Vibrio infection in freshwater fish in Poland.

Vibrio species are common inhabitants of aquatic environments and have been described in connection with fish and human diseases. Six Vibrio species were isolated from diseased freshwater and ornamental fish in Poland. The strains were identified based on morphological and biochemical characteristics and confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) as V. albensis (n=3) from Gymnocephalus cernua, Sander lucioperca, Paracheirodon innesi, and Xiphophorus hellerii; V. mimicus (n=1) from Xiphophorus maculatus; and V. vulnificus (n=1) from Nematobrycon palmeri. This is the first time that Vibrio species have been isolated and described from ornamental fish in Poland. The isolates were resistant to ampicillin (83.3%), gentamicin (16.6%), ciprofloxacin (16.6%), sulfamethoxazole-trimethoprim (16.6%), and chloramphenicol (16.6%). The multiple antibiotic resistance (MAR) index was 0.00-0.08 for V. albensis, 0.17 for V. mimicus, and 0.33 for V. vulnificus. Our study confirmed the presence of potentially pathogenic Vibrio species in freshwater and ornamental fish. Therefore, further monitoring of the presence of Vibrio species, mainly in ornamental fish, is necessary.

Combined antimicrobial action of streptomycin and terpenes against atypical mycobacteria isolated from fish.

The aim of this study was to investigate the antimycobacterial activity of 39 free terpenes and their activity in combination with streptomycin. Antimicrobial activity was first evaluated by screening 39 free terpenes at concentrations from 1.56 to 400 µg/mL. None of these exhibited positive effects against any of the nontuberculous mycobacteria (NTM) strains tested. However, six of the 39 terpenes (isoeugenol, nerol, (+)-α-terpineol, (1R)-(-)-myrtenol, (+)-terpinen-4-ol, and eugenol) were shown to enhance the activity of streptomycin against the NTM strains isolated from diseased ornamental fish.

Evaluation of the anti-mycobacterial activity and composition of Carlina acaulis L. root extracts.

Nontuberculous mycobacteria (NTM) have recently emerged as important bacterial pathogens of animals and humans. Of particular concern is the high level of antimicrobial resistance displayed by these organisms, which complicates treatment and potential successful outcomes. Here, we evaluated the potential of Carlina acaulis L. as a source of novel anti-mycobacterial agents. Our goal was to measure the activity of aqueous, ethanol, and chloroform C. acaulis root extracts against 99 NTM strains. GC-MS spectroscopy analyses were performed to deliver qualitative and quantitative data on the composition of C. acaulis extract. In our study, we have shown for the first time the activity of C. acaulis extracts against NTM. The highest activity was exhibited by the chloroform extract, which inhibited the growth of more than 90% of the strains at the dose of 100 µg/mL (MIC90 = 100 µg/mL). The results of the GC-MS analysis of the C. acaulis chloroform extract contributed to the identification of 37 compounds, with carlina oxide as the most representative compound (69.52%) followed by 3,4-dihydro-2H-phenanthren- -1-one (6.54%) and stigmast-5-en-3-ol (4.14%). Our results indicate that C. acaulis chloroform and ethanol extracts have potential for treatment of NTM infections and that this plant contains anti-mycobacterial compounds.

Effect of alkaloid berberine on the susceptibility of nontuberculous mycobacteria to antibiotics.

Nontuberculous mycobacteria (NTM) have recently emerged as important bacterial pathogens of both animals and humans. In this study, we aimed to evaluate the effect of a combination of ten antibiotics with an inhibitor of efflux pumps (EPI), i.e. berberine (BER), against 6 strains of NTM. Our results showed that the BER potentiated the anti-mycobacterial activities of the antibiotics. Overall, our findings show the importance of BER in increasing the efficacy of antibiotics in NTM.

Parasiticidal effects of Tanacetum vulgare extract against Ichthyophthirius multifiliis.

Ichthyophthiriasis, which is caused by Ichthyophthirius multifiliis (Ich) infections, has a severe impact on productivity in freshwater aquaculture. These infections were previously treated effectively with malachite green, a compound that is now banned on fish farms due to its carcinogenicity. To find efficacious drugs to control Ich, flowers of tansy Tanacetum vulgare were evaluated for their antiprotozoal activity. Tanacetum vulgare extract significantly reduced the survival of Ich trophonts and theronts. In vitro, the extract killed all trophonts at 3200 mg l-1, terminated tomont reproduction at 50 mg l-1, and caused mortality of all theronts at 100 mg l-1. T. vulgare extract may be a new and efficacious drug for the control of Ich.

Anti-babesial potential and chemical composition of essential oil from yarrow Achillea millefolium.

Essential oils from plants used in traditional medicine are known as a rich source of chemically diverse compounds with specific biological activities. Achillea millefolium essential oil (AEO) was screened for in vitro activity against Babesia canis. The AEO was obtained by hydrodistillation and analysed by gas chromatography coupled to mass spectrometry (GC-MS). GC-MS revealed the presence of 47 compounds in the essential oil. Those present in the highest concentrations were chamazulene (34.45%), ß-caryophyllene (8.93%), (E)-germacrene D (7.55%), patchoulene (7.27%), ß-guaiene (4.62%), α-humulene (4.59%), santolina epoxide (4.41%), ethyl iso-allocholate (2.97%), aromadendrene (2.62%), and neoclovenoxid-alkohol (2.46%). AEO was found to be active in vitro against B. canis, with 50% inhibitory concentration (IC50) values of 0.06 mg/mL, as compared to imidocarb, with IC50 = 0.007 mg/mL. The study confirms that essential oil from A. millefolium has anti-babesial properties in vitro.

Parasiticidal effects of Eclipta alba and Arctium lappa extracts against Ichthyophthirius multifiliis.

Ichthyophthiriasis, commonly known as white spot disease, occurs in both wild and cultured fish and is responsible for heavy economic losses to the aquaculture industry. In past decade, several chemical therapeutants were used to treat ichthyophthiriasis, but the effective drugs, such as malachite green, have been banned for use in food fish due to its genotoxic and carcinogenic properties. To find efficacious drugs to control Ichthyophthirius multifiliis (Ich), whole Eclipta alba plants and dried root of Arctium lappa were evaluated for their antiprotozoal activity. E. alba and A. lappa extracts significantly reduced the survival of Ich trophonts and theronts. In vitro, the E. alba and A. lappa methanol extracts killed all trophonts at 3200 mg l-1. All trophonts were killed after exposure to E. alba aqueous extract at 3200 mg l-1. The methanol extracts of E. alba and A. lappa killed 100% of I. multifiliis theronts at 400 mg l-1 and 800 mg l-1, respectively. The aqueous extract of E. alba and A. lappa killed 100% of I. mulitifiliis theronts at 1600 mg l-1 and 3200 mg l-1, respectively. E. alba and A. lappa extracts may be new and efficacious drugs for the control of ichthyophthiriasis.

Inhibitory activities of essential oils against Babesia canis.

The in vitro anti-Babesia canis activities of nine essential oils were investigated. Among the tested essential oils Achillea millefolium, Eugenia caryophyllus and Citrus grandis were the most active (IC50 values of 51.0, 60.3 and 61.3 µg/mL, respectively). The oils from Abies sibirica, Rosmarinus officinalis, Eucalyptus globulus, Cinnamonum zeylanicum, Mentha piperita and Pinus sylvestris were less active (IC50 values of 134.3, 237.3, 239.3, 367.9, 837.5 and 907.3 µg/mL, respectively). The results support the concept that some essential oil constituents may be useful in the clinical management of babesiosis.

The concentration of urea in hemolymph as a marker of health in Lissachatina fulica and Cornu aspersum edible snails - a preliminary study.

Edible snails are kept in farms in many countries worldwide. As farm animals, they are an object of interest of veterinary studies and applied biology. There is a large demand for tests which would help identify their health and well-being. The objective of this study was to assess the usefulness of determining the concentration of urea in hemolymph as a marker of health of the Lissachatina fulica and Cornu aspersum edible snails. The observation covered snails from four farms marked from A to D, in which numerous deaths (farm A) and decreased body weight gain (farms B and C) were observed. In experimental farm D we observed a group of snails subjected to stress and a control group maintained in correct conditions. High concentrations of urea were found in the hemolymph of all farm animals from farms A, B and C, as well as in those subjected to food deprivation in farm D (on average from 96 mg/dl in farm D to 320 mg/dl in farm A). On the other hand, in controls from group D, the concentration of the parameter in question was much lower (⟨ 2.0 mg/dl). The results obtained indicate that the urea concentration is a non-specific marker of pathological conditions in snails, and that the continuous monitoring of this parameter makes it possible to demonstrate irregularities in farming and introduce appropriate and early measures to eliminate such disturbances.

In vitro antioxidant and antibabesial activities of the extracts of Achillea millefolium.

Despite many phytochemical and pharmacological investigations, to date, there are no reports concerning the antibabesial activity of extracts of A. millefolium against B. canis. This study was aimed at investigating the biological activities of A. millefolium against the Babesia canis parasite and to identify its chemical ingredients. The water (WE), ethanol (EE) and hexane/acetone (H/AE) extracts of plant aerial parts were screened for total phenolic content (TPC), total flavonoid compound (TFC), DPPH free radical-scavenging activity and its antibabesial activity assay. In this study, imidocarb diproprionate was used as a positive control. The H/AE and EE extracts were analysed using gas chromatography-mass spectroscopy (GC-MS). In the EE extract, the main compounds were 17.64% methyl octadec-9-ynoate, 16.68% stigmast-5-en-3-ol(3α,24S) and 15.17% hexadecanoic acid. In the H/AE extract, the main compounds were 34.55% 11-decyldocosane, 14.31% N-tetratetracontane, 8.22% ß-caryophyllene, and 7.69% N-nonacosane. Extract of EE contained the highest content of phenolics followed by H/AE and WE. The concentration of flavonoids in EE, H/AE and WE extracts showed that TFC was higher in the EE samples followed by H/AE and WE. The antioxidant activities were highest for AA, followed by EE, WE and H/AE. The antibabesial assay showed that the WE, EE and H/AE extracts of A. millefolium were antagonistic to B. canis. At a 2 mg/mL concentration, it showed 58.7% (± 4.7%), 62.3% (± 5.5%) and 49.3% (± 5.1%) inhibitory rate in an antibabesial assay, respectively. Considering these results, the present findings suggest that A. millefolium extracts may be a potential therapeutic agent and that additional studies including in vivo experiments are essential.

Study on establishing normal ranges of chosen biochemical parameters of haemolymph of Cornu aspersum maxima and Cepaea nemoralis gastropods.

The aim of the study was to establish normal ranges for chosen biochemical parameters of haemolymph of snails (Gasropoda: Mollusca), in the light of the use of these animals as experi- mental models in various types of studies. The study was conducted on 100 specimens of Cornu aspersum maxima (CAM) and 100 specimens of Cepaea nemoralis (CN). The haemolymph col- lected from the animals was analysed using colorimetry to assay aspartate transaminase (AST) activity, alanine transaminase (ALT) activity, amylase activity and the concentrations of urea and triglycerides. In the further part of the study, the influence of administering doxycycline with feed on the change of AST and ALT activity in snail haemolymph has been studied. The normal values established for CAM are as follow: AST activity: 26-38 u/l, ALT activity: 0-11 u/l, amylase activity 9-16 u/l, concentration of urea: 3-6 mg/dl, concentration of triglycerides: 16-20 mg/dl. For CN, the following data have been obtained: AST activity: 30-80 u/l, ALT activity: 0-15 u/l, amylase activity 12-15 u/l, concentration of urea: 5-8 mg/dl, concentration of triglycerides: 18-24 mg/dl. It has been shown that doxycycline presents a high workload on the hepatopancreas of snails, which is reflected by a statistically significant (p ⟨ 0.05) increase of AST and ALT activity in the haemolymph of the specimens which obtained doxycycline in feed, as compared to the groups with antibiotic-free feed. The haemolymph activity of both studied parameters increased together with study time and tetracycline administration time.

Size effect of ZnO nanorods on physicochemical properties of plasticized starch composites.

This work demonstrates that the size of ZnO nanorods (ZnONR) with similar aspect ratio determines several physicochemical and microbiological properties of thermoplastic starch composites (TPS/ZnONR) at a given concentration of ZnONRs. A combination of sol-gel and hydrothermal methods was developed to synthesize ZnONR with different sizes but similar aspect ratios. Starch composites containing 1wt.% of ZnONR were prepared by casting. Composites with smaller size nanorods (ZnONR-S) showed more efficiency in shielding UVA radiation and had a higher solubility and water vapor permeability than those with larger nanorods (ZnONR-L). Mechanical properties, biodegradability and antibacterial activity were also influenced by the size of the ZnONR. X-ray diffraction analysis showed that composites with ZnONR-S maintained the typical B-V type starch structure, intensifying the V-type starch structure peaks, while composite with ZnONR-L induced the formation of an amorphous structure, preventing starch retrogradation during storage. Properties affected by nanorods size are fundamental in determining composite applications.

Effect of dietary supplementation with Echinacea purpurea on vaccine efficacy against infection with Flavobacterium columnare in zebrafish (Danio rerio).

The effect of dietary Echinacea purpurea (EP) on the response of zebrafish (Danio rerio) to a Flavobacterium columnare vaccine was investigated. Two hundred D. rerio with an average weight of 290 ± 40 g were selected and fed different levels of E. purpurea (5 g kg(-1) diet--group 1, 10 g kg(-1) diet--group 2, 20 g kg(-1) diet--group 3, 30 g kg(-1) diet--group 4, and 0 g kg(-1) diet--group 5). Experimental feeding was begun 3 weeks prior to bath immunization and continued until the end of the experiment. Twenty-eight days after immunization the fish were challenged by bath immersion with F. columnare at a concentration of 1 x 10(6) CFU/ml. The relative percent survival of the experimental groups (1, 2, 3, 4 and 5) was 5.0, 6.0, 30.0, 36.0 and 5.0, respectively. In conclusion, diet supplementation with E. purpurea may effectively enhance the response of zebrafish to a F. columnare vaccine.

Poly(ADP-ribose) glycohydrolase in bovine retained and not retained placenta.

Poly(ADP-ribose) glycohydrolase (PARG) is the enzyme which degrades poly(ADP-ribose) polymers synthesized by poly(ADP-ribose) polymerase (PARP). Both enzymes are activated in response to different stimuli like oxidative stress and are involved in DNA repair processes. The retention of bovine foetal membranes (RFM) is supposed to be connected with oxidative stress conditions. The aim of the study was to detect the presence of PARG protein in bovine placenta in order to find the relationship between the process of releasing, retaining placenta and DNA repair. Placentomes, collected alter spontaneous delivery or caesarian section were divided into maternal as well as foetal part of placenta, homogenized and subjected to electrophoresis. Animals were divided into six groups as follows: A--caesarian section before term with RFM; B--caesarian section before term without RFM; C--spontaneous delivery at term with RFM; D--spontaneous delivery at term without RFM; E--caesarian section at term with RFM; F--caesarian section at term without RFM. PARG protein was detected in nitrocellulose membranes using commercially available bovine anti-PARG antibody and Western blotting technique. Single bands referred to bovine PARG standard were observed in all examined tissues as well as in human placenta used as the control of procedure. In addition, the intensity of staining was stronger in retained than properly released term placenta and in foetal than in maternal part of the placenta. These results may suggest the differences in enzyme protein content and careful conclusions can be drawn that the activities of PARG may be altered between compared groups of animals. It may confirm the presence of oxidative stress conditions and their consequences on metabolic pathways, the content of biologically active substances and processes of proper releasing placenta. Further experiments on PARG activity in bovine foetal membranes with respect to proper and improper placental release are necessary.

The effect of various Aeromonas bestiarum vaccines on non-specific immune parameters and protection of carp (Cyprinus carpio L.).

Aeromonas bestiarum is one of the causal agents of motile aeromonad infection/motile aeromonad septicemia (MAI/MAS) in fish. Infections of the bacterium is an increasing problem in commercial carp (Cyprinus carpio L.) farmed in Poland. Non-specific immune response of the fish, vaccinated with oil-emulsified experimental vaccines containing formalin killed whole cells (WCs), formalin killed whole culture (WCt) or crude LPS (50 or 1250 microg per fish) were studied on days 7 and 30 after vaccination. Fish vaccinated for 30 days were challenged with the pathogen and mortalities recorded over 14 days. The cumulative mortalities were 10%, 0%, 20% and 20% in WCs, WCt, LPS-1250 and LPS-50 groups, respectively, whereas 70% fish died in the control group. Vaccinated fish showed significant increase of phagocytic activity (PA) and phagocytic index (PI). The total serum Ig (TSIg) level was significantly higher in most vaccinated fish groups than in control. Moreover, WCs and WCt induced significant increase of mucus lysozyme level in vaccinated fish.

Is poly(ADP-ribose) polymerase involved in bovine placental retention?

Poly(ADP-ribose) polymerase (PARP) is the enzyme which utilises NAD to synthesise poly(ADP-ribose) polymers. This process appears in response to DNA lesions. Oxidative stress, which might be involved in bovine placental retention, is the reason for oxidative DNA injury. In this mini-review, the relationship between PARP activity and bovine placental retention is discussed. The results of our experiments on PARP activity in placental tissues showed that the enzyme of 113 kDa and its cleavage products were present in retained as well as released fetal membranes. Western blotting technique showed different intensities in the staining of bands which might suggest different activities of the enzyme.

Humoral antibody response of Atlantic salmon, Salmo salar L., vaccinated against Moritella viscosa.

Immunization of Atlantic salmon, Salmo salar L., with experimental vaccine for protection against M. viscosa was tested. The antigen preparation used for the comparative studies was sonicate formalin killed M. viscose unwashed bacteria. Vaccination was carried out by intraperitoneal injection of bacterin in Biojec mineral oil as an adjuvant. When accumulated mortality in the control group with PBS of fish challenged by i.p. injection of M. viscose reached 100% with a challenge dose of 2.3 x 10(6) CFU, mortality in the experimental group was 71.23%. A positive correlation between the survival rates and corresponding median antibody levels of each group was found at the challenge time.

Influence of temperature on the immune response of carp to lipopolysaccharide and whole cell Aeromonas bestiarum vaccines.

Immunisation of carp, Cyprinus carpio, with four different experimental vaccines for protection against A. bestiarum was tested. Carp immunized with different antigens of A. bestiarum P1s, and kept at 18 degrees C responded immunologically by relatively high titers of antibodies. Antibody production was inhibited at 12 degrees C.