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Abstract Background Toxoplasma gondii is a zoonotic protozoan parasite with a heteroxenus life cycle that involves felids as the definitive hosts and any warm-blooded animal, including humans, as intermediate hosts. Cats are key players in parasite transmission as they are capable of shedding high numbers of oocysts in their feces that contaminate the environment. Methods The study was performed on 31 domestic cats (31.23 ± 27.18 months old) originating from rural and urban areas (5.17:1) in the center and north-west Romania. Feces (n = 31), blood (n = 28), and heart samples (n = 27) were collected. Fecal samples were analyzed by flotation technique, and PCR (529 bp repetitive element). Fecal samples with T. gondii oocysts were bioassayed in mice. Serum samples were analyzed by modified agglutination test and ImmunoComb for the detection of specific anti-T. gondii IgG antibodies. Heart samples were bioassayed in mice, and analyzed by PCR. Toxoplasma gondii positive samples were genotyped by nPCR-RFLP targeting eleven genetic loci (SAG1, SAG2, alt-SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico). Results Toxoplasma gondii oocysts were found in 2 out of 31 fecal samples collected from a 3-months old stray kitten, and a 4-years old female. In total, 17 out of 27 sera were positive for T. gondii IgG antibodies. The antibody titers in MAT ranged from 1:6 to 1:384. Toxoplasma gondii DNA was detected in 7 out of 27 heart samples, and four of them were positive also by bioassay. Six T. gondii DNA samples from bioassayed mice could be assigned to ToxoDB PCR-RFLP genotype #1 or #3 (Type II) and one T. gondii DNA from heart digest to genotype #2 (Type III). Both of these genotypes are common in Europe. Conclusions Our results revealed that the infection with T. gondii is still high in cats from Romania. The oocysts shedded by these cats represent an important source of infection for intermediate hosts, including humans. Further studies on a wider range of cases are necessary for a more exhaustive definition of the T. gondii genotypes circulating in Romania.

Abstract Background Toxoplasma gondii is a protozoan parasite that infects a large spectrum of warm-blooded animals, including humans. Small rodents and insectivores play an important role in the epidemiology of T. gondii and may serve as a source of infection for both, domestic and wild definitive felid hosts. Factors influencing the occurrence of T. gondii in wild small mammals are unknown, despite the fact that many intermediate host species are identified. We have used small mammals (Rodentia and Lipotyphla) captured over two years in various habitats, both in urbanised and in natural landscapes. We assessed the importance of land-use, season and host ecology on T. gondii infection. Results We examined 471 individuals belonging to 20 small mammal species, collected at 63 locations spread over wide altitude, habitat and land-use ranges from Romania. Heart tissue samples were individually analysed by PCR targeting the 529 bp repetitive DNA fragment of T. gondii. The overall prevalence of infection was 7.3%, with nine species of rodents and two species of shrews being found to carry T. gondii DNA. Five species showed high frequency of infection, with the highest prevalence found in Myodes glareolus (35.5%), followed by Spermophilus citellus (33.3%), Sorex minutus (23.1%), S. araneus (21.7%) and Micromys minutus (11.1%). Adults seemed more often infected than young, however when controlling for season, the difference was not significant, as in spring both adults and young showed higher infection rates, but more adults were sampled. Contrary to our expectations, urban/rural areas (with their implicit high density of domestic feline presence) had no effect on infection prevalence. In addition, neither habitat, nor land-use at sampling sites was important as only geographical location and host species were contributing factors to the infection risk. Conclusions High prevalence of T. gondii infection showed a highly localised, patchy occurrence, with long living and higher mobility host species being the most common carriers, especially during autumn.

Abstract Background Toxoplasmosis is a widespread zoonosis caused by the intracellular protozoan parasite Toxoplasma gondii. Limited epidemiological information is available about the prevalence of T. gondii in sheep in Romania, and a high incidence would have implications for both the economy and public health. To our knowledge, no studies are available about the T. gondii strains circulating in lambs. The objective of this study was to assess the prevalence of T. gondii in sheep (serology), lambs (serology, bioassay, PCR) and sheep abortions (PCR) in Romania. Moreover, the study aimed to perform the genetic characterization of T. gondii isolates from lambs. Methods Serum samples collected from 2650 sheep (2067 adults and 583 lambs) were tested for anti-T. gondii antibodies (IgG) using a commercial ELISA kit. Likewise, 328 pairs of diaphragmatic muscle-serum samples were collected from lambs aged between 2 and 4 months. Lamb serum samples were analyzed using MAT for anti-T. gondii antibody detection. The diaphragm tissue samples from MAT-positive lambs (at a dilution ≥ 1:25) were bioassayed in mice. The T. gondii strains were genotyped using 15 microsatellites markers. Additionally, brain and heart samples from 76 sheep abortions were analyzed for T. gondii DNA by polymerase chain reaction (PCR) targeting the 529-bp repeat region (REP529). Results The results showed that more than half of the tested sheep were T. gondii seropositive (53.5%). The seroprevalence was significantly higher in adults (61.1%) than in lambs (26.4%). The seroprevalence of T. gondii infection in slaughtered lambs, by MAT, was 37.5% (123/328). There were bioassayed in mice 56 diaphragmatic tissues from 123 seropositive lambs. Toxoplasma gondii strains were isolated from 18 (32.1%) lambs intended for human consumption. All T. gondii strains were confirmed by PCR. Six strains were genotyped using 15 microsatellite markers and belonged to genotype II. Toxoplasma gondii DNA was detected in 11.8% (9/76) of sheep abortions. Conclusions The present study showed the presence of T. gondii in sheep in all the regions considered in the study. The high prevalence of T. gondii infection in sheep and lambs, demonstrated by serology, molecular analysis and bioassay, highlighted that there is an important risk of human infection in consuming raw or undercooked sheep/lamb meat. Graphical Abstract

Abstract Background During the last decades, filarial infections caused by Dirofilaria spp. have spread rapidly within dog populations of several European countries. Increasing scientific interest in filariasis, and the availability of new diagnostic tools, has led to improved knowledge of the biology, morphology, and epidemiology of different species of filarial worms. However, data are still scarce for a number of countries, including the Republic of Moldova. Thus, we assessed the epidemiological status of canine filariasis in the Republic of Moldova to address part of this knowledge gap. Methods A total of 120 blood samples were collected between June 2018 and July 2019 from dogs originating from the cities of Cahul and Chişinău. The samples were examined microscopically, and multiplex polymerase chain reaction was performed to evaluate filarioid species diversity. Results Microscopic examination revealed that 12 dogs (10.0%) were positive for circulating microfilariae. The molecular test showed that one dog was positive for Acanthocheilonema reconditum (0.8%), one for Dirofilaria immitis (0.8%), six for Dirofilaria repens (5.0%), and four (3.3%) harboured a co-infection with D. immitis and D. repens. Prevalence was significantly higher in dogs aged ≥ 2 years. Conclusions The epidemiological survey presented here for the Republic of Moldova confirmed the presence D. immitis, D. repens and A. reconditum in dogs that had not received any heartworm preventive. Graphical abstract

Abstract Background Leporacarus gibbus is a highly specific acarian parasitizing in rabbits, with a proven zoonotic potential. While the majority of cases of L. gibbus infestation are asymptomatic, several cases of pruritic cutaneous condition in both laboratory and pet rabbits were reported. Up to date, L. gibbus has not been linked with clinical signs in any other species than rabbits and humans. Case presentation This case report described the clinical case of a 14-month-old cat with a dermatitis linked to L. gibbus. Mites specimens were collected by brushing, followed by light microscopy examination and species identification. To the best of our knowledge, this is the first report of L. gibbus-related dermatitis in cat. Conclusions L. gibbus infestation should be considered as a possible differential diagnosis of pruritic skin conditions in cat.

This article describes two cases of notoedric mange concurrent with aelurostrongylidosis in two domestic-owned cats from a rural environment in Romania. Two European shorthair cats originating from the same litter were referred to our clinic, at 2 months apart, with a history of skin lesions, pruritus, weight loss, and respiratory signs. Cats lived mainly outdoor together with the queen and a third littermate. The latter two expressed the same clinical signs and had died before the presentation of the first cat. None of the four cats was vaccinated, dewormed, or treated for external parasites. Coat brushing, skin scrapings, skin cytology, earwax direct microscopic examination, and coproparasitological techniques (flotation and Baermann methods) were used as laboratory procedures. A blood sample was also collected for hematology, blood biochemistry, and feline immunodeficiency virus/feline leukemia virus (FIV/FeLV) test. Notoedres cati mites in high numbers were identified by all complementary tests, alongside fleas and Aelurostrongylus abstrusus first-stage larvae. The blood analysis revealed neutrophilia, and the FIV/FeLV fast test was negative. The cats were successfully treated off-label with selamectin spot-on formulation (Stronghold®, Zoetis) three times at 1- or 4-week intervals. Furthermore, they were treated with amoxicillin trihydrate/clavulanic acid, housed indoor, and fed with a commercial diet. Before presentation to the clinic, the female cat was unsuccessfully treated with a combination of fipronil, S-methoprene, eprinomectin, and praziquantel. During this period, the female cat remained outdoor and fed with home wastes. The cats become negative for A. abstrusus L1 larvae after 2–4 months of treatment. The owners developed pruritic skin lesions 1 month after introducing the first cat in the house. In conclusion, notoedric mange and aelurostrongylidosis can be treated successfully with selamectin as a spot-on formulation and the treatment must be continued until no parasite will be detected through specific techniques. The success of treatment depends on improving the quality of animal life (nutrition and hygiene) and treatment of secondary complications.

(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.

Abstract Background Foodborne toxoplasmosis in humans can be due to the exposure to tissue cysts of Toxoplasma gondii through the consumption of meat, including pork, of infected animals. Traditional Romanian food habits include pork as the preferred meat, while backyard pig rearing remains a common practice in many rural areas of Romania. The aims of the present study were to estimate the prevalence of T. gondii infection in naturally infected backyard pigs slaughtered for familial consumption and to genetically characterize the T. gondii strains obtained. Methods Paired blood and heart samples were collected from 94 backyard pigs, home slaughtered for private consumption. Serum samples were analyzed using the immunofluorescence antibody test (IFAT) for anti-T. gondii antibody detection. Heart samples were screened by polymerase chain reaction (PCR) targeting the 529-bp repeat region (REP529) for T. gondii detection. In addition, heart samples from IFAT positive animals were bioassayed in mice. The T. gondii isolates were genotyped by the analysis of 15 microsatellite markers. Results The results showed that almost half of the pigs investigated were T. gondii seropositive (46.8%, 95% confidence interval (CI): 36.4–57.4%) and in more than a quarter of the pigs (26.6%, 95% CI: 18.0–36.7%), the parasite was detected by PCR. Three (3/44) T. gondii strains were isolated from hearts of seropositive pigs and they all belonged to genotype II. Conclusions The present study showed the presence of T. gondii infection in backyard pigs in Romania, which suggests that consumption of pork from animals reared and slaughtered at home may pose a potential threat to human health and should be given attention. In addition, to our knowledge, this is the first study to provide data concerning T. gondii strains circulating in pigs from Romania.

Abstract Background Coccidiosis represents a serious threat to the poultry industry, affecting production and causing high morbidity, mortality and significant costs resulting from treatment and prophylaxis. In-feed anticoccidials have been used for decades for managing avian coccidiosis and were very effective until drug resistance emerged. The use of natural remedies has become a promising alternative in combating coccidiosis in chickens. Therefore, the purpose of the present study was to assess the efficiency of a commercial herbal formula (H), as oral liquid preparations, in experimental chicken coccidiosis. Methods Two independent controlled battery experiments (BE1 and BE2) were designed and the product was tested in 3 different formulas (H1, H2 and H3): H1 contained a propylene glycol extract of Allium sativum and Thymus serpyllum; H2 contained Origanum vulgare, Satureja hortensis and Chelidonium majus; and H3 contained Allium sativum, Urtica dioica, Inula helenium, Glycyrrhiza glabra, Rosmarinus officinalis, Chelidonium majus, Thymus serpyllum, Tanacetum vulgare and Coriandrum sativum. Chickens were divided into five groups for each BE as follows: (i) uninfected untreated control (UU1, UU2); (ii) infected untreated control (IU1, IU2); (iii) infected treated with amprolium (ITA1, ITA2); and (iv, v) two experimental groups infected treated with H1 (ITH1) and H2 (ITH2) formulas in the BE1 and with H3 (ITH3-5 and ITH3-10) formula in the BE2. The chickens from infected groups were challenged with 5000 (BE1) and 50,000 (BE2) sporulated oocysts of Eimeria spp. (E. acervulina, E. tenella and E. maxima), respectively. The anticoccidial efficacy was assessed by recording the following: oocysts output (OPG), lesion score (LS), weight gain (WG), feed conversion ratio (FCR) and anticoccidial index (ACI). Additionally, polyphenolics and flavonoids (caffeic-chlorogenic acid, apigenin, kaempferol, luteolin, quercitin, quercitrin) from herb extracts found in H3 formula were determined by the liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. Results H1 and H2 reduced the WG, and increased the FCR and OPG compared with controls. H1 reduced the duodenal lesions, whilst H2 reduced the caecal lesions, compared with control. H3 decreased the OPG of Eimeria spp., reduced the total lesion score and improved the zootechnical performance (weight gain and feed conversion ratio). According to ACI value, H1 and H2 had no efficacy on Eimeria spp. infection, but H3 had good to marked anticoccidial effect, the ACI being slightly greater in the group ITH3-5. According to the results of LC-MS/MS, the concentration of polyphenols in H3 formula was the highest, the sum of chlorogenic acid and caffeic acid being 914.9 µg/ml. Conclusions H3 formula is a promising natural anticoccidial and field trials are recommended in order to validate the obtained data.

The aim of this study was to determine the normal and pathological behavior of captive primates in a zoo in Tirgu-Mures. The biological material consisted of 59 monkeys belonging to 11 species. The ethological research involved the following instruments: observation, experimentation, ethogram, spectrum analyzer, video, respectively the causal analysis. The obtained results revealed that the captive monkeys sheltered in isolated cages, with a similar development to what is found in their natural environment and placed at a tolerable distance away from visitors’ activity show no behavioral changes. On the other hand, in the case of those primates sheltered in cages which are exposed to visitors’ noise, we observed the presence of oral and motor stereotypies but also an attitude of lethargic disappointment and preoccupation. The collected data allow to define the investigated primates’ needs and also to get an idea of their welfare in zoo in Tirgu-Mures.

Canine leishmaniasis (CanL) is a zoonotic disease considered endemic in the Mediterranean region. Romania is traditionally regarded as a non-endemic country. Considering the zoonotic character of the disease, this study aims to increase the knowledge on diagnostic aspects of CanL. A 2-year-old, mix breed male dog, recently returned from Italy had a history of progressive weight loss and skin lesions. The clinical examination was followed by hematology and serum biochemistry, fine-needle aspiration of lymph nodes, impression smears, VetExpert® rapid test Leishmania Ab, and PCR. The clinical examination revealed muscle atrophy, non-pruritic crusting dermatitis, ulcers, and lymphadenopathy. Hematology showed severe anemia. The serum biochemistry revealed hyperproteinaemia, hypoalbuminemia, hyperglobulinemia. Cytological exams evidenced the intracellular amastigotes in macrophages, confirmed by rapid test and PCR. In Romania, under the light of the new case reports, leishmaniasis should be reconsidered from both veterinary and public health perspective.

Abstract Background Giardia duodenalis is a parasitic protist that infects a large number of species, being localized in the small intestine. Two of the eight recognized assemblages have zoonotic potential, but studies regarding their distribution in less important pet or farm species are scarce. Of these species, the long-tailed chinchilla is a host for Giardia spp., although data on the spread of infection and assemblages involved are confined. The present work aimed to determine the prevalence of Giardia infection and assemblage identification in farmed chinchillas in Romania. A total of 341 fecal samples were collected from 5 farms and microscopically examined using flotation test based on saturated sodium chloride solution. DNA from all positive samples was extracted and identified by PCR targeting the gdh gene. Results The overall prevalence of Giardia infection was 55.7% (190/341); there was no statistically significant difference (P = 0.25) in prevalence between young animals (58.8%) and adults (52.6%). Assemblages B (151/190), D (33/190) and E (6/190) were identified. Among assemblage B, sub-assemblages BIII (6/151) and BIV (145/151) were determined. Conclusions This study demonstrates that Giardia spp. infection is highly prevalent in farmed chinchillas from Romania, and the sub-assemblages identified are potentially zoonotic.

Intestinal parasites are involved in the health and wellbeing of cats and some of them, due to their zoonotic potential, represent a problem for public health. This study aimed to assess the prevalence of parasitic infections in cats with gastrointestinal signs. Fecal samples collected from 137 cats were analyzed by the flotation method using a sodium chloride saturated solution. The overall prevalence of intestinal parasites was 50.4%. Intestinal parasites were significantly (p < 0.0001) more common in cats with digestive clinical signs (66.7%) than in clinically healthy ones (19.2%). Toxocara cati (40.2%) was the most frequently identified intestinal parasite, followed by Cystoisospora spp. (10.2%), hookworms (3.7%), Taeniidae (2.2%), Giardia duodenalis (2.2%), and Toxoplasma gondii (0.7%). Toxocara cati (53.3%, p < 0.0001) and Cystoisospora spp. (15.6%, p < 0.001) were significantly more frequently diagnosed in cats with clinical signs. A lack of deworming in the last three months (OR: 15.9), outdoor access (OR: 13.8), the presence of digestive symptoms (OR: 5.4), and young age (OR: 4.2) were identified as risk factors for T. cati infection by logistic regression analysis. Regardless of age, gastrointestinal signs like vomiting, diarrhea, and inappetence were positively associated with T. cati.

Background: Lungworms are recognized as important agents in the pathology of the respiratory system in domestic cats. While Aelurostrongylus abstrusus is worldwide known and studied, Troglostrongylus brevior has gained the attention of the scientific community only in the last decade. The pathogenicity of this species seems to be higher than A. abstrusus, causing more severe clinical presentations and being potentially fatal, especially in young animals. Methods: In this study, 371 DNA isolates of faecal samples were tested by multiplex polymerase chain reaction for the presence of A. abstrusus, T. brevior, and Angiostrongylus chabaudi. Results: The results showed that 30.2% and 6.7% of the investigated domestic cats were positive for A. abstrusus and T. brevior respectively, stressing out the importance of these parasites as agents of respiratory conditions in domestic cats from Romania. None of the samples were positive for A. chabaudi. The age, the outdoor access, and the lack of deworming were identified as significant risk factors for infection with A. abstrusus. Conclusions: This paper represents the first report of T. brevior in domestic cats from Romania. Moreover, it presents the most northern localization in Europe of T. brevior.

Abstract Canine leishmaniasis is a vector-borne zoonotic disease caused by the protozoan parasite Leishmania infantum. In Romania between 1955 and 2013, no cases of human autochthonous visceral leishmaniasis were reported. Data regarding canine leishmaniasis is similarly scarce. Since the first report of clinical autochthonous canine leishmaniasis in 1935, there were only three sporadic reports of positive dogs all without any clinical signs. Our study reports the first clinical case of autochthonous canine leishmaniasis in the last 80 years, stressing the importance of a targeted surveillance of Leishmania infection, as infected dogs act as the primary reservoir for zoonotic visceral leishmaniasis.

ABSTRACT In the present study, we aimed to assess the toxicity of artemisinin on the haematological system and its effect on the productive performance of broiler chickens. Eighteen-day-old chickens were randomly divided into four groups of 30 chickens (three replicates of 10 broilers/group): control group and three experimental groups: ART5 - diet with 5 ppm of artemisinin; ART50 - diet with 50 ppm of artemisinin; and ART500 - diet with 500 ppm of artemisinin. Artemisinin enhanced the productive performances of broiler chickens at the lowest concentration (5 ppm), but at the highest concentration (500 ppm), it negatively affected weight gain and the feed conversion ratio. The performance characteristics of the chickens whose diets were supplemented with 50 ppm artemisinin were similar to those of the control group. Additionally, 5 ppm artemisinin in feed did not significantly affect the haematological parameters of the chickens, but 50 and 500 ppm artemisinin induced a gradual decline of the total leukocytes, lymphopenia, monocytosis, and eosinopenia, and the highest concentration caused anaemia. Artemisinin at a low concentration could be used as a feed additive in the poultry industry to improve organic broiler production performance with no serious side effects.

Abstract  The first identification of Neospora caninum infection in aborted bovine foetuses in Romania is reported. Nine aborted foetuses were collected from a dairy farm. The foetal age of foetuses was between 3 and 7 months. A 7-month-old foetus was mummified. N. caninum DNA Nc-5 region was amplified from samples extracted from brain tissues of three (33.3%) aborted foetuses. The foetal ages of PCR positive foetuses were 3, 4 and 7 months (mummified). No specific lesions or tissue cysts were found to the histological examination, because of advanced autolysis of brains. [ABSTRACT FROM AUTHOR]

Few studies were conducted to investigate the pathogenesis and to characterize the histopathological alterations in the intestinal mucosa of kids with cryptosporidiosis. In our study, 58 kids were presented for necropsy, and the faeces of another 83 goat kids were studied. Animals were aged between 1 day and 3 weeks. Infection was confirmed by oocyst identification in the faeces (Ziehl-Neelsen stain modified by Henriksen). Gross examination was followed by examination of the tissue using histopathological procedures (Hematoxylin-Eosin stain), scanning electron microscopy (SEM) and immunofluorescence. Pathological changes occurred mainly in the ileum, but also jejunum and caecum of the infected animals, at the apical surface of intestinal epithelial cells. Oocysts appeared as round or oval formations, with a diameter ≤4.5 µm, sometimes covering the atrophic and denudated free border of intestinal villi. Most histological and morphological characteristics of infection with C. parvum are common on the whole to other Cryptosporidium spp. Massive dentification of cryptospodiosis in kids indicates the possible role of these animals as disease reservoirs for other animals and for humans. This is the first study to characterize the Cryptosporidium infection by confocal technique and SEM in Romania. [ABSTRACT FROM AUTHOR]