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Abstract Background Dictyocaulosis is a parasitic disease caused by pulmonary nematodes from genus Dictyocaulus affecting various ungulate hosts. It can cause verminous bronchopneumonia and for heavily infected individuals, fatal outcomes can occur. Case presentation The study describes the case of a male European bison which died three months after relocation from Slovakia to one of the reintroduction areas in Nucșoara village, Făgăraș Mountains, Romania. Necropsy revealed the presence of pulmonary nematodes in the respiratory tract. Morphology and molecular diagnosis was performed and Dictyocaulus viviparus was identified. Conclusion Rigorous health monitoring is essential to assure the success of reintroduction programs, understanding and preventing infectious diseases and limiting their impact on population health.

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

Thelaziosis caused by Thelazia callipaeda is an emerging disease in Europe. Only two reports of naturally infected lagomorphs have been published so far. The aim of this study was to evaluate the status of the Romanian populations of European brown hares, Lepus europaeus as reservoir hosts for T. callipaeda. Between November 2019 and November 2021, the eyes of 326 L. europaeus carcasses were examined for the presence of ocular parasites. Nematodes were stored in plastic vials with physiological saline, followed by morphological and molecular identification. QGis 3.20 and EpiInfoTM 7 were used for mapping and statistical analysis. Four (1.23%) hares harbored T. callipaeda infection, with a total of 84 nematodes collected (mean intensity 21 nematodes/host), with 45 males, 39 females (two sexually immature, seven with only eggs, and 30 with eggs and larvae). One specimen from each host was successfully sequenced resulting in a 100% similarity with several other sequences of T. callipaeda haplotype 1. Statistical analysis revealed no significant results. The current study represents a first report of T. callipaeda in the European brown hare in Romania, and the second in Europe, also reiterating the role of lagomorphs as reservoir hosts for this zoonotic ocular nematode.

Background: Platyhelminthes are common parasites of diurnal birds of prey, including common buzzard (Buteo buteo). They are widely distributed in Europe. Despite the extensive study of flatworms across the continent, this study represents the first report for Romania and extends the biogeographic knowledge of Platyhelminthes in diurnal raptors. It also identifies common buzzards as a new host of Parastrigea flexilis in Europe. Materials and methods: Between 2017 and 2020, 63 common buzzards (Buteo buteo) found road killed or dead due to other causes were collected from Romania. All specimens were necropsied and examined for internal helminths, which were preserved in ethanol for molecular identification and formaldehyde for morphological identification. Results: Species identified included Cestoda: Cladotaenia globifera (33.3%) and five species of Trematoda: Neodiplostomum attenuatum (12.7%), Strigea falconis (6.4%), Parastrigea flexilis (3.2%), Neodiplostomum spathoides (3.2%) and Brachylaima fuscata (1.6%). Conclusions: This study provides new data about diversity, phylogenetics and geographical distribution of Platyhelminthes parasitizing Buteo buteo in Romania. To our best knowledge, it identifies B. buteo as a new host of Parastrigea flexilis and reports the first genetic sequence of Neodiplostomum spathoides.

Abstract Background Ethno-veterinary practices could be used as a sustainable developmental tool by integrating traditional phytotherapy and husbandry. Phytotherapeutics are available and used worldwide. However, evidence of their antiparasitic efficacy is currently very limited. Parasitic diseases have a considerable effect on pig production, causing economic losses due to high morbidity and mortality. In this respect, especially smallholders and organic producers face severe challenges. Parasites, as disease causing agents, often outcompete other pathogens in such extensive production systems. A total of 720 faecal samples were collected in two farms from three age categories, i.e. weaners, fatteners, and sows. Flotation (Willis and McMaster method), modified Ziehl–Neelsen stained faecal smear, centrifugal sedimentation, modified Blagg technique, and faecal cultures were used to identify parasites and quantify the parasitic load. Results The examination confirmed the presence of infections with Eimeria spp., Cryptosporidium spp., Balantioides coli (syn. Balantidium coli), Ascaris suum, Oesophagostomum spp., Strongyloides ransomi, and Trichuris suis, distributed based on age category. A dose of 180 mg/kg bw/day of Allium sativum L. and 90 mg/kg bw/day of Artemisia absinthium L. powders, administered for 10 consecutive days, revealed a strong, taxonomy-based antiprotozoal and anthelmintic activity. Conclusions The results highlighted the therapeutic potential of both A. sativum and A. absinthium against gastrointestinal parasites in pigs. Their therapeutic effectiveness may be attributed to the content in polyphenols, tocopherols, flavonoids, sterols, sesquiterpene lactones, and sulfoxide. Further research is required to establish the minimal effective dose of both plants against digestive parasites in pigs.

Internal parasitic diseases of swine constitute a major welfare and health concern in low-input livestock farming. Due to an increase in chemical resistance, phytotherapeutic remedies have become an alternative for the prophylaxis and therapy of digestive parasitosis, albeit few remedies have been subjected to scientific validation. Low-input swine farming in Romania has adopted the traditional use of phytotherapy for controlling pathogens in livestock. The current study aimed to assess the antiparasitic potential of Calendula officinalis and Satureja hortensis against digestive parasites of swine in two low-input farms. The fecal samples were collected from sows, fatteners, and weaners, and were tested using the following coproparasitological methods: centrifugal sedimentation, flotation (Willis, McMaster egg counting technique), Ziehl–Neelsen stain modified by Henricksen, modified Blagg method, and in vitro nematode larvae/protozoan oocyst cultures. Six species of digestive parasites were diagnosed, namely Ascaris suum, Trichuris suis, Oesophagostomum spp., Balantioides coli, Eimeria spp., and Cryptosporidium spp., in various combinations, dependent on the swine category. A dose of 140 mg/kg bw/day of C. officinalis and 100 mg/kg bw/day of S. hortensis powders administered for 10 consecutive days revealed a strong antiprotozoal and anthelmintic activity on the aforementioned parasites. The curative efficacy can be attributed to the presence of polyphenols, sterols, tocopherols, and methoxylated flavones. In conclusion, our results indicate that S. hortensis and C. officinalis are promising alternatives to the commercially available antiparasitics, enabling their use as natural antiparasitic products against gastrointestinal parasites in pigs.

Parasitic diseases are responsible for substantial losses in reproduction and productivity in swine, creating a major impairment to efficient and profitable livestock management. The use of phytotherapeutic remedies has notably increased over the past decade due to their bioavailability, decreased toxicity, non-polluting nature, and to some extent due to their antiparasitic effect. The aim of this study was to evaluate the antiparasitic potential of Cucurbita pepo L. and Coriandrum sativum L. against protozoa and nematodes found in swine. The samples were collected from weaners, fatteners, and sows and examined via flotation (Willis and McMaster), active sedimentation, Ziehl-Neelsen staining as modified by Henricksen, a modified Blagg method, and eggs/oocyst culture. The parasite species detected were Ascaris suum, Trichuris suis, Oesophagostomum spp., Balantioides coli (syn. Balantidium coli), Eimeria spp., and Cryptosporidium spp., depending on age category. A dose of 500 mg/kg bw/day of C. pepo and 170 mg/kg bw/day of C. sativum powders, administered for ten consecutive days, demonstrated a pronounced anthelmintic (pumpkin) and antiprotozoal (coriander) effect against the aforementioned parasites. Future studies are required to ascertain the optimal dose that maximizes their antiparasitic effectiveness. The current study represents the first Romanian report on the in vivo antiparasitic activity of these two plants tested on digestive parasites in swine.

Coccidiosis in pigs seldom results in important economic losses. However, it can influence growth rates in weaners and it is an important hygiene indicator in swine farms. Certain herbs, along with their extracts, have been used over the course of history in traditional medicine. The aim of this study was to evaluate the in vitro anticoccidial effects of Allium sativum L. (garlic), Artemisia absinthium L. (wormwood), Coriandrum sativum L. (coriander), Cucurbita pepo L. (pumpkin), Satureja hortensis L. (summer savory), and Calendula officinalis L. (marigold) against Eimeria suis and Eimeria debliecki oocysts. The stock solution of oocysts (58% E. suis + 42% E. debliecki) was incubated for three days, before adding the tested solutions. The unsporulated Eimeria spp. oocysts were then placed in a 3 mL well plate and incubated for 96 h at 27 °C, in a suspension containing serial dilutions of alcoholic plant extracts (5%, 2.5%, 1.25%, 0.625%, and 0.312%). The percentage of sporulated and destroyed oocysts was recorded every 24 h for 96 h. All extracts had a good in vitro anticoccidial effect against oocysts of Eimeria spp. compared with the control groups. Oocyst sporulation was significantly (p = 0.05) inhibited by the 5% marigold extract (3.6% sporulated oocysts). The same extract had the highest lytic effect on oocysts (65.2% destroyed oocysts). Our results prove that the most effective alcoholic plant extract is the marigold extract, followed, in order of efficiency, by the wormwood, coriander, garlic, pumpkin, and summer savory extracts. This study represents a preliminary contribution for establishing a new generation of natural disinfectants aimed at destroying Eimeria oocysts in the context of swine contamination.