African swine fever virus DNA is present in non-biting flies collected from outbreak farms in Romania.

BACKGROUND: African swine fever (ASF) is a highly contagious and severe haemorrhagic disease of Suidae, with mortalities that approach 100 percent. Several studies suggested the potential implication of non-biting dipterans in the spread of ASFV in pig farms due to the identification of the ASFV DNA. However, to our knowledge, no study has evaluated the viral DNA load in non-biting dipterans collected in outbreak farms and no risk factors have been analysed. In this context, our study aimed to analyse the risk factors associated with the presence of non-biting dipterans collected from ASF outbreaks in relation to the presence and load of viral DNA. METHODS: Backyard farms (BF), type A farms (TAF), and commercial farms (CF), were targeted for sampling in 2020. In 2021, no BF were sampled. Each farm was sampled only once. The identification of the collected flies to family, genus, or species level was performed based on morphological characteristics using specific keys and descriptions. Pools were made prior to DNA extraction. All extracted DNA was tested for the presence of the ASFV using a real-time PCR protocol. For this study, we considered every sample with a CT value of 40 as positive. The statistical analysis was performed using Epi Info 7 software (CDC, USA). RESULTS: All collected non-biting flies belonged to five families: Calliphoridae, Sarcophagidae, Fanniidae, Drosophilidae, and Muscidae. Of the 361 pools, 201 were positive for the presence of ASFV DNA. The obtained CT values of the positive samples ranged from 21.54 to 39.63, with a median value of 33.59 and a mean value of 33.56. Significantly lower CT values (corresponding to higher viral DNA load) were obtained in Sarcophagidae, with a mean value of 32.56; a significantly higher number of positive pools were noticed in August, mean value = 33.12. CONCLUSIONS: Our study brings compelling evidence of the presence of the most common synanthropic flies near domestic pig farms carrying ASFV DNA, highlighting the importance of strengthening the biosecurity measures and protocols for prevention of the insect life cycle and distribution.

Diversity of Thelazia spp. in domestic cattle from Romania: epidemiology and molecular diagnosis by a novel multiplex PCR.

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.

Thelazia lacrymalis in horses from Romania: epidemiology, morphology and phylogenetic analysis.

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.

European Hares, Lepus europaeus, Represent a Reservoir Host for Thelazia callipaeda in Romania.

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.