African swine fever (ASF) is a pressing economic problem in a number of Eastern European countries. It has also depleted the Chinese sow population by 50%. Managing the disease relies on culling infected pigs or hunting wild boars as sanitary zone creation. The constraints on the development of an efficient vaccine are mainly the virus’ mechanisms of host immune response evasion. The study aimed to adapt a field ASFV strain to established cell lines and to construct recombinant African swine fever virus (ASFV) strain.
Material and Methods
The host immune response modulation genes A238L, EP402R, and 9GL were deleted using the clustered regularly interspaced short palindromic repeats/caspase 9 (CRISPR/Cas9) mutagenesis system. A representative virus isolate (Pol18/28298/Out111) from Poland was isolated in porcine primary pulmonary alveolar macrophage (PPAM) cells. Adaptation of the virus to a few established cell lines was attempted. The plasmids encoding CRISPR/Cas9 genes along with gRNA complementary to the target sequences were designed, synthesised, and transfected into ASFV-infected PPAM cells.
The reconstituted virus showed similar kinetics of replication in comparison to the parent virus isolate.
Taking into account the usefulness of the developed CRISPR/Cas9 system it has been shown that modification of the A238L, EP402R, and 9GL genes might occur with low frequency, resulting in difficulties in separation of various virus populations.
The experiments reported in this research paper were aimed at assessing the genetic responses of a Holstein cow population, as a response to the variations in environmental temperature, through the analysis of the effects resulting from the genotype by environment interaction (GEI), based on reaction norms. Therefore, milk production data was collected from the database of the Paraná Holstein Breeders Association in Brazil for 67,360 primiparous cows born between 1990 and 2015, with the purpose of evaluating the temperature effect, considered as an environmental variable, distinguished under six gradients (17 ºC to 19.5 ºC) over the region. A random regression model was adopted, utilizing the fourth order under the Legendre polynomials, applying the mixed models of analysis by the REML method, and using the WOMBAT software. Additionally, the breeding value of the 15 most representative bulls was assessed, in response to the changes in the temperature gradient. The total milk production on average was estimated at 8,412.83 ± 2,012.08 kg. The heritabilities estimates were found in the low to moderate range, from 0.18 to 0.23, displaying a decline with a rise in the temperature, highlighting the influence it exerted on the heritabilities. Variations in the genetic expression of some bulls were noted to show differences of up to 289 kg of milk in response to the increase in the temperature from 17 ºC to 19.5 ºC. However, all the genetic correlations between the gradients for milk yield were above 0.80, in the range of 0.873 to 0.998, revealing no remarkable interaction between the genotype and environment. This result indicates that the application of the temperature variable in the models of genetic analysis in southern Brazil is not required.
In the Balkan region, the state of sheep welfare is little known. In this context, the assessment of sheep welfare is important for the identification of health and behavioral problems that affect these animals, along with the deficiencies present in the environment. The study was conducted during the winter season on a farm of 220 autochthonous breed Vlašićka Zackel sheep reared in extensive systems on the mountain regions of Bosnia and Herzegovina. A total of 65 ewes, aged 2–4 years, were observed. The welfare assessment was performed according to the AWIN protocol for sheep. The authors found the following issues: dry ewes were significantly fat (p<0.001) in regard to pregnant and lactation ewes; 42.11% of pregnant ewes were thin while 31.25% of dry ewes were fat; most of the assessed animals (78.46%) had dry or damp, slightly soiled fleece; 32.31% of animals had lesions on face/muzzle while ocular discharge, respiratory problems, mild mastitis and minor lameness were present in 16.92%, 4.62%, 15.38% and 7.69% sheep, respectively. All animals showed a negative relationship with humans. Using this methodology, the welfare problems in animals may be detected, and thus certain segments in the production cycle may be improved. The on-farm welfare assessment is highly recommended for emphasizing the key points in improving sheep welfare in Balkan countries.
This study was aimed at the investigation into the influence of polyphenols on fermented sausages produced with and without nitrite addition, during storage which lasted for 280 days. Three types of sausages were produced and formed the three experimental groups: C – the control – sausages of usual composition containing nitrites; N+P - sausages with nitrites and polyphenols; and P - nitrite-free sausages with added polyphenols. The proximate chemical composition of all groups was in the range with that of dry fermented sausages. P sausages contained 0.3 mg nitrites per kg, while C and N+P contained 54.8 mg/kg and 52.2 mg/kg, respectively. Polyphenol-enriched sausages had significantly lower peroxide and TBARS values than C sausages. In all sausages lactic acid bacteria counts reached 8.9-9.9 log cfu/g, but decreased during storage to 4.3-4.8 log cfu/g at the end of the storage period. Micrococcaceae counts remained stable: 3.5-3.9 log cfu/g. In P and N+P sausages a significantly lower number of Pseudomonadaceae was observed than in the control. The lightness of C and P sausages was similar (L=50.2 and L=49.5, respectively), while N+P sausages were darker (L=42.5). C and N+P sausages had similar redness (a*=14.5 and a*=13.2, respectively) and yellowness (b*=5.9 and b*=6.4, respectively), but the values which correspond to redness and yellowness were lower in P sausages (a*=8.0 and b*=4.6). Sensory characteristics of all products were found to be very similar. The flavour of polyphenol-enriched sausages was considered to be better. The most dominant polyphenol in sausages was kaempferol-3-O-glucoside followed by quercetin, luteolin-7-O-glucoside, catechin and syringic acid. Nitrite-free polyphenol-enriched sausages reached the same shelf life as conventional sausages containing nitrites did, which is a promising result implying that polyphenols might be used as natural preservatives and nitrite substitutes. Simultaneous use of nitrite and polyphenols is questionable due to their interactions which should be further studied.
The aim of this study was to determine the effect of Virginia fanpetals (Sida hermaphrodita) silage on carcass and beef quality characteristics. Forty Polish Holstein-Friesian bulls aged 16 months were assigned to 4 dietary treatments (n=10) and were fed different types of silage during a 7-month fattening period. The proportion (g/kg dry matter) of silage in the diets was as follows: (1) grass silage (GS) (600); (2) Sida silage (SS) (600); (3) SS (300) and GS (300); and (4) SS (300) and maize silage (MS) (300). Silage was supplemented with concentrate at 400 g/kg DM in each diet. The animals were slaughtered at the end of the fattening period. Silage type had no significant effect on BWG or feed to gain ratio. The carcasses of bulls fed Sida silage and maize silage received higher scores for conformation than the carcasses of bulls fed grass silage (P<0.05). The meat of bulls fed Sida silage had the lowest value of Warner-Bratzler shear force. Meat from SS+MS group bulls had the highest intramuscular fat (IMF) content and was lightest in color, whereas meat from bulls fed Sida silage and grass silage received the highest scores for color uniformity, aroma, taste and overall acceptability.
Recent studies have demonstrated a significant role of mitochondrial DNA (mtDNA) defects in the pathogenesis of many human and some canine tumours. The aim of this study was to identify mutations in the ND2 and ND4 mitochondrial genes in canine mast cell tumours and determine their association with the process of neoplastic transformation and the phenotypic traits of dogs. In total, 136 gene sequences from 68 biological samples, including blood and neoplastic tissue samples from 34 dogs with diagnosed MCTs, were analysed. The study consisted in DNA sequencing of the ND2 and ND4 genes as well as bioinformatics and statistical analyses. For the first time, mutations in NADH-dehydrogenase genes were detected in dogs with MCTs. In total, 22 polymorphic loci and 19 mutations in the ND2 and ND4 genes were identified. The majority of the identified mutations were homoplasmic, and tumour heteroplasmy was detected in eight nucleotide positions in three dogs. Seven of the ND2 mutations and two of the ND4 mutations caused an amino acid change. The changes in non-synonymous protein-coding SNPs did not exert an adverse effect on proteins. A statistically significant correlation of the presence of mutations/polymorphisms with the sex, age, and size of the dogs and the tumour location was demonstrated. Polymorphisms and mutations in NADH-dehydrogenase genes, including mastocyte-specific changes, in canine mast cell tumours that had not been reported earlier in the literature were identified. Some of these changes may imply that these are the hotspot mutations in canine mast cell tumours. It cannot be excluded that the molecular changes are directly associated with the development of mast cell tumours, and further investigations are needed to verify whether they can become molecular markers of MCTs in the future.
The aim of the study was to determine the effect of the method of fragmentation of hybrid rye and maize grain on digestibility parameters for ruminants. Varying degrees of fragmentation – none (whole grains; WG), crushed (CG) or ground to pass through a 4.0 (GG4.0) or 1.5 mm (GG1.5) screen – were estimated by the in sacco nylon bag technique, in vitro true digestibility (IVTD), and the in vitro gas production (GP) technique. WG, CG, GG4.0 and GG1.5 were categorized as none, slight, moderate and extensive fragmentation of the grain kernel, respectively. Three non-lactating cows fitted with ruminal and duodenal cannulas were used to determine the effective rumen degradability (ERD) and intestinal and total tract digestibility (ID and TTD, respectively) of dry matter (DM), crude protein (CP) and starch. IVTD was determined with an ANKOM DaisyII Incubator, and GP and in vitro organic matter digestibility were determined with an ANKOMRF Gas Production System. Dry matter, CP and starch of hybrid rye grain were rapidly degraded in the rumen, and this degradability as well as ID and TTD was only marginally affected by the method of kernel fragmentation; however, among the methods used, the ERD of GG4.0 rye was the lowest. On the other hand, the greater the degree of kernel fragmentation, the higher ERD, ID and TTD were obtained for the DM, CP and starch of maize grain. In summary, rye grain is more susceptible to fermentation in the rumen than maize grain, but the means of grain processing may alter the rate, extent and site of their digestion, particularly for maize grain.
Bacillus cereus, usually ingested by food, can cause two types of disease due to the presence of toxins: vomiting and diarrhea syndrome. Systemic infections can also occur. The aim was to detect genes for enterotoxins (hblA, entFM) and emetic toxin (cer) and to investigate the genetic heterogeneity of B. cereus isolates from food, environment and human stool. Identification of B. cereus was performed by means of selective medium, classical biochemical test and polymerase chain reaction (PCR). Toxin genes were detected by PCR. Typing was performed by random amplified polymorphic DNA (RAPD). EntFM gene was present in all stool and food samples and in 28/30 environmental isolates. HblA gene was present in 29/30 stool, 23/30 food and 24/30 environmental isolates. Cer gene was present in 30/30 stool, 28/30 food and 25/30 environmental isolates. The RAPD results show high heterogeneity among the isolates from each group. In the cumulative dendrogram, representative isolates from all three groups formed two clusters with a difference of 53%. The detection of toxin genes in all B. cereus isolates indicated these bacteria as potentially pathogenic and a serious threat for human health. The presence of isolates from all three groups in the same cluster suggests the existence of similar strains in the environment, food and patients, which is in line with the circulation of strains in nature through the food chain.
Vector-borne diseases are one of the main causes of morbidity and mortality in small animals in Europe. Many of these diseases are well-known among veterinary practitioners and some of them are called emerging diseases as prevalence, temporal and spatial distribution seem to increase in Europe. The number of newly recognized pathogens, transmitted by a variety of arthropod vectors, that are relevant for dogs and cats, is also increasing every year. The prevalence among infected vectors and hosts is a hot topic in veterinary science throughout the entire continent, as well as the development of efficient diagnostic procedures, therapy and prophylactic measures. Companion animal vector-borne diseases comprise a large group of pathogens including viruses, bacteria, protozoa and helminths. These pathogens are mainly transmitted by bloodsucking arthropods (ticks, fleas, mosquitos, sand flies), and more seldom by direct transmission between vertebrate hosts. Vector prevalence and activity is influenced by local climate conditions, host species density, changes in landscape and land use. Human parameters such as poverty and migration affect the use of prophylactic measures against pathogen transmission and infection as well as increasing the zoonotic risk to introducing pathogens by infected humans. Small animal associated factors such as pet trade and pet travel spread infection and certain vectors such as ticks and fleas. All these factors pose several complex and significant challenges for veterinarians in clinical practice to decide on efficient laboratory work-up and constructive diagnostic procedures.
Female fertility is an important trait that contributes to cow’s profitability and it can be improved by genomic information. The objective of this study was to detect genomic regions and variants affecting fertility traits in Iranian Holstein cattle. A data set comprised of female fertility records and 3,452,730 pedigree information from Iranian Holstein cattle were used to predict the breeding values, which were then employed to estimate the de-regressed proofs (DRP) of genotyped animals. A total of 878 animals with DRP records and 54k SNP markers were utilized in the genome-wide association study (GWAS). The GWAS was performed using a linear regression model with SNP genotype as a linear covariate. The results showed that an SNP on BTA19, ARS-BFGL-NGS-33473, was the most significant SNP associated with days from calving to first service. In total,  significant SNPs were located within 27 candidate genes. Novel potential candidate genes include OSTN, DPP6, EphA5, CADPS2, Rfc1, ADGRB3, Myo3a, C10H14orf93, KIAA1217, RBPJL, SLC18A2, GARNL3, NCALD, ASPH, ASIC2, OR3A1, CHRNB4, CACNA2D2, DLGAP1, GRIN2A and ME3. These genes are involved in different pathways relevant to female fertility and other characteristics in mammals. Gene set enrichment analysis showed that thirteen GO terms had significant overrepresentation of genes statistically associated with female fertility traits. The results of network analysis identified CCNB1 gene as a hub gene in the progesterone-mediated oocyte maturation pathway, significantly associated with age at first calving. The candidate genes identified in this study can be utilized in genomic tests to improve reproductive performance in Holstein cattle.