Molecular identification of Sarcocystis species in wild boar (Sus scrofa) and pigs (Sus scrofa domesticus) in Brazil.

Sarcocystis spp. are protozoan parasites that form cysts in the organs and musculature of various animal species. The species Sarcocystis miescheriana and Sarcocystis suihominis are pathogenic to pigs and wild boars (Sus scrofa), acting as intermediate hosts, while humans are the definitive host for S. suihominis. To date, there have been no reports of the identification of these coccidian species in Sus scrofa in Brazil. Therefore, in this study, we conducted the first molecular identification of Sarcocystis species using PCR-RFLP and sequencing. A total of 210 samples were analyzed, of this total, 67 tested positive for Sarcocystis spp., representing 31.9% of the total samples assessed. Out of the total positive samples, 55 (82.1%) were identified as S. miescheriana and 8 (11.9%) as S. suihominis, a zoonotic species. Additionally, other species related to bovines, such as S. cruzi and zoonotic S. hominis, were detected in 3.0% of the samples, serving as contaminants in the pork products. The presence of S. suihominis in swine and wild boar samples is concerning due to the zoonotic risk and potential environmental contamination, as humans act as definitive hosts, also for the presence of S. hominis as a bovine contaminant in pork sausages. Furthermore, we confirmed the efficacy of the PCR-RFLP technique as a reliable tool for the identification of Sarcocystis species, demonstrating its potential use in laboratories for molecular diagnosis and rapid identification of these parasites, aiming to protect public health and ensure food safety.

Sarcocystis in horses from Rio Grande do Sul, Brazil: Molecular identification of Sarcocystis bertrami and Sarcocystis neurona in muscle tissues.

Horses are intermediate hosts of Sarcocystis spp. capable of forming cysts in their musculature. This study aimed to detect sarcocysts and investigate the presence of nucleic acids from Sarcocystis spp. in samples of striated muscles from horses in the State of Rio Grande do Sul, Brazil, necropsied at the Veterinary Pathology Laboratory of the Federal University of Santa Maria. A total of 108 samples were collected from 24 horses and examined through direct examination. Microscopic tissue cysts were observed in three samples: tongue (2) and esophagus (1) from two animals. Extractions were performed on the found cysts and tissues, even though sarcocystosis detection was not present. DNA samples were subjected to Nested-PCR using Tg18s primers, and the amplified products were subjected to Restriction Fragment Length Polymorphism (RFLP) using DdeI and HpaII enzymes. DNA belonging to Sarcocystis spp. was amplified in tissues from 91.7% (22/24) of the equines, and 67.6% (73/108) of the samples tested positive in the Nested-PCR reaction. The tissues with the highest detection frequency were: diaphragm 92.3% (12/13), gluteal muscle 77.2% (17/22), and esophagus 66.7% (4/6). In RFLP, Sarcocystis spp. was detected in 21 tissues from 11/22 equines, and cysts, identified through nucleotide sequencing, were determined to be S. bertrami. S. neurona was identified in 11 samples from 7/22 animals, with co-infection detected in 5/22 cases. The high detection rate indicates a concerning circulation of the protozoan, particularly the zoonotic S. bertrami found in all tissues, which are commonly exported for human consumption.

Effect of dietary supplementation with grape residue flour on weight gain, metabolic profile, leukogram, proteinogram and antioxidant response in suckling lambs.

The objective of this study was to determine whether the addition of grape residue flour (GRF) in the diet of suckling lambs would improve their health and consequently enhance their growth. We used 48 lambs, 30 days of age divided into four treatments with four repetitions each, with each repetition consisting of three animals. The groups were identified as follows: G-0%, used as a control (without GRF) and G-0.5%, G-1% and G-2% referring to treatment with grape residue flour in doses of 0.5%, 1%, and 2% of inclusion in the concentrate, respectively. Lambs in groups G-1% and G-2% had higher final body weights, weight gain and average daily gain (ADG) compared to the other groups. GRF dietary supplementation had good antioxidant potential, being able to stimulate glutathione S-transferase (GST) activity and consequently reduce levels of reactive oxygen species (ROS) in lambs that consumed the highest dose of GRF (G-2%). GRF supplementation improved humoral responses, with increased serum levels of heavy-chain and light-chain immunoglobulins; however, there was a reduction in serum ceruloplasmin levels in these lambs. We observed higher concentrations of glucose and triglycerides in lambs in the 2% group. There were lower lymphocyte counts in lambs that received GRF. Lambs supplemented with the highest doses of GRF (G-1% and G-2%) had lower total bacterial counts in the feces. We conclude that the use of GRF in the supplementation of suckling lambs improved animal health, as it stimulated the antioxidant and immune systems and consequently favored their growth.

Experimental infection with Escherichia coli in broilers: Impacts of the disease and benefits of preventive consumption of a stimulator of homeopathic immunity.

Colibacillosis is a disease caused by Escherichia coli that manifests itself when there are homeostatic imbalances or in the context of increased exposure, in which case the organism displays opportunistic behavior. To control this problem in poultry, antibiotics are used in the feed, because E. coli is component of the intestinal microbiota of birds. However, because of the changing dietary habits of the human population that seeks out healthier foods without antimicrobial residues, there have been many studies of alternatives to replace conventional antimicrobials as performance enhancers. Thus, the objective of the present study was to determine whether daily consumption of a homeopathic product (immune stimulator) by broilers stimulates immune responses and thereby minimizes the negative effects of experimental E. coli infection. We used 320 1-day-old Cobb 500 chicks, distributed in two groups with eight repetitions each, and 20 birds per repetition: control (CG) and homeopathy (HG). HG birds consumed doses of 0.02 mL/bird/day (1-7 d) via water, 0.01 ml/bird (8-21 d), 0.02 ml/bird (22-28 d), 0.01 mL/bird (29-35 d), and 0.02 mL/bird (35-45 d), as recommended by the manufacturer. At day 22 of the birds' life, the two groups were divided into four subgroups, with four repetitions per subgroup. On day 22, birds in CG1 and HG1 groups were infected intraperitoneally with 0.5 mL of inoculum containing 1.0 × 108 CFU of E. coli/mL. During the experimental period, data were collected for analysis of performance. On days 21 and 45 of age, we collected blood and feces. During the first 21 days of the experiment, we found that birds that consumed the immunostimulator had lower neutrophil counts and higher levels of globulins, however without significant difference between groups in terms of performance. Uninfected birds that consumed the homeopathic product in the water had less feed conversion (HG2) between days 1-35 and 1 to 45 compared to the other treatments. Mortality was higher in groups experimentally infected with E. coli (HG1 and CG1) from 22 to 35 days of life. There were greater numbers of lymphocytes in the HG2 group on day 45 than in CG1 and CG2; while numbers of neutrophils were lower at 42 days in birds of groups HG1 and HG2 than in CG1. Lower total bacterial counts, total coliforms and E. coli were observed in the feces of birds in the HG2 group compared to the other groups. Taken together, these findings suggest that inclusion of homeopathic product in the water of broilers had positive effects on the modulation of the immune response and on feed conversion in birds not challenged with E. coli. But the preventive protocol used in this study was not able to minimize the negative effects caused by the experimental E. coli intraperitoneal infection in broilers, featuring a substantial infectious challenge.

Curcumin addition in diet of laying hens under cold stress has antioxidant and antimicrobial effects and improves bird health and egg quality.

Curcumin is an herbal component with several biological properties, and we highlight here the thermal stability, antioxidant and anti-inflammatory activity. We determined whether curcumin supplementation in the diets of laying hens under cold stress and naturally infected with Escherichia coli would control infection, and would have positive effects on overall health, as well as egg production and quality. We allocated 36 chickens that had been naturally infected with E. coli into two groups, with six replicates and three chickens per repetition: control group and curcumin group (200 mg curcumin/kg). The experimental period lasted 42 days, and fecal, blood and egg samples were collected at 1, 21 and 42 days We found that feces and eggs had lower total bacterial counts, E. coli counts and total coliform counts in the curcumin group at 21 and 42 days. In fresh eggs, the brightness and yellow intensity (b+) were significantly higher in the curcumin group. In stored eggs, higher specific gravity, albumen height and lower yolk pH were observed in the curcumin group. Fresh eggs collected and stored on day 42 showed lower levels of lipid peroxidation in the curcumin group, while the total antioxidant capacity in the stored eggs was significantly higher in the curcumin group. The curcumin group showed lower total leukocyte counts as a result of lower numbers of neutrophils and lymphocytes, as well as lower levels of total protein, alkaline phosphatase and alanine aminotransferase. Lower serum lipoperoxidation at 42 days was observed in the curcumin group, probably because of the higher activity of glutathione peroxidase and glutathione transferase in other words, because of antioxidant stimulation. Taken together, our findings suggest that curcumin supplementation in laying hens under cold stress and with colibacillosis has positive effects on infection control because of antioxidant stimulation.

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.

Impacts of Escherichia coli infection in young breeder chicks on the animal behavior and cerebral activity of purinergic and cholinergic enzymes involved in the regulation of molecules with neurotransmitter and neuromodulator function.

The objective of this study was to evaluate if infection by Escherichia coli in juvenile breeder chicks alters the activity of enzymes involved in neurotransmission and cerebral immunomodulation, including acetylcholinesterase (AChE), nucleoside triphosphate diphosphohydrolase (NTPDase), 5'-nucleotidase (5'NT) and adenosine deaminase (ADA), as well as their effects on the pathogenesis of the disease. We divided 20 growing breeder chicks into two groups (n = 10 per group). One group was experimentally infected with 1 mL of culture medium containing 1 × 108 CFU of E. coli intraperitoneally. The other was the negative control. On the tenth day after infection, the animals were euthanized and brain samples were collected. Macroscopically, pericarditis and hepatic congestion were observed in the birds, but without histopathological lesions in the encephalon although the bacterium was present in the cerebral cortex of all animals in the infected group (i.e., they were PCR-positive). The activity of AChE, NTPDase, 5'-NT and ADA were evaluated in the cerebral homogenates of the birds after 10 days of infection. AChE activity in the cerebral cortex was lower in the infected group than in the control; there was an increase in the activity of NTPDase, 5'-nucleotidase and ADA, possibly indicating greater hydrolysis of ATP (P < 0.001), ADP (P < 0.01) and AMP (P < 0.01), followed by increased adenosine deamination (P < 0.001). Despite these changes, no apparently diseased animals were observed throughout the experimental period. Therefore, such changes in enzymatic activity may affect the functioning of the central nervous system because these enzymes are responsible for extracellular regulation of molecules that act on neurotransmission and immunomodulation such as acetylcholine, ATP and adenosine.

Impact of colibacillosis on production in laying hens associated with interference of the phosphotransfer network and oxidative stress.

The objective of this study was to evaluate the productive impact of colibacillosis on laying hens and to investigate whether energetic metabolism and oxidative stress were involved in the pathogenesis of the disease. An experimental shed containing 270 laying hens of the Hy-Line lineage (32 weeks old) presented approximately 40% daily laying, and many birds presented with diarrhea and apathy followed by death. Necropsy revealed macroscopic lesions compatible with colibacillosis and infectious agent Escherichia coli was isolated from fecal samples of all birds in the infected group, as well as from tissue (ovary, liver and peritoneum). Sixteen chickens were selected for this study, divided into two groups: Control (animals without clinical alterations) and infected (with diarrhea and apathetic). E. coli isolates were subjected to the antimicrobial susceptibility testing according to the methodology approved by CLSI, 2018. This testing showed sensitivity to gentamicin, amoxicillin, norfloxacin and colistin. It was then determined that laying hens would be treated with norfloxacin (15 mg/kg) diluted in water offered at will to the birds for three days. Blood collections were performed via brachial vein after the diagnosis of E. coli (before starting treatment) and seven days after treatment. Three debilitated chickens died on the second day after initiating therapy. Before treatment, birds with clinical signs had higher levels of lipoperoxidation (LPO) and activities of antioxidant enzymes superoxide dismutase (SOD) and glutathione peroxidase (GPx) than in the control group (asymptomatic animals). After treatment, LPO levels remained higher in birds that had clinical disease (infected group), whereas the activity of SOD and GPx enzymes did not differ between groups. Activity levels of creatine kinase (CK) and pyruvate kinase (PK) were higher in the group of chickens with clinical disease before treatment. Post-treatment, no differences were observed between groups in terms of CK; however, PK activity remained high in these animals. In the hens that died, there were lesions characteristic of avian colibacillosis, with ovary involvement, explaining the low laying activity of the birds at their peak of production. For 10 days after starting treatment, the percentage of laying increased to 90%. Therefore, we conclude that colibacillosis interferes with the phosphotransfer network by stimulating ATP production, in addition to causing oxidative stress of the birds during laying, that negatively affects health and productive efficiency.