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.

Efficacy of Artemisia annua against Coccidiosis in Broiler Chickens: A Field Trial.

(1) Background: Various studies on artemisinin and its derivatives have shown that Artemisia annua may be of therapeutic interest for different diseases, including chicken coccidiosis. This study aimed to evaluate the effects of Artemisia annua on farm-reared broiler chickens by analyzing both the anticoccidial efficacy and its effect on the intestinal microbiota of poultry. (2) Methods: The experiment was performed within three houses on a broiler chicken farm located in Romania. House 1 was the experimental group and received a diet with an addition of A. annua. Houses 2 and 4 were the control groups and received anticoccidials. The prophylactic efficacy of A. annua against coccidiosis was evaluated by recording the weight gain, feed conversion rate, number of oocysts per gram of feces, lesion score, and mortality rate. (3) Results: The chickens fed with A. annua showed a decreasing trend in the number of oocysts per gram of faeces, and their lesion score was 80% lower than in the control group. The weight gains of the chickens treated with A. annua was lower, whilst the feed conversion rate was better than in controls. (4) Conclusions: Artemisia annua showed promising results in the prophylaxis of coccidiosis. Overall, the broiler chickens that received A. annua presented promising zootechnical performances and medical data related to coccidiosis and gut microbiota.

Seroprevalence Rates against West Nile, Usutu, and Tick-Borne Encephalitis Viruses in Blood-Donors from North-Western Romania.

INTRODUCTION: West Nile virus (WNV), Usutu virus (USUV), and the tick-borne encephalitis virus (TBEV) are all arboviruses belonging to Flaviviridae family. All are characterized by vectorial transmission and sometimes associated with neuroinvasive infections. The circulation of these viruses is considered endemic in parts of Europe, with human cases reported in many countries. Among hosts, the viruses are vectored by hematophagous arthropods, such as mosquitoes (WNV, USUV) and ticks (TBEV). Considering the currently outdated knowledge regarding the epidemiology of these viruses in Romania, the aim of our study was to assess the seroprevalence rates of WNV, USUV, and TBEV among healthy blood donors in north-western Romania. METHODS: Human blood samples from healthy donors were collected between November 2019 and February 2020 in six counties from the north-western region of Romania. The samples were serologically tested by ELISA and serum neutralization test. RESULTS: Overall, we obtained a seroprevalence of 3.17% for WNV, 0.08% for TBEV, and 0% for USUV. CONCLUSION: Despite the low seroprevalence of WNV, USUV, and TBEV in our study, we highlight the need for continuous nationwide vector and disease surveillance and implementation of control measures. Further research is required for an optimal overview of the epidemiological status of the Romanian population regarding these flaviviruses together with countrywide awareness campaigns.

Spotted Fever Group Rickettsia spp. Diversity in Ticks and the First Report of Rickettsia hoogstraalii in Romania.

Tickborne bacterial pathogens have been described worldwide as risk factors for both animal and human health. Spotted fevers caused by Rickettsiae may cause non-specific symptoms, which make clinical diagnosis difficult. The aim of the current study was to evaluate and review the diversity of SFG Rickettsiae in ticks collected in 41 counties in Romania. A total of 2028 questing and engorged ticks collected in Romania belonging to five species were tested by PCR amplification of Rickettsia spp. gltA and 17-D gene fragments: Ixodes ricinus (n = 1128), Dermacentor marginatus (n = 507), D. reticulatus (n = 165), Rhipicephalus rossicus (n = 128) and Haemaphysalis punctata (n = 100). Five Rickettsia species were identified following DNA sequence analysis: R. helvetica, R. monacensis, R. slovaca, R. raoultii, and R. hoogstraalii. The most common species detected was R. monacensis. Moreover, R. hoogstraalii was detected for the first time in Romania and in R. rossicus ticks. The detection of R. raoultii and R. monacensis in questing larvae of Hae. punctata suggests the possible transovarial transmission of these Rickettsia species in ticks. The detection of R. hoogstraalii for the first time in Romania increases the reported SFG Rickettsia diversity in the country.

Antiparasitic Action of Lactobacillus casei ATCC 393 and Lactobacillus paracasei CNCM Strains in CD-1 Mice Experimentally Infected with Trichinella britovi.

Nematodes of the genus Trichinella are among the most widespread parasites of domestic and wild omnivores and predatory animals. The present study aimed to evaluate the antiparasitic effect of Lactobacillus casei ATCC 393 (original) and L. paracasei CNCM in CD-1 mice experimentally infected with Trichinella britovi. Four groups of 20 mice (10 females and 10 males/group) were used, with two control (C) groups and two experimental (E) groups, in which each animal received a daily oral dose of 100 µL of 105 CFU/mL probiotics in Ringer's solution. On day 7, all mice (except the negative control group) were infected orally with Trichinella (100 larvae/animal) as well as the two probiotics. On day 9 post-infection (p.i.), 10 mice/group were euthanized, and the presence of adult parasites in the intestinal content and wall was tested. On day 32 p.i., 10 mice/group were euthanized, then trichinoscopy and artificial digestion were performed to assess the muscle infection with T. britovi. On day 9 p.i., the experimental group pretreated with L. casei ATCC 393 (6.3 ± 3.03) showed a significantly lower number of adult parasites in the intestinal wall compared with the positive control group (24.6 ± 4.78). Additionally, a significantly lower adult parasite count in the intestinal wall was registered in female mice pretreated with L. paracasei CNCM (7.4 ± 4.71) compared to female mice from the positive control (29.0 ± 5.17). No statistically relevant results were obtained concerning the male mice or the data obtained at 32 days p.i., irrespective of mice gender.

Recreational behaviour, risk perceptions, and protective practices against ticks: a cross-sectional comparative study before and during the lockdown enforced by the COVID-19 pandemic in Romania.

BACKGROUND: Gaining insight into the risk perceptions and the knowledge evolution of the public about emerging or changing health risks is vital for the improvement of health promotion activities. Currently, scientific evidence regarding the attitudes of the Romanian public towards ticks is scanty. This study aimed to identify how the lockdown enforced by the COVID-19 pandemic in Romania impacted the recreational behaviour, risk perceptions, and protective practices of the Romanian population regarding ticks. METHODS: A cross-sectional, nationwide web-based questionnaire was designed and distributed via social media to evaluate if, and how, the COVID-19 lockdown impacted the behaviour of the Romanian public concerning ticks. The survey was available online from 6 May until 15 May 2020, which marked the last day of the travel ban in Romania. The collected data were processed by applying both uni- and multivariate methods. RESULTS: Respondents reported a higher frequency of finding ticks on themselves and their dogs during the lockdown. Bathing/showering and checking the body for ticks were the two most used protective behaviours both before and during the lockdown. Nevertheless, an overall lower usage rate of protective measures was registered during the lockdown. Almost all dog owners used a form of ectoparasite control for their dogs, and only three stopped due to lockdown-associated reasons. Respondent characteristics that were found to be positively associated with risk perceptions were being female and living in peri-urban/suburban/rural environments. CONCLUSIONS: Despite spending less time outdoors during the lockdown, more respondents reported finding ticks on themselves or their dogs. Changes in the preferences for recreational locations, rates of protective practices usage, amount of time spent in specific areas, or tick seasonal activity might have contributed to this outcome. Concerning risk groups, men of all ages, senior citizens, and rural inhabitants should be targeted by the relevant Romanian authorities when promoting local or nationwide tick awareness campaigns.