Identification and Molecular Characterization of Giant Liver Fluke (Fascioloides magna) Infection in European Fallow Deer (Dama dama) in Romania-First Report.

Fascioloidosis is a parasitic disease of primary wild and domestic ruminants, caused by giant liver fluke, Fascioloides magna. The definitive host of the liver fluke in its area of origin (North America) is the white-tailed deer (Odocoileus virginianus). In Europe, the red deer (Cervus elaphus) and European fallow deer (Dama dama) are definitive hosts and the most sensitive hosts to F. magna infection, on which the parasite exerts serious pathogenic effects. In this study, we analyzed fecal samples and livers of 72 D. dama from 11 hunting grounds in Arad County, Romania. Of the 72 fecal samples and livers from D. dama, trematodes of the genus Fascioloides were identified in four (5.56%). Sequencing revealed that the trematodes identified in the samples were similar to the sequence of F. magna (GenBank no. EF534992.1, DQ683545.1, KU232369.1). The sequence obtained from the molecular analysis has been deposited in GenBank® under accession number OQ689976.1. This study describes the first report of giant liver fluke (F. magna) infection in D. dama in Romania.

First study in the frequency of isolation and phenotypic antimicrobial resistance profiles of pig and cattle origin Campylobacter strains in Romania.

Campylobacter spp. is recognized as one of the most common pathogens involved in the development of gastrointestinal infections in humans. The current study aimed to enhance the knowledge on the occurrence and molecular characterization of Campylobacter spp. in pigs and cattle origin caecum samples (n = 56) collected in one year, from nine Romanian slaughterhouses, and to determine the antimicrobial resistance profile of the isolated strains. All Campylobacter spp. strains (n = 41) isolated from swine and cattle caecum samples were analyzed in terms of antimicrobial resistance, in accordance with the EURL protocol and with the Commission Implementing Decision No. 2020/1729. The prevalence rate for C. coli. in pig caecum samples was 92.3% (36/39), and the prevalence of C. jejuni, in cattle origin samples was 29.4% (5/17). C. coli strains isolated from pigs proved resistant to tetracycline 75% (27/36), ciprofloxacin 69.4% (25/36), erythromycin 8.3% (3/36), ertapenem 2.8% (1/36) and gentamicin 2.8% (1/36), but no resistance was observed towards chloramphenicol. C. jejuni strains originating from cattle expressed resistance to ciprofloxacin 60.0% (3/5) and tetracycline 20.0% (1/5), but they were susceptible to chloramphenicol, erythromycin, ertapenem and gentamicin. In the present study, 19.5% (7/36) C. coli strains isolated from pigs were identified as multidrug-resistant (MDR) bacteria. The obtained results demonstrated that pigs especially, but cattle to, can be considered important natural reservoirs for zoonotic multidrug-resistant Campylobacter strains, having a stimulating effect for further studies aiming at the molecular screening of the genotypic antimicrobial resistance processing of a higher number of samples.

First Molecular Data of Gongylonema pulchrum (Rhabditida: Gongylonematidae) in European Fallow Deer Dama dama from Romania.

Due to its adaptive versatility to numerous types of habitats, extremely diverse both in terms of composition and specificity, developed in various areas of the Western Plains of Romania, the European fallow deer (Dama dama) is a species with high ecological plasticity. In this area, the D. dama interacts with other species of wild fauna but also with numerous domestic animals, an important aspect in terms of the sanitary-veterinary status of animal populations, as well as the existence of a potential risk of infection with various species of parasites that can cause the D. dama specimens to obtain certain diseases and even zoonoses. A total of 133 esophagi from D. dama have been examined for helminths. Of the 133 esophagus samples from D. dama, nematodes of the genus Gongylonema were identified in 25 (18.80%). Sequencing revealed that the nematode identified in the samples was 99% similar to the sequence of Gongylonema pulchrum (GenBank no. LC026018.1, LC388754.1, AB646061). The present research is the first report of the nematode G. pulchrum from D. dama in Romania.

Opuntia Ficus-Indica Peel By-Product as a Natural Antioxidant Food Additive and Natural Anticoccidial Drug.

The present study was carried out to valorize the Opuntia ficus-indica (OFI) by-products by extracting and identifying their biochemical compounds and evaluating their antioxidant potential by in vitro activities (DPPH radical and FRAP), as well as their capacity to stabilize margarine oxidation (rancimat test). In addition, their in vitro anticoccidial effect on the destruction of Eimeria oocysts isolated from naturally infected chickens was also targeted. Microwaves and response surface methodology tools were used to extract the maximum amount of phenolic compounds (42.05 ± 0.46 GAE mg/g DW of total phenolic compounds in 90 s at 400 watts). Moreover, the effect of extraction factors was also studied. Eight phenolic compounds, including isorhamnetin, dihydrokaempferol, and kaempferol-3-O-rutinoside, were identified. The findings confirmed that OFI peel extract has strong antioxidant activities (DPPH radical, ferric reducing power). The rancimat test shows that OFI peel extract improves margarine stability by 3.2 h. Moreover, it has a notable destruction rate of Eimeria oocysts (30.06 ± 0.51%, LC50: 60.53 ± 0.38 mg/mL). The present investigation offers promise for the reuse of food waste as natural margarine additives, protection of the environment, and substitution of anticoccidial synthetic treatments.

Causes of Post-Mortem Carcass and Organ Condemnations and Economic Loss Assessment in a Cattle Slaughterhouse.

The study was undertaken to investigate the main causes of carcass and organ condemnations, as well as to estimate the financial losses suffered by a cattle slaughterhouse. In this regard, an active abattoir survey, based on standard post-mortem inspection procedures for meat, was conducted on 151,741 cattle, from January 2021 to December 2022. Overall, 13.27% (n = 20,125) of the carcasses expressed lesions or pathological conditions and, out of them, 1.15% (n = 1738) were totally confiscated, while another 12.12% (n = 18,387) were partially admitted for human consumption. In the case of organs, the general inspection data reveal that 12.28% (n = 18,630), 7.56% (n = 11,477), 1.89% (n = 2862), and 0.27% (n = 412) of the examined liver, lung, heart, and kidney specimens presented one or more types of abnormalities. In addition, regarding the types of specific pathological findings, dystrophies/anomalies (69.8%), circulatory disorders (40.6%), fecal contamination (60.9%), and suspected bacterial/viral infections showed a dominant occurrence in the liver, lung, heart, and kidneys, respectively. Consequently, the total direct financial losses resulting from edible part condemnation over the two years was estimated at EUR 4,021,717.3, which represents 1.17% of the total achievable net revenue without carcass and organ condemnation. Of this, EUR 3,661,400.4 (1.07%) and EUR 360,316.9 (8.73%) was related to carcass and organ condemnation, respectively. The study results demonstrate that the post-mortem inspection of meat at the slaughterhouse level plays a crucial role in identifying pathological lesions, in addition to some other issues, such as fecal contamination or non-compliant laboratory results, relevant to both public health and economic factors.

First Molecular Identification of Calicophoron daubneyi (Dinnik, 1962) and Paramphistomum leydeni (Nasmark, 1937) in Wild Ruminants from Romania.

Rumen flukes are geographically widespread trematodes affecting wild and domestic ruminants. The juvenile forms, which are found in the small intestine, are more pathogenic compared to the adults. Severe diarrhoea and weight loss are the major clinical signs, and the disease might be fatal in severely infested individuals. In the last decade, paramphistomosis has been described as an emerging parasitic disease in Europe. This study aimed to identify the rumen fluke species in wild ruminants from western Romania. Fifty-two pre-stomachs obtained from roe deer (Capreolus capreolus) that were hunted on 14 hunting grounds from Timiș and Arad counties were examined for the presence of paramphistomes. Three (9.09%) out of 33 samples were positive in Timiș County, and one (5.26%) out of 19 samples was positive in Arad County. Subsequent PCR testing revealed that three samples were positive for Calicophoron daubneyi and one for Paramphistomum leydeni. The presence of C. daubneyi and P. leydeni in roe deer has not been previously reported in Romania. Two Paramphistomum species-C. daubneyi and P. leydeni-were revealed as the main species of rumen flukes in roe deer from forests in Romania.

Epidemiological Surveillance of Hypodermosis in Cattle from Romania.

Hypodermosis, or warble fly, is an endemic parasitic disease, common in countries from the northern hemisphere. The use of effective insecticides has decreased the frequency of this parasitic disease, with untreated cattle remaining to act as reservoirs. This study focused on assessing the status of hypodermosis in northwestern Romania by means of clinical examination (skin inspection and palpation performed in order to identify nodules) conducted on a number of 11.741 cattle. The study was carried out from March until June 2021. The identified larvae were subject to molecular assays for species identification and genotyping, followed by comparison with data available in the GenBank database. The average prevalence rate of parasitism caused by Hypoderma spp. was 0.31%, with values ranging from a minimum of 0.11% to a maximum of 1.32%. The dominant age group among positive animals was the 1-3 years old category and in terms of breed distribution, most positive cases were seen in cows belonging to an indigenous breed: Bruna de Maramures. April was the most prolific month in terms of nodule count/animal. The species identified in our study by means of molecular assays was H. bovis with two haplotypes: HB3 and HB8.

Characterization of the Tongue Worm, Linguatula serrata (Pentastomida), Identified from Hares (Lepus europaeus) in Romania.

Linguatula serrata (Frölich, 1789) is a widespread parasite known as the tongue worm belonging to the family Linguatulidae. The adult form of the parasite is usually located in the upper respiratory tract of domestic and wild carnivores while the larval forms are located in the visceral organs of intermediate hosts (various herbivorous mammals). Twenty-four European brown hares (Lepus europaeus) were examined in this study, of which two were positive with L. serrata nymphs. The collected nymphs were examined morphologically using electron-microscopic analysis and molecularly by amplification of 18S rRNA and COX1 genes. Lung tissue samples were also collected and histopathological examination was performed. Histopathological examination revealed the following lesions: generalized inflammatory oedema, granulomas with necrosis, calcification and fibrosis in the bronchial tree. The results of molecular sequencing for L. serrata specimens collected from the European brown hares are deposited in GenBank. This study presents the first report on Linguatula serrata nymphs collected from L. europaeus in Romania, using molecular and morphological characterization simultaneously.

Bioactive Compounds and In Vitro Antioxidant and Anticoccidial Activities of Opuntia ficus-indica Flower Extracts.

The objective of the present study is to identify the biochemical compounds extracted from OFI flowers using ultra-high-performance liquid chromatography-electrospray ionization quadrupole time-of-flight mass spectrometry and to evaluate their in vitro antioxidant activities and anticoccidial effects on the destruction of Eimeria oocysts isolated from naturally infected chickens. A domestic microwave was used with a refrigerant to condense the vapors generated during the extraction. The flavonoid and phenolic compound contents of the OFI flowers were determined according to standard methods. DPPH radical and H2O2 scavenging capacities were used to assess the antioxidant activity. Regarding the anticoccidial activity, the Eimeria spp. oocysts used were isolated from the fresh feces of infected broilers and were determined in triplicate by incubation at an ambient temperature for 24 h. The results highlighted the considerable influence of the optimized acetone concentration, ratio, irradiation time, and microwave power parameters on the phenolic content and antioxidant activities. Our results revealed significant matches between the predicted and experimental values of the models. Molecular analysis revealed the presence of several biophenol classes such as quercetin, isorhamnetin 3-O-rutinoside, and quercetin-3-O-rutinoside. OFI flower extracts inhibited sporulation and damaged the morphology of Eimeria oocysts compared with normal sporulated Eimeria oocysts containing sporocysts. In conclusion, the optimized conditions were validated and found to fit very well with the experimental values. These findings suggest that the flowers of OFI should be considered sources of antioxidants. The results of the present study revealed that OFI flower extracts have anticoccidial activities against Eimeria-spp.-induced infection in broiler chickens.

Epidemiology of Cryptosporidium Infection in Romania: A Review.

Since 1983, when the first report of a human Cryptosporidium spp. infection was published in Romania, and until now, many studies on cryptosporidiosis have been published in our country, but most of them are in the Romanian language and in national journals less accessible to international scientific databases. Although the infection was first recognized as a problem in children or immunocompromised people or more of a problem in low-income or underdeveloped global countries, we have shown in this review that it can also occur in people with normal immunological function and that the epidemiology of our country can provide a theoretical basis for the formulation of a Cryptosporidium spp. prevention strategy. In addition, 9.1% of healthy children and 73% of immunocompromised children were observed to have Cryptosporidium spp. infections. Higher rates have also been reported in immunocompromised adults (1.8-50%). Analyzing the prevalence of Cryptosporidium spp. infection in animals, we found values of 28.52% in cattle, 18% in buffalo calves, between 27.8 and 60.4% in pigs, 52.7% in dogs, and 29.4% in cats. Furthermore, in Romania, the burden of cryptosporidiosis, including acute infections and long-term sequelae, is currently unknown.

First Morphological and Molecular Identification of Demodex injai in Golden Jackal (Canis aureus Linnaeus, 1758) in Romania.

Demodicosis is one of the most important external parasitic diseases found in carnivores. Three species of the Demodex mite inhabit the skin of dogs and related species, D. canis being the most prevalent. This paper describes the first case of infestation with D. injai in a golden jackal in Romania. An emaciated golden jackal female body found in Timiș County, western Romania, was examined at Parasitology Department of Faculty of Veterinary Medicine, Timișoara. The gross lesions were present on different regions of the body: feet, tail, axillary and inguinal areas, and skin folds as well, consisting of erythema, extensive severe alopecia with lichenification, seborrhea, and scaling. In order to establish diagnosis, microscopic examination of skin scrapes, trichogram (hair plucking), acetate tape test (impression), fungal culture, and PCR were performed. Both microscopic measurements and PCR analysis have confirmed the presence of D. injai.

Occurrence of Campylobacter spp. and Phenotypic Antimicrobial Resistance Profiles of Campylobacter jejuni in Slaughtered Broiler Chickens in North-Western Romania

Campylobacteriosis is recognized as one of the most common food-borne zoonoses, with worldwide distribution, having undercooked poultry meat and other cross-contaminated foodstuffs as the main sources of human infections. The current study aimed to provide data on the occurrence of the thermophilic Campylobacter spp. in seven broiler chicken flocks, from three north-western Transylvanian counties of Romania, as well as to determine the antimicrobial resistance profile of the isolated C. jejuni strains. A total of 324 fresh cecal samples were collected during the slaughtering process, and screened for the presence of Campylobacter spp., using routine microbiological and molecular diagnostic tools. Overall, 85.2% (276/324; 95% CI 80.9-88.6) of the tested samples expressed positive results for Campylobacter spp., with dominant occurrence of C. coli towards C. jejuni (63.4% vs. 36.6%). From the six tested antimicrobials, the 101 isolated C. jejuni strains were resistant against ciprofloxacin (79.2%), nalidixic acid (78.2%), tetracycline (49.5%), and streptomycin (7.9%), but total susceptibility was noticed against erythromycin and gentamicin. Seven (6.9%) isolates exhibited multidrug resistance. The study results emphasize the role of broiler chicken as reservoir of Campylobacter infections for humans, as well as strengthen the necessity of the prudent using of antimicrobials in the poultry industry.

Investigation on Descriptive Epidemiology, Geographical Distribution, and Genotyping of Echinococcus granulosus s.l. in Bovine from Romania.

Bovine echinococcosis is a zoonotic disease with worldwide distribution, causing significant economic losses in the affected animals and important public health concerns. The aim of the current study was to investigate the presence and geographical distribution of cystic echinococcosis, and molecular characterization of Echinococcus granulosus s.l. from slaughtered cattle in Romania. In the period 2020−2021, a total of 2693 cattle (aged 2−21 years) slaughtered from the breeds Bruna de Maramureș, Balțata Româneasca, and a mixed breed were examined to identify hydatid cysts. Cysts were identified in 66 cattle (2.45%). The predominant location of hydatid cysts was the lungs. Most cysts were non-fertile. By age categories, hydatidosis was reported in the age categories 2−12 years (6/2341; 0.25%), 13−16 years (11/244; 4.5%), 17−20 years (41/85; 48.23%), and over 21 years (8/23; 34.78%). Following the PCR analysis of 10 samples from the lungs (protoscoleces/proliferative membrane/hydatid liquid), DNA fragments were identified in 5 samples. Following sequencing, the identified species was Echinococcus granulosus sensu stricto (s.s.). The study results indicate that the disease is still present in several of the investigated regions of Romania, highlighting a potential public health risk.

Natural Strategies as Potential Weapons against Bacterial Biofilms.

Microbial biofilm is an aggregation of microbial species that are either attached to surfaces or organized into an extracellular matrix. Microbes in the form of biofilms are highly resistant to several antimicrobials compared to planktonic microbial cells. Their resistance developing ability is one of the major root causes of antibiotic resistance in health sectors. Therefore, effective antibiofilm compounds are required to treat biofilm-associated health issues. The awareness of biofilm properties, formation, and resistance mechanisms facilitate researchers to design and develop combating strategies. This review highlights biofilm formation, composition, major stability parameters, resistance mechanisms, pathogenicity, combating strategies, and effective biofilm-controlling compounds. The naturally derived products, particularly plants, have demonstrated significant medicinal properties, producing them a practical approach for controlling biofilm-producing microbes. Despite providing effective antibiofilm activities, the plant-derived antimicrobial compounds may face the limitations of less bioavailability and low concentration of bioactive molecules. The microbes-derived and the phytonanotechnology-based antibiofilm compounds are emerging as an effective approach to inhibit and eliminate the biofilm-producing microbes.

Paraclinical Changes Occurring in Dairy Cows with Spontaneous Subacute Ruminal Acidosis under Field Conditions.

This study was undertaken to investigate the changes in the blood and milk biochemical parameters found in naturally occurring and long-lasting spontaneous subacute ruminal acidosis (SARA), with the aim of identifying the patterns of paraclinical changes and providing valuable data for more accurately identifying SARA in cows under field conditions. The study was conducted on a dairy herd with a history of the occurrence of SARA-associated clinical signs. Twelve cows, between 20 and 150 days in milk, were randomly selected and subsequently subjected to venous blood, milk, and ruminal fluid collection. The mean pH value of the ruminal fluid was 5.56 ± 0.32, and 58% (7/12) of the tested cows were SARA positive (ruminal pH ≤ 5.5). The albumin, calcium, and phosphorus serum concentration values were significantly lower (p < 0.05) in the SARA group than in the group of healthy cows. Serum aspartate aminotransferase (AST) and glutamate dehydrogenase (GLDH) activity were significantly higher in the SARA cows (p < 0.05) than in the group of healthy cows. The mean values of milk fat, milk protein content, and milk fat-to-protein ratio were significantly lower (p < 0.05) in the tested cows of the SARA group than in the healthy group of cows. In conclusion, the results of the current study indicate that long-term SARA triggered by a high-concentrate diet is associated with clinically significant changes in both the blood composition (hypoalbuminemia, hypocalcemia, and increased serum AST and GLDH activity) and the milk composition (decreased fat and protein percentage and milk fat-to-protein ratio). Altogether, the obtained results provide a more reliable pattern of paraclinical changes and useful insights for detecting SARA in dairy cows under field conditions.

Prevalence of Multidrug-Resistant Salmonella enterica Serovars in Buffalo Meat in Egypt.

The current study aimed to investigate the presence of Salmonella spp. prevalence in buffalo meat in Egypt, along with studying the antimicrobial susceptibility of the recovered isolates. Salmonella spp. was detected in 25% of tested buffalo meat. A total of 53 (100%) isolates were genetically verified by PCR as Salmonella, based on the detection of the invA gene. The stn and hilA genes were detected in 71.7% (38/53), and 83.0% (44/53) of the recovered isolates, respectively. Salmonella Enteritidis (11/53; 20.7%) was the most commonly isolated serovar, followed by S. Typhimurium (9/53; 17%), S. Montevideo (6/53; 11.3%), meanwhile, S. Chester, S. Derby, S. Papuana, and S. Saintpaul were the least commonly identified serovars (a single strain for each; 1.9%). Among the 16 antimicrobials tested, amikacin, imipenem, gentamicin, cefotaxime, meropenem, ciprofloxacin, and enrofloxacin were the most effective drugs, with bacterial susceptibility percentages of 98.1%, 94.3%, 92.5%, 86.8%, 83.0%, 73.6%, and 69.8%, respectively. Meanwhile, the least effective ones were erythromycin, streptomycin, clindamycin, cefepime, and nalidixic acid, with bacterial resistance percentages of 100%, 98.1%, 88.7%, 77.4%, and 66%, respectively. Interestingly, the high contamination level of Egyptian buffalo meat with multidrug-resistant Salmonella (79.2%; 42/53) can constitute a problem for public health. Therefore, programs to control Salmonella contamination are needed in Egypt.

Occurrence, Pathogenic Potential and Antimicrobial Resistance of Escherichia coli Isolated from Raw Milk Cheese Commercialized in Banat Region, Romania.

The aim of the present study was to investigate the presence, pathogenic potential and antimicrobial susceptibility profile of Escherichia coli isolated from raw milk cheese, traditionally produced by farmers and marketed directly to the consumer in Banat region, Romania. A total of 81.1% (43/53) of the processed samples expressed positive results for E. coli, with a distribution of 83.8% (31/37), and 75.0% (12/16) in the cow- and sheep-milk-origin assortments, respectively. Overall, 69.8% (30/43) of the specimens had a contamination level ≤10 CFU/g. Molecular tests showed that, from the total number of E. coli isolates, 9.3% (4/43) harbored the stx2, and 2.3% (1/43), the stx1 virulence genes. The E. coli O157 (including H7) biovariety was identified in 7% (3/43) of the samples by the Vidas equipment. From the 27 antimicrobials tested with the Vitek2 automated system, the E. coli isolates displayed resistance to enrofloxacin (100%, 15 out of 15 tested isolates), ampicillin (39.5%, 17/43), norfloxacin (28.6%, 8/28), fosfomycin (25%, 7/28), amoxicillin/clavulanic acid (23.3%, 10/43), cefalexin (20%, 3/15), cefalotin (13.3%, 2/15), tetracycline (13.3%, 2/15), trimethoprim-sulfamethoxazole (9.3%, 4/43), piperacillin-tazobactam (7.1%, 2/28), cefotaxime (7.1%, 2/28), cefepime (7.1%, 2/28), ticarcillin/clavulanic acid (6.7%, 1/15), florfenicol (6.7%, 1/15), ceftazidime (3.6%, 1/28), and ertapenem (3.6%, 1/28). Ten (23.3%) strains were multidrug-resistant. The obtained preliminary results indicated hygienic-sanitary deficiencies throughout the cheese production process, and demonstrated that these products can harbor virulent and multidrug-resistant E. coli strains, which constitute a public health risk. However, future investigations, processing a higher number of samples, are still necessary to draw comprehensive conclusions.

Diversity and Antibiotic Resistance Profiles of Listeria monocytogenes Serogroups in Different Food Products from the Transylvania Region of Central Romania.

ABSTRACT: This study used molecular serogrouping to assess the presence and antimicrobial susceptibility profile of Listeria monocytogenes isolates from food products of different animal origin, collected from a county situated in the historical region of Transylvania in central Romania. Seventeen (7.7%) of 221 screened samples were positive for L. monocytogenes; these included 8 (6.2%) of 130 ready-to-eat products (i.e., sausages, ham, and smoked specialties), 6 (12.8%) of 47 raw meat samples (i.e., minced pork, pork organs, and snails), and 3 (6.8%) of 44 dairy samples (i.e., assortment of cheeses). The identified L. monocytogenes serogroups were 1/2a-3a (47.1%), 4b-4d-4e (29.4%), 1/2c-3c (11.8%), and 4a-4c (11.8%). All isolates were resistant to benzylpenicillin and fusidic acid. Resistance was also detected toward oxacillin (88.2%), fosfomycin (82.4%), clindamycin (76.5%), imipenem (52.9%), ciprofloxacin (41.2%), rifampin (41.2%), trimethoprim-sulfamethoxazole (29.4%), and tetracycline (29.4%). On the other hand, all isolates proved susceptible to gentamicin, moxifloxacin, teicoplanin, vancomycin, tigecycline, erythromycin, and linezolid. All tested strains exhibited multidrug resistance, resulting in the expression of a total of 12 resistance profiles. These findings extend the understanding of the spread of an important pathogen in Romanian food products, highlighting a substantial public health issue and medical concern, especially for consumers with a compromised health status.

Identity of Microfilariae Circulating in Dogs from Western and South-Western Romania in the Last Decade.

Dirofilaria infections in dogs are recognized as widespread mosquito-borne diseases with zoonotic potential, that are caused by the filarioid nematode (Onchocercidae) species Dirofilaria immitis and Dirofilaria repens. The long-term survey conducted in western and south-western Romania was undertaken in order to provide valuable data on the occurrence of Dirofilaria infections in dogs. Blood samples from 1088 dogs, originating from 73 localities of 11 western and south-western counties in Romania, were collected and examined using the modified Knott test. Subsequently, all of the microscopically positive samples were subjected to molecular analysis for confirmation. Altogether, the data obtained showed the percentage of dogs with circulating microfilariae to be 21.42% (233/1088) of dogs tested in the investigated region. The identified species, in cases of monoinfections, were D. immitis, D. repens, and Acanthocheilonema reconditum in 106 (9.74%) samples, 102 (9.38%) samples, and 1 (0.09%) sample, respectively. Twenty-four (2.21%) samples were simultaneously positive for D. immitis and D. repens. There was no association (p > 0.05) between infection status and breed; however, sex and lifestyle were positively associated (p < 0.05) with the percentage of dogs with circulating microfilariae and might be regarded as risk factors for infection. The results of the present investigation indicate potential zoonotic risks for humans living in the screened area and support the imperative to increase awareness among both veterinarians and physicians, regarding the continuous spread of these zoonotic filariae.

Feline Demodicosis Case Report-First Molecular Characterization of Demodex Mites in Romania.

Cat demodicosis is uncommon to rare, and is caused by Demodex cati, Demodex gatoi and another unnamed species. The investigated patient was a mix-breed, 10-year-old feline with no dermatological history. Alopecia, erythema, minor erosions and ulcerations and crusts, associated with pruritus and self-trauma, were observed on the head. Dark, agglutinated cerumen was also present in the external ear canal. The agent causing the skin condition in the feline patient was identified as being a Demodex genus mite, based on the specific, morphological characteristics noticed upon the microscopic examination of deep skin scrapes. Biological samples were collected from the patient with to perform a PCR assay for clear species-determination and morphological assessment. PCR amplification of DNA extracted from the Demodex mites produced a single band of ~330 bp, indicating the presence of the D. cati species. The acaricidal treatment consisted of topical treatment using a fluralaner and moxidectin-based spot-on. Upon follow-up appointments, scheduled three times at a monthly interval, the patient failed to provide a positive result upon deep skin scrapes. The negative scrapes were also accompanied by the complete resolution of the existing lesions. In conclusion, this is the first molecular study to highlight the presence of Demodex cati within the feline population of Romania, and the fluralaner-moxidectin spot-on therapy has led to a complete recovery of the feline patient affected by feline demodicosis.