the 273 animals sampled, 12 (4.39%) were positive for Thelazia spp. infection. Eighty-seven nematodes were recovered, all morphologically identified as T. lacrymalis. The intensity of infestation varied between one and 33 nematodes/animal while five animals presented a bilateral infestation and seven a unilateral one. The highest prevalence was encountered in Pannonian ecoregion (12.12%) while the lowest was in the Alpine ecoregion (0%). Seventy-five intact specimens underwent detailed morphometric analysis, of the 18–20 parameters, resulting in notable differences in striation lengths compared to the data available in other reports. BLAST analysis identified a 96.46–98.60% similarity to the only other COI gene sequence available for T. lacrymalis. Conclusions The current study represents the first report of T. lacrymalis in horses in Romania. The low prevalence rates are probably linked to the wide use of macrocyclic lactones. Graphic Abstract