Volume 64 (2020): Issue 4 (December 2020)

Repeated incursions of highly pathogenic avian influenza virus (HPAIV) H5 subtype of Gs/GD lineage pose a serious threat to poultry worldwide. We provide a detailed analysis of the spatio-temporal spread and genetic characteristics of HPAIV Gs/GD H5N8 from the 2019/20 epidemic in Poland.

Samples from poultry and free-living birds were tested by real-time RT-PCR. Whole genome sequences from 24 (out of 35) outbreaks were generated and genetic relatedness was established. The clinical status of birds and possible pathways of spread were analysed based on the information provided by veterinary inspections combined with the results of phylogenetic studies.

Between 31 December 2019 and 31 March 2020, 35 outbreaks in commercial and backyard poultry holdings and 1 case in a wild bird were confirmed in nine provinces of Poland. Most of the outbreaks were detected in meat turkeys and ducks. All characterised viruses were closely related and belonged to a previously unrecognised genotype of HPAIV H5N8 clade 2.3.4.4b. Wild birds and human activity were identified as the major modes of HPAIV spread.

The unprecedentedly late introduction of the HPAI virus urges for re-evaluation of current risk assessments. Continuous vigilance, strengthening biosecurity and intensifying surveillance in wild birds are needed to better manage the risk of HPAI occurrence in the future.

Canine parvovirus type-2 (CPV-2) causes acute infectious diseases in puppies, which show high morbidity and mortality. Better effect of vaccination against these diseases could be achieved with deeper knowledge of CPV-2 genotype dissemination and mutation history. This study investigated CPV-2–positive samples collected recently over a wide region of China.

A total of 118 faecal samples from dogs identified as CPV-positive were collected from veterinary clinics in central and eastern China. Overall, 16 strains collected from Anhui, 29 from Henan, and 16 from Zhejiang Province were sequenced to determine the genotypic composition of CPV-2 and mutational complexity of CPV-VP2.

The CPV-2a, CPV-2b, and CPV-2c genotypes were detected in Anhui and Henan Provinces, while CPV-2c alone was detected in Zhejiang Province. Sequence analysis of all strains showed 98.5%–99.8%, 98.3%–99.9%, and 98.7%–99.8% identity among the 16 Anhui, 29 Henan, and 16 Zhejiang strains, respectively. Strains collected from Anhui and Henan Provinces showed lower identity (97.0%), suggesting greater genetic divergence in central China. The mutation rates of Henan and Anhui strains were lower than that of Zhejiang strains. Major amino acid mutations occurred at sites 5, 370, 426, and 440. Epitope and entropy analyses implied these sites’ likely conformance to the principles of mutation tendency, complexity, and diversity.

The findings for the evolutionary structure of CPV-2 strains collected from three provinces in central and eastern China advance trend monitoring of the genetic variation in canine parvovirus and point to its implications in the development of novel vaccines.

Three strains of the FMD virus (A, O, and SAT 2) were recognised as causes of the FMD circulating in Egypt. The aims of this study were to trace the FMDV isolates from outbreaks in Egypt to understand their epidemiology and evolution and to understand the situation of the vaccine strains compared with the circulating serotypes.

A meta-analysis was carried out by using the data available for FMD outbreaks in Egypt from GenBank and the World Reference Laboratory for Foot-and-Mouth Disease (WRLFMD); a comparison was done with both data sets for the three serotypes. MEGA-X was used for the evolution analysis, through constructions of phylogenetic trees for all sequences recorded in GenBank for each serotype in different Egyptian outbreaks in different years and also within the same year. Additionally, nucleotide substitution rate, molecular clock, and mean evolutionary rates were estimated for the three serotypes to understand and compare their evolution.

Absence of some records of certain serotype outbreaks from the WRLFMD database was noted as were subsequent missing appropriate vaccine programmes. Genetic variation was recorded among the virus isolates within the same years and also the vaccine strain was associated with up to 26 amino acid substitutions. The evolution rate of the SAT2 strain was the highest of the circulating strains. SAT2 had high amino acid substitution per year at an important immunogenic site (130–170), serotype A had less, and serotype O the least.

The need for different strategies for vaccine serotype selection is indicated.

Avian polyomavirus (APV) and psittacine beak and feather disease virus (PBFDV) induce contagious and persistent diseases that affect the beaks, feathers, and immune systems of companion birds. APV causes hepatitis, ascites, hydropericardium, depression, feather disorders, abdominal distension, and potentially death. PBFDV can induce progressive beak deformity, feather dystrophy, and plumage loss. We conducted the first prevalence survey of both APV and PBFDV infections in companion birds in eastern Turkey.

A total of 113 fresh dropping samples from apparently healthy companion birds were collected in a random selection. The dropping samples were analysed for PBFDV and APV by PCR. Positive samples were sequenced with the Sanger method. The sequence was confirmed through alignment and the phylogenetic tree generated through the maximum likelihood method computationally.

PBFDV and APV were detected in a respective 48.7% and 23.0% of samples. Coinfection was found in 12.4% of the samples, these all being from budgerigars (Melopsittacus undulatus). APV and PBFDV were detected in budgerigar and cockatiel (Nymphicus hollandicus) samples.

This report provides a foundation for future studies on the influence of these viruses on the health of companion birds. These high positive rates for both pathogens emphasise that healthy M. undulatus and N. hollandicus in eastern Turkey may be prone to the emergence and spread of APV and PBFDV with subclinical potential.

Marek’s disease (MD) is a tumourous disease caused by Marek’s disease virus (MDV) and most commonly described in poultry. The aim of the study was to determine the occurrence of Marek’s disease virus infections in Poland and analyse clinical cases in the years 2015–2018.

The birds for diagnostic examination originated from 71 poultry flocks of various types of production. Birds were subjected to anatomopathological examination post mortem, during which liver and spleen sections and other pathologically changed internal organs were taken. These sections were homogenised with generally accepted methods, then total DNA was isolated and amplified with a real-time PCR. A pair of primers complementary to the MDV genome region encoding the meq gene were used.

MDV infection was found predominantly in broiler chicken flocks (69.01%), and also in layer breeder (9.85%) and commercial layer flocks (7.04% each).

The results of research conducted in the years 2015–2018 clearly indicate that the problem of MDV infections is still current.

Gastrointestinal parasites are some of the most common pathogens which are seriously harmful to the camel’s health. The infection status of gastrointestinal parasites in camels (Camelus bactrianus) in the Tianshan Mountains pastoral area in China is still unclear. The aim of this study was to investigate the species and infection intensity of gastrointestinal tract parasites in local camels.

A total of 362 fresh faecal samples were collected and examined for parasite eggs using the saturated saline floating and natural sedimentation method. The parasite eggs were subjected to morphological and molecular examination and identification, and the infection rate and mean intensity of the parasites were analysed.

A total of 15 gastrointestinal tract parasite species’ eggs were identified, with a detection rate of 100%. Ostertagia spp. (100%) and Trichostrongylus spp. (98.1%) were dominant. Camels were often coinfected by 5–14 species. The average number of eggs per gram of faeces was higher for Ostertagia spp. (298), Haemonchus contortus (176) and Nematodirus spp. (138). The number of species of parasites infecting young camels was significantly lower than that of adult camels, but the infection intensity in young camels was significantly higher.

Gastrointestinal parasites were highly prevalent in camels from the Tianshan Mountains pastoral area in China. This finding provides important epidemiological data for the prevention and control of associated infections in camels.

Candida species are a natural component of the intestinal tract microflora, but in favourable conditions they can cause superficial, mucosal, or even systemic candidiasis. Poultry production might be a source of human drug-resistant yeast infections, including Candida spp. The limited data concerning the antifungal susceptibility of poultry Candida isolates prompted us to carry out research to determine the susceptibility of isolates from turkey intestinal tracts.

The beak cavity, crop and cloaca were swabbed of 580 turkeys from 58 flocks in western Poland. The susceptibility tests were conducted using the E-test method with amphotericin B, fluconazole, itraconazole, and voriconazole on 52 isolates of C. albicans, C. catenulata, C. glabrata, C. palmioleophila, C. rugosa, C. krusei and C. lusitaniae.

All isolates were susceptible to voriconazole. According to the MIC values obtained for amphotericin B and fluconazole, all Candida spp. isolates were classified as susceptible according to the described breakpoints except for C. krusei, which was the only isolate that was amphotericin B-, fluconazole- and itraconazole-resistant. The susceptibility to itraconazole varied: 11 of the Candida isolates were susceptible (21.1%), 29 were dose-dependently susceptible (55.8%), and 12 isolates were resistant (23.1%).

There are few resistant strains of Candida in turkeys, and the drug resistance varies. When Candida passes from turkeys to humans, there is a wide range of antifungal treatment options.

Bovine mastitis is an inflammatory disease of the udder that causes important economic losses in the animal breeding and dairy product industries. Nowadays, the conventional livestock antibiotic treatments are slowly being replaced by alternative treatments. In this context, the main aim of this study was to evaluate the efficacy of natural products in alternative treatment of bovine mastitis.

Two natural formulations with previously suggested in vitro antimicrobial effect were tested in vivo on mastitic cows. Animals with a positive diagnosis for mastitis (n = 20) were divided into three treatment groups: two groups (n = 8) were administered formulations of propolis, alcoholic extracts of Brewers Gold and Perle hops, plum lichen, common mallow, marigold, absinthe wormwood, black poplar buds, lemon balm, and essential oils of oregano, lavender, and rosemary designated R4 and R7 (differing only in the latter being more concentrated) and one group (n = 4) a conventional antibiotic mixture. In vivo efficacy of treatments was evaluated by somatic cell and standard plate counts, the treatment being considered efficacious when both parameters were under the maximum limit.

R7 was effective in the most cases, being therapeutically bactericidal in six out of eight cows, while R4 gave good results in three out of eight cows, and conventional antibiotics cured one out of four.

These results suggest the possible therapeutic potential of these natural products in bovine mastitis.

The influence of inflammation on the patterns of muscarinic 2 and 3 receptor subtypes (M2R and M3R), and α-7 nicotinic acetylcholine receptor (α-7 nAChR) expression in the porcine uterus was investigated.

On day three of the oestrous cycle of gilts aged 7–8 months with body weight 90–120 kg, either an E. coli suspension (E. coli group, n = 5) or saline (Sal group, n = 5) was administered into the uterine horns via laparotomy or only laparotomy was performed on control swine (Ctrl group, n = 5). After eight days, and the onset of severe acute endometritis in the E. coli group, the uterine mRNA and protein receptor expression levels were determined using real-time RT-PCR and Western blotting, respectively, with receptor localisation by immunofluorescence.

The studied receptors were in the luminal epithelium, glands, blood vessels, and myometrial muscle cells of all gilts. The M2R mRNA level was lower in the inflamed endometrium compared to the Ctrl and Sal groups. Also in this tissue, the expression of M3R mRNA and protein was lower than in the Ctrl and Sal groups. The M3R protein level in the bacterially challenged myometrium was found to be increased compared to unadministered groups. In the endometrium of the E. coli group, the α-7 nAChR protein level was lower than in the Sal group, and in the myometrium it was reduced in relation to both the other groups. P values were ≤ 0.05 in all cases.

Inflammation causes alterations in the M2R, M3R, and α-7 nAChR expression in the pig uterus, suggesting their significance in the course and repercussions of uterine inflammation.

Snail eggs can be the raw material for the production of a caviar substitute. The substitute varies from the original in caloric value and nutrient content which determine the nutritional value of every foodstuff. The present study aimed to determine and compare the nutritional value and protein quality of eggs from two subspecies of edible snail.

The chemical composition of the snail eggs i.e. Cornu aspersum maxima and Cornu aspersum aspersum was determined in accordance with international standards. In order to evaluate the protein quality of the eggs of the two studied snail subspecies, the chemical score (CS), and a reference protein were used.

Significant differences in the content of water, ash, and carbohydrates, but comparable protein and fat contents and caloric values were found.

The protein in the eggs of the snails was complete by the measure of the model adopted for this study, however, meeting the daily essential amino acid requirements of an adult would require an immense supply of both species’ eggs. Snail eggs of the Cornu genus were characterised by much lower nutritional value in comparison with caviar and caviar substitutes.

Silage quality deteriorates with Clostridium spp. contamination, and if consumed, such silage jeopardises herd health and productivity. Minimising its occurrence reduces economic and animal welfare risks. The study investigated the influence of environmental and technological determinants on the Clostridium genus’ occurrence in silage.

Analyses were conducted on 305 silage samples directly collected from farms located in all Polish provinces. Cultures and isolates were evaluated phenotypically and examined for occurrence of Clostridium spp., particularly C. perfringens and C. botulinum using PCR techniques. The results were statistically analysed using the ᵡ² test for continuous and Student’s t-test for non-continuous values.

The most influential effect on Clostridium spp. occurrence is exerted by factors potentially associated with primary production, like the type of fertilisation and the contamination level of the ensiled feed material. Clostridium spp. was detected in 232 (76%) samples, and C. perfringens strains, predominantly toxinotype A, in 79 (26%). C. botulinum occurrence was not detected.

Deterioration of silage by clostridia could be prevented by a properly conducted ensiling process with the addition of starter cultures, but the presence of spores mainly depends on primary production and the extent of contamination of the feed material.

An effective way of preventing undesirable boar taint in pork meat caused by the presence of androstenone, skatole and indole is surgical castration of piglets. This, however, arouses growing social opposition. An alternative method of inhibiting the development of unpleasant odour is immune castration. The aim of the study was to compare the effectiveness of both methods of castration for the elimination of the compounds responsible and to assess the suitability of oral fluid for pre-slaughter predictive testing for boar taint.

The research material was pooled oral fluid and fat samples taken from gilts and surgically and immunologically castrated piglets. The samples were tested with a liquid chromatography– tandem mass spectrometry method developed in this research.

The compounds giving rise to boar taint were found only sporadically above the accepted limits; only one sample of oral fluid contained skatole at a concentration above 200 μg L⁻¹ and one contained indole more concentrated than 100 μg L⁻¹. Indole above the limit value was also detected in one fat sample. In none of the tested samples was androstenone found.

The results indicate the similar effectiveness of both methods of piglet castration on the reduction of compounds generating boar taint. The usefulness of testing oral fluid for the ante-mortem prediction of boar taint has not been fully confirmed and further investigation is needed.

Early detection of pregnancy is vital for appropriate reproductive management programmes to facilitate the rapid re-insemination of non-pregnant females and reduce the calving interval.

A barium chloride test was compared with a commercial progesterone ELISA to detect pregnancy in non-descriptive cows and investigate if it could be applied as an alternative to ELISA in the field. Blood and urine samples were collected from 74 cows with recorded insemination dates. The progesterone ELISA and barium chloride assay were implemented to detect progesterone (P4) in blood and urine specimens, respectively. The cows' reproductive systems were examined after they were slaughtered to determine the uterus's status. Macroscopic examination of the uterus was used as a reference standard for both tests.

The sensitivity rates of the P4 ELISA and barium chloride test to detect pregnant cows were 100.0% and 79.4%, and to detect the corpus luteum (CL) were 83.0% and 87.0%, respectively, their sensitivity increasing in the presence of the CL. The ELISA and barium chloride tests were 79.7% and 52.7% accurate in the diagnosis of pregnancy. The accuracy of the barium chloride test in CL detection increased to 81.0%, and that of the ELISA to 86.4%. There were no significant differences (P = 0.052) between the barium chloride assay and ELISA when they were utilised for the identification of the CL.

The barium chloride test can be an inexpensive and time-saving alternative to ELISA in pregnancy diagnosis when the insemination date is known.

The aim of the study was to determine the effect of the vitamins, omega-3 polyunsaturated fatty acid and minerals in the supplement Toryum administered before and during oestrus synchronisation on some fertility parameters of ewes during the non-breeding season.

The experimental animals were clinically healthy Pirlak ewes, 55–75 days postpartum, aged 2–4 years and weighing 40–50 kg. A sponge was inserted into the vagina for 10 d (G1, n = 30; G2, n = 30) or 14 d (G3, n = 30; G4, n = 30) for oestrus synchronisation, and on the day of removal, 400 IU equine chorionic gonadotropin was injected. Toryum soft capsules were administered individually (1 capsule/ewe p.o.) to G1 and G3 ewes seven days before the sponge was inserted and on the day it was removed. Oestrus detection was started 12 h after sponge removal. Pregnancy was diagnosed by transrectal ultrasonography on the 30^th day after mating.

The pregnancy rate was statistically different between G1 and G4 (P < 0.05). The onset of oestrus was statistically different (P < 0.001) between the 10-d groups (G1 and G2) and the 14-d groups (G3 and G4). The litter size and oestrus, conception, lambing, multiple birth, and survival rates were not significantly different between the groups (P > 0.05).

Toryum administered to Pirlak ewes during progesterone-based oestrus synchronisation protocols during the non-breeding season may increase pregnancy rates. The relationship between Toryum and fertility parameters in ewes would be better understood by comprehensive studies.

To date, there have been no panoramic studies of the serum metabolome in feline mammary carcinoma. As the first such study, metabolomics techniques were used to analyse the serum of cats with these tumours. Three important metabolic pathways of screened differential metabolites closely related to feline mammary carcinomas were analysed to lay a theoretical basis for further study of the pathogenesis of these carcinomas.

Blood in a 5–8 mL volume was sampled from twelve cats of the same breed and similar age (close to nine years on average). Six were feline mammary carcinoma patients and six were healthy. L glutamate, L alanine, succinate, adenine, hypoxanthine, and inosine were screened as were alanine, aspartate, and glutamate metabolism, the tricarboxylid acid (TCA) cycle, and purine metabolism. Data were acquired with LC-MS non-target metabolomics, multiple reaction monitoring target metabolomics, and multivariate statistical and bioinformatic analysis.

Expression of five of the metabolites was upregulated and only inosine expression was downregulated. Up- and downregulation of metabolites related to glycometabolism, potentiation of the TCA cycle, greater content of lipid mobilisation metabolites, and abnormality of amino acid metabolism were closely related to the occurrence of the carcinomas.

These findings provide a new direction for further study of the mechanisms associated with cat mammary neoplasms.

Clinoptilolite has antiviral, antibacterial, anti-inflammatory, antidiabetic, and anticancer properties due to its biological activities. In various cancer cell culture studies, it has been reported effective against tumour cells and gave positive results in treatment of various tumours in dogs. No study was found on the effects of the nanoparticulate form, nanoclinoptilolite, on cancer cells. The aim of this study was to determine its cytotoxic and apoptotic effects in canine osteosarcoma (OSA) cell culture.

Doses at 50% inhibitory concentration were determined by measuring the dose- and duration-dependent cytotoxicity of nanoclinoptilolite on canine D-17 osteosarcoma cells by methylthiazol tetrazolium (MTT) test for 24 h, 48 h, and 72 h. Murine caspase-3 and -7 activity and expression levels of the BAX and BCL2 genes were measured using RT-PCR to investigate the apoptotic effect.

Nanoclinoptilolite decreased cell viability and induced caspase-3- and -7-mediated apoptosis in treated canine OSA cells. Furthermore, its application to canine OSA cells downregulated the expression of BCL2 and upregulated the expression of proapoptotic BAX.

Clinoptilolite, which was previously demonstrated to have anticancer properties, decreased cell viability effectively and rapidly and increased the apoptotic cell ratio in a novel use in nanoparticle form, exhibiting this effect by increasing the BAX/BCL2 ratio.

Sunset Yellow FCF (SY), used frequently in ready-made foods, cosmetics, and the pharmaceutical industry, may cause many health problems. This study is intended to evaluate the morphological and cellular effects of SY on the embryonic chicken immune system throughout incubation.

Babcock white leghorn eggs were randomly divided into four groups. Besides a control group, there were three treatment groups which received a single injection of 200, 1,000, or 2,000 ng of SY into the air sac just before incubation. The eggs were opened on the 10^th, 13^th, 16^th, and 21^st days of incubation. Samples of the bursa of Fabricius, thymus, and spleen were taken from embryos. Serial sections of 5 μm thickness were stained with histological methods and routine histological procedures were performed.

An increase in the spleen volume was determined as the hatching time of the chicks approached. The highest eosinophil ratio was found in the SY_1,000 and SY_2,000 groups (P < 0.05), where the most significant change was developmental retardation in the thymus. In the bursa of Fabricius, there was less lymphocyte accumulation and eosinophilic cell infiltration with increasing doses.

It was concluded that in ovo administered SY has undesired effects on embryonic development of the bursa of Fabricius, spleen, and thymus, and on spleen volume.

Due to the widespread occurrence of acrylamide in the environment, its likely carcinogen status, and the suitability of the pig model as a human analogue, the authors decided to evaluate the impact of high and low doses of this compound on the processes of erythropoiesis in swine bone marrow.

The experiment was carried out on Danish Landrace pigs at the age of eight weeks and body weight about 20 kg. The animals were divided into three equal groups consisting of five pigs in each. Control animals received empty gelatin capsules (placebos). Animals from the first experimental group received a low dose of acrylamide of 0.5 μg/kg b.w./day (> 99% purity; Sigma-Aldrich, Poland), and animals from the second experimental group received a dose 10 times higher. Placebos and acrylamide capsules were administered with feed every morning for 28 days. After anaesthetisation of the animals, bone marrow from the femur was collected into tubes without an anticoagulant on days 0 and 28. After drying and staining, bone marrow smears were subjected to detailed cytological evaluation using a light microscope.

This study showed that high and low doses of acrylamide affected the process of porcine erythropoiesis. The cytotoxic effect of acrylamide on this process was demonstrated in a change of the polychromatic erythroblasts/normochromatic erythroblasts ratio.

Both doses of acrylamide caused a decrease in the number of ortho- and polychromatic erythroblasts.