An Insight into Practices Associated with the Control of Internal Parasites in the Dairy Goat Herds of Romania: A Questionnaire Survey.

The widespread and uncontrolled use of anthelmintic products has contributed to the emergence of anthelmintic resistance (AR). This phenomenon globally threatens the productivity and welfare of small ruminants. A questionnaire consisting of 34 questions was handed to 234 goat farmers across Romania to gain insight into control practices against internal parasites and the farmers' perception of the parasitic infections present in their herds and the efficacy of anthelmintic treatments. The majority of farmers (88.5%) admitted they had never submitted fecal samples for parasitological laboratory analysis, and 77.4% had treated the animals on their own. In general, the farmers dewormed their goats based on visual body weight estimation. Prophylactic anthelmintic treatment was practiced by more than 85% of the farmers. A traditional control approach based on treating the entire herd at fixed time intervals is widespread among Romanian goat and sheep farmers. The most commonly used anthelmintic drugs in the previous 3 years (2021-2023) were benzimidazoles (85.5%) and macrocyclic lactones (81.6%). Poor anthelmintic efficacy was suspected by 14.5% of farmers, and the minority (18.0%) considered internal parasites as a problem in their herds. Regarding the farmers' perception of the presence of parasites, there was a significant level of uncertainty. This is the first survey carried out in Romanian goat herds, and it provides up-to-date information on practices aimed at controlling internal parasites.

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.

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.

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.

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.

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.

Highly Sensitive Detection of PQS Quorum Sensing in Pseudomonas Aeruginosa Using Screen-Printed Electrodes Modified with Nanomaterials.

The rapid diagnosis of Pseudomonas aeruginosa infection is very important because this bacterium is one of the main sources of healthcare-associated infections. Pseudomonas quinolone signal (PQS) is a specific molecule for quorum sensing (QS) in P. aeruginosa, a form of cell-to-cell bacterial communication and its levels can allow the determination of the bacterial population. In this study, the development of the first electrochemical detection of PQS using screen-printed electrodes modified with carbon nanotubes (CNT-SPE) is reported. The electrochemical fingerprint of PQS was determined using different electrode materials and screen-printed electrodes modified with different nanomaterials. The optimization of the method in terms of electrolyte, pH, and electrochemical technique was achieved. The quantification of PQS was performed using one of the anodic peaks in the electrochemical fingerprint of the PQS on the CNT-SPE. The sensor exhibited a linear range from 0.1 to 15 µM, with a limit of detection of 50 nM. The sensor allowed the selective detection of PQS, with low interference from other QS molecules. The sensor was successfully applied to analysis of real samples (spiked urine and human serum samples, spiked microbiological growth media, and microbiological cultures).

Label-Free Electrochemical Aptasensor for the Detection of the 3-O-C12-HSL Quorum-Sensing Molecule in Pseudomonas aeruginosa.

Pseudomonas aeruginosa, an opportunistic Gram-negative bacterium, is one of the main sources of infections in healthcare environments, making its detection very important. N-3-oxo-dodecanoyl L-homoserine lactone (3-O-C12-HSL) is a characteristic molecule of quorum sensing-a form of cell-to-cell communication between bacteria-in P. aeruginosa. Its detection can allow the determination of the bacterial population. In this study, the development of the first electrochemical aptasensor for the detection of 3-O-C12-HSL is reported. A carbon-based screen-printed electrode modified with gold nanoparticles proved to be the best platform for the aptasensor. Each step in the fabrication of the aptasensor (i.e., gold nanoparticles' deposition, aptamer immobilization, incubation with the analyte) was optimized and characterized using cyclic voltammetry, differential pulse voltammetry, and electrochemical impedance spectroscopy. Different redox probes in solution were evaluated, the best results being obtained in the presence of [Fe(CN)6]4-/[Fe(CN)6]3-. The binding affinity of 106.7 nM for the immobilized thiol-terminated aptamer was determined using surface plasmon resonance. The quantification of 3-O-C12-HSL was performed by using the electrochemical signal of the redox probe before and after incubation with the analyte. The aptasensor exhibited a logarithmic range from 0.5 to 30 µM, with a limit of detection of 145 ng mL-1 (0.5 µM). The aptasensor was successfully applied for the analysis of real samples (e.g., spiked urine samples, spiked microbiological growth media, and microbiological cultures).

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.

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).