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 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.

Research on the Control of Gastrointestinal Strongyles in Sheep by Using Lotus corniculatus or Cichorium intybus in Feed.

The general practice of sheep farmers in gastrointestinal helminth control is based on the use of commercial drugs, making chemoresistance very common. Considering this, our study focused on the biological control of gastrointestinal parasitism using high-tannin plant hay. Three groups of 30 animals each were formed. The control group was additionally fed meadow hay, while the other two groups received chicory (group 2) and bird's foot trefoil hay (group 3). The number of gastrointestinal strongyle eggs, shed through faeces (EPG), was surveyed for 28 days for all animals. The amounts of total tannins for meadow, chicory, and Lotus corniculatus hay supplements were 13.92 mg/g, 78.59 mg/g, and 94.43 mg/g, while their condensed tannin contents were 2.58 mg/g, 29.84 mg/g, and 15.94 mg/g, respectively. Compared to experimental day 0, there was an increase in EPG of 80.83% in the control group, a decrease of 24.72% in group 2, and a 20% decrease in group 3, by day 28. The p-value was <0.05 between group 1 and the other groups, showing significant differences between the control and experimental groups. The decrease in EPG rates in the experimental groups compared to the control group demonstrates an antiparasitic effect of Lotus corniculatus and chicory.

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.

Surveys on Pet-Reptile-Associated Multi-Drug-Resistant Salmonella spp. in the Timișoara Metropolitan Region-Western Romania.

The number of reptiles owned as pets has risen worldwide. Additionally, urban expansion has resulted in more significant human encroachment and interactions with the habitats of captive reptiles. Between May and October 2022, 48 reptiles from pet shops and 69 from households were sampled in the Timișoara metropolitan area (western Romania). Three different sample types were collected from each reptile: oral cavity, skin, and cloacal swabs. Salmonella identification was based on ISO 6579-1:2017 (Annex D), a molecular testing method (invA gene target), and strains were serotyped in accordance with the Kauffman-White-Le-Minor technique; the antibiotic susceptibility was assessed according to Decision 2013/652. This study showed that 43.28% of the pet reptiles examined from households and pet shops carried Salmonella spp. All of the strains isolated presented resistance to at least one antibiotic, and 79.32% (23/29) were multi-drug-resistant strains, with the most frequently observed resistances being to gentamicin, nitrofurantion, tobramycin, and trimethoprim-sulfamethoxazole. The findings of the study undertaken by our team reveal that reptile multi-drug-resistant Salmonella is present. Considering this aspect, the most effective way of preventing multi-drug-resistant Salmonella infections requires stringent hygiene control in reptile pet shops as well as ensuring proper animal handling once the animals leave the pet shop and are introduced into households.

Thermographic Image of the Hoof Print in Leisure and Cross-Country Warmblood Horses: A Pilot Study.

BACKGROUND: The field of veterinary medicine lacks information on equine thermal hoof printing, and few data on the same subject are available in dogs. In human medicine, thermography is used to detect heat emitted by the foot when it comes in contact with a flat surface to detect the abnormalities of the foot balance. The hypothesis states that the thermal pattern of the hoof print in Warmblood horses is detectable and it does not vary among the four limbs in leisure and cross-country Warmblood horses in terms of mean temperature of the hoof print surface. A pilot study was conducted to investigate the accuracy of thermography in temperature detection of the hoof print and to investigate the occurrence of possible differences in the mean value of six selected areas and whether there are any differences in the mean temperature of the hoof print between leisure and cross-country Warmblood horses. METHODS: The study included sixty non-lame Warmblood horses with all limbs taken into consideration (n = 240). The selection criteria for the horses were: no alterations in posture and no muscle group asymmetry during visual examination, no lateral or medial deviation of the carpus or hock, no reaction to the flexion tests, negative reactions to the hoof tester, no lameness during walking, trotting or lunging, no anti-inflammatory medication in the last three weeks prior to examination and rectal temperature between 37 °C and 38 °C. The hoof print of each hoof was measured with the horse in the standing position, all four limbs on the ground, using a FLIR E50 thermal camera. Six areas of temperature from the hoof print were taken into consideration, and for each of them, the mean value was identified using FLIR Tools software for photo interpretation. The One-Way ANOVA test was used to test the differences between the mean temperatures obtained for each selected area from all limbs and to compare the hoof print temperature values between the leisure horses and cross-country horses. Data were statistically processed using SAS Studio. RESULTS: Thermography can detect the temperature emitted by the hoof but the thermal patterns of the hoof print show no difference for all four studied limbs. No significant statistical differences were noticed between the mean temperatures identified for each studied area. Also, there were no statistical differences between the mean temperature of the selected areas from the forelimbs and hindlimbs from the horses used for leisure and those used for cross-country. Based on this aspect, the mean temperature of one selected area can be determined in any of the four limbs, without visible variations. CONCLUSIONS: Thermography can detect the hoof print on a flat surface and the mean temperature for each studied area can be proposed as a reference temperature value. There were no differences in the mean temperature of the hoofprint between leisure and cross-country Warmblood Horses. Further investigations are required to clarify whether there are any differences in the thermal pattern of hoof prints from other breeds or from horses with musculoskeletal conditions.

Evaluation of Thermal Changes of the Sole Surface in Horses with Palmar Foot Pain: A Pilot Study.

BACKGROUND: Horses with palmar foot pain do not show a typical increase in temperature in the palmar aspect of the hoof and heel due to low blood flow. The objectives of the current study were to determine the changes and differences in the thermographic pattern of the sole surface in horses with unilateral palmar foot pain and non-lame horses before and after training. We hypothesized that the thermal pattern and the local temperature of the frog area and toe area would be similar, with an increased local temperature after training in both lame and non-lame horses. A pilot study was conducted to investigate the accuracy of thermography used to detect changes in local temperature and to compare the thermal patterns observed on the sole surface after training. METHODS: The study group included eight horses with unilateral forelimb palmar foot pain (n = 8) and healthy contralateral limbs (n = 8). Four additional non-lame horses served as controls. The horses were selected for the study based on the following criteria: forelimb with unilateral chronic progressive lameness and positive reactions when performing the hoof test and frog wedge test, degenerative findings of the navicular bone, and improvement in lameness after perineural analgesia of the medial and lateral palmar digital nerve. The local temperatures of the frog and toe areas were measured before and after training in the affected and contralateral limbs in the study group and both forelimbs in the control group using an FLIR E50 thermal camera. Receiver operating characteristic (ROC) curve analysis was applied to check the sensitivity and specificity of the results for the studied groups. RESULTS: The thermal patterns of the hoof presented differences between the three groups of horse limbs. After training, the temperature of the sole surface increased, and its area increased in the limbs affected with palmar foot pain compared with the non-lame limbs in the study group and the limbs in the control group. The temperature of the frog area did not increase after training in the limbs affected with palmar foot pain compared with the same area in the other groups. The ROC curve analysis demonstrated the medical applicability of this tool and showed that thermography is a reliable diagnostic test to correctly discriminate between non-lame horses and those with palmar foot pain. CONCLUSIONS: We recommend thermographic scanning of the toe and frog area to detect changes in the local temperature of the sole surface after training to discriminate the horses with palmar foot pain from non-lame ones. Further investigation is required to clarify whether the observed thermographic imaging features of the sole surface are characteristic for horses with palmar foot pain.

The Variation of Serotonin Values in Dogs in Different Environmental Conditions.

Serotonin is considered to be the neurotransmitter that controls several types of behavior: aggressiveness, impulsivity, food selection, stimulation, sexual behavior, reaction to pain, and emotional manifestations. The aim of this study was to determine the serotonin values in 43 dogs, divided into three different experimental variants: (1) between two groups of medium (n = 6) and small (n = 4) breed shelter dogs; (2) in dogs with (n = 15) and without (n = 10) owners after administration of pre-spaying/neutering anesthesia; (3) in different behavioral states (n = 8) classified as follows: M1-happy, M2-aggressive, M3-calmed status, post-exposure to a stressful situation, compared to the reference time referred to as M0. There were no significant differences (p ≥ 0.05) regarding the serotonin values between the two groups of medium and small breed shelter dogs. Following anesthesia, the average mean serotonin values were significantly lower (p ≤ 0.003), by 63.85 ng/mL, in stray dogs compared to dogs with owners. No significant differences (p ≥ 0.05) were found when comparing the reference time M0 to M1, M2, and M3. The differences decreased significantly (p ≤ 0.05), by 89.61 ng/mL, between M1 and M2 and increased significantly (p ≤ 0.008), by 112.78 ng/mL, between M2 and M3.

Evaluation of Russian sturgeon (Acipenser gueldenstaedtii) Semen Quality and Semen Cryopreservation.

The alarming decline in sturgeon populations doubled by growing demands for sturgeon products, urge us to prevent the loss of these species. Fish stocking in natural habitats and developing fish farms are viable solutions, which can be successfully implemented with the help of reproductive biotechnologies. Despite the fact that semen cryopreservation is admittedly an important step for saving the Russian sturgeon, a reproducible standard method with good results has yet to be identified. Sperm quality assessment is essential for quantifying the impact of cryopreservation on spermatozoa. The purpose of our study was to provide additional information regarding semen cryopreservation and semen quality evaluation for the Russian sturgeon. Our study method is based on the use of two yolk-free extenders (with different cryoprotectants: DMSO and methanol) for freezing semen, using a simple freezing protocol. Parameters such as volume, concentration, motility, morphology and membrane integrity were evaluated. In conclusion, cryopreservation of Russian sturgeon spermatozoa using an extender containing methanol as cryoprotectant led to high egg fertilization percentages (72.67 ± 5.4%) even if the total motility values recorded for thawed semen were quite low (18-25%). Additionally, we identified two optimal stains for morphological studies and morphometry (Spermac stain kit and Trypan Blue Solution).

Seroprevalence of Anti-Theileria equi Antibodies in Horses from Three Geographically Distinct Areas of Romania.

Equine piroplasmosis (EP) is an endemic tick-borne disease found in most countries around the world. It affects all species of Equidae, and it is caused by Theileria equi, Babesia caballi and T. haneyi. The research herein is the second study on the prevalence of piroplasms in Romania conducted in the past two decades. The aim of this study was to assess the seroprevalence of anti-Theileria equi antibodies and the geographical distribution of this disease in the southwest, west, and northwest regions of Romania in order to obtain a more thorough understanding of the parasitological status of horses in this country. This study included 522 apparently healthy, mixed-breed horses from three different counties. The serum samples were analysed using the cELISA Theileria equi Antibody Test Kit. The overall seroprevalence rate was 12.84%. From the total number of positive horses, 13.96% were females and 11.21% were males. Based on the distribution of positive cases into age groups, the following values were obtained: 0−60 months: 16.26%, 60−180 months: 10.03%, and >180 months: 15.83%. There was no statistically significant difference between samples, based on age or gender. The positivity percentage in the localities included in the study ranged from 8.33 to 100%. In the population under study, the seroprevalence rate was high, indicating a possible exposure risk in this area of Romania, which could have severe effects on equids in the case of clinical manifestations of the disease. EP represents a serious threat for equine health in Romania; therefore, close and continuous monitoring of the situation is required.

The Use of Pythium oligandrum in the Biological Control of Roundworm Infection in Dogs and Cats.

Pythium oligandrum is an oomycete used in veterinary medicine to treat dermatophytosis in animals. The ovicidal potential against various types of parasite eggs has not been investigated. Ascarids are very common in young animals and the resistance of eggs in the external environment is very high. A commercial product containing P. oligandrum was used in the present study. Its ovicidal action against Toxocara spp. eggs was investigated. Thus, two categories of media (soil and sandstone) were used, on which the ascarid eggs were dispersed. The commercial product was prepared as a solution and was applied according to the manufacturer. The treatment scheme indicated in case of animals with dermatophytosis was used. Thus, the external natural conditions were recreated in the laboratory, in order to establish whether there is any applicability for this oomycete in the biological control of some parasitic diseases. The results indicated an ovicidal action of Pythium oligandrum, supporting the prospects of its use in the decontamination methods of various surfaces or environments where ascarid eggs from carnivores are found.

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.