The effect of Limosilactobacillus fermentum 2i3 and 0.6% addition of humic substances on production parameters and the immune system of broilers.

The widespread use of antibiotics in the poultry industry as growth promoters has led to the emergence of bacterial resistance, which poses a significant health risk to humans and animals. Substances of natural origin, such as probiotic bacteria and humic substances, can be a promising solution. The aim of this experiment was to study the effect of the administration of a probiotic strain of Limosilactobacillus fermentum 2i3 and/or a new formula of humic substances specifically designed for detoxification on the production parameters, including gene expression of myogenic growth factors and selected parameters of the immune response. We found that production parameters such as feed conversion ratio and weekly weight gain, as well as gene expression of mucin-2 and immunoglobulin A, were positively influenced mainly by the administration of L. fermentum 2i3. Similarly, the percentage of active phagocytes and their absorption capacity as well as the proportions of CD8+ and CD4+CD8+ T-lymphocyte subpopulations were significantly increased. The addition of humic substances, either alone or in combination with probiotics, significantly reduced the aforementioned parameters compared to the control. On the other hand, the relative gene expression for all myogenic growth factors was the highest in the humic group alone. Based on the results obtained, we can confirm the immunostimulating effect of L. fermentum 2i3 administered in drinking water, which also had an impact on important production parameters of broiler meat. On the other hand, in the combined group there was no expected potentiation of the positive effects on the observed parameters.

Polystyrene microparticles can affect the health status of freshwater fish - Threat of oral microplastics intake.

Plastic waste pollution is considered one of the biggest problems facing our planet. The production and use of these materials has led to huge amounts of plastic waste entering the aquatic environment and affecting aquatic life. In our experiment, the effect of polystyrene microparticles (PS-MPs; 52.5 ± 11.5 µm) on individual juvenile rainbow trout (Oncorhynchus mykiss) was tested at three different dietary concentrations of 0.5, 2 and 5 % for six weeks. At the end of the experiment, various health parameters of exposed organisms were compared with the control group. The haematological profile revealed an immune response by a decrease in lymphocyte count with a concurrent increase in the number of neutrophil segments at the highest concentration of PS-MPs (5 %). Biochemical analysis showed significant reductions in plasma ammonia in all tested groups, which may be related to liver and gill damage, as determined by histopathological examination and analysis of inflammatory cytokines expression. In addition, liver damage can also cause a significant decrease in the plasma protein ceruloplasmin, which is synthesized in the liver. PS-MPs disrupted the antioxidant balance in the caudal kidney, gill and liver, with significant changes observed only at the highest concentration. In summary, PS-MPs negatively affect the health status of freshwater fish and represent a huge burden on aquatic ecosystems.

Effects of plastic particles on aquatic invertebrates and fish - A review.

This review summarises the current knowledge on the effects of microplastics and their additives on organisms living in the aquatic environment, particularly invertebrates and fish. To date, microplastics have been recognised to affect not only the behaviour of aquatic animals but also their proper development, causing variations in fertility, oxidative stress, inflammations and immunotoxicity, neurotoxicity, and changes in metabolic pathways and gene expression. The ability of microplastics to bind other xenobiotics and cause combined toxicity along side the effect of other agents is also discussed as well. Microplastics are highly recalcitrant materials in both freshwater and marine environments and should be considered extremely toxic to aquatic ecosystems. They are severely problematic from ecological, economic and toxicological standpoints.

Non-steroidal anti-inflammatory drugs caused an outbreak of inflammation and oxidative stress with changes in the gut microbiota in rainbow trout (Oncorhynchus mykiss).

One of the main contributors to pharmaceutical pollution of surface waters are non-steroidal anti-inflammatory drugs (NSAIDs) that contaminate the food chain and affect non-target water species. As there are not many studies focusing on toxic effects of NSAIDs on freshwater fish species and specially effects after dietary exposure, we selected rainbow trout (Oncorhynchus mykiss) as the ideal model to examine the impact of two NSAIDs - diclofenac (DCF) and ibuprofen (IBP). The aim of our study was to test toxicity of environmentally relevant concentrations of these drugs together with exposure doses of 100× higher, including their mixture; and to deepen knowledge about the mechanism of toxicity of these drugs. This study revealed kidneys as the most affected organ with hyalinosis, an increase in oxidative stress markers, and changes in gene expression of heat shock protein 70 to be signs of renal toxicity. Furthermore, hepatotoxicity was confirmed by histopathological analysis (i.e. dystrophy, congestion, and inflammatory cell increase), change in biochemical markers, increase in heat shock protein 70 mRNA, and by oxidative stress analysis. The gills were locally deformed and showed signs of inflammatory processes and necrotic areas. Given the increase in oxidative stress markers and heat shock protein 70 mRNA, severe impairment of oxygen transport may be one of the toxic pathways of NSAIDs. Regarding the microbiota, an overgrowth of Gram-positive species was detected; in particular, significant dysbiosis in the Fusobacteria/Firmicutes ratio was observed. In conclusion, the changes observed after dietary exposure to NSAIDs can influence the organism homeostasis, induce ROS production, potentiate inflammations, and cause gut dysbiosis. Even the environmentally relevant concentration of NSAIDs pose a risk to the aquatic ecosystem as it changed O. mykiss health parameters and we assume that the toxicity of NSAIDs manifests itself at the level of mitochondria and proteins.

Do foodborne polyethylene microparticles affect the health of rainbow trout (Oncorhynchus mykiss)?

Due to the fact that plastic pollution is a global environmental problem of modern age, studies on the impact of these synthetic materials on aquatic, and especially fish organisms, are an important part of the ecosystem and human nutrition. In our study, the toxicity of pristine polyethylene (PE) microparticles (approx. 50 µm) on rainbow trout (Oncorhynchus mykiss) was tested in three different dietary concentrations - 0.5%, 2% and 5%. After six weeks of exposure, various health indices were evaluated. Electron microscopy of the intestine revealed the disintegration of PE particles to <5 µm in size, and thus we concluded that microplastics are able to reach tissues. The haematological profile revealed changes in total red blood cells count and haematocrit (5% PE) which could be associated with spleen congestion observed histologically. The marker of lipid peroxidation was increased in gills suggesting the disruption of balance in antioxidant enzymes capacity and histopathological imaging revealed inflammation in higher PE concentrations. In addition, ammonia was decreased and calcium elevated in biochemical profile, confirming the gill damage. Electron microscopy of the gills showed lesions of lamellae and visible rings around the mucinous cell opening indicating their higher activity. Another injured was the liver tissue, as confirmed by hepatodystrophies and increased expression of pro-inflammatory genes in 2% PE. Impaired innate immunity was confirmed by an increased presence of mucinous cells and a decrease in leukocytes. Kidney damage manifested itself by higher expression of pro-inflammatory cytokines and histopathology. The damage in gills, liver and kidney together correlated with the increased antioxidant capacity of plasma. In conclusion, PE microparticles are able to affect health indices of O. mykiss. The potential problem for aquatic ecosystems and even human consumption should be considered.

Platelet-rich plasma, platelet-rich fibrin, and enamel matrix derivative for oral mucosal wound healing.

Different approaches to enhance healing of hard or soft tissues include the use of cytokines and growth factors to modify cellular behaviour. Numerous growth factors are found in autologous blood concentrates - platelet-rich plasma (PRP) and platelet-rich fibrin (PRF). Enamel matrix derivative (EMD) may improve tissue healing via amelogenins. Bilayered collagen matrix (CM) is used for soft tissue augmentation. The aim of the present study was to assess potential benefits of PRP, PRF and EMD in combination with bilayered collagen matrix or CM alone in treatment of oral mucosal defects in rabbits. Twenty-seven New Zealand white rabbits were included in this randomized controlled trial. Artificial oral mucosal defects were treated with one of these five approaches: PRP+CM, PRF+CM, EMD+CM, CM alone, or left untreated as a negative control - CO. The animals were euthanized 1 day, 7 days, or 28 days after surgery and necropsies were harvested. Histological and molecular biological analyses were performed. All defects were healed by day 28. No differences between PRP+CM, PRF+CM, CM alone and CO groups were recorded at any time point. Slower angiogenesis and a higher presence of inflammatory infiltrate were observed in the EMD+CM group 28 days after surgery. Molecular biological analyses did not reveal any statistically significant changes. In conclusion, no improvement in mucosal healing of wounds covered with a collagen membrane and PRP, PRF, or EMD was observed, compared with CM alone or untreated controls.

Could the musk compound tonalide affect physiological functions and act as an endocrine disruptor in rainbow trout?

In the present study, the effect of polycyclic musk compound tonalide (AHTN) in two concentrations was studied in male rainbow trout (Oncorhynchus mykiss, Walbaum 1792). A feeding trial was conducted with AHTN incorporated into feed granules. One concentration was environmentally relevant (854 µg/kg); the second one was 10× higher (8699 µg/kg). The fish were fed twice a day with the amount of feed at 1 % of their body weight. After an acclimatization period, the experimental phase in duration of six weeks followed. At the end of the experiment, fish were sampled and the biometrical data were recorded. Subsequently, hematological and biochemical tests, histopathological examination, analysis of oxidative stress markers and evaluation of endocrine disruption using plasma vitellogenin were performed. In conclusion, an increase of hematocrit for both AHTN concentrations was found, but no significant changes were observed in biochemical profile. Moreover, AHTN caused lipid peroxidation in caudal kidney tissue, which was confirmed by histopathological images. The long-lasting AHTN exposure could thus be harmful for maintaining homeostasis in the rainbow trout organism. However, the vitellogenin concentration seemed not to be affected by AHTN.

High resolution mass spectrometry analysis of the sertraline residues contained in the tissues of rainbow trout reared in model experimental conditions.

The growing consumption of pharmaceuticals in the human population and the insufficient efficiency of their elimination in waste water has a long-term negative impact on the environment of aquatic ecosystems, including the organisms that inhabit them. A significant contributor is the consumption of anti-depressants from the SSRI group, which corresponds to their increasing concentration in the environment. The aim of this work was to determine if antidepressant sertraline is able to be stored in fish organisms and to evaluate the content of residues in various body tissues. Rainbow trout (Oncorhynchuss mykkis) was selected as the test organism and was artificially exposed to the antidepressant for 1 month (concentrations 0; 4.2; 44 and 400 ng.g-1 sertraline in the feed). Liver, kidney, brain and muscle tissue biopsies samples were taken for analysis. Detection was performed using an Accela 1250 LC pump and an Accela autosampler coupled with a high-performance mass analyzer with a heated electrospray ionization source Q-Exactive Orbitrap, operating in positive ionization mode and in PRM mode (m/z 306.08108->275.03888 and 309.009991->275.03888 for sertraline and internal standard, respectively). The limit of quantification of the method was 0.1 ng.g-1 of sertraline and the calibration curve showed a good linearity up to 20 ng.g-1. From the collected data, amount of residues was found in the liver, kidney and brain. In contrast, the incidence of residues in muscle tissue was not detected in all groups, which is favorable from the point of view of fish meat consumption, by humans.

Detection of IgM-rheumatoid factor and anti-citrullinated protein antibodies in healthy horses and their comparison.

Rheumatoid factor (RF) is used in human and veterinary medicine in the form of IgM RF traditionally to support the diagnosis of rheumatoid arthritis (RA). In the latest diagnostic criteria, presence of anti - citrullinated protein antibodies (ACPA) was added to the grading system for the diagnosis of RA in humans. A change which is not integrated or routinely used in veterinary medicine. The criteria changed partly because of RF's diagnostic shortcomings, which include its increased titer detection in humans with non-rheumatoid diseases, inability to predict the disease and increased titers over the limit in the older population. Clinical signs similar to human RA were reported in horses in a condition known as idiopathic polysynovitis. Similarities in the clinical presentation to RA raised a question to the usability of RF and ACPA in horses. In our study, sixty clinically healthy horses, ranging from 3 days to 30 years of age, were evaluated for their serum levels of IgM RF. 55 of these horses were tested for ACPA, using methods of ELISA measuring Anti - CFG (Anti citrullinated fibrinogen antibody). The results of the study demonstrated the existence of an age-dependent increase in the level of IgM RF up to the age of about 9 years and ACPA's independence of the horse's age as well as both markers independence of the horse sex.

Effect of dietary fish oil on selected inflammatory markers in pigs.

The present study tested a hypothesis that dietary fish oil (eicosapentaenoic acid+docosahexaenoic acid) in a commonly achievable dose ameliorates a systemic inflammation in pigs. Two groups of pigs of 16 animals each were fed a diet with either 2.5% of fish oil (F) or a control diet with 2.5% of palm oil (P). After 70 days of fattening, eight F and eight P pigs were challenged (F+; P+) i.v. by lipopolysaccharide. After 3 h, all pigs were sacrificed and blood, liver and visceral adipose tissue (VAT) samples were taken. No significant effect (P>0.05) of dietary oil on the feed intake and daily weight gain was found out. Less neutrophils (16.8% v. 28.8%; P0.05) between F+ and P+ pigs in the peroxisome proliferator-activated receptor γ, GPR120, Adipor1 and Adipor2 (adiponectin receptor) gene expression, respectively, was established; plasma adiponectin was the same (21.1 ng/ml) in F+ and P+ pigs. In comparison with the P+ pigs, increased expression of the lipopolysaccharide-binding protein (LBP) gene and intercellular adhesion molecule 1 (ICAM1) gene was found out in the liver of the F+ pigs; expression of the tumor necrosis factor α (TNFα) gene was higher in the liver but lower in the VAT of the F+ pigs (P<0.05). The F+ pigs had higher (P<0.05) plasma concentration of both anti-inflammatory cytokine interleukin-4 (0.46 v. 0.04 ng/ml) and pro-inflammatory TNF-α (13.41 v. 7.72 ng/ml). It was concluded that dietary fish oil at the tested amount had a negligible effect on expression of the evaluated receptor genes and plasma adiponectin, and had an ambiguous effect on expression of cytokine genes and plasma cytokine levels.

Effect of dietary fish oil on fatty acid deposition and expression of cholesterol homeostasis controlling genes in the liver and plasma lipid profile: comparison of two animal models.

The objective of the present study was to compare hepatic fatty acid deposition, plasma lipid level and expression of cholesterol homeostasis controlling genes in the liver of rats (Wistar Albino; n = 32) and pigs (Large White × Landrace; n = 32) randomly assigned into two groups of 16 animals each and fed 10 weeks the diet with either 2.5% of fish oil (F; source of eicosapentaenoic and docosahexaenoic acid, EPA+DHA) or 2.5% of palm oil (P; high content of saturated fatty acids; control). F-rats deposited in the liver three times less EPA, but 1.3 times more DHA than F-pigs (p < 0.05). Dietary fish oil relative to palm oil increased PPARα and SREBP-2 gene expression much strongly (p < 0.01) in the pig liver in comparison with the rat liver, but expression of Insig-1 and Hmgcr genes in the liver of the F-pigs relative to the expression of these genes in the liver of the P-pigs was substantially lower (p < 0.01 and p < 0.05 respectively) as compared to rats. When plasma lipid concentration in the F-animals was expressed as a ratio of the plasma concentration in the P-counterparts, dietary fish oil decreased HDL cholesterol less (p < 0.01), but LDL cholesterol and triacylglycerols more (p < 0.05 and p < 0.001 respectively) in rats than in pigs: more favourable effect of fish oil on rat plasma lipids in comparison with pigs can therefore be concluded. Concentration of total cholesterol and both its fractions in the rat plasma was negatively correlated (p < 0.01) with hepatic DHA, but also with unsaturated myristic and palmitic acid respectively. It has been concluded that regarding the similarity of the plasma lipid levels to humans, porcine model can be considered superior; however, using this model, dietary fish oil at the tested amount (2.5%) was not able to improve plasma lipid markers in comparison with saturated palm oil.

The effects of live yeast Saccharomyces cerevisiae on postweaning diarrhea, immune response, and growth performance in weaned piglets.

The effects of live yeast Saccharomyces cerevisiae (strain CNCM I-4407, 10(10) cfu/g; Actisaf; Lesaffre Feed Additives, Marcq-en-Baroeul, France) on the severity of diarrhea, immune response, and growth performance in weaned piglets orally challenged with enterotoxigenic Escherichia coli (ETEC) strain O149:K88 were investigated. Live yeast was fed to sows and their piglets in the late gestation, suckling, and postweaning periods. Sows were fed a basal diet without (Control; n = 2) or with (Supplemented; n = 2) 1 g/kg of live yeast from d 94 of gestation and during lactation until weaning of the piglets (d 28). Suckling piglets of the supplemented sows were orally treated with 1 g of live yeast in porridge carrier 3 times a week until weaning. Weaned piglets were fed a basal starter diet without (Control; n = 19) or with (Supplemented; n = 15) 5 g of live yeast/kg feed for 2 wk. Significantly lower daily diarrhea scores (P < 0.05), duration of diarrhea (P < 0.01), and shedding of pathogenic ETEC bacteria (P < 0.05) in feces was detected in the supplemented piglets. Administration of live yeast significantly increased (P < 0.05) IgA levels in the serum of piglets. Evidence indicates that decreased infection-related stress and severity of diarrhea in yeast-fed weaned piglets positively affected their growth capacity in the postweaning period (P < 0.05). The results suggest that dietary supplementation with live yeast S. cerevisiae to sows and piglets in the late gestation, suckling, and postweaning periods can be useful in the reduction of the duration and severity of postweaning diarrhea caused by ETEC.

γδ T lymphocytes are recruited into the inflamed uterus of bitches suffering from pyometra.

Very little is known about the occurrence of immune system cells in the canine uterus. The aim of this study was to generate information about lymphocyte subsets that are present in the healthy canine uterus and that are recruited under inflammatory conditions caused by pyometra. Using immunohistochemistry and flow cytometry, a significant influx of γδ T lymphocytes was found in pyometra samples mainly due to recruitment of γδ(+)/CD8(-) T lymphocytes. The relative expression of genes encoding selected cytokines/chemokines was evaluated in samples from healthy and pyometra-affected uteri. Expression of pro-inflammatory cytokines (including IL-1ß, TNF-α, IL-8, IL-17 and IFN-γ) and chemokines (including CXCL10, CCL4 and CCL5) was upregulated in pyometra samples confirming the presence of inflammation. In contrast, the expression of the homeostatic chemokine CCL25 and of the anti-inflammatory cytokine IL-10 was downregulated and unchanged, respectively.

Characterization of CD34⁺ thymocytes in newborn dogs.

Using two-color flow cytometry, we characterized CD34(+) cells in the newborn canine thymus. CD34(+) thymic cells comprised approximately 5% of cells recovered by thymus tissue teasing and both large and small thymocytes have been present in this population, the former being 7-12 times more frequent. All CD34(+) cells expressed the pan-leukocyte antigen CD45. The expression of CD44 profile on the large and small CD34(+) thymocytes differed: almost all large CD34(+) cells were CD44(+), while only 75% of small CD34(+) thymocytes co-expressed the CD44 antigen. We have previously described that CD172α is present on the surface of CD34(+) bone marrow cells in dogs. In the thymus, CD172α was expressed on 5-10% and less than 5% of large and small CD34(+) cells, respectively. Some CD34(+) thymocytes also co-expressed T-lineage-specific markers like CD3, CD4, CD8, TCR1 and TCR2. Their expression increased during the large-to-small thymocyte transition. Based on our findings we suggest that thymocyte progenitors enter their primary differentiation center as large CD34(+), CD44(+), CD45(+) and CD172α(+) cells. T-cell specific markers appear on their surface at early stages of differentiation. As the size of progenitors decreases with terminal primary differentiation, the CD34, CD44, and CD172α surface markers are down-regulated.

Salmonella enterica serovar Typhimurium and Enteritidis infection of pigs and cytokine signalling in palatine tonsils.

Pigs are considered as one of the major sources of zoonotic strains of Salmonella enterica for humans. Out of many S. enterica serovars, S. Typhimurium dominates in pigs, however, in several countries in Central Europe, S. Enteritidis is also quite frequent in pig herds. In this study we therefore compared the colonisation of pigs with S. Typhimurium and S. Enteritidis. We found that 3 weeks after infection S. Enteritidis 147 colonised the intestinal tract in higher quantities but was shed in faeces in lower quantities than S. Typhimurium 17C10. In a second experiment we found out that S. Enteritidis 147 and its SPI-1 and SPI-4 mutants increased proinflammatory cytokine (IL-1ß and IL-8) signalling in the ileum 5 days post infection. On the other hand, independent of SPI-1 or SPI-4, S. Enteritidis 147 suppressed expression of IL-18, MCP1, TLR2, CD86, IL-7, IL-10 and IL-15 in the palatine tonsils. The suppression of cytokine signalling may facilitate the initial colonisation of the palatine tonsils by Salmonella. Moreover, immune suppression may also influence pig resistance to opportunistic pathogens and Salmonella infection in pigs thus may become an issue not only in terms of pork contamination but also in terms of affecting the immunological status of pig herds.

Passive immunisation of post-weaned piglets using hyperimmune serum against experimental Haemophilus parasuis infection.

The protective role of hyperimmune serum in the prevention of Haemophilus parasuis infections in post-weaned piglets was assessed by experimental challenge. The hyperimmune serum was obtained from a pig vaccinated with a commercial vaccine against Glässer's disease. Thirty-eight weaned piglets were divided into four groups: three groups were immunised intramuscularly with 10 ml of hyperimmune serum and one group consisted of unimmunised control animals. All piglets were subsequently infected intraperitoneally with H. parasuis serotype 5 at different times after immunisation. The use of hyperimmune serum provided the piglets with partial protection against experimental infection. The levels of protection indirectly depend on time between serum inoculation and challenge infection. The best protection of piglets against experimental infection was obtained in the group immunised 1 week before inoculation; the same group in which the highest levels of antibodies were detected at the time of challenge.

Experimental oral and ocular Encephalitozoon cuniculi infection in rabbits.

Encephalitozoon cuniculi is an obligate intracellular pathogen that has a wide host distribution, but primarily affects rabbits. The aim of this study was to characterize both the cell-mediated and the antibody response in rabbits after experimental infection using 2 different infection routes: oral and ocular. SPF rabbits were infected with low (10³ spores) and high (107 spores) infection doses. Monitored parameters included clinical signs, detection of spores in urine, antibody response detected with ELISA, and cell-mediated immunity detected by antigen-driven lymphocyte proliferation. At week 13 post-infection, half of the rabbits in each group were suppressed by intramuscular administration of dexamethasone. At week 18 post-infection, animals were euthanized. Clinical signs were mild with exacerbation after immunosuppression. Spores in urine and antigen-specific cell-mediated immunity were detected from weeks 5 and 4 post-infection, respectively. Specific IgM was detected 1 week after infection, and IgG antibodies followed 1 week later in rabbits infected with the high dose. Immunological responses were dose dependent. The authors can conclude that both oral and ocular experimental infection with E. cuniculi resulted in an immune response of the infected animals. Rabbits could be used as an experimental model for the study of ocular microsporidiosis.

Lactoferrin in canine sera: a pyometra study.

The concentration of lactoferrin was measured in canine sera from groups of healthy male dogs as well as pregnant and non-pregnant female dogs and was compared with that of bitches with pyometra. Lactoferrin concentrations were higher in bitches with pyometra. The role of elevated lactoferrin concentrations in the suppression of lymphocyte activity was examined in sera from bitches with pyometra in a series of investigations. Although the sera from bitches with pyometra were capable of suppressing lymphocyte activity, lactoferrin was not found to be involved in this action.

Age-related changes in selected haematology parameters in rabbits.

Even though there is an abundance of information on the reference values of haematological parameters in adult rabbits, a little is known about the changes in haematology in newborn rabbits or during their postnatal development. Therefore, the aim of our study was to investigate changes in red blood cells (RBC), white blood cells (WBC) and differential leukocyte counts in SPF New Zealand White rabbits from the age of one day to 20 weeks. Significant age-related changes during the first four weeks of life were detected. These included an increase of RBC and WBC, reversal of the neutrophil/lymphocyte ratio and increase of total counts of eosinophils and basophils. From the age of six weeks of life, all of the studied haematological parameters were comparable to those of adult rabbits.

Comparison of different doses of antigen for intradermal administration in pigs: the Actinobacillus pleuropneumoniae model.

The intensity of antibody responses and level of protection against challenge infection induced by Actinobacillus pleuropneumoniae (APP) were compared in piglets vaccinated intramuscularly with different vaccination doses of a subunit vaccine. Secondary antibody responses to APP exotoxins induced by the intradermally administered diluted vaccines did not differ from those induced by undiluted vaccines administered either intradermally or intramuscularly. The level of protection measured by the clinical course of challenge infection to the extent of lung lesions was significantly higher in animals vaccinated with a three-time diluted vaccine in comparison with animals that were administered intramuscularly or intradermally with the most concentrated or the most diluted vaccines.