In vivo assessment of the antiparasitic effects of Allium sativum L. and Artemisia absinthium L. against gastrointestinal parasites in swine from low-input farms.

BACKGROUND: Ethno-veterinary practices could be used as a sustainable developmental tool by integrating traditional phytotherapy and husbandry. Phytotherapeutics are available and used worldwide. However, evidence of their antiparasitic efficacy is currently very limited. Parasitic diseases have a considerable effect on pig production, causing economic losses due to high morbidity and mortality. In this respect, especially smallholders and organic producers face severe challenges. Parasites, as disease causing agents, often outcompete other pathogens in such extensive production systems. A total of 720 faecal samples were collected in two farms from three age categories, i.e. weaners, fatteners, and sows. Flotation (Willis and McMaster method), modified Ziehl-Neelsen stained faecal smear, centrifugal sedimentation, modified Blagg technique, and faecal cultures were used to identify parasites and quantify the parasitic load. RESULTS: The examination confirmed the presence of infections with Eimeria spp., Cryptosporidium spp., Balantioides coli (syn. Balantidium coli), Ascaris suum, Oesophagostomum spp., Strongyloides ransomi, and Trichuris suis, distributed based on age category. A dose of 180 mg/kg bw/day of Allium sativum L. and 90 mg/kg bw/day of Artemisia absinthium L. powders, administered for 10 consecutive days, revealed a strong, taxonomy-based antiprotozoal and anthelmintic activity. CONCLUSIONS: The results highlighted the therapeutic potential of both A. sativum and A. absinthium against gastrointestinal parasites in pigs. Their therapeutic effectiveness may be attributed to the content in polyphenols, tocopherols, flavonoids, sterols, sesquiterpene lactones, and sulfoxide. Further research is required to establish the minimal effective dose of both plants against digestive parasites in pigs.

Delivery of florfenicol in veterinary medicine through a PLGA-based nanodelivery system: improving its performance and overcoming some of its limitations.

As is the case with other veterinary antibiotics, florfenicol (FFC) faces certain limitations, such as low solubility in water, or the fact that it is reported to interfere with the immune response after some immunoprofilactic actions in livestock. Aiming to improve its efficacy and overall performance, FFC was loaded into a polymeric nanobased delivery system by succesfully using the emulsion-evaporation technique. The poly(lactic-co-glycolic acid) (PLGA) nanoparticles loaded with FFC were characterized in terms of size (101 ± 0.52 nm), zeta potential (26.80 ± 1.30 mV) and poly-dispersity index (0.061 ± 0.019). The achieved loading was 2.24 µg FFC/mg of NPs, with an entrapment efficiency of 7.9%. The antimicrobial effect, the anti-biofilm formation and the cytotoxicity properties of the NPs were evaluated. The results indicated a MIC decreased by ~97.13% for S. aureus, 99.33% for E.coli and 64.1% for P. aeruginosa when compared to free FFC. The minimum inhibitory concentration (MIC) obtained indicated the potential for using a significantly lower dose of florfenicol. The delivery system produced biofilm inhibition while showing no cytotoxic effects when tested on porcine primary fibroblasts and horse mesenchymal stem cells. These findings suggest that florfenicol can be improved and formulations optimized for use in veterinary medicine through its incorporation into a nanobased delivery system designed to release in a controlled manner over time.

Satureja hortensis L. and Calendula officinalis L., Two Romanian Plants, with In Vivo Antiparasitic Potential against Digestive Parasites of Swine.

Internal parasitic diseases of swine constitute a major welfare and health concern in low-input livestock farming. Due to an increase in chemical resistance, phytotherapeutic remedies have become an alternative for the prophylaxis and therapy of digestive parasitosis, albeit few remedies have been subjected to scientific validation. Low-input swine farming in Romania has adopted the traditional use of phytotherapy for controlling pathogens in livestock. The current study aimed to assess the antiparasitic potential of Calendula officinalis and Satureja hortensis against digestive parasites of swine in two low-input farms. The fecal samples were collected from sows, fatteners, and weaners, and were tested using the following coproparasitological methods: centrifugal sedimentation, flotation (Willis, McMaster egg counting technique), Ziehl-Neelsen stain modified by Henricksen, modified Blagg method, and in vitro nematode larvae/protozoan oocyst cultures. Six species of digestive parasites were diagnosed, namely Ascaris suum, Trichuris suis, Oesophagostomum spp., Balantioides coli, Eimeria spp., and Cryptosporidium spp., in various combinations, dependent on the swine category. A dose of 140 mg/kg bw/day of C. officinalis and 100 mg/kg bw/day of S. hortensis powders administered for 10 consecutive days revealed a strong antiprotozoal and anthelmintic activity on the aforementioned parasites. The curative efficacy can be attributed to the presence of polyphenols, sterols, tocopherols, and methoxylated flavones. In conclusion, our results indicate that S. hortensis and C. officinalis are promising alternatives to the commercially available antiparasitics, enabling their use as natural antiparasitic products against gastrointestinal parasites in pigs.

The Effects of Coriandrum sativum L. and Cucurbita pepo L. against Gastrointestinal Parasites in Swine: An In Vivo Study.

Parasitic diseases are responsible for substantial losses in reproduction and productivity in swine, creating a major impairment to efficient and profitable livestock management. The use of phytotherapeutic remedies has notably increased over the past decade due to their bioavailability, decreased toxicity, non-polluting nature, and to some extent due to their antiparasitic effect. The aim of this study was to evaluate the antiparasitic potential of Cucurbita pepo L. and Coriandrum sativum L. against protozoa and nematodes found in swine. The samples were collected from weaners, fatteners, and sows and examined via flotation (Willis and McMaster), active sedimentation, Ziehl-Neelsen staining as modified by Henricksen, a modified Blagg method, and eggs/oocyst culture. The parasite species detected were Ascaris suum, Trichuris suis, Oesophagostomum spp., Balantioides coli (syn. Balantidium coli), Eimeria spp., and Cryptosporidium spp., depending on age category. A dose of 500 mg/kg bw/day of C. pepo and 170 mg/kg bw/day of C. sativum powders, administered for ten consecutive days, demonstrated a pronounced anthelmintic (pumpkin) and antiprotozoal (coriander) effect against the aforementioned parasites. Future studies are required to ascertain the optimal dose that maximizes their antiparasitic effectiveness. The current study represents the first Romanian report on the in vivo antiparasitic activity of these two plants tested on digestive parasites in swine.

Old Antibiotics Can Learn New Ways: A Systematic Review of Florfenicol Use in Veterinary Medicine and Future Perspectives Using Nanotechnology.

Florfenicol is a broad-spectrum bacteriostatic antibiotic used exclusively in veterinary medicine in order to treat the pathology of farm and aquatic animals. It is a synthetic fluorinated analog of thiamphenicol and chloramphenicol that functions by inhibiting ribosomal activity, which disrupts bacterial protein synthesis and has shown over time a strong activity against Gram-positive and negative bacterial groups. Florfenicol was also reported to have anti-inflammatory activity through a marked reduction in immune cell proliferation and cytokine production. The need for improvement came from (1) the inappropriate use (to an important extent) of this antimicrobial, which led to serious concerns about florfenicol-related resistance genes, and (2) the fact that this antibiotic has a low water solubility making it difficult to formulate an aqueous solution in organic solvents, and applicable for different routes of administration. This review aims to synthesize the various applications of florfenicol in veterinary medicine, explore the potential use of nanotechnology to improve its effectiveness and analyze the advantages and limitations of such approaches. The review is based on data from scientific articles and systematic reviews identified in several databases.

A large-scale study on the seroprevalence of small ruminant lentiviral infection in the Polish goat population.

A large-scale study was carried out in a Polish goat population in 2014-2022 to determine the herd-level (between-herd) and within-herd seroprevalence of small ruminant lentivirus (SRLV) infection. A total of 8354 adult goats (aged >1 year) from 165 herds located in various regions of Poland were serologically tested using a commercial ELISA. One hundred twenty eight herds were randomly selected while 37 were enrolled based on convenience non-random sampling. At least 1 seropositive result was obtained in 103 / 165 herds. For all these herds the probability that they were truly positive (herd-level positive predictive value) was calculated. It was ≥ 90% in 91 seropositive herds and 73% to < 90% in 12 herds in which only 1-4 goats were seropositive (22 goats in total). The seropositive goats in the latter herds were retested using a different commercial ELISA and 14 goats (9 males and 5 females) from 9 herds were confirmed to be seropositive (serial testing). The true herd-level seroprevalence was estimated at 61% (95% confidence interval [CI 95%]: 53%-68%). It differed significantly between herd size classes (p = 0.003): the highest prevalences were found in the medium (51 - 100 adult goats) and large herds (>100 adult goats) - 72% (CI 95%: 56-84%) and 86% (CI 95%: 67%-95%), respectively, while prevalences in very small (≤ 20 adult goats) and small herds (21 - 50 adult goats) were 46% (CI 95%: 34%-59%) and 57% (CI 95%: 43%-70%), respectively. The true herd-level seroprevalence differed significantly also between geographical regions of Poland (p = 0.003), with the highest values in the north-western and the lowest in the southern region of the country. The true within-herd seroprevalence estimated using a Bayesian approach ranged from 0.7% to 100% with the median (IQR) of 42% (17%-84%), and did not vary significantly between herd size classes (p = 0.393) or geographical regions of Poland (p = 0.570). Concluding, SRLV infection is widespread in the Polish goat population, the north-western region of Poland is most extensively infected, and herds counting > 50 adult goats are more often infected.

Enhanced Bioactive Potential of Functionalized Injectable Platelet-Rich Plasma.

Injectable platelet-rich fibrin (iPRF) is a frequently used platelet concentrate used for various medical purposes both in veterinary and human medicine due to the regenerative potential of hard and soft tissues, and also because of its antimicrobial effectiveness. This in vitro study was carried out to assess the cumulative antimicrobial and antibiofilm effect of iPRF functionalized with a multifunctional glycoprotein, human lactoferrin (Lf). Thus, the ability to potentiate cell proliferation was tested on keratinocytes and evaluated by the CCK8 test. The combinations of iPRF and Lf induced an increase in the proliferation rate after 24 h. The average cell viability of treated cultures (all nine variants) was 102.87% ± 1.00, and the growth tendency was maintained even at 48 h. The highest proliferation rate was observed in cultures treated with 7% iPRF in combination with 50 µg/mL of Lf, with an average viability of 102.40% ± 0.80. The antibacterial and antibiofilm activity of iPRF, of human lactoferrin and their combination were tested by agar-well diffusion (Kirby-Bauer assay), broth microdilution, and crystal violet assay against five reference bacterial strains. iPRF showed antimicrobial and antibiofilm potential, but with variations depending on the tested bacterial strain. The global analysis of the results indicates an increased antimicrobial potential at the highest concentration of Lf mixed with iPRF. The study findings confirmed the hypothesized enhanced bioactive properties of functionalized iPRF against both Gram-positive and Gram-negative biofilm-producing bacteria. These findings could be further applied, but additional studies are needed to evaluate the mechanisms that are involved in these specific bioactive properties.

The In Vitro Anticoccidial Activity of Some Herbal Extracts against Eimeria spp. Oocysts Isolated from Piglets.

Coccidiosis in pigs seldom results in important economic losses. However, it can influence growth rates in weaners and it is an important hygiene indicator in swine farms. Certain herbs, along with their extracts, have been used over the course of history in traditional medicine. The aim of this study was to evaluate the in vitro anticoccidial effects of Allium sativum L. (garlic), Artemisia absinthium L. (wormwood), Coriandrum sativum L. (coriander), Cucurbita pepo L. (pumpkin), Satureja hortensis L. (summer savory), and Calendula officinalis L. (marigold) against Eimeria suis and Eimeria debliecki oocysts. The stock solution of oocysts (58% E. suis + 42% E. debliecki) was incubated for three days, before adding the tested solutions. The unsporulated Eimeria spp. oocysts were then placed in a 3 mL well plate and incubated for 96 h at 27 °C, in a suspension containing serial dilutions of alcoholic plant extracts (5%, 2.5%, 1.25%, 0.625%, and 0.312%). The percentage of sporulated and destroyed oocysts was recorded every 24 h for 96 h. All extracts had a good in vitro anticoccidial effect against oocysts of Eimeria spp. compared with the control groups. Oocyst sporulation was significantly (p = 0.05) inhibited by the 5% marigold extract (3.6% sporulated oocysts). The same extract had the highest lytic effect on oocysts (65.2% destroyed oocysts). Our results prove that the most effective alcoholic plant extract is the marigold extract, followed, in order of efficiency, by the wormwood, coriander, garlic, pumpkin, and summer savory extracts. This study represents a preliminary contribution for establishing a new generation of natural disinfectants aimed at destroying Eimeria oocysts in the context of swine contamination.

Serological testing of an equal-volume milk sample - a new method to estimate the seroprevalence of small ruminant lentivirus infection?

BACKGROUND: In cattle attempts to evaluate within-herd prevalence of various infectious and parasitic diseases by bulk-tank milk (BTM) testing with ELISA have been made with moderate success. The fact that BTM is composed of variable and unknown volumes of milk from individual lactating animals weakens the relationship between numerical result of the ELISA and the within-herd prevalence. We carried out a laboratory experimental study to evaluate if a pooled milk sample created by mixing an equal volume of individual milk samples from seropositive and seronegative goats, henceforth referred to as an equal-volume milk sample (EVMS), would allow for accurate estimation of within-herd seroprevalence of caprine arthritis-encephalitis (CAE) using 3 different commercial ELISAs. By mixing randomly selected milk samples from seronegative and seropositive goats, 193 EVMS were created - 93 made of seronegative samples and 100 with the proportion of seropositive individual milk samples (EVMS%POS) ranging from 1 to 100%. EVMS%POS could be considered as a proxy for the within-herd seroprevalence. Then, OD of EVMS (ODEVMS) of the 193 EVMS was measured using 3 commercial ELISAs for CAE - 2 indirect and 1 competitive. RESULTS: The cut-off values of ODEVMS indicating SRLV infection were determined. The regression functions were developed to link ODEVMS with EVMS%POS. A significant monotonic relationship between ODEVMS measured with 2 commercial indirect ELISAs and EVMS%POS was identified. Two regression models developed on this basis described approximately 90% of variability and allowed to estimate EVMS%POS, when it was below 50%. High ODEVMS indicated EVMS%POS of > 50%. CONCLUSION: Our study introduces the concept of serological testing of EVMS as a method of detecting SRLV-infected herds and estimating the proportion of strongly seropositive goats. Further field studies are warranted to assess practical benefits of EVMS serological testing.

Prevalence of Swine Gastrointestinal Parasites in Two Free-Range Farms from Nord-West Region of Romania.

Parasitic diseases cause significant economic losses in swine, including free-range swine farms, the number of which in Romania has increased in the last decades. The current study aimed to identify the parasitic profile of swine raised on two free-range (low-input) farms from Transylvania. Nine hundred sixty samples collected from weaners, fatteners, and sows were investigated by flotation, centrifugal sedimentation, modified Ziehl-Neelsen stained fecal smear, modified Blagg technique, and oocyst/egg cultures. The number of oocysts (OPG), cysts (CPG), and eggs (EPG) were counted per gram of fecal matter. The examination revealed parasitic infections with Balantidium coli, Eimeria spp., Ascaris suum, Trichuris suis, Oesophagostomum spp., Strongyloides ransomi and Cryptosporidium spp. Prevalence (P) and the mean intensity (MI) of the infections varied according to age, swine category, farm, and season. The overall prevalence in both free-range farms according to the age category was 63.2%­Eimeria spp., 70.31%­B. coli, 9.38%­Oesophagostomum spp., 3.75% S. ransomi, and 18.12% Cryptosporidium spp. in weaners. In fatteners Eimeria spp. revealed a prevalence of 50.93%, B. coli­72.5 %, A. suum­63.13%, T. suis­39.06%, and in sows Eimeria spp.­39.06%, B. coli­62.19%, A. suum­34.06%, Oesophagostomum spp.­27.19%, S. ransomi­1.56% and Cryptosporidium spp.­9.38%. The study revealed statistically significant (p < 0.05) differences between age groups, seasons, and farms for all diagnosed parasites. Further research is required to better understand the epidemiology of these infections in swine from Transylvania.

The Effects of Allium sativum L., Artemisia absinthium L., Cucurbita pepo L., Coriandrum sativum L., Satureja hortensis L. and Calendula officinalis L. on the Embryogenesis of Ascaris suum Eggs during an In Vitro Experimental Study.

Ascaris suum is present in traditionally managed indoor pig herds and in industrialized farms, especially in older fatteners and sows. The increasing resistance to common antihelminthic drugs redirected research towards alternative and traditional therapies, which also include medicinal plants. This study comparatively evaluated the in vitro antiparasitic effects of Allium sativum L., Artemisia absinthium L., Cucurbita pepo L., Coriandrum sativum L., Satureja hortensis L. and Calendula officinalis L. against A. suum egg hatching and larval development. A. suum eggs were sampled from randomized fecal specimens collected from traditionally raised swine. The egg suspension (ES, 12 × 103/mL) was divided into two controls (C) (1C-1 mL ES + 1 mL distilled water, 2C-five plates of 1 mL ES + 1 mL ethanol of 70%, 35%, 17.5%, 8.75%, and 4.375%, respectively) and six experimental groups, and placed in 3 mL cell plates. The experimental groups (EG, 1-6) included ES + each alcoholic plant extract (10%, 5%, 2.5%, 1.25%, 0.625%). Both C and EG were performed in quintuplicate. All variants were incubated at 27 °C for a total of 21 days. A. suum eggs were examined after 2, 14 (L1), and 21 (L2/L3) days of incubation. The efficacy of all tested plant extracts increased with concentration. Anti-embryogenic effects on A. suum eggs were expressed by all plants. A superior influence was observed in A. sativum L., A. absinthium L., C. pepo L. and S. hortensis L. extracts, at all concentrations tested. A. sativum L. and A. absinthium L. extracts showed the strongest antihelminthic activity, while C. sativum L. and C. officinalis L. were the weakest ascaricids. Future in-depth phytochemical studies are required to identify the compounds responsible for the anthelminthic properties of these plant species.

A Peek into the Bacterial Microbiome of the Eurasian Red Squirrel (Sciurus vulgaris).

Sciurus vulgaris (the Eurasian red squirrel) is native to Europe and Asia, but due to habitat destruction or fragmentation, interspecific competition, and infectious diseases, especially in European island areas the species finds itself at the brink of extinction. The repopulation of such bare habitats requires healthy squirrel specimens, either translocated from other wild habitats or reintroduced to the wilderness following captive breeding. Captivity, nonetheless, has shown an immense capacity to reshape the structure of wild species' microbiota, adapting it to the less diverse diet and fewer environmental challenges. Therefore, assessing the differences between "wild" and "captive" microbiota in this species could elucidate if special living conditions are needed in order to augment the survival rate of specimens reintroduced into the wild. Furthermore, the microflora profile of the normal flora of healthy red squirrels raised in captivity could support clinicians in addressing infectious diseases episodes and also raise awareness on the zoonotic risk. Hence, this study aimed at documenting the bacterial species carried by S. vulgaris, disclosing overall similarities and variability patterns of the microbiota identified in individuals from two different living environments. We anticipated that the bacterial community would be less diverse in individuals raised in captivity, owing to their restrictive diet and to unchanging conditions in the enclosure. We also hypothesized that there would be a higher prevalence of zoonotic microorganisms in the captive animals, due to the proximity of humans and of other domestic species. To test this, samples (n = 100) were taken from five body regions of 20 red squirrels, both free-ranging and bred in captivity, processed by classical microbiology techniques, and further identified by biochemical assay (VITEK®2 Compact System). A relatively poor bacterial community, comprising 62 bacterial strains belonging to 18 species and 8 different genera, was identified. Most of these microorganisms were reported for the first time in S. vulgaris. With no discrimination between living environments, the highest prevalence (p < 0.001), was registered in Staphylococcus sciuri (60%; 12/20), followed by Escherichia coli (45%; 9/20) and Bacillus cereus (35%; 7/20). The results suggest unremarkable differences in diversity and richness of the resident aerobic microbiota of S. vulgaris, in relation to the living environment.

First Report of Anthelmintic Resistance in Gastrointestinal Nematodes in Goats in Romania.

Currently, there are three classes of anthelmintics most commonly used in small ruminants: the benzimidazoles (BZs), macrocyclic lactones (MLs) and cholinergic agonists (especially levamisole; LEV). The widespread use of those products has led to the emergence of drug-resistant parasite strains which represents a serious threat to the livestock industry. In the present study, we describe for the first time a case of resistance to anthelmintics in goats in Romania. The study was carried out in 2021 in a dairy goat herd from the Transylvania region. Two types of diagnostic methods were used to confirm anthelmintic resistance (AR). First, the faecal egg count reduction test (FECRT), an in vivo AR diagnostic method, was used to evaluate the efficacy of eprinomectin (EPM). The results of this test were analysed applying two different calculative methods that are used only in treated animals (without the control group). Furthermore, two in vitro methods were used: the egg hatch test (EHT) for the detection of resistance to BZs, and the larval development test (LDT) for detection of resistance to all three classes of anthelmintics. The results of FECRT indicate the resistance of gastrointestinal nematodes (GINs) to EPM in both calculative methods (FECR1 = -88% and FECR2 = -202%). In addition, the results obtained for ivermectin aglycone (IVM-AG) in LDT also indicate resistance to drugs from MLs group, especially avermectins. Similarly, the results of in vitro methods (EHT and LDT) indicate resistance to BZs in this herd. LEV was the only drug that stopped the development of L3 larvae 100% (LDT). H. contortus was the only nematode species found in coproculture after EPM treatment. Furthermore, H. contotus L3 larvae was the only species found in the wells with the highest concentrations of thiabendazole (TBZ) and IVM-AG in LDT. This suggests that resistance to both BZs and MLs was present for that species.

Pigeons as Carriers of Clinically Relevant Multidrug-Resistant Pathogens-A Clinical Case Report and Literature Review.

Pigeons are widespread bird species in urban regions (Columba livia forma urbana) and may carry pathogens with zoonotic potential. In recent years, more and more data indicate that these zoonotic pathogens are multidrug resistant. Our results confirmed that global trend. Three different multidrug-resistant pathogens were isolated from an oral cavity of a racing pigeon with lesions typical for pigeon pox virus infection. Staphylococcus aureus was recognized as methicillin resistant, thus resistant to all beta-lactams. Additionally, it was also resistant to many other classes of antibiotics, namely: aminoglycosides, tetracyclines, phenicols, lincosamides, and macrolides. Escherichia coli showed resistance to all antimicrobials tested, and it was classified as intermediate to amikacin. Moreover, Candida albicans resistant to clotrimazole, natamycin, flucytosine, and amphotericin and intermediate to ketoconazole, nystatin, and econazole was also isolated. This raises the question how pigeons acquire such highly resistant strains. Therefore, more data are needed concerning the resistance to antibiotics in strains from domestic and wild pigeons in Poland. Until the problem is fully understood, it will be challenging to implement adequate planning of any control measures and check their effectiveness.

Diagnostic accuracy of three commercial immunoenzymatic assays for small ruminant lentivirus infection in goats performed on individual milk samples.

Caprine arthritis-encephalitis (CAE) caused by small ruminant lentivirus (SRLV) infection is one of the most widespread and devastating diseases of goats. Serological methods, mainly immunoenzymatic assays (ELISA), are the mainstay of CAE diagnostics. Even though blood is still the most commonly tested material, animal welfare issues and increasing costs of veterinary service prompt the development of serological methods based on milk testing. Several different types of ELISAs for CAE are available on the market. All of them perform well on serum, however their diagnostic accuracy for testing milk has not been so far compared. Therefore, we carried out the study in 5 dairy goat herds in Poland whose previous epidemiological situation regarding CAE was known. Paired serum and milk samples were collected from all adult females (n = 420) and tested with 3 commercial ELISAs - indirect ELISA based on the whole-virus antigen (wELISA), indirect ELISA based on the recombined transmembrane and capsid protein (TM/CA-ELISA), and competitive ELISA based on the surface glycoprotein (SU-ELISA). Milk was tested as lactoserum at dilution of 1/2 in wELISA and TM/CA-ELISA, and undiluted in SU-ELISA. The true status of goats was based on the composite reference standard comprising the results of all three ELISAs done on serum and the true prevalence of SRLV infection in the herd of origin. 243 (57.9 %) goats were classified as truly positive and 177 (42.1 %) goats as truly negative. Diagnostic accuracy was evaluated using the area under the ROC curve (AUROC) as well as sensitivity (Se) and specificity (Sp) for a range of cut-off values. AUROC was 98.8 % (CI 95 %: 97.5 %, 100 %) for wELISA, 97.9 % (CI 95 %: 96.5 %, 99.2 %) for TM/CA-ELISA, and 91.7 % (CI 95 %: 88.9 %, 94.5 %) for SU-ELISA. At the cut-off values recommended by the manufacturers both indirect ELISAs were highly sensitive (89.3 % and 91.4 %, respectively) and highly specific (98.3 % and 95.5 %, respectively), whereas SU-ELISA had only moderate Se (71.2 %) at comparably high Sp (96.6 %). Nevertheless, the optimal cut-off values were lower than those recommended by manufacturers for serum - sample-to-positive control serum ratio (S/P%) of 10 % for wELISA, S/P% of 80 % for TM/CA-ELISA, and percentage inhibition of 23 % for SU-ELISA. Concluding, the study shows that wELISA and TM/CA-ELISA may be interchangeably used for testing individual goat milk samples for SRLV infection. Diagnostic sensitivity and specificity of these ELISAs appear not to be lower on milk than on serum. SU-ELISA is considerably less sensitive on milk samples than indirect ELISAs.

Assessment and Antibiotic Resistance Profiling in Vibrio Species Isolated from Wild Birds Captured in Danube Delta Biosphere Reserve, Romania.

Antimicrobial and multidrug-resistant bacteria are a major problem worldwide and, consequently, the surveillance of antibiotic-resistant bacteria and assessment of the dissemination routes are essential. We hypothesized that migratory birds, coming from various environments, would carry more numerous Vibrio strains than sedentary species, with increased risk to be passed to their contacts or environment in habitats they transit or nest in. Similarly, we presumed that strains from migratory birds will show multidrug resistance. A total of 170 oral and rectal swabs were collected from wild birds captured in different locations of the Danube Delta (Malic, Sfantu-Gheorghe, Letea Forest) and processed using standardized selective media. V. cholerae strains were confirmed by serology and molecular methods and, subsequently, their susceptibility was evaluated. The prevalence of Vibrio species by host species, habitat type, and location was interpreted. The isolated Vibrio species were identified as Vibrio cholerae 14.33%, V. fluvialis 13.33%, V. alginolyticus 12%, V. mimicus 17.33%, V. vulnificus 10.88%, with V. parahaemolyticus and V. metschnikovii (16%) also being prevalent. Of the 76 Vibrio spp. isolates, 18.42% were resistant towards at least three antimicrobials, and 81.57% demonstrated a multidrug resistance phenotype, including mainly penicillins, aminoglycosides, and macrolides. The results of the present study indicate higher numbers of Vibrio strains in migratory (74.66%) than in sedentary birds (25.33%), confirming our hypothesis. Furthermore, the increased pathogenicity of Vibrio spp. strains, isolated from wild migratory and sedentary birds, was confirmed by their increased multiple antibiotic resistance (MAR) index (0.09-0.81).

Trueperella pyogenes Isolates from Livestock and European Bison (Bison bonasus) as a Reservoir of Tetracycline Resistance Determinants.

Determinants of tetracycline resistance in Trueperella pyogenes are still poorly known. In this study, resistance to tetracycline was investigated in 114 T. pyogenes isolates from livestock and European bison. Tetracycline minimum inhibitory concentration (MIC) was evaluated by a microdilution method, and tetracycline resistance genes were detected by PCR. To determine variants of tetW and their linkage with mobile elements, sequencing analysis was performed. Among the studied isolates, 43.0% were tetracycline resistant (MIC ≥ 8 µg/mL). The highest MIC90 of tetracycline (32 µg/mL) was noted in bovine and European bison isolates. The most prevalent determinant of tetracycline resistance was tetW (in 40.4% of isolates), while tetA(33) was detected only in 8.8% of isolates. Four variants of tetW (tetW-1, tetW-2, tetW-3, tetW-4) were recognized. The tetW-3 variant was the most frequent and was linked to the ATE-1 transposon. The tetW-2 variant, found in a swine isolate, was not previously reported in T. pyogenes. This is the first report on determinants of tetracycline resistance in T. pyogenes isolates from European bison. These findings highlight that wild animals, including wild ruminants not treated with antimicrobials, can be a reservoir of tetracycline-resistant bacteria carrying resistance determinants, which may be easily spread among pathogenic and environmental microorganisms.

Discovery and genetic characterization of a novel orthonairovirus in Ixodes ricinus ticks from Danube Delta.

Different arthropod species are vectors of a wide array of arboviruses (arthropod-borne viruses) and have likely been central to viral evolution. To better understand the extent of arthropod-borne pathogens, as well as their origin and evolutionary history, it is crucial to uncover the full range of microbial agents, including viruses associated with arthropods. In this study, a collection of ticks obtained in 2016 directly from mammal and bird hosts from several rural and natural sites of Danube Delta was subjected to transcriptome sequencing and amplification assays. Vector surveillance revealed the presence of a novel orthonairovirus species, designated Sulina virus, in Ixodes ricinus ticks. Phylogenetic clustering of each viral protein consistently placed the new virus in the Orthonairovirus genus as a new genogroup closely related to Tamdy orthonairovirus, a genogroup comprising both pathogenic and tick-associated orthonairoviruses. The serological testing of engorged ticks and blood of infected hosts, along with the inoculation of vertebrate cells and mice found no specific antibodies or viral replication, suggesting that Sulina virus is an orthonairovirus associated with the virome of Ixodes ricinus. Finally, the characterization of a novel orthonairovirus identified using high throughput sequencing will advance our knowledge of interactions between viruses and tick vectors, expanding our perspective on fundamental questions regarding orthonairovirus evolution, diversity, ecology and potential of emergence as pathogens.

Prevalence of anthelmintic resistance of gastrointestinal nematodes in Polish goat herds assessed by the larval development test.

BACKGROUND: Helminthic infections, in particular those caused by gastrointestinal nematodes (GIN), are found worldwide and are among the most economically important diseases of goats. Anthelmintic resistance (AR) in GIN of goats is currently present worldwide, and single- or multidrug resistant species are widespread. The aim of this study was to determine the prevalence of AR to benzimidazoles (BZ), macrocyclic lactones (ML) and imidazothiazoles represented by levamisole (LEV) in the Polish goat herds by using an in vitro larval development test, which is useful especially in large-scale epidemiological surveys. RESULTS: This cross-sectional study was conducted from September 2018 to June 2019 and enrolled 42 dairy goat herds scattered over the entire country. The most commonly used anthelmintic class in goat herds in Poland were BZ (92%), followed by ML (85%) and LEV (13%). BZ-resistant GIN populations were found in 37 herds (88%, CI 95%: 75 to 95%), ML-resistant GIN populations in 40 herds (95%, CI 95, 84 to 99%), and LEV-resistant GIN populations in 5 herds (12%, CI 95%: 5 to 25%). Multidrug resistance involving all three anthelmintic classes was found in 5 herds (12%, CI 95, 5 to 25%). Based on the morphological features of stage 3 larvae the main resistant GIN turned out to be Haemonchus contortus and Trichostrongylus spp. The use of BZ and frequency of anthelmintic treatments were significantly related to the presence of AR to BZ in Polish goat herds. CONCLUSIONS: This cross-sectional study demonstrates the existence of AR to BZ, ML and LEV on Polish goat farms. Resistance to BZ and ML is widespread, while AR to LEV is currently at a low level. A considerable proportion of herds harbours multidrug resistant GIN, which requires further consideration. An effective anthelmintic treatment strategy, reasonable preventive measures and better understanding of the resistance-related management practices by farmers and veterinarians may delay further development of AR.