Volume 62 (2018): Issue 2 (June 2018)

Bovine Norovirus (BoNeV) which has been confirmed in Asia, America, and Europe, seems to be distributed worldwide, even though only reported from a number of countries. Bovine noroviruses are predominantly detected in diarrhoeic animals rather than neboviruses. The study reveals the importance of noro- and neboviruses in early age diarrhoea of calves.

A total of 127 stool samples were collected from three provinces located in the central region of Turkey. Samples were subjected to nucleic acid isolation and reverse transcription and polymerase chain reaction (PCR). Positive samples were sequenced and analysed.

According to PCR, five samples (3.93%) were found to be positive for bovine norovirus while 32 (25.19%) samples were found to be positive for bovine nebovirus. Phylogenetic analysis indicated that the novel Turkish norovirus strains were found to be of genotype III.2 and all novel neboviruses were substituted under Nebraska-like strains.

Although predominantly bovine noroviruses are detected worldwide, the study indicated that bovine neboviruses were more prevalent in the studied area. We suggest that bovine neboviruses are more frequently responsible for calf diarrhoea than supposed by virologists. This is also the first report of neboviruses other than Kirklareli virus which is distantly related to neboviruses detected in Turkey.

Diarrhoetic shellfish poisoning (DSP), an alimentary intoxication known to lead to intestinal symptoms, and caused by toxins produced by some dinoflagellates (including several Dinophysis), represents a serious threat to public health. The aim of this paper was to provide information about the occurrence and abundance of potentially toxic harmful algal species causing DSP, and the associated concentration of okadaic acid (OA) toxins. The departing assumption was that in the study area there was an increase in the presence both of Dinophysis species and OA and its derivates that could result in a risk to the health of seafood consumers.

During 2015–2016, water and shellfish samples were collected in the Mediterranean area (Sardinia, Italy). Dinophysis cells were counted according to Utermöhl’s method from water samples, while mass spectrometry was used to identify lipophilic toxins in molluscs.

A total of 46 non-compliant samples of Mytilus galloprovincialis were observed. Their non-compliance concerned their OA levels above the legal limit. Among toxic dinoflagellates, D. acuminata and D. sacculus were the species found mostly during DSP events.

No cases of human intoxication have been reported, but continuous surveillance of toxic phytoplankton is necessary to predict and prevent its harmful effects on human health.

The purpose of this study was to determine the microbiological quality of food fish and its safety for consumers.

The study included 24 fish representing grass carp, bighead carp, Siberian sturgeon, and wels catfish. Specimens were collected in winter. Aerobic bacteria, psychrophilic, Enterobacteriaceae, Staphylococcus spp., and E. coli counts were made, and the presence of Salmonella spp., L. monocytogenes, S. aureus, and other coagulase-positive staphylococci was investigated.

The microbiological analysis showed a similar level of aerobic, psychrophilic, and Staphylococcus spp. contamination of the four fish species. The Enterobacteriaceae count was higher in the muscles of grass carp and bighead carp than S. sturgeon and wels catfish. No pathogenic bacteria such as Salmonella spp., E. coli, L. monocytogenes, Staphylococcus aureus, or other coagulase positive staphylococci were found in samples of the examined fish species.

The fresh fish examined in this study were of good microbiological quality and there was no health risk for consumers.

Health, religious, and commercial aspects justify the need for meat species identification. The lack of officially approved methods prompts the undertaking of research on validation of isoelectric focusing of proteins (IEF) for official purposes.

Samples were prepared from pigs (Sus scrofa ferus domestica), cattle (Bos taurus), and poultry (Gallus gallus domesticus). Meat mixtures were made by blending 50%, 25%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.2% meat of other species. Samples were examined on ultrathin polyacrylamide gels with pH 3–9 gradient.

The results of the study confirmed the stable and reproducible pattern of meat protein bands. The detection limit of raw meat admixtures from pigs, cattle, and poultry mostly ranged from 2% down to 0.2% (0.2% for poultry). However, the IEF method can be used to detect the addition of pig meat to bovine meat in an amount higher than 3%. At the significant mixture level (i.e at least 5% addition of meat of another species) IEF proves itself with 100% specificity, sensitivity, and accuracy.

The achieved detection limits provide a basis for recommending the IEF method for routine tests in laboratories detecting the species origin of meat.

The major difficulty in analysis of nitrofurans in feed, feed water, and food of animal origin is that nitrofurans have low molecular weights and fast metabolism. The principal goal of this study was to prepare a procedure for the determination of nitrofurans and their metabolites by a single method in different types of feed, feed water, and food of animal origin.

Two-gram samples were subjected to hydrolysis and derivatisation processes by addition of hydrochloric acid and 2-nitrobenzaldehyde. After incubation the sample was purified by solid phase extraction technique. Nitrofurans were analysed using ultra-high-pressure liquid chromatography-MS/MS (UHPLC-MS/MS).

The results of validation fulfil the requirement of the confirmatory criteria according to the European Commission Decision 2002/657/EC regarding apparent recoveries (88.9%–107.3%), repeatability (2.9%–9.4%) and within-laboratory reproducibility (4.4%–10.7%).

The method can be successfully applied to monitor nitrofurans and their metabolites in different matrices.

A mini-study of 20 raw milk samples was conducted to examine the spectrum of fungal metabolites in sheep milk from the first spring milking.

Samples were collected from randomly selected ewes in two animal flocks from the Bieszczady Mountains and analysed using liquid chromatography-tandem mass spectrometry.

Out of ~700 bacterial, fungal, and plant metabolites tested for, only one mycotoxin – Enniatin B – was detected in sheep milk samples (18/20; 0.0055–0.0121 μg/kg; 0.0078 μg/kg average).

The results indicated that there was no high-level exposure to fungal metabolites via consumption of raw sheep milk during the sample collection period.

Pyrrolizidine alkaloids (PAs) are probably the most widespread toxins of natural origin. More than 6,000 plant species produce these toxic compounds. Bees can forage on flowers of plants producing PAs, which leads to contamination of honey with the toxic compounds. To determine the contamination of honey with PAs, a sensitive method based on liquid chromatography coupled with mass spectrometry has been developed.

PAs were extracted with 0.05 M sulphuric acid and purified with MCX cartridges. A solvent mixture consisting of ethyl acetate, methanol, acetonitrile, ammonia, and triethylamine (8:1:1:0.1:0.1, v/v) was used to wash alkaloids from the cartridges. After evaporation the residues were reconstituted in water and methanol mixture and subjected to LC–MS analysis.

The developed method was validated according to SANTE/11945/2015 requirements. The recovery was from 80.6% to 114.5%. The repeatability ranged from 2.3% to 14.6%, and the reproducibility was from 4.9% to 17.7%.

A new method for the determination of PAs in honey has been developed and validated. All evaluated parameters were in accordance with the SANTE/11945/2015 guidance document. Out of 50 analysed honey samples, 16 (32%) were positive for the content of at least one PA.

Pyrrolizidine alkaloids (PAs) are secondary metabolites produced by many plant species. Due to their toxicity PAs can pose a risk to human and animal health. To detect the toxic compounds in feed materials a sensitive method based on liquid chromatography coupled with mass spectrometry has been developed.

PAs were extracted with sulphuric acid and purified with cation exchange cartridges. A newly developed solvent mixture consisting of ethyl acetate, methanol, acetonitrile, ammonia, and triethylamine was used to wash alkaloids from the cartridges. After evaporation the residues were reconstituted in water and methanol mixture and subjected to LC-MS analysis.

The developed method was validated according to SANTE/11945/2015 guidelines. The recovery was from 84.1% to 112.9%, the repeatability ranged from 3.0% to 13.6%, and the reproducibility was from 4.8% to 18.9%.

A sensitive and selective method for determination of PAs in feed materials has been developed and validated. All evaluated validation parameters were in accordance with EU Reference Laboratories document no. SANTE/11945/2015. Almost 41% of the analysed feed samples were positive for the presence of at least one PA.

Lameness is a painful and debilitating condition that affects dairy cows worldwide. The aim of this study was to determine the plasma concentration of norepinephrine, β-endorphin, and substance P in dairy cows with lameness and different mobility scores (MS).

A total of 100 Friesian and Jersey cows with lameness (parity range: 1–6; weight: 400–500 kg; milk yield: 22–28 L a day, and lactation stage less than 230 days) were selected. Animals were selected and grouped according to MS (MS 0–3; n = 25), and plasma concentration of norepinephrine, substance P, and β-endorphin was measured using ELISA.

Cows with MS 3 had higher plasma concentrations of norepinephrine and substance P and lower plasma concentrations of β-endorphins when compared to MS 0 cows.

Variations in plasma concentration of norepinephrine, substance P, and β-endorphin could be associated with intense pain states in dairy cows with lameness, but are insufficient to differentiate these states from the mildest pain states. Further studies are necessary in order to evaluate the potential use of these biomarkers in the detection of chronic bovine painful conditions.

The aim of this study was to investigate whether the type and form of oil (raw/non-oxidised (N) or post-frying/oxidised (O)) consumed in high-fat diets affect the oxidative status of an organism, as observed by malondialdehyde (MDA) concentration as an oxidative factor and antioxidant enzyme activity.

Fats in the diet came from rapeseed oil (R) and olive oil (O).

The applied diet caused a decrease in MDA concentration (μmol/L) in serum in group RN from 2.94 ± 0.87 to 1.76 ± 0.13, in group ON from 2.45 ± 0.62 to 1.50 ± 0.10, and in group OO from 2.70 ± 1.16 to 1.84 ± 0.36. Meanwhile, MDA concentration (mmol/L) increased in blood haemolysate in group RO from 0.15 ± 0.07 to 0.22 ± 0.03 and in group OO from 0.17 ± 0.02 to 0.22 ± 0.02. The observed changes caused a response of the enzymatic antioxidant system in both models, especially followed by an increase in activities of total superoxide dismutase and its mitochondrial isoenzyme in all experimental groups, while its cytosolic isoenzyme activity increased only in ON and OO groups. Increased activity of glutathione peroxidase (GPX) in groups RN and RO and of catalase (CAT) in groups ON and OO was observed. Significant differences in responses to the different types and forms of oils were probably caused by the different oxidative stability of the studied oils.

This diet disturbed the body’s oxidative status; however, during the six-month study the enzymatic antioxidant system remained effective.

Inosine pranobex (Methisoprinol, ISO, Isoprinosine) is an immuno-modulatory antiviral drug that has been licensed since 1971 in several countries worldwide. In humans, the drug is approved for the treatment of viral infections, and it might also have therapeutic use in animals. The aims of the presented work were to investigate the genotoxicity of inosine pranobex on BALB/3T3 clone A1 and HepG2 cell lines and to elucidate its mutagenicity using the Ames test.

The BALB/3T3 clone A1 and HepG2 cells were incubated with inosine pranobex at concentrations from 0.1 to 1,000 μg/mL. The genotoxicity was determined by comet and micronucleus assays, and the mutagenicity was determined by Ames assay.

Inosine pranobex did not induce a significant dose-related increase in the number of comets or micronuclei in BALB/3T3 clone A1 and HepG2 cells. Moreover, based on the results of the Ames test, it was concluded that inosine pranobex is not mutagenic in the Salmonella typhimurium reverse mutation assay.

Based on the results of a comet assay, micronucleus assay, and Ames test, it was concluded that inosine pranobex is neither genotoxic nor mutagenic.

Inosine pranobex (Isoprinosine) stimulates cell-mediated immune responses to viral infections in humans and might have also therapeutic use in animals. The aim of this study was to compare three in vitro cytotoxicity assays on mouse embryo fibroblasts and liver cancer cells and determine their ability to detect early cytotoxic effects for inosine pranobex.

BALB/3T3 clone A31and HepG2 cells were incubated with inosine pranobex at concentrations from 0.1 to 1,000 μg/mL. Cell viability was determined with the MTT reduction, the LHD release, and the NRU tests.

A decrease in the cell viability was observed after incubating the BALB/3T3 clone A31and HepG2 cells with inosine pranobex.

Based on the cytotoxicity endpoints measured in these investigations in BALB/3T3 clone A31cells, it can be concluded that the cell membrane may be the first part of the cell to be affected by inosine pranobex. The disintegration of lysosomes and mitochondria follows mitochondria damage. In HepG2 cells likewise, the cell membrane may be the first part of the cell to be affected by inosine pranobex. Also in liver cancer cells, the disintegration of mitochondria (assessed with the MTT reduction assay) and next of lysosomes (assessed with the NRU assay) follows mitochondria damage.

p63 is a homologous molecule of p53 and was recently identified as playing important roles in several key cellular processes, including epithelial development and proliferation. Since then, several studies have demonstrated altered p63 expression in various cancers of epithelial origin. Canine perianal gland tumour is one of the most common skin neoplasms in dogs; however, the molecular characteristics of this tumour remain poorly understood. The objective of the present study was to analyse and compare the expression of p63 in canine perianal gland adenomas and carcinomas.

Haematoxylin and eosin-stained slides were examined and immunohistochemistry was conducted for a total of 65 samples. Immunohistochemical data for p63 expressions were compared between groups using the Mann-Whitney U test.

The p63 expression level was increased in perianal gland carcinomas compared to that in the adenoma samples (P < 0.0001). The percentage of cells expressing p63 was higher in perianal gland carcinomas than in adenomas, although the intensity of immunostaining did not differ significantly between the two groups.

p63 is a candidate factor contributing to the malignant transformation and progression of canine perianal gland tumours.

Glucagon-like peptide-1 (GLP-1) is a polypeptide that is mainly produced by intestinal L cells and is encoded by the proglucagon gene. In this study, GLP-1 localisation was investigated in the ileum of healthy and diabetic mice by immunohistochemistry and proglucagon gene expression was assayed by reverse transcription-polymerase chain reaction.

This study included 18 male Balb/c mice that were divided into diabetic, sham, and control groups. Mice in the diabetic group received 100 mg/kg of streptozotocin. Immunohistochemical expression of GLP-1 was determined using the avidin–biotin–peroxidase complex technique, and proglucagon gene expression was determined by RT-PCR.

Analysis of GLP-1 immunohistochemical localisation showed that GLP-1-immunopositive cells (L cells) were present between epithelial cells in the intestinal crypts. The intensity and localisation of GLP-1 immunoreactivity were similar among the mice in all the groups. Proglucagon gene expression levels were also statistically similar among the mice in all the groups.

No difference was demonstrated among the mice in the diabetic, sham, or control groups with respect to proglucagon gene expression and GLP-1 localisation in the ileum, suggesting that diabetes does not affect proglucagon gene expression in the ileum.

Measurement of intraocular pressure (IOP) in dogs has high diagnostic value because of the possibility of detecting ocular and systemic diseases. Various types of tonometers are available for this measurement in small animal practice. The aim of the study was to compare the IOP values measured with Schiotz and Tono-Pen Vet tonometers in healthy dogs. Clinical diagnostic usefulness of both models was also evaluated.

The examination was performed in 62 eyes in 31 clinically healthy dogs of different races, gender, and ages.

The values for intraocular pressure obtained with Schiotz tonometer were in the range of 12 to 24 mmHg, with the mean of 16.3 ± 2.1 mmHg. The intraocular pressure measured with Tono-Pen Vet tonometer was in the range of 11–25 mmHg, with a mean of 18.1 ± 3.8 mmHg. The mean results of measurements taken using the two tonometers differed statistically significantly, the difference being 1.79 mmHg and the higher values being read from the Tono-Pen Vet tonometer.

Correlation coefficients calculated for the results obtained in the right and left eyes using two tonometers indicated highly correlative relationships between the results. The study shows that both tonometers can be advantageously used in clinical practice to measure intraocular pressure in dogs.