Volume 66 (2022): Edizione 3 (September 2022)

In May 2020, an outbreak of rabbit haemorrhagic disease 2 (RHD2) caused by the rabbit haemorrhagic disease virus 2 (RHDV2, GI.2) occurred in Sichuan, China. The acute onset and short disease course resulted in rabbit mortality as high as 42.86%. Currently, basic research on the aetiology and genetic characteristics of GI.2 is lacking in China.

Pathological changes in various tissues from infected rabbits were investigated and the viral genome was characterised. This study used RT-PCR, histopathology and scanning electron microscopy to identify the pathogen in samples from infected rabbits that had died. Phylogenetic trees were constructed based on whole genome sequence analysis, and recombination events were analysed.

RT-PCR identified the presence of GI.2. Histopathology revealed liver cell necrosis and haemorrhaging into lung alveoli. Electron microscopy demonstrated spherical GI.2 particles that were 40 nm in size. The gene sequence length of the isolate was 7,445 bp (GenBank accession number MW178244). A phylogenetic analysis based on the genome of the isolated strain and 60 reference strains showed that the isolate was grouped together with GI.2 strain MT586027.1 in a relatively independent sub-branch. The results of the recombination analysis showed that the strain was recombined from the MT586027.1 (major parent) and MN90145.1 (minor parent) strains, and recombination breakpoints were at locations in the 2858–5137 nt range.

The results of this study extend our understanding of the molecular epidemiology of GI.2.

The single member of the Asfarviridae family is African swine fever virus (ASFV). This double-stranded DNA virus infects wild and farmed swine and loses the pig industry large sums of money. An inner envelope, capsid, and outer envelope are parts of the ASFV particle containing structural proteins playing different roles in the process of infection or host immune defence evasion. When expressed by the baculovirus system, the p22 protein from the inner envelope was found to induce partial protection against a virulent virus strain. This study aimed to express a part of this protein in a different system and evaluate its immunogenicity.

We designed two proteins, the extracellular (C terminal) part of the p22 protein (p22Ct) and its fusion with the heat-labile enterotoxin B subunit from Escherichia coli (LTB-p22Ct), which is supposed to be a potent enhancer of the immune response. Both proteins were produced in the E. coli expression system and subsequently used for mice immunisation to analyse their safety and immunogenicity.

The protein fused with LTB did not show the expected adjuvant properties and did not prove safe, because abscess formation was observed after immunisation. In contrast, immunisation with the p22Ct protein alone induced a higher antibody titre but caused no adverse symptoms.

These results show the high potential of the p22Ct region as an immunogenic protein for ASFV serological detection purposes.

Bovine leukaemia virus (BLV) is the retroviral causative agent of enzootic bovine leukosis, the most common neoplastic disease of cattle and a serious problem worldwide. Its diagnosis is commonly by tests for antibodies recognising the p24 capsid protein and structural glycoprotein (gp) 51. With flow cytometry recently having come to veterinary immunology, applications for it may now include BLV. The study determined BLV gp51 expression in blood and milk lymphocytes of naturally infected cows by flow cytometry.

Nineteen Polish Black and White Lowland breed cows aged 4–9 years and naturally infected with BLV and ten uninfected counterparts had blood and milk sampled and cultured. The immunological status of the animals was confirmed with ELISA and PCR. Dual-colour flow cytometry analysis was performed with specific monoclonal antibodies for lymphocyte cluster of differentiation (CD) markers and gp51 viral envelope protein and conjugates labelled with fluorescein isothiocyanate or phycoerythrin. Bovine leukaemia virus gp51 was confirmed in lymphocytes by immunofluorescence with anti-gp51 monoclonal antibodies.

The gp51 antigen was detected in blood and milk lymphocytes of infected cows, but the percentage of cells expressing it in milk was much lower than in blood. A depleted number of CD4+ lymphocytes, an augmented number of CD8+ lymphocytes, a lower ratio of CD4+ to CD8+ and a proliferation of CD19+ immunoglobulin M+ cells were also found.

These proliferated cells were immature, gave no sign of a tendency to differentiation and were characterised by prolonged vitality.

Porcine epidemic diarrhoea virus (PEDV) is an enteric pathogen causing porcine epidemic diarrhoea and acute gastroenteritis in pigs of all ages. Previous analysis of the viral genome of PEDV in Poland was only based on the spike protein (S) gene sequences and no analysis of other genes has been performed. The aim of this study was to analyse the envelope (E), membrane (M) and nucleocapsid (N) protein and open reading frame 3 (ORF3) gene sequences.

Viral RNA from 18 Polish pig faecal samples that were quantitative reverse transcription PCR-positive for PEDV was analysed in four genomic regions (E, M, N and ORF3).

Phylogenetic analysis based on these regions’ sequences revealed that Polish PEDV isolates were highly related and were clustered into group G2a across the four genes compared. Moreover, the Polish strains were located in distinct subclusters on the phylogenetic trees, which suggests the presence of at least three independently evolving PEDV genetic lines circulating in Poland. The occurrence of unique mutations in the sequences of Polish PEDV strains suggests that PEDV continues to undergo evolutionary processes, accumulating the mutations necessary for viral fitness in its natural hosts. The Polish PEDV strains differed genetically from the CV777 vaccine strain, suggesting the risk of relatively low vaccine efficacy if this strain is used.

Our results promote a better understanding of the genetic diversity of PEDV field isolates in Poland and highlight the importance of molecular characterisation of PEDV field strains for the development of an effective vaccine against PEDV.

Kasba virus belongs to the Palyam serogroup of the Orbivirus genus and Reoviridae family. Kasba virus is the causative agent of many reproductive disorders in infected animals, which result in considerable economic losses, mainly in the cattle industry. The epidemiology of Kasba virus infection is poorly defined and remains unclear in South Korea.

This study investigated the prevalence of antibodies against Kasba virus in sheep and goats in South Korea. Individual, management, and regional risk factors associated with seropositivity were also evaluated. In addition, a retrospective serosurvey was conducted.

Serum samples from 28 out of 441 sheep or goat flocks (6.3%, 95% confidence interval (CI): 4.4–9.0%) and 115 out of 1003 animals (11.5%, 95% CI 9.6–13.6%) were positive for antibodies against Kasba virus. According to our results, a history of reproductive problems increased the probability of Kasba virus positivity. Preventive measures such as routine insecticide application decreased this probability. We observed significant differences in the prevalence of seropositivity between southern provinces and northern provinces and between western provinces and eastern provinces at the individual level.

The virus was widely distributed among sheep and goats in South Korea, with seropositivity ranging from 6.8% in 2004 to 13.7% in 2008. The current study represents the first assessment of factors associated with Kasba virus seroprevalence in sheep and goats in South Korea.

Bovine tuberculosis is one of the most dangerous zoonotic diseases. Despite the near-complete elimination of the disease from cattle breeding in Poland achieved in 2009, its re-emergence is now observed. Globally, the number of human cases is underestimated and the importance of free-living animals as reservoirs of tuberculosis is growing. As a species highly susceptible to Mycobacterium tuberculosis complex infection, the European bison (Bison bonasus) has a role in the transmission of the disease in Poland. The purpose of the investigation was to assess the epidemiological situation of tuberculosis in Polish European bison serologically.

A total of 460 serum samples were collected from 436 European bison from 15 out of 26 national populations between 2013 and 2020. An M. bovis ELISA was used, and its sensitivity and specificity were assessed with an eyelid tuberculin skin test (TST) and interferon gamma release assay (IGRA).

Mycobacterium bovis antibodies were detected in nine serum samples. The presence of antibodies was found in two animals from the Białowieża Forest (1.2% of the population), and one each from the Borecka Forest (2.4%) and the Warsaw Zoo (14.3%). One European bison among the 14 sampled (7.1%) from Smardzewice was positive on five occasions. Other samples from Smardzewice and the Bieszczady Mountains, where tuberculosis had previously been reported, were negative.

ELISA testing is an effective, easy and cost-efficient tool for monitoring of tuberculosis-naïve populations. Serological testing in tuberculosis control programmes can significantly improve the detection of infected herds. Antibody ELISAs may supplement TST and IGRA, but cannot replace them.

Contagious agalactia of ruminants is an endemic disease caused by Mycoplasma agalactiae in flicting significant losses on farms in deaths and forced slaughter of sick animals, abortions, births of sick young animals, and reduced milk and wool production. The aim of the study was to determine the influence of hydrometeorological conditions on the distribution and forms of contagious agalactia in sheep in Bessarabia, Ukraine.

The epizootic situation regarding contagious agalactia was studied during 2011–2021 on sheep farms in the south of the Odesa region in Bessarabia. Over two million blood samples from sheep aged 1–6 years were serologically investigated and the prevalence of agalactia was correlated with Selyaninov’s hydrothermal coefficient for each sampling year.

High rates of infection of sheep with contagious agalactia (from 13.1% to 14.4%) were registered in 2012, 2016 and 2021, years which according to the hydrothermal coefficient of 1.0 were sufficiently moist. The lowest incidence rates, from 6.5% to 7.4%, were registered in the very dry 2013, 2014 and 2019 with hydrothermal coefficients of 0.5‒0.6. In sufficiently moist years, contagious agalactia of sheep manifested itself in the mastitic form, while in the dry period the mastitic form was half as prevalent, and the mixed, articular and ocular forms of the disease proliferated.

The results indicate the circulation of Mycoplasma agalactiae among small ruminants in Bessarabia, and that the prevalence and the course of the associated disease depend on the humidity of the climate, i.e. the value of the hydrothermal coefficient.

The widespread occurrence of drug-resistant bacteria has increased interest in alternatives to antibiotics for combatting bacterial infections, among which bacteriophages play an important role. The ability of phage proteins to induce an anti-phage immune response can significantly limit the effectiveness of treatment, which was the basis for the study described in this article. The aim of the study was to assess the effects of bacteriophages on the induction of an anti-phage humoral response in calves.

The study was conducted using phage components of experimental preparations and sera from calves treated and not treated with phages. Levels of G, M and A immunoglobulins were analysed by ELISA. The assay plates were coated with whole Escherichia coli and Mannheimia haemolytica phages and selected phage proteins obtained in sodium dodecyl sulphate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis. Neutralisation of phages by immunoglobulins was assessed by determining phage titres using double-layer plates.

The results confirmed an increased anti-phage response affecting all immunoglobulin classes in the calf sera. The highest significant (P ≤ 0.05) level of antibodies was observed for IgG in the sera of calves receiving phages. The phage neutralisation test showed a significant differences (P ≤ 0.05) in the reduction of phage titres in comparison to untreated calves.

Despite the induction of an anti-phage response, no significant negative effect on the antibacterial activity of phages was observed in vitro.

The current study characterises Staphylococcus bacteria recovered from dead free-living birds and captive capercaillies kept in south-eastern Poland. The results provide novel information about the antimicrobial resistance phenotype/genotype and the virulence profile of these bacteria.

Samples of internal organs were taken from dead birds. Staphylococcus strains were identified by matrix-assisted laser desorption/ionisation–time-of-flight mass spectrometry. Susceptibility to 13 antibiotics was tested using a standard disc diffusion method on Mueller–Hinton agar. All isolates were screened for the presence of antibiotic resistance genes and staphylococcal enterotoxins (A to E), toxic shock syndrome toxin 1, exfoliative toxins A and B and Panton–Valentine leukocidin.

A total of 129 bacterial strains belonging to 19 species of the Staphylococcus genus were isolated. A relatively high percentage of them resisted fluoroquinolones, tetracyclines, macrolides and β-lactams to a significant degree and harboured the tetK, tetM, ermC, mphC and mecA genes. Strains of the coagulase-negative S. sciuri, S. xylosus and S. cohnii were isolated with genes encoding enterotoxin A and toxic shock syndrome toxin.

Both coagulase-positive and coagulase-negative staphylococci isolated from aviary capercaillies and free-living birds have significant pathogenic potential, and greater attention must be paid to the coagulase-negative species, which are still often considered mere contaminants. Virulence factors associated with resistance to antimicrobials, this being multiple in some strains, seem most important because they can be easily transferred between animals, especially those living in a given area.

Avian malaria caused by Plasmodium and the malaria-like parasites of the genus Haemoproteus has been regularly described in multiple regions worldwide. These parasites significantly affect many avian taxa, including domestic chickens and fighting cocks. There are limited epidemiological studies of these blood parasites in vertebrate hosts, especially in Thailand.

This study used microscopic examination of blood samples and PCR amplification exploiting primers for nucleotide sequences of Plasmodium or Haemoproteus species based on the cytochrome b gene to determine the occurrence of Plasmodium spp. in fighting cocks.

Examination of 249 blood samples of fighting cocks revealed that 41.37% (103/249) were positive for malaria by microscopic examination and 88.76% (221/249) were positive by DNA amplification. Sequencing and DNA analysis of 61 PCR products revealed that infection by Plasmodium juxtanucleare was the most common avian malaria in fighting cocks in Thailand followed by infections by Plasmodium gallinaceum; however, Haemoproteus infection was not discovered.

This study indicated that plasmodiasis is widespread in fighting cocks in Thailand although the prevalence was not clearly determined; therefore, prevention and control strategies for these protozoa should be improved, especially those for avoiding vector exposure and eliminating mosquito breeding sites.

Fascioloides magna is a parasite of high veterinary importance due to its pathogenicity for wild and domestic ruminants. The aim of our study was to describe the presence of trematode infection in the red deer population in the Lower Silesian Wilderness, one of the established fascioloidosis foci in Central Europe, and to assess the overall prevalence of F. magna in the studied area. In order to achieve this, a coprological study of different cervid species was performed.

The livers of 99 red deer were collected over three years and examined for the presence of trematodes. Prevalence and infection intensity was estimated and a histopathological analysis was performed. In addition, 172 faecal samples from red deer, fallow deer and roe deer were examined.

By year, Fascioloides magna was isolated from the livers of 2/30 (6.7%), 9/34 (26.5%) and 10/35 (28.6%) red deer. Severe hepatic lesions, including massive tissue damage, extensive fibrosis, and cirrhotic changes in the liver parenchyma were observed. Faecal examination revealed the presence of F. magna eggs, with a prevalence of approximately 40%, 50% and 53% in roe deer, fallow deer and red deer, respectively.

The eggs of F. magna may be commonly excreted in the faeces of roe deer, as well as those of red deer and fallow deer. The presence of F. magna throughout the cervid population in the Lower Silesian Wilderness favours the risk of the trematode’s transmission to livestock or farmed deer.

The alpaca (Vicugna pacos) is a camelid native to South America, but the species has wide distribution outside its natural habitat and is found in various countries on other continents, Poland being one in Europe. Parasitic infections affect the productivity and health of alpacas. The aim of the study was to estimate the parasite loads in alpacas kept in Poland with the use of two direct centrifugal flotation methods.

A total of 248 faecal samples from alpacas from 12 provinces in Poland were examined for parasite eggs and oocysts with a modified Willis method (WM), and 59 samples were examined simultaneously with WM and a modified Stoll method (SM).

The WM detected eggs of Trichostrongylidae, and the SM oocysts of Eimeria spp. as the respective most prevalent parasite material. The eggs of Nematodirus sp., Nematodirus battus, Aonchotheca sp., and Trichuris sp. were detected in lower numbers in both methods. Oocysts of E. macusaniensis and eggs of Moniezia sp. were the rarest in WM, the former also being so in SM; eggs of Moniezia were absent in SM. The prevalence of Eimeria spp. was significantly higher as detected by SM than as detected by WM; however, the prevalence of eggs of Nematodirus spp. and trichostrongyles was significantly higher as detected by WM than as detected by SM.

WM is more accurate at detecting heavy gastro-intestinal nematode eggs, including those of trichostrongyles and Nematodirus, whereas SM is more accurate at detecting smaller coccidia from the genus Eimeria.

Escherichia coli is present in the normal intestinal flora but some strains can cause intestinal and extraintestinal diseases, and research on its presence in food of animal origin is in the interests of public health. This study was designed to characterise E. coli strains according to their origin, their carriage of virulence genes specific for certain pathogroups, and phylogenetic group affiliation.

The study was carried out on 100 E. coli strains isolated from food samples of various animal origin as well as pig and cattle carcass swabs. Isolation of the strains was performed using two methods. One method included colony count and the other an overnight enrichment of the samples. Isolation was followed by DNA extraction and detection of virulence genes and phylogenetic group with conventional and multiplex PCRs.

In this study, the most prevalent gene was EAST1 (20%) and strains which carried it were identified as enteroadherent E. coli. Other pathogroups were represented in lower incidences. Phylogenetic group analysis revealed the prevalence of the A and B1 groups, with B1 mainly present in game and cattle strains, while the majority of pig and poultry strains were assigned to group A.

This study provides an overview of the presence of potentially pathogenic strains and E. coli phylogenetic groups in Croatia, for which the data are limited. Further microbiological and molecular research is required to examine the epidemiological situation in the country.

The use of insects and their processed animal proteins (PAPs) for animal nutrition creates the need for research into methods useful for routine surveillance for their presence. The aim of this study was to evaluate a modified microscopic method for the detection of particles of insects in poultry feed.

A total of 90 samples including PAP of insects (Hermetia illucens and Tenebrio molitor), poultry feeds produced with different levels (0–27%) of insect PAP content, and other poultry feeds spiked with insect PAP at 1% were investigated using a modified microscopic method with a double sedimentation protocol.

Characteristic features of insects including cuticulae, muscles, bristles and tracheoles were determined in the microscopic images obtained. In all spiked samples, characteristic fragments of insects were detected. The fragments of muscle and tracheoles only indicated the presence of material from members of the insect class but could not facilitate identification of organisms to species level.

The results obtained with this double sedimentation protocol for the isolation of insect PAP from feed for poultry have shown that the method can be used in routine analysis.

The use of probiotics and phytobiotics has attracted interest because of their protective effect against acidosis. Ferula elaeochytris (FE) is considered a good source of bioactive compounds, mainly monoterpene α-pinene. This study aimed to investigate the effect of a direct-fed microbial blend (Pro) and FE on rumen fermentation parameters in vitro under normal and acidosis conditions.

An in vitro experiment using the Hohenheimer Futterwerttest (HFT) gas production system was conducted. An acidosis challenge was made to compare the effectiveness of the probiotics blend and FE extract on ruminal pH regulation. To generate different ruminal fermentation parameters, the design of the trial considered the 2 additives (Pro and FE) × 6 incubation times (2, 4, 8, 12, 24 and 48 h) × 2 conditions (acidosis and normal) × 2 incubation runs for each feedstuff (barley, alfalfa and straw).

An acidosis challenge was successfully induced. The Pro and FE additives had no impact on the observed rumen fermentation parameters such as volatile fatty acid concentration or ammonia (P = 0.001). The acidosis condition decreased total in vitro degradability (IVD) by 3.5% and 21.9% for barley and straw, respectively (P < 0.001). The additives had different significant effects on the IVD of nutrients during both normal and acidosis conditions. In alfalfa samples, FE supplementation significantly decreased the IVD of all observed nutrients under the ruminal acidosis condition, although it had no effect during the normal condition.

An acidosis challenge was successfully induced and the effect of additives was varied on fermentation parameters and rumen degradability of different feeds either under normal or acidosis conditions.

The aim of this study was to investigate changes in haematological parameters and the antioxidant system in the early and late pregnancy of red deer (Cervus elaphus).

Blood samples were collected from 30 red deer females 50 days after impregnation and 40 days before calving. Complete blood counts and stained blood smears were assessed. Paraoxonase 1 (PON1), superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR) activities, glutathione disulphide (GSSG), total glutathione, total bilirubin, total protein, albumin, uric acid, malondialdehyde (MDA), beta-hydroxybutyrate (BHB), low-density lipoprotein (LDL), high-density lipoprotein (HDL), triglycerides, cholesterol levels and total antioxidant status (TAS) were measured.

The haematological characteristics of blood collected in the third trimester showed higher haemoglobin levels, haematocrit, mean corpuscular volume (P < 0.05) and a lower mean cell haemoglobin concentration (P < 0.05) in comparison to samples collected on the 50^th day after mating. Activity of CAT and MDA, LDL, and triglyceride levels were lower while GR, GSSG, total glutathione, total protein and BHB levels were increased in blood samples drawn in the third trimester compared with the first trimester blood samples. There were no changes in SOD, PON1 activities, TAS, bilirubin, albumin, HDL, cholesterol or uric acid concentrations.

Red deer’s compensatory mechanisms facilitate its optimal adaptation to seasonal changes evidenced by mild pronounced haematological disturbances and an effective antioxidant system during pregnancy.

An analysis of samples originating from domestic and exotic animals from Lower Silesia but also from other parts of Poland was carried out based on research conducted in 2014–2017.

The materials used for the study were 11,338 tumour samples taken in vivo during surgery and as biopsies and posthumously during necropsies. They were sent to the Department of Pathology at Wrocław University of Environmental and Life Sciences for histopathological diagnosis.

The largest group were canine tumours, of which there were 9,745 (85.95%), followed by feline tumours, totalling 1,397 (12.32%), tumours from exotic animals (186; 1.64%), from horses (7; 0.06%), and from cows (2; 0.02%). A significant number of cases of animals suffering from more than one tumour were also found, which had not been frequently diagnosed previously.

The number of neoplasms diagnosed in pets and exotic animals is increasing every year. The average animal age at diagnosis continues to fall. The greatest number of neoplasms were diagnosed in mixed-breed dogs and cats, and the number of tumours in a pure breed strictly correlated with breed’s popularity in the research period. Mesenchymal tumours are still the most prevalent type of tumours in dogs, while in cats epithelial tumours predominate. The neoplasm case pattern in animals conforms to that in humans in the same area.

Acute myocardial infarction (MI) is one of the most common causes of death in humans in highly developed countries. Among its most frequent complications affecting the patient’s prognosis are cardiac arrhythmias: ventricular tachycardia (VT) and ventricular fibrillation (VF).

The study aimed to characterise arrhythmias in 19 pigs subjected to experimentally induced MI obtained by occlusion of the proximal left anterior descending (LAD) coronary artery using an angioplasty balloon. The anaesthetic protocol was modified to reduce mortality by including procedures stabilising haemodynamic disorders which develop during episodes of ischaemia and arrhythmia. During 30 min of experimentally induced ischaemia, the heart rhythm was recorded using a 12-lead ECG. The time, frequency, and type of arrhythmias were analysed.

Ventricular arrhythmias were found in 94.74% of the treated pigs. The most common were ventricular premature complexes, reported in 88.89% of pigs with arrhythmia. Ventricular tachycardia was recorded in 66.67% and ventricular fibrillation in 50% of pigs with arrhythmias.

Myocardial infarction due to proximal LAD occlusion is characterised by a high incidence of ventricular arrhythmias, especially VT and VF. Because of the high survival rate, this MI porcine model may serve as a model for research on acute ischaemic ventricular arrhythmias in humans. Additionally, it reduces the total number of animals required for testing while yielding meaningful results, which is in line with the 3R principle.