Microencapsulated phytogenic in dog feed modulates immune responses, oxidative status and reduces bacterial (Salmonella and Escherichia coli) counts in feces.

Improving the health and immunity of pets is a concern of the guardians, while maintaining the health of the animals directly impacts the owners' health and public health. The objective of this study was to produce a dog feed containing a microencapsulated phytogenic blend, as well as determine the impact of the additive on animal health and its intestinal microbiota. The composition included microencapsulated thymol, carvacrol, and cinnamaldehyde, at 300 mg/kg of feed. Ten male beagle dogs were divided into two groups, identified as follows: the control group (C; ingested the feed without the additive) and the treated group (T; consumed feed containing the phytogenic blend). The dogs received 300 g of feed/day divided into two meals. Greater neutrophil counts in group T and lymphocyte counts were observed at the end of the experiment, as well as levels of α2-globulins and γ-globulin, while ß1 and ß2-globulins levels were lower in group T. Serum nitrogen oxide levels were higher in group T. Levels of reactive oxygen species were lower in group T at days 30 and 45, unlike activity of glutathione peroxidase that was higher. We found less bacterial contamination in the feces of dogs in group T, i.e., total bacterial count, total coliform counts, and counts of Salmonella and Escherichia coli were lower on days 30 and 45. Phytogenic blend intake reduces bacterial counts in stool and improves antioxidant/oxidative status and immune responses.

Leptospira spp. in horses in southern Brazil: Seroprevalence, infection risk factors, and influence on reproduction.

Leptospirosis in horses is often associated with reproductive disorders. In the southern states of Brazil, horses are used for various jobs and cultural practices; nevertheless, serological surveillance for Leptospira is rare. Therefore, the objective of this study was to determine the seroprevalence of Leptospira spp. in horses in southern Brazil, as well as to identify the risk factors for infection and its impacts on reproduction. We performed microscopic agglutination tests for 12 serovars that corresponding 9 serogroup (Sejroe, Icterohaemorrhagiae, Australis, Pyrogenes, Pomona, Canicola, Grippotyphosa, Tarassovi and Ballum) in 595 samples from 60 herds. A brief history was obtained to analyze risk factors for reproductive disorders. A total of 45.9% of the tested horses were seropositive, of which the most frequent serogroups were Icterohaemorrhagiae (Icterohaemorrhagiae and Copenhageni serovars) and Ballum (Ballum serovar). Simple infections were found in 45.4% of seropositive animals, while mixed infections occurred in 54.6% of horses. There was a correlation between seropositivity and age and sex, that is, seropositivity was more frequent in animals over 6 years old and in females. There was no correlation between seropositivity and reproductive disorders. We conclude that there is a high seroprevalence of Leptospira spp. in southern Brazil with predominance of Icterohaemorrhagiae serogroup, mainly in older animals. Location, breeds, contact with dogs or other domestic animals are not risk factors, whereas gender is a risk factor. Reproductive disorders are not due to leptospirosis in the study region.

Daily consumption of a homeopathic product decreases intestinal damage and stool bacterial counts in mice challenged with Escherichia coli.

Escherichia coli is a bacterium normally found in the gastrointestinal tract of domestic animals that can usually control the infection. Nevertheless, some factors (high exposure, stress conditions, animal category, among others) can favor the exacerbation of E. coli infection and cause of disease. Because it is a zoonotic bacterium, it is important to control the infection, avoiding contamination of home interiors in the case of pets. There are various forms of treatment for E. coli; nevertheless, there are few options for prevention. In the present study, we evaluated homeopathy. Thus, the objective of this study was to determine whether administration of a prophylactic homeopathic in water would minimize the negative effects of E. coli infection, as well as reducing bacterial counts in the feces of a experimental model. Forty mice were divided into four experimental groups (n = 10/group). Groups NC (negative control) and PC (positive control) were not treated; in group T1, the animals received 0.002 mL/day/animal of the homeopathic in water, and animals in group T2 0.004 mL/day/animal. The experiment lasted 54 days, and on the 31st day, mice of T1, T2 and PC groups were infected orally a 0.2 mL inoculum of 1.5 × 108 CFU of E. coli. Euthanasia and sample collection were performed on the 40th and 54th days of the experiment (n = 5/group/time point). Blood, liver, spleen, intestine, and feces samples were collected from the final portion of the intestine. There was no significant difference in animal weight between groups at the end of the experiment. Neutrophil count was lower in PC group animals on day 40, while on day 54, the counts were lower in T2 and PC. Lymphocyte counts were lower only in the PC group than in the NC group on day 54. Globulins were lower in the NC and PC groups than in T1 and T2 on day 40, remaining lower the PC group and higher in T1 on day 54; levels of immunoglobulin IgG and IgM were higher in groups T1 and T2, which differed from PC and NC. TNF-α levels were higher in the T1 and T2 groups at 40 and 54 days. INF-γ levels were higher in T1, T2, and PC compared to NC on day 40, remaining higher than NC in groups T1 and T2 on day 54. Total bacterial count, total coliforms and E. coli counts were lower in group T1 and higher in NC and PC on days 40 and 54, when they were lower for T1 and T2. Histologically, no lesions were observed in extra-intestinal tissues; however the height of intestinal crypts in the PC group was smaller than the others on day 40. On day 54, villi and crypts of all infected groups were larger in T1 and T2 than in NC; sizes in the PC group were higher than those of all other groups. These data suggest that the homeopathic agent in the drinking water improved health of the mice.

Changes in cardiac and hepatic energetic metabolism in gerbils infected by Listeria monocytogenes.

Energy metabolism is a sensitive indicator of cellular disorders. Therefore, the objective of this study was to investigate changes in cardiac and hepatic energy metabolism during listeriosis using an experimental model. We divided gerbils into two groups: Control (n = 11) and orally Infected (n = 12) with 5 × 109 CFU/mL of Listeria monocytogenes. Euthanasia and sampling were performed on days 6 and 12 post-infection (PI). Histopathological lesions were not found in the heart; however, the liver showed pyogranuloma. In the hearts of infected animals, cytosolic creatine kinase activity was lower on day 6 and 12 PI; mitochondrial creatine kinase/pyruvate kinase (PK), and sodium potassium pump (Na+/K+-ATPase) activities were lower on day 12 PI. Hepatic PK and Na+/K+-ATPase activities were lower in the infected group on day 12 PI. Lipoperoxidation was higher in the livers and hearts of infected animals on day 12 PI, and antioxidant capacity against peroxyl radicals (ACAP) was also higher in this group. These data suggest that subclinical listeriosis alters hepatic and cardiac energy metabolism, possibly related to decreased activity of phosphotransferases and ATPase. Subsequent antioxidant responses are not sufficient to correct alterations in lipid peroxidation and bioenergetics, possibly leading to important cellular pathological mechanisms.

Experimental listeriosis: A study of purinergic and cholinergic inflammatory pathway.

The cholinergic, purinergic and oxidative stress systems were related to nervous system damage in some pathologies, as well as being involved in pro-inflammatory and anti-inflammatory pathways. The objective was to investigate changes in purinergic, cholinergic systems and oxidative stress related to the neuropathology of listeriosis. Gerbils were used as experimental models. The animals were divided in two groups: control and infected. The animals were orally infected with 5 × 108 CFU/animal of the pathogenic strain of Listeria monocytogenes. Collected of material was 6 and 12th days post-infection (PI). Infected animals showed moderate mixed inflammatory infiltrates in the liver. The spleen and brain was used for PCR analyses, confirming infection by L. monocytogenes. Increase in number of total leukocytes because of an increase in lymphocytes in infected (P < 0.001). ATP and ADP hydrolysis by NTPDase was lower at 6 and 12th days PI in infected animals than in the control group. ADA (adenosine deaminase) activity was higher on the 6th day PI (P < 0.05) and decreased on the 12th day PI (P < 0.05) in infected animals. AChE (acetylcholinesterase) activity did not differ between groups on the 6th day PI; however, activity decreased in infected group on the 12th day PI (P < 0.05). On the 12th day PI, an increase of oxygen-reactive species levels and lower catalase and superoxide dismutase activities in the infected group was observed, characterizing a situation of cerebral oxidative stress. The inflammatory and oxidative mechanisms are present in listeriosis in asymptomatic animals, and that ectonucleotidases and cholinesterase's are involved in immunomodulation.

Low-dose curcumin-loaded Eudragit L-100-nanocapsules in the diet of dairy sheep increases antioxidant levels and reduces lipid peroxidation in milk.

The main objective of this study was to evaluate whether the addition of curcumin-loaded nanocapsules (prepared and characterized) in the diets of dairy sheep improved milk quality. The nanocapsules were prepared using two polymers: poly-ε-caprolactone (PCL) and Eudragit L-100. The nanocapsules contained 0.25 mg/ml (Nano-Eudragit L-100) and 2 mg/ml (Nano-PCL) of curcumin. Dairy sheep were divided into four groups: A (control), B (30 mg free curcumin/kg concentrate), C (3 mg Nano-PCL/kg concentrate), and D (3 mg Nano-Eudragit/kg concentrate). We observed that the number of total leukocytes and serum globulin levels were lower in Group D than in the control (Group A) (p < 0.05). Antioxidant capacity against peroxyl radicals (ACAP) and catalase enzymes was elevated in Group D, with consequently reduced lipid peroxidation (LPO; p < 0.05). In milk, there were no differences in production and composition between groups during the experimental period (p > 0.05); however, ACAP increased and LPO decreased in milk. PRACTICAL APPLICATIONS: Curcumin is a functional molecule with potent antioxidant, anti-inflammatory, and antimicrobial actions, used frequently and with medical indications in human food. Free curcumin in sheep diets improves milk quality and increases its shelf life. This study showed that curcumin nanocapsules produced from the Eudragit L-100 polymer potentiated the anti-inflammatory and antioxidant actions of dairy sheep when used in the diet daily, at doses 10 times lower than that of free curcumin. These positive effects were reflected in higher total antioxidant capacity and lower lipid peroxidation in milk in sheep-fed curcumin-loaded Eudragit L-100 nanocapsules, generating desirable milk properties. In practice, the use of nanotechnology enhances the beneficial effects of curcumin in milk, possibly creating a nutraceutical food desirable to consumers.

Effect of free and nano-encapsulated curcumin on treatment and energetic metabolism of gerbils infected by Listeria monocytogenes.

Bacterial infections require special care since the indiscriminate use of antibiotics to treat them has been linked to the emergence of resistant strains. In this sense, phytoterapeutic alternatives such as curcumin and its nanocapsules have emerged as a promising supplement in optimizing availability of bioactives and reducing the development of antimicrobial resistance. Thus, the aim of this study was to verify the effects of pure and nanoencapsulated curcumin in the treatment of experimental listeriosis in gerbils regarding many aspects including antibacterial effect, antioxidant mechanisms involved and the energetic metabolism. Four groups were used containing 6 animals each: T0 (control), T1 (infected), T2 (infected and treated with free curcumin - dose of 30 mg/kg/day) and T3 (infected and treated with nanocapsules containing curcumin - a dose of 3 mg/kg/day). Treated animals received curcumin for 6 consecutive days starting 24 h after Listeria monocytogenes infection. All animals were euthanized on the 12th day after L. monocytogenes infection. Quantitative polymerase chain reaction (qPCR) identified L. monocytogenes DNA in the spleens of all animals of the T1 group, as well as T2 (2 out of 6) and T3 (5 out of 6). The weight of the spleens confirmed the infection, since it was larger in the T1 group, differing statistically from T0, and similarly to T2 and T3. Hepatic histopathological examination showed mild infiltration of neutrophils and macrophages, except for the T3 group (only 1/6). In the liver, the pyruvate kinase activity was higher in T1 and T2 compared to T0 and T3. The adenylate kinase activity did not differ between groups. The Na+/K+ATPase activity was lower in T1 group compared to T0 and T3. Lipoperoxidation was lower in the T3 group compared to groups T0, T1 and T2. The antioxidant capacity against peroxyl radicals was higher in T1, T2 and T3 groups compared to T0. In conclusion, free curcumin showed potent antibacterial effects; however, the nanoencapsulated form was able to minimize the effects caused by L. monocytogenes regarding tissue injury, changes on enzymes of the energetic metabolism, in addition to an antioxidant effect against lipoperoxidation.

Cholinesterase's activities of infected mice by Brucella ovis.

The role of cholinesterase in inflammatory reactions has been described in several infectious diseases. However, in Brucella spp. this has not yet been studied. Therefore, the objective of this study was to evaluate whether experimental infection by Brucella ovis alters the cholinergic activity in pro- or anti-inflammatory responses to the disease. For the study 48 mice were used, 24 infected by B. ovis and 24 non-infected. We collected samples of whole blood on days 7, 15, 30 and 60 post-infection (PI) by B. ovis. Acetylcholinesterase (AChE) activity in the blood increased on days 15 and 60 PI (P < 0.05). Butyrylcholinesterase (BChE) activity in serum increased on days 7 and 60 PI (P < 0.05). An increase in serum free radical levels occurred on days 7, 15 and 60 PI (P < 0.05), and consequently superoxide dismutase activity increased on day 15 PI (P < 0.05). A reduction in catalase activity occurred when the infection became chronic (60 PI). The increase in AChE and BChE characterized a pro-inflammatory response, since these enzymes regulate levels of acetylcholine (ACh) and butyrylcholine (BuSCh), molecules with anti-inflammatory properties. Therefore, with the increase of cholinesterase activity, there was an extracellular reduction of ACh, an inhibitor of several inflammatory mediators. This proinflammatory response of B. ovis infection leads to oxidative stress, and consequently to cellular damage.

Involvement of the phosphoryl transfer network on cardiac energetic metabolism during Staphylococcus aureus infection and its association to disease pathophysiology.

Evidences have suggested that the phosphoryl transfer network by the enzymatic activities of creatine kinase (CK), adenylate kinase (AK), pyruvate kinase (PK), and lactate dehydrogenase (LDH), shows new perspectives to understand some disturbances in the energy metabolism during bacterial infections. Thus, the aim of this study was to evaluate whether Staphylococcus aureus infection in mice could alter serum and cardiac activities of these enzymes and their association to disease pathophysiology. For that, we measured total leukocytes, lymphocytes and neutrophils (just 48 h of infection) that were lower in infected animals after 48 and 72 h in infected mice compared with negative control, while total protein and globulin plasma levels were higher after 72 h of infection. The serum CK activity was higher in infected animals 48 and 72 h post-infection compared to the control group, as well as observed for mitochondrial cardiac CK activity. The serum PK activity was higher in infected animals after 72 h of infection compared to the control group, and lower in the cardiac tissue. The cardiac AK activity was lower in infected animals 48 h and 72 h post-infection compared to the control group, while serum and cardiac LDH activities were higher. Based on these evidences, it is possible to conclude that the stimulation of CK activity exerts a key role as an attempt to maintain the bioenergetic homeostasis by the production of phosphocreatine to avoid a rapid fall on the concentrations of total adenosine triphosphate. In summary, the phosphoryl transfer network can be considered a pathway involved in the improvement on tissue and cellular energy homeostasis of S. aureus-infected mice.

Effects of phytogenic feed additive based on thymol, carvacrol and cinnamic aldehyde on body weight, blood parameters and environmental bacteria in broilers chickens.

The aim of this study was to evaluate whether a phytogenic feed additive (PFA) based on essential oils such as carvacrol, thymol and cinnamic aldehyde, could be considered a replacement for antimicrobials used as growth promoters in broiler chickens, as well as to investigate its effect on total bacterial count, biochemical profiles, meat quality and meat fatty acid profile. A total of 240 broiler chicks were randomly distributed into 4 groups with 4 replicates of 15 animals each, as follow: T1 (basal diet only; the control group), T2 (basal diet supplemented with zinc bacitracin), T3 (basal diet with 0.5% of the PFA), T4 (basal diet with 1.0% of the PFA). The addition of 0.5% of the PFA improved live body weight of supplemented birds compared to the control group at 35 and 42 days of age, while the total bacterial count in the environment was reduced when 1.0% of the PFA was used. In addition, intestinal villi height and crypt depth suffered variations during the entire experiment in birds treated with both concentrations of the PFA and zinc bacitracin. Total erythrocyte counts were higher on days 14, 28 and 42 in both treated groups (PFA) compared to the control group, as well as hemoglobin content on days 28 and 42. On the other hand, leukocyte counts were lower on days 14, 28 and 42 due to reduced lymphocyte counts in both PFA treated groups compared to the control group. Serum levels of alanine aminotransferase (ALT) were lower in broilers fed with either concentration of PFA on day 14 of life, and the same was observed regarding aspartate aminotransferase (AST) in broiler treated with 0.5% of the PFA. Also, total protein and globulin levels were lower on days 14 and 28 in groups fed with phytogenic compared to the control group. Regarding meat quality, breast meat showed higher red intensity and shear force in groups fed with both concentrations of phytogenic compared to the control group, while weight loss by cooking was lower. Finally, 1.0% of phytogenic showed lower docosadienoic acid (C22:2) content in breast meat. In conclusion, results showed that the use of PFAs based on carvacrol and thymol may be considered an interesting alternative to increase broilers performance, replacing the use of antimicrobials as growth promoters, as well as an interesting alternative to reduce the total bacterial count in the environment of broiler chickens. Moreover, the diet containing phytogenic also showed hepaprotective effects but deserves attention regarding possible alterations on the immune response.

Listeria monocytogenes impairs enzymes of the phosphotransfer network and alters antioxidant/oxidant status in cattle brain structures.

Several evidences have suggested the involvement of enzymes belonging to the phosphotransfer network, formed by creatine kinase (CK), pyruvate kinase (PK) and adenylate kinase (AK), as well the oxidative stress on the pathogenesis of infectious diseases associated with the central nervous system (CNS). Thus, the aim of this study was to evaluate whether listeriosis alters the brain energy metabolism and/or causes oxidative stress in different brain structures of cattle experimentally infected by Listeria monocytogenes. The cytosolic CK activity was inhibited in the cerebral cortex, cerebellum, brainstem and hippocampus of infected animals compared to uninfected animals, while the mitochondrial CK activity was increased. The PK activity was inhibited in all brain structures of infected animals, while the AK activity was unchanged. Na+, K+-ATPase activity decreased in the cerebral cortex, cerebellum and hippocampus of animals infected by L. monocytogenes. Regarding the oxidative strees variables, the cerebellum and brainstem of infected animals showed increased thiobarbituric acid reactive substances, while the catalase activity was inhibited. Glutathione S-transferarase was inhibited in the cerebral cortex and brainstem of infected animals, and it was increased in the cerebellum. L. monocytogenes was quantified in the liver (n = 5/5) and cerebral cortex (n = 4/5) of the infected cattle. Based on these evidences, the nucleocytoplasmic communication between CK isoenzymes was insufficient to avoid an impairment of cerebral bioenergetics. Moreover, the inhibition on brain PK activity caused an impairment in the communication between sites of ATP generation and ATP utilization. The lipid peroxidation and alteration on antioxidant status observed in some brain structures were also involved during the disease. In summary, these alterations contribute to disease pathogenesis linked to CNS during cattle listeriosis.

Experimental infection of cattle with Listeria monocytogenes: Participation of purinergic metabolism in disease pathogenesis.

The objective of this study was to evaluate whether experimental infection with Listeria monocytogenes alters the activity of triphosphate diphosphohydrolase (NTPDase), 5'-nucleotidase, and adenosine deaminase (ADA) in cattle. Ten male Holstein breed cattle were divided in two groups of five animals each: a control group, and a group infected with a pathogenic strain of L. monocytogenes. We drew blood for platelets on days 0, 7 and 14 of the experiment. On the 14th day post infection (PI), the animals were euthanized. Brain, spleen and liver were processed for histopathological examination and measurement of enzyme activities. The five (n = 5/5) bovines experimentally infected by L. monocytogene were positive-PCR in hepatic tissue. In the brain, only four (n = 4/5) of these animals were positive-PCR for listeriosis. There were no differences in platelet counts between groups (P > 0.05). In platelets, NTPDase activity (with ATP and ADP as substrates) were higher on the 7th PI day in the infected group, whereas the activities of 5'-nucleotidase and ADA were higher on the 7th and 14th PI. In serum and liver, ADA activity was higher in infected animals, but was lower on day 14 PI in spleen. NTPDase activity (with ATP as substrate) was higher in the cerebellum of infected animals, but was lower in the cerebral cortex and medulla oblongata. NTPDase activity (with ADP as substrate) was lower in the cerebellum and cerebral cortex of infected animals, whereas 5'-nucleotidase was higher. ADA activity was lower in the cerebellum, cerebral cortex and medulla oblongata in infected animals compared with controls. In conclusion, there appears to be a protective immunomodulatory response in spleen and brain structures of cattle infected with L. monocytogenes.

Changes on the activity of cholinesterase's in an immunomodulatory response of cattle infected by Listeria monocytogenes.

The aim of this study was to evaluate whether Listeria monocytogenes infection alters the activity of cholinesterases in cattle to module their inflammatory response and neurotransmission. Thus, ten male bovines (Holstein) were divided into two groups of five animals each: uninfected (control) and L. monocytogenes infected. Blood samples were collected on days 0, 7 and 14 post-infection (PI) to evaluate the activity of acetylcholinesterase (AChE) in the blood, and seric butyrylcholinesterase (BChE) activity, as well as total protein, albumin, globulin and C-reactive protein (CPR) levels in serum. The AChE activity and acetylcholine (ACh) levels were measured in the central nervous system on day 14 PI, and histopathological analyses were also performed. The infected animals did not show apparent clinical signs of listeriosis, however histopathological alterations were seen in the intestines and spleens. On days 7 and 14 PI, AChE activity in the blood was lower in infected animals, as well the seric BChE activity on day 7 PI. In the cerebral cortex and cerebellum, AChE activity was lower in infected animals compared to the control group, while the ACh levels were higher in the cerebral cortex compared to uninfected animals. Moreover, seric levels of total protein, globulin and CRP were higher in infected animals on days 7 and 14 PI compared to the control group. Therefore, we concluded that acute infection by L. monocytogenes alters the cholinergic system through the reduction of cholinesterase enzymes in the blood, serum and cerebral tissues as an adaptive response to an anti-inflammatory effect in order to increase the ACh levels, an anti-inflammatory molecule with an important role in the host immunomodulation.

Vertical transmission of Trypanosoma evansi in experimentally infected rats.

Many reproductive problems has been described in male and female animals infected by Trypanosoma evansi. Thus, the aim of this study was to evaluate the occurrence of vertical (Experiment I) and venereal (Experiment II) transmission of T. evansi in rats experimentally infected. In the experiment I, eight female Wistar rats were used: three animals as negative controls, and five rats were infected by T. evansi on day ten of gestation. Out of these eight females, half puppies were used for molecular analysis (polymerase chain reaction - PCR) for T. evansi. Two infected females showed delivery problems, such as stillbirth, and fetal death that also led to female death. Three female rats infected had normal delivery of stunted offspring at term that died 2 days after birth. Rats from the control group had normal delivery of healthy offspring. T. evansi PCR was positive for 80% (12/15) of pups in the infected group. For the experiment II, five male rats were infected by T. evansi, and monitored by blood smears to check the parasitemia level. When the male rats showed parasitemia between 2 and 5 parasites per field, they were individually housed with one female adult rat. After approximately 21 days, the females delivered their offspring. Blood sample was collected from the females for blood smears and T. evansi PCR tests, which revealed negative results. Therefore, we were able to prove the occurrence of transplacental transmission of T. evansi and its negative effect on female rats and their offspring.