Volume 66 (2022): Edition 4 (December 2022)

Many countries have reported severe acute respiratory syndrome-coronavirus 2 (SARS-CoV-2) infections in mink, and transmission back to humans has raised the concern of novel variants emerging in these animals. The monitoring system on Polish mink farms detected SARS-CoV-2 infection first in January 2021 and has been kept in place since then.

Oral swab samples collected between February 2021 and March 2022 from 11,853 mink from 594 farms in different regions of Poland were screened molecularly for SARS-CoV-2. Isolates from those with the highest loads of viral genetic material from positive farms were sequenced and phylogenetically analysed. Serological studies were also carried out for one positive farm in order to follow the antibody response after infection.

SARS-CoV-2 RNA was detected in mink on 11 farms in 8 out of 16 Polish administrative regions. Whole genome sequences were obtained for 19 SARS-CoV-2 strains from 10 out of 11 positive farms. These genomes belonged to four different variants of concern (VOC) – VOC-Gamma (20B), VOC-Delta (21J), VOC-Alpha (20I) and VOC-Omicron (21L) – and seven different Pango lineages – B.1.1.464, B.1.1.7, AY.43, AY.122, AY.126, B.1.617.2 and BA.2. One of the nucleotide and amino acid mutations specific for persistent strains found in the analysed samples was the Y453F host adaptation mutation. Serological testing of blood samples revealed a high rate of seroprevalence on the single mink farm studied.

Farmed mink are highly susceptible to infection with SARS-CoV-2 of different lineages, including Omicron BA.2 VOC. As these infections were asymptomatic, mink may become an unnoticeable virus reservoir generating new variants potentially threatening human health. Therefore, real-time monitoring of mink is extremely important in the context of the One Health approach.

African swine fever (ASF) is a lethal haemorrhagic disease of Suidae, present in Poland since 2014. The natural reservoir of ASF in Europe is the wild boar (Sus scrofa); however, human activity facilitates long-distance introductions of the disease. In ASF control it is important to identify areas at increased risk of infection. Such identification and estimation of the disease’s progress and subsequent spread will help to identify the specific preventive action needs in given zones. Serving this purpose, this study is a spatial and statistical analysis of ASF spread through noted outbreak data.

The spatial-temporal analysis was conducted on the basis of data including the time and location of all ASF outbreaks both in wild boars and domestic pigs in Poland in 2014–2021.

The analysis indicates possible routes and directions for further ASF spread in Poland, estimates the annual increase of the affected area (approx. 25,000 km² every year since 2017) and marks trends. The strong method-independent correlation between the year and the surface area affected by African swine fever indicated a near-linear generalised trend.

Given the growth trend, we can expect ASF to expand further into new territories of the country; however, it is important to realise that there is still a significant area to protect, because 60% of Poland remains ASF-free.

Rabies as a zoonosis threatens public health worldwide. Several thousand people die each year of infections by the rabies virus (RABV). Oral rabies vaccination (ORV) of wildlife was successfully implemented in many European countries and led to rabies being brought under control there. In Poland, ORV was introduced in 1993 using vaccines containing an attenuated strain of the rabies virus. However, attenuated rabies viruses may have residual pathogenicity and cause the disease in target and non-target animals.

A red fox carcass was tested as part of national rabies surveillance, and its brain was screened for RABV infection using two conjugates and a fluorescent antibody test (FAT). The rabies virus was isolated in mouse neuroblastoma cells by rabies tissue culture infection test (RTCIT), and viral RNA was detected by heminested reverse transcriptase PCR (hnRT-PCR) as well as by quantitative real-time RT-PCR (rtRT-qPCR). An amplicon of 600 bp was subjected to Sanger sequencing. To differentiate between vaccine and field RABV strains, PCR-restriction fragment length polymorphism (PCR-RFLP) using the Dra I, Msp I, Nla IV and Mbo II restriction endonucleases was performed.

The rabies virus was detected in the fox’s brain using FAT, RTCIT and molecular tests. The PCR-RFLP revealed of vaccine-induced rabies, and full-length genome analysis showed 100% nucleotide sequence identity of the isolate with the reference sequences of Street Alabama Dufferin Bern (SAD Bern) vaccine strains and other vaccine-induced rabies virus isolates detected in animals and deposited in GenBank.

We detected vaccine-induced rabies for the first time in Poland in a fox during routine rabies surveillance.

Bovine coronavirus (BCoV) is a causative agent of enteric and respiratory diseases in cattle. Despite its importance for animal health, no data is available on its prevalence in Poland. The aim of the study was to determine the virus’ seroprevalence, identify risk factors of BCoV exposure in selected cattle farms and investigate the genetic variability of circulating strains.

Serum and nasal swab samples were collected from 296 individuals from 51 cattle herds. Serum samples were tested with ELISA for the presence of BCoV-, bovine herpesvirus-1 (BoHV-1)- and bovine viral diarrhoea virus (BVDV)-specific antibodies. The presence of those viruses in nasal swabs was tested by real-time PCR assays. Phylogenetic analysis was performed using fragments of the BCoV S gene.

Antibodies specific to BCoV were found in 215 (72.6%) animals. Seropositivity for BCoV was more frequent (P>0.05) in calves under 6 months of age, animals with respiratory signs coinfected with BoHV-1 and BVDV and increased with herd size. In the final model, age and herd size were established as risk factors for BCoV-seropositivity. Genetic material of BCoV was found in 31 (10.5%) animals. The probability of BCoV detection was the highest in medium-sized herds. Polish BCoVs showed high genetic homology (98.3–100%) and close relatedness to European strains.

Infections with BCoV were more common than infections with BoHV-1 and BVDV. Bovine coronavirus exposure and shedding show age- and herd density-dependence.

Bovine immunodeficiency virus (BIV) is found worldwide in cattle under natural conditions. However, the effect of BIV infection on immune functions has not been fully characterised.

Transcriptome analysis of BoMac cells after in vitro infection with BIV was performed using BLOPlus bovine microarrays. Genes identified as differentially expressed were subjected to functional analysis with the Ingenuity Pathway Analysis software (IPA).

Out of 1,743 genes with altered expression, 1,315 were mapped as unique molecules. In total, 718 genes were identified as upregulated and 597 genes as downregulated. Differentially expressed genes were involved in 16 pathways related to immune response. The most enriched canonical pathway was leukocyte extravasation signalling. Interleukin-15 (IL-15) production was indicated as the most activated pathway and the 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 4 (PFKFB4) signalling pathway was the most inhibited one. In addition, the study showed that the inflammatory response was decreased during BIV infection.

This is the first report to describe the microarray analysis of changes in gene expression upon BIV infection of bovine macrophages. Our data indicated how BIV influences the expression of genes and signalling pathways engaged in the immune response.

Previous gag and env sequence studies placed Polish small ruminant lentiviruses (SRLVs) isolated from sheep and goats in subtypes B1, B2, A1, A5, A12, A13, A16–A18, A23, A24 and A27. This study extended the genetic/phylogenetic analysis of previously identified Polish SRLV strains by contributing long terminal repeat (LTR) sequences.

A total of 112 samples were analysed. Phylogenetic analyses were carried out on the LTR fragment using the neighbour-joining, maximum likelihood, and unweighted pair group method with arithmetic mean methods.

Polish caprine and ovine LTR sequences clustered within group A and grouped in at least 10 clusters (subtypes A1, A5, A12, A13, A16–A18, A23, A24 and A27). Most of the Polish strains (78%) belonged to the same subtype by the indication of the gag, env and LTR genomic regions. Discrepancies in affiliation depending on the particular sequence were observed in 24 (21%) strains, most of which came from mixed-species flocks where more than one SRLV genotype circulated. Sequences of the LTR reflected subtype-specific patterns. Several subtype-specific markers were identified, e.g. a unique substitution of T to A in the fifth position of the TATA box in A17, A27, A20 and B3.

This study provides valuable insights into the genetic diversity of SRLV field strains in Poland, their phylogenetic relationships and their position in the recently established SRLV classification. Our results confirmed the existence of the ten subtypes listed and the readier emergence of new SRLV variants in mixed-species flocks.

Small ruminant lentivirus (SRLV) causes caprine arthritis-encephalitis in goats and maedi-visna disease in sheep. Transmission is via ingestion of colostrum and milk from infected dams or long-term direct contact between animals. Lifelong seroconversion can occur several weeks after infection via ingestion. However, sub-yearling lambs that ingest contaminated colostrum may be able to clear the infection and become seronegative. Whether a similar phenomenon occurs in goats remains unknown. Therefore, the serological status of goats was studied longitudinally from the moment of natural exposure to colostrum and milk of SRLV-positive dams through the age of 24 months.

Between February 2014 and March 2017 a dairy goat herd was studied which had been infected with SRLV for more than 20 years and carried maedi-visna virus-like genotype A subtype A17. Thirty-one kids born to dams seropositive for SRLV for at least a year beforehand were followed. They ingested colostrum immediately after birth and then remained with their dams for three weeks. The goats were tested serologically every month using two commercial ELISAs. The clinical condition of the goats was also regularly assessed.

Out of 31 goats, 13 (42%) seroconverted at the age ranging from 3 to 22 months with a median of 5 months. Two goats seroconverted in the second year of life. The other eleven did so before the age of one year; two of these reverted to seronegative status. Only 9 out of 31 goats (29%) seroconverted in the first year of life and remained seropositive. They were early and stable seroreactors to which SRLV was transmitted lactogenically. The age at which they seroconverted ranged from 3 to 10 months with a median of 5 months. In 8 of the 18 persistently seronegative goats, a single isolated positive result occurred. No goats showed any clinical signs of arthritis. The level of maternal antibodies at the age of one week did not differ significantly between the stable seroreactors and the remainder.

Seroconversion appears to occur in less than 50% of goats exposed to heterologous SRLV genotype A via ingestion of colostrum and milk from infected dams and is delayed by 3–10 months. The natural lactogenic route of transmission of SRLV genotype A in goats appears to be less effective than this route of genotype B transmission reported in earlier studies.

Proventricular dilatation disease (PDD) is caused by avian bornavirus (ABV) has been identified in psittacine, non-psittacine birds and waterfowl. Birds may show signs of gastrointestinal tract deficit or neurological dysfunction or even both. The objectives of this study were to determine the molecular prevalence, risk factors and public awareness of ABV and PDD among captive and non-captive birds in Peninsular Malaysia.

A total of 344 cloacal swabs or faeces were collected and subjected to detection using the RT-PCR assay. Meanwhile, KAP questionnaires were distributed by using the Google forms platform.

Molecular prevalence studies revealed that 4.5% (9/201) of the pet birds were ABV-positive, whereas 0% (0/143) in waterfowl. Nine positive pet birds were identified to be PaBV-2, which is closest to ABV isolates EU781967 (USA). Among the risk factors analysed, category, age and, location, were found to show an association with the ABV positivity. The KAP survey result showed: the respondents have low knowledge (32.9%), however, they showed positive attitude (60.8%) and good practice (94.9%). The association between knowledge, attitude and practice showed that there was a significant association between knowledge-attitude and also attitude-practice (P<0.05).

This study proved that avian bornavirus (ABV) causes proventricular dilatation disease (PDD) among a group of pet birds of Psittaciformes, but it is present in Peninsular Malaysia with a low prevalence rate. Furthermore, in addition to the useful databases obtained from this study, the level of public awareness on the importance of avian bornavirus that causes fatal disorders among a wide range of bird species is satisfactorily raised.

Haemorrhagic enteritis virus (HEV) is a common turkey pathogen which suppresses the immune function. The immunosuppressive potential of both field and vaccine strains of HEV makes it necessary to seek substances which can limit or prevent this phenomenon. The aim of the presented work was to investigate the effect of two immunomodulators in the immune response of HEV-infected turkeys. The immunomodulators were synthetic methisoprinol and a natural preparation containing 34.2% β-glucans (β-1,3/1,6) and 12% mannan oligosaccharides (MOS).

The synthetic immunomodulator was administered to female Big 6 turkey chicks at a dose of 200 mg/kg b.w. in drinking water i) for 3 days before, ii) for 5 days after, or iii) for 3 days before, on the day of infection, and for 5 days after experimental HEV infection in turkeys. The natural counterpart was also given to female Big 6 turkey chicks at a dose of 500 g/tonne of feed i) for 14 days before, ii) for 5 days after, or iii) for 14 days before, on the day of infection, and for 5 days after infection. Their effect was evaluated on the synthesis of interferon gamma (IFN-γ) by splenic CD4+ and CD8α+ T cells in response to mitogen stimulation in vitro. Samples were taken 3, 5 and 7 days after infection and analysed by intracellular cytokine staining assay.

Methisoprinol was shown to increase the CD4⁺IFN-γ⁺ and CD8α⁺IFN-γ⁺ T cell count in these birds over the same cell count in control turkeys. A similar effect was obtained in turkeys that received the natural immunomodulator.

The evaluated immunomodulators may be used to attenuate the effects of immunosuppression in HEV-infected turkeys.

The intracellular bacterium Coxiella burnetii is the aetiological agent of Q fever, a zoonosis affecting many animal species worldwide. Cattle and small ruminants are considered the major reservoirs of the bacteria and they shed it through multiple routes.

A total of 2,180 sera samples from 801 cattle herds in all Polish voivodeships were tested by ELISA for the presence of specific antibodies. Milk samples were obtained from seropositive cows in 133 herds as part of a separate study. The milk samples were examined by ELISA and real-time PCR tests.

Seroprevalence at the animal level was 7.06% and true positive seroprevalence was 6.0% (95% confidence interval (CI) 1.1–9.4). Seroprevalence at the herd level was estimated at 11.1% and true positive seroprevalence was 10.5% (95% CI 3.2–15.8). Shedding of the pathogen in milk was detected by real-time PCR in 33 out of 133 tested herds (24.81%, 95% CI 17.74–33.04%) and the presence of C. burnetii antibodies was confirmed in 85 of them (63.9%, 95% CI 55.13–72.05%). The highest level of conformity between ELISA and real-time PCR results was obtained for bulk tank milk samples.

Coxiella burnetii infections are quite common in cattle herds across the country, which emphasises the crucial roles of surveillance and adequate biosecurity measures in the prevention and limitation of Q fever spread in Poland.

Brucellosis is a widespread zoonosis of great economic importance for livestock farming in many areas of the world. It is a highly infectious disease which is diagnosed using conventional serological and microbiological methods. The aim of this study was to assess the efficiency of a specific real-time PCR in combination with broth cultivation in detecting Brucella spp. in organs of infected cattle, in order to compare the sensitivity of the two approaches and the time needed in them until a correct diagnosis is made.

We examined 67 organs collected from 10 cattle slaughtered following a brucellosis outbreak which occurred in February 2016 in southern Italy. The research was carried out by enrichment broth cultivations in combination with a real-time PCR every week for six weeks.

Brucella strains were isolated by cultivation from 44 enrichment broths of organs. All isolates were later identified as Brucella abortus by real-time PCR. Using this method in combination with cultivation made it possible to identify the same percentage of infected animals faster than by cultivation alone. Moreover, the same diagnostic results were obtained, on average two weeks before they would have been using only cultivation. In almost all cases, Brucella was detected by real-time PCR after the first week of cultivation in pre-enrichment Brucella broth, while the bacterial growth was evident usually after 2 or 3 weeks.

Real-time PCR has allowed results to be obtained faster than in the classical microbiological method, reducing the response times to identify positive animals by half.

Raccoons are an invasive alien species widely distributed in the Madrid region of Spain. These animals can carry a variety of enteric bacteria with associated antimicrobial resistance, which can infect humans and livestock. However, to our knowledge, the presence of non-E. coli Enterobacteriaceae in raccoons has not been previously studied.

We conducted a study to examine the species distribution of Enterobacteriaceae isolates other than E. coli, as well as their antimicrobial resistance, in the faeces of 83 raccoons in the Madrid region.

We detected 12 Enterobacteriaceae isolates other than E. coli belonging to seven different species: Citrobacter freundii (1 isolate), Citrobacter gillenii (3 isolates), Citrobacter murliniae (1 isolate), Citrobacter portucalensis (2 isolates), Enterobacter hormaechei subsp. hoffmannii (1 isolate), Hafnia paralvei (2 isolates) and Raoultella ornithinolytica (2 isolates). These isolates were found in 7 of the 83 (8.4%) animals studied. To our knowledge, this study is the first report of the presence of non-E. coli Enterobacteriaceae in raccoon faeces. All isolates but one were resistant to at least one of the 14 antimicrobials tested. Resistance to ampicillin (83.3%), amoxicillinclavulanic acid (50%) and cefoxitin (33.3%) was the most frequent.

Our study indicates that raccoons are a potential source of infection with Enterobacteriaceae other than E. coli for humans and livestock in the Madrid region.

Escherichia coli is a widespread environmental pathogen frequently causing dairy cow mastitis. This bacterium is particularly capable of acquiring antimicrobial resistance, which can have severe impacts on animal food safety and human health. The objective of the study was to investigate antimicrobial resistance and genetic correlations of E. coli from dairy cow mastitis cases in northern China.

Forty strains of E. coli from 196 mastitis milk samples were collected, susceptibility to 13 common antibiotics and the prevalence of resistance genes were tested in these strains, and the genetic characteristics were identified by multilocus sequence typing.

The results showed that most isolates were multidrug resistant (MDR) (75%), and the resistance rates to cefazolin, trimethoprim-sulfamethoxazole and ampicillin were 77.5%, 55.0%, and 52.5%, respectively. The representative genes of the isolates were aadA (62.5%) and tet(B) (60.0%). Multilocus sequence typing showed 19 different sequence types (STs) and 5 clonal complexes (CCs) in the 40 isolates, mainly represented by ST10 and CC10. The strains of the same ST or CC showed a high level of genetic relatedness, but the characteristics of their antimicrobial resistance were markedly different.

Most E. coli isolates in the study were MDR strains. Some strains of the same ST or CC showed diverse resistance characteristics to common antimicrobials. Therefore, E. coli from dairy cow mastitis in northern China should be investigated to elucidate its antimicrobial resistance and genotypes.

Streptococcus agalactiae is an important zoonotic pathogen that affects milk production and quality and poses a threat to public health. Treatment of infections with this bacterium exploits antimicrobials, to which the resistance of S. agalactiae is a growing problem. Addressing the possibility of a correlation between this pathogen’s genetic factors for antimicrobial resistance and virulence, this study attempted to identify the relevant genes.

Antimicrobial resistance of S. agalactiae isolated from 497 Chinese bovine mastitic milk samples was detected by the broth microdilution method. Eight drug resistance genes and eleven virulence genes were detected using PCR.

Streptococcus agalactiae was 100% susceptible to rifampicin and vancomycin, 93.33% susceptible to sulfisoxazole and sulfamethoxazole, but 100% resistant to ≥3 of the 16 antimicrobial agents, thereby being multidrug resistant, with resistance to oxacillin, tetracycline, erythromycin, clindamycin, and gentamicin being common. The ermB, ermA and lnuA genes were carried by 73.33%, 66.67% and 60.00% of the strains, respectively. The carriage rates of the glnA, clyE, hylB, bibA, iagA, and fbsA virulence genes were greater than 40%, lmb and bac were not observed in any strain, and glnA+hylB+bibA+iagA+fbsA+clyE combined virulence gene patterns were the most commonly detected.

Antimicrobial resistance of S. agalactiae is still a great concern for cattle health in China, and multidrug resistance coupled with the high positive rates of this bacterium’s strains for virulence genes indicates the importance of S. agalactiae surveillance and susceptibility tests.

Nematodes of the Trichuris genus are commonly reported parasites that can cause trichuriasis in many animals, which leads to inflammation, intestinal bleeding and reductions of productivity in livestock. Knowledge of the prevalence of Trichuris infestation in the Tianshan ovine population and of the nematode species parasitising the population is not exhaustive, and this study aimed to expand the knowledge.

A total of 1,216 sheep slaughtered in five pasture areas in the Tianshan Mountains of Xinjiang were investigated and a phylogenetic analysis based on the mitochondrial cox1 gene was performed to clarify the genetic relationships of the various Trichuris species.

Sheep totalling 1,047 were infected with Trichuris spp. establishing the rate at 86.1%. Using a morphological protocol, six documented and one undefined species were identified, namely T. gazellae, T. lani, T. ovina, T. longispiculus, T. concolor, T. discolor and Trichuris sp. Among them, T. gazellae and T. lani were the dominant species, accounting for 34.5% and 31.0% of Trichuris spp., respectively. Phylogenetic analysis divided the detected species of Trichuris spp. into two genetic clades (clade I and clade II). The six documented species that can infect sheep and the undefined species were clustered into clade I, with inter- and intra-species genetic diversity apparent.

This survey described in detail the morphological characteristics of six known and one undefined species of Trichuris, which not only enriched the taxonomic information on record regarding Trichuris spp., but also provided valuable epidemiological data for the prevention and control of trichuriasis in sheep.

Histamine intoxication, known as scombroid fish poisoning, is caused by the consumption of foods with high levels of histamine. This biogenic amine is formed as a result of histidine decarboxylation by bacterial decarboxylases present in food, including fish and fish products. The aim of this study was to investigate the content of histamine at different production stages of canned, marinated and smoked fish.

Raw fish, semi-finished fish products, and the final products of the same production batches were collected between 2019 and 2022 from different fish production facilities in Poland. A total of 133 raw fish samples and 76 smoked fish, 54 brined fish, 39 canned fish and 18 marinated fish final products were analysed using high performance liquid chromatography with a diode array detector.

Histamine was identified in 55 (17.2%) out of 320 tested samples, including 8 samples of raw fish with a histamine level above 100 mg/kg. However, no samples of fish products had histamine content above the European Union Commission limit.

The obtained results show that fish products on the Polish market are generally safe for consumers in regard to histamine intoxication risk.

Heat treatment is indispensable in fish canning to provide an acceptable shelf life. Its optimisation reduces the risk of the presence of Clostridium botulinum spores, which could potentially cause botulism cases. This study evaluated canned fish samples for botulism neurotoxin (BoNT)-producing clostridia contamination and can bulging through microbiological contaminant growth. A new analytical approach was developed for detection of such clostridia and phenotypically similar species.

A total of 70 canned fish samples suspected of exhibiting bulging features were analysed. Culture methods were used to detect clostridia. The isolates obtained were evaluated on the basis of the exhibited phenotypic characteristics. Also, PCRs were used for the detection of genes determining BoNT production (non-toxic non-haemagglutinin (ntnh) genes) and the amplification of conservative 16S rDNA genes, which were Sanger sequenced. The obtained sequences were analysed using the Basic Local Alignment Search Tool.

Clostridium genus species were isolated from 17 (24%) bulging and organoleptically changed samples. No ntnh genes were present in these isolates; however, sequencing confirmed the presence of C. sporogenes, a species with close affinity to C. botulinum.

To eliminate the threat of foodborne botulism, laboratory diagnostic techniques must detect species of the Clostridium genus and elucidate their ability to produce BoNTs. Although Clostridium botulinum is the most common cause of botulism, the possibility may not be ignored that non-pathogenic Clostridium species may acquire botulinum toxigenicity. The similarity between the isolated strains of C. sporogenes and C. botulinum should be incorporated in the optimisation of heat treatment to guarantee a sterilised, microbiologically safe product.

Carvacrol is an essential oil extracted from oregano which can be used as a natural additive in poultry litter and could have a positive impact not only on production rates but also on the quality of poultry meat. The aim of this study was to evaluate the effect of the addition of carvacrol to litter on weight gain and the occurrence of residues in chicken tissues.

One-day-old Ross 308 chicks were used for the study and were randomly divided into two experimental groups. For 42 days, one group was kept in a room with litter enriched with carvacrol and the second group was kept in a room with litter without carvacrol. After 42 days, the birds were sacrificed and necropsied. Carvacrol content was determined in homogenised organ tissue samples by liquid chromatography–mass spectrometry.

Weekly weighing results showed that exposure to carvacrol in litter had no impact on chicken body weight. The analysis of plasma, muscle, liver and lung tissue after 42 days’ exposure clearly indicated that there were residues of carvacrol in the analysed matrices.

Exposure of chickens to carvacrol left residues but did not affect body weight.

Cadmium and zinc are often found in aquatic environment and may accumulate in living organisms. The aim of this study was to evaluate the genotoxic effect of Cd, Zn, and their binary mixture on the peripheral blood erythrocytes of Prussian carp (Carassius gibelio B.).

The fish were exposed to 4.0 mg/L Cd, 4.0 mg/L Zn or a mixture of 4.0 mg/L Cd and 4.0 mg/L Zn for a period of 14, 21 or 28 days. Genotoxic effects were investigated in peripheral blood cells using the comet assay and the erythrocyte micronucleus assay.

The results demonstrated that the frequencies of micronuclei (MN) and both nuclear and cellular abnormalities in erythrocytes were significantly higher in all exposure groups as compared to the control group. The fish exposed to the mixture of Cd and Zn presented the highest frequency of MN. Furthermore, there was a decrease in the frequency of MN and an increase in the occurrence of DNA integrity defects (DNA damage) with longer time of exposure to the metals studied.

Erythrocyte micronucleus and comet assays confirmed the genotoxicity of Cd and Zn. The results of the tests applied (which showed considerable variability) suggest the involvement of various toxicity mechanisms. Therefore, an integrative and comprehensive approach, using a set of assays for toxicity profile determination, should be adopted during ecotoxicological studies and environmental risk assessment pertaining to these elements.

Diabetic retinopathy (DR) is the leading cause of blindness in human and animal patients. Early detection and treatment of the disease are important and can be facilitated by proteomic approaches providing biomarkers.

Tear films were collected on Schirmer strips from 32 canine patients (12 diabetic dogs without changes in the retina, 8 diabetic dogs with signs of DR, and 12 control dogs). Two-dimensional electrophoresis was used to separate tear film proteins prior to their identification with matrix-assisted laser desorption/ionisation–tandem time-of-flight mass spectrometry and interrogation of protein function databases to find matches.

Five significantly differentially expressed proteins were identified; of those, one was downregulated (2ʹ-5ʹ-oligoadenylate synthase 3) and four were upregulated in the tear film of two diabetic groups (Ras-related protein RAB-13; aldo-keto-reductase family 1 member C3; 28S ribosomal protein S31, mitochondrial; and 60S ribosomal protein L5). The differentially expressed proteins identified in the tear film were involved in signalling pathways associated with impaired protein clearance, persistent inflammation and oxidative stress.

The results of our study confirm that the pathological process in the retina in the course of diabetes mellitus causes changes in the tear film proteome.