Hypericum Perforatum L. Hairy Root Extracts – Regulation of Glycemic, Metabolic, Serum Enzyme and Lipid Profile in Stz - Induced Diabetic Rats

Apart from currently available therapeutics for the treatment of diabetes mellitus, much attention has been paid to discover phytochemicals from natural resources, mainly due to their low side-effects. Hypericum perforatum hairy root (HR) transformed with Agrobacterium rhizogenes A4 represent prospective experimental system enriched in xanthones, known as potent antidiabetic agents. Thus, the aim of this study was to evaluate HR extracts for their potential antihyperglycemic activity in streptozotocin (STZ)-induced diabetic rats, also compared to the effects of wild-growing Hyperici herba (HH). We conducted an acute-toxicity study, multiple dose study, and 24h blood glucose measurements after a single dose administration of HH and HR (200 mg/kg) in diabetic rats. Furthermore, we examined the effects of 14-days administration of HH and HR extracts on blood glucose levels, metabolic parameters, enzyme, and lipid status in healthy and diabetic rats. Both extracts produced a fall of about 70% in blood glucose level after 24h of administration. Two-week treatment with HH and HR induced a significant decrease (70-72%) in blood glucose levels. Moreover, we found an improvement of the dysregulated metabolic parameters (body weight, food, and water consumption and urine output). Serum enzyme (AST, ALT, and γ-GT) and lipid profile parameters (CHOL, TAG, and HDL) were also improved by both extracts. These findings might provide a new insight for managing diabetic hyperglycemia and dysregulated serum enzyme and lipid profile, using extracts from transgenic roots cultures from H. perforatum.

Rnase A Enzyme Modification of Optimized SDS Protocol for DNA Extraction Suitable for Real-Time PCR Screening of GMOs

As the number of genetically modified crops increases rapidly, their accurate detection is significant for labelling and safety assessment. Currently, real-time PCR is the “golden standard” method for GMO detection. Hence, extraction of high quality DNA represents a crucial step for accurate and efficient DNA amplification. For GMO presence evaluation in the extracted DNA from raw corn kernels and roasted soybean, we used real-time PCR method, in consistent with the ISO17025 accreditation standards. As for DNA extraction, modified basic SDS protocol by adding RNase A enzyme in different steps of the protocol, with different time and temperature of incubation was used. The results showed as most suitable, the protocol where 10 µl of RNase A enzyme was added together with the lysis buffer at 65 °C for 30 minutes. Data for DNA yield and purity for roasted soybean was 469.6±3.3 µg/ml with A260/280 absorbance ratio 1.78±0.01. Suitability of DNA extracts for GMO analysis was assessed by screening for the presence of 35S promotor and Tnos terminator. Diluted extracts in concentrations 10, 1, 0.1, 0.01 and 0.0027 ng/µl, were tested in six replicates. Positive signal of amplification (LOD) was detected in all concentrations for both genetic elements in both matrices. The LOQ for 35S and Tnos for both matrices was 0.1 ng, while for Tnos in raw corn kernels was 0.01 ng. This in-house developed DNA extraction method is simple and obtains high-quality DNA suitable for GMO screening of 35S promotor and Tnos terminator in both raw and processed matrices.

Importance and Impact of the Poultry Red Mite (Dermanyssus Gallinae) in Layer Farms - Analysis of Farmers’ Perception

Poultry red mite (PRM), Dermanyssus gallinae, causes egg drop production, anemia and can be a vector in transmitting diseases. The PRM control mainly focuses on usage of the conventional chemical biocides. The objective of this study was to analyze the farmers’ perception regarding the impact, management and control of PRM in Macedonian layer farms. The data were collected with direct on-site visits using a unified questionnaire. In total, 29 poultry farms (28% of farms in the country), all with conventional cages, were part of this study. The data were analyzed by descriptive statistics, inferential statistics and Naïve Bayes Classifier technique. In 30% of the poultry farms the farmers had observed that the flock was infested with PRM. In total, 32 different treatments against PRM were reported from the farmers, and three of them were non-biocide treatments. The most used biocides (17% of the farms) were crude oil, Formalin, Neopitroid® and disinfectants. The highest agreement regarding biocides application among the farms (38%) was before the production starts. Most of the farmers applied biocides routinely, before the infestation is evident (75%). The median costs for PRM treatment were 175€ per flock, higher in the infested farms 493±677€ compared to non - infested 100±71€, p<0.05. None of the Macedonian farmers included in the study was using monitoring method for PRM infestation, contributing to poor data records. This study highlights the need of developing unified strategy for PRM control included in the Integrated Pest Management in poultry layer farms.

The Effect of Alkaline Water and Sodium Ascorbate on Glucose and Cortisol Levels During Acute Hyperthermic Stress in White Laboratory Rats

Stress can be a reason for some physiological and biological disorders in the body. The antioxidative defense system is necessary for the maintenance of redox homeostasis in the organisms. Alkaline water (AW) is in the focus of the scientific interest due to its antioxidative effect. The treatment with AW and sodium ascorbate (SA) is expected to have potential preventive effect on the organism to hyperthermic stress. The aim of this study was to investigate the effect of AW and SA on glucose and cortisol levels during acute hyperthermic stress, in white female Wistar laboratory rats. The rats were divided into three groups, each having 10 subjects. They were exposed in hyperthermic conditions (41˚C) for 80 min, in 21 consecutive days in order to induce oxidative stress. The first group received drinkable water (control group), the second AW, and the third, AW and SA. Plasma glucose levels were determined by colorimetric method. Cortisol level was measured by the enzyme-linked immunosorbent assay method (ELISA). The means were compared using the Tukey test. Differences were considered significant at a level of p<0.05. Our results showed that levels of glucose and cortisol were significantly higher in the group treated with AW on the 21st day after treatment (p<0.0001), but not on the 7th and 14th day as compared to the control group. Also, co-treatment of animals with AW and SA had significantly increased the levels of glucose and cortisol on the 21st day after treatment, indicating a synergistic effect. In conclusion, the individual action of AW or in synergism with SA caused a high protective effect on oxidative damage in white Wistar laboratory rats.

The Safety Profile of the Anesthetic Effect of Alfaxalone and Its Interaction with Xylazine and Ketamine in Chick’s Model (Gallus gallus domesticus)

The objective of our research was to estimate the therapeutic index and assess the interaction of alfaxalone (IP) with ketamine or xylazine (IM) in chicks by using isobolographic analysis. The up-and-down technique was involved to calculate the median effective anesthetic dosages (ED50) of alfaxalone, xylazine, and ketamine given separately or at the same time in young chicks. Then the up-and-down technique was involved to estimate the median lethal dosage (LD50) of alfaxalone (IP) to determine the safety profile. The ED50 of all anesthetics was evaluated isobolographically to assess the type of interaction between alfaxalone and xylazine or alfaxalone and ketamine. The alfaxalone ED50 was 32.88 mg/kg (IP), whereas the LD50 was 102.40 mg/kg (IP). The ED50 values for alfaxalone, ketamine, and xylazine were 32.88, 12.24, and 2.45 mg/kg, respectively. The ED50 values of alfaxalone with ketamine or xylazine (25:25 ED50 values) were: 7.39+2.35, and 8.61+0.63 mg/kg, respectively. ED50 values were decreased when the combinations of alfaxalone/ketamine or alfaxalone/xylazine were administered by 22-21% and 26-25%, respectively. The anesthesia of chicks with alfaxalone is safe, produces a surgical stage of anesthesia, and can be used for minor surgical procedures. The use of alfaxalone with ketamine or xylazine has been shown to have a synergistic effect and these findings may be of clinical relevance in poultry or may be extended to mammals following further clinical trials.

Biosecurity and Antimicrobial Use Practices in Live Bird Markets Within Abeokuta Metropolis, Southwest, Nigeria: A Preliminary Survey

The development of an antimicrobial stewardship plan (AMSP) for live bird sellers (LBS) requires an understanding of the current biosecurity status, antimicrobial use (AMU) and the practices involved in live bird selling (e.g., drivers, sellers, etc.) which is a direct source of poultry meat for human consumption. Seven Live Bird Markets (LBMs) within Abeokuta, Ogun State were surveyed using a semi-structured questionnaire. Data on LBMs characteristics, LBS demographics, biosecurity, and AMU practices, awareness on Antimicrobial Resistance (AMR), as well as preferred channels of information on antimicrobial stewardship were gathered. A total of 40 consenting LBS with 82.5% female and 17.5% male participants were included in the study. The participants’ mean age was 45.3 years (SD±11.9, range: 23-70 years). Laying hens, broilers, and cockerels were the main poultry types sold by LBS. Antimicrobials (AMs) were used for growth promotion (57.5%), therapeutic (40.0%), and prophylactic (2.5%) purposes. Tetracycline, metronidazole, and chloramphenicol were the most frequently used AMs. The majority of the participants (90.0%) have treated birds based on their empirical experience, with little or no inputs from veterinarians. Biosecurity and AMU practices were generally low (54.0% and 34.0%, respectively). The contact with veterinarians was associated with satisfactory biosecurity practices (p=0.049). No significant factors were found to be linked with AMU. This study has provided recent evidence-based data on practices in poultry management among LBS in Abeokuta, Ogun state. The findings would be useful for policy decisions and the development of AMSP on prudent AMU among LBS.

Laparoscopic Retrieval of Abdominal Cysts in Sheep and Goat

The study was planned to evaluate the use of laparoscopy for the diagnosis and retrieval of abdominal cysts in sheep and goats. The abdominal cysts were located in 10 of 135 adult, healthy small ruminants by using ultrasonography (USG). Percutaneous Puncture-Aspiration-Injection-Reaspiration (PAIR) technique was used for six animals. Thirty animals, including four diagnosed with abdominal cysts by USG, were subsequently subjected to laparoscopy under diazepam sedation (0.1 mg/kg, IV) and lumbosacral epidural regional anesthesia using 2% lignocaine hydrochloride (1.0 ml/5 kg). The animals were restrained in dorsal recumbency and two-port paramedian laparoscopy was performed. Abdominal cysts were located in seven animals (one cyst/animal). The cysts were grasped carefully, lifted close to the abdominal wall, and evacuated percutaneously under laparoscopic vision. The collapsed cysts were then retrieved. In two animals enlarged ports were sutured, treated with an antibiotic and an analgesic, resulting in uneventful healing. Five cysts were nonparasitic, two were parasitic, one extracted from a sheep, and one from a goat. In conclusion, laparoscopy has higher diagnostic accuracy in detecting unattached abdominal cysts in sheep and goats compared to USG. It is also a reliable, minimally invasive, and safe procedure for cyst retrieval. However, a larger-scale study is necessary for ascertaining long-term complications and the recurrence rate.

The Microscopic Detection of Animal Proteins in Animal Feed Regarding Bovine Spongiform Encephalopathy

Due to the actuality of spongiform encephalopathies and their proven spreading by means of animal feed containing meat and bone meal, the description and measurement of osteocytic lacunae contributes to more easily distinguish bone fragments in meat and bone meal. Transmissible spongiform encephalopathies (TSEs) have attracted a lot of attention, especially after 1986, when the first case of BSE (bovine spongiform encephalopathy) was detected. Since the outbreak of spongiform encephalopathy (BSE), the use of animal protein including bone meal as an ingredient in animal feed has been controlled by several regulations including Regulation (EC) 999/2001, Regulation (EC) 1774/2002, and Regulation (EC) 1234/2003. The classical microscopic method is the only official method for detecting animal protein in animal feed in the European Union (Commission Regulation (EC) 152/2009). By applying the microscopic method to the animal feed samples, we performed detection in order to determine the presence of animal proteins that originate from mammals and fish. The microscopic analysis of all 421 samples, of which 115 were raw materials for the production of animal feed, 230 were concentrates for ruminant nutrition and 76 were concentrates for non-ruminant nutrition (32 concentrates for laying hens and 44 concentrates for pigs), did not provide positive results, that is, no remains of animal tissues of mammalian origin were found in any specimen. Whereas in 10 out of 32 (31.25%) concentrates intended for non-ruminant nutrition (laying hens), pieces of fish tissue were found. In these samples, we usually detected the presence of fish bones, gills and scales.

Isolation Rate of Campylobacter Spp. and Detection of Virulence Genes of Campylobacter jejuni Across the Broiler Chain

The aim of the study was to identify the isolation rate of thermotolerant campylobacters in a small-scale broiler-meat production farm over a one-year period. The second deliverable of the study was to determine the potential virulence markers. The laboratory investigation was performed on 283 samples (cloacal swabs, caeca, carcass swabs) collected on three sampling points (farm, slaughter line, and cold storage). The isolates obtained with the conventional microbiological method were confirmed with multiplex PCR for identification of campylobacters. The presence of 10 virulence genes was analyzed in the C. jejuni isolates ( flaA, racR, virB11, dnaJ, wlaN, cadF, ciaB, cdtA, cdtB, cdtC). Out of 283 samples, 169 (59.7%) were confirmed as Campylobacter spp., 111 (39.2%) C. jejuni, and 43 (15.2%) C. coli. C. jejuni was the most prevalent in all sampling points. Campylobacter spp. showed a characteristically seasonal prevalence with the highest isolation rate during the warmer period of the year. We detected the cadF and ciaB genes in all C. jejuni isolates. The flaA gene was present in 50% of the examined strains. The cdt genes (cdtA, cdtB, and cdtC) were confirmed in 52.8%, 52.8%, and 47.2% of the C. jejuni strains, respectively. C. jejuni showed 15 profiles of virulence patterns with four predominant profiles.

Morphological Peculiarities of Parasitic (Trichosomoides crassicauda) Infection in Rat Urinary Bladder

Trichosomoides crassicauda (T. crassicauda) is a parasite commonly localized in the urinary bladder (UB) of laboratory and wild rats. The presence of these helminths can influence the prediction of pathological changes in the UB. Therefore, the purpose of this research was to make a comprehensive study of the features of the morphological changes in the UB wall of white laboratory rats as a result of T. crassicauda infestation. The study was performed on male rats using histological (Hematoxyline-Eosin and Alcian Blue staining) and immunohistochemical (Ki-67, Hsp70, Hsp90α, CD3 and CD20) methods. T. crassicauda was detected in both urine and UB samples. Morphological changes were observed as disruption in urothelial cell stratification and insignificant proliferative and immune responses in the UB. Increased heat shock protein levels were observed which may suggest a natural body’s resistance to this parasite.