Necropsy Day Research Articles

HISTOPATOLOGICAL OF WHITE RATS' KIDNEY CAUSED BYAPPLICATION OF MIMOSIN FROM SIMPLISIA OF THE LEUCAENA LEAF

Mimosine is a toxic substance contained in Leucaena leucocephala leaves. The toxic effects of mimosine can disrupt several tissues and organs, especially the kidneys. This study aims to determine the histopathology of white rat kidney exposed to mimosine with different doses. Male white rats with Wistar strain, 2 months old and 300-350 g body weight were being used in this study. The 20 rats used were divided into four treatment groups, namely P0 (negative control), P1 (positive control, given standard mimosine 15 mg/head/day), P2 given leucena leaf simplisia at a dose of 50 mg/head/day orally, P3 given leucaena leaf simplisia at a dose of 150 mg/head/day. On the 16th day of necropsy, the skin organs were taken and placed in 10% NBF. After the kidney organs were fixed, histopathology preparations were made using HE staining. Histopathological examination was performed including three lesion variables, namely congestion, bleeding, and inflammation. The severity of the lesions were scored as 0, 1, 2 and 3 respectively for normal, mild, moderate and severe lesions. Data were analyzed using non-parametric Kruskal-Wallis and Mann- Whitney tests. The results of the research showed that the mimosine substance in leucaena leaf simplicia caused some histopathological changes in the kidneys of white rats like congestion, bleeding and inflammation and there was a difference in dose effect for each giving of mimosine from leucaena leaf simplicia, between the dose of 50 mg/head/day and the 150 mg/head/day. Administration of mimosine from leucaena leaf simplicia can cause histopathological changes in the kidneys of white rats and there is an effect of the difference in the dose of mimosine from leucaena leaf simplicia between a dose of 50 mg/head/day and a dose of 150 mg/day. tail/day on changes in kidney pathology of white rats, especially in the form of bleeding and inflammation. With this research, it hoped that in the future further research can be carried out on leucaena leaf simplicia with a longer administration period so that the results obtained are more accurate.

HISTOPATOLOGICAL CHANGES OF WHITE RATS' SKIN CAUSED BY APPLICATION OF MIMOSIN FROM SIMPLISIA OF THE LAMTORO LEAF

Mimosine is one of the substances contained in lamtoro (Leucaena leucocephala) leaves. The toxic effect of mimosine on livestock is highly dependent on the concentration of mimosine in feed ingredients and the length of time livestock consume feed high in mimosine. This study aims to determine the histopathology of white rat skin exposed to mimosine compounds with different doses. This study used male white rats with Wistar strain, 2 months old and 300-350 g body weight. The 20 rats used were divided into four treatment groups, namely P0 (negative control, P1 (positive control, given standard mimosine 5 mg/head/day), P2 (given lamtoro leaf simplisia at a dose of 50 mg/head/day orally), P3 (given lamtoro leaf simplisia at a dose of 150 mg/head/day). On the 15th day of necropsy, the skin organs were taken and fixed using 10% NBF. After the skin organs were fixed, histopathology preparations were made using HE staining. Histopathologic examination was performed including three lesion variables: hair follicle necrosis, congestion, and inflammation. The severity of the lesions was scored as 0, 1, 2, and 3 for normal, mild, moderate and severe lesions, respectively. Data were then analyzed using Kruskal-Wallis and Mann-Whitney non-parametric tests. The results showed that exposure to mimosine from lamtoro leaf simplisia did not cause hair loss in experimental animals as it does in cattle. However, exposure to mimosine in P1 (5 mg/head/day) and P3 (150 mg/head/day) caused histopathological skin lesions in the form of congestion, necrosis, and mild inflammation. The results showed that exposure to mimosine from lamtoro leaf simplisia did not cause hair loss in experimental animals as it does in cattle. However, exposure to mimosine in P1 (5 mg/head/day) and P3 (150 mg/head/day) caused histopathological skin lesions in the form of congestion, necrosis, and mild inflammation. The conclusion of the study is that mimosine compounds from lamtoro leaf simplisia cause histopathological changes in white rat skin tissue, especially congestion lesions. However, there was no difference in the effect of mimosine administration from lamtoro leaf simplisia between a dose of 50 mg/head/day and a dose of 150 mg/head/day on the histopathology of rat skin tissue. Further research needs to be done on the effect of exposure to mimosine from lamtoro leaf simplisia with higher concentrations.

Ninety-day repeated dose toxicity of Ashwagandha (Withania somnifera) root extract in Wistar rats

Many pharmacological studies have been carried out to describe multiple biological properties of Ashwagandha (Withania somnifera) and the additional safety information on repeated dose toxicity is limited. Therefore, the aim of this study was to obtain safety data for KSM-66 Ashwagandha Root Extract (ARE) through repeated-dose toxicity in Wistar rats according to the Organisation for Economic Co-operation and Development (OECD) test guideline (TG 408). ARE was orally administered to rats at doses of 0, 500, 1000, and 2000 mg/kg body weight/day for 90-day and reversibility of effects of 0 and 2000 mg/kg body weight/day was assessed for 14 days. All the animals from treated, control, recovery control and recovery groups were observed for clinical signs of toxicity once daily, detailed clinical examination every week after dosing and before necropsy day. Mortality/Morbidity was observed twice daily. In addition, observations were noted in the detailed sensory reactivity, functional assessments, body weight, food consumption, ophthalmological examination, hematological parameters, biochemical parameters, organ weights, histopathological findings. The present results show that the no observed adverse effect level (NOAEL) of KSM-66 Ashwagandha Root Extract was considered to be 2000 mg/kg body weight/day in rats after repeated oral administration for 90-day under the present study conditions.

Assessment of Escherichia coli-derived Recombinant Human Bone Morphogenic Protein-2 on Fertilization and Early Embryonic Development in Rats

ObjectiveBone morphogenetic protein-2 (BMP-2) impacts fertility in women by affecting the menstrual cycle and embryonic development. We aimed to determine the reproductive toxicity of Escherichia coli (E. coli)-derived recombinant human BMP-2 (rhBMP-2) by measuring changes in the reproductive performance and organs in rhBMP-2-treated rats.MethodsOverall, 88 male and female rats each were categorized into one control and three experimental groups. rhBMP-2 was intravenously administered to the experimental groups at 0.05, 0.15, and 0.50 mg/kg/day, respectively. The male rats were administered rhBMP-2 daily, starting from 28 days before mating until the day of necropsy (48 days), after which they were euthanized and necropsied. The female rats were administered rhBMP-2 daily, starting from 14 days before mating until 7 days after fertilization (22–36 days), after which they were necropsied 13 days after fertilization.ResultsNo rhBMP-2-related death occurred throughout the study period. All rhBMP-2-treated groups showed swelling in the tail at the site of rhBMP-2 administration. In the high-dose rhBMP-2 group, the male rats showed a slight reduction in body weight and food consumption, whereas the female rats showed a reduction in the weights of the ovary and oviduct. Examining the fertilization status and necropsy showed no effect of rhBMP-2 on fertility and early embryonic development. The no-observed-adverse-effect level of rhBMP-2 was 0.50 mg/kg/day in all rats.ConclusionrhBMP-2 had no reproductive toxicity on the reproductive performance and organs in female and male rats. Therefore, these results provide new toxicology information on E. coli-derived rhBMP-2 as a therapeutic protein.

Vaccination against swine influenza in pigs causes different drift evolutionary patterns upon swine influenza virus experimental infection and reduces the likelihood of genomic reassortments.

Influenza A viruses (IAVs) can infect a wide variety of bird and mammal species. Their genome is characterized by 8 RNA single stranded segments. The low proofreading activity of their polymerases and the genomic reassortment between different IAVs subtypes allow them to continuously evolve, constituting a constant threat to human and animal health. In 2009, a pandemic of an IAV highlighted the importance of the swine host in IAVs adaptation between humans and birds. The swine population and the incidence of swine IAV is constantly growing. In previous studies, despite vaccination, swine IAV growth and evolution were proven in vaccinated and challenged animals. However, how vaccination can drive the evolutionary dynamics of swine IAV after coinfection with two subtypes is poorly studied. In the present study, vaccinated and nonvaccinated pigs were challenged by direct contact with H1N1 and H3N2 independent swine IAVs seeder pigs. Nasal swab samples were daily recovered and broncho-alveolar lavage fluid (BALF) was also collected at necropsy day from each pig for swine IAV detection and whole genome sequencing. In total, 39 swine IAV whole genome sequences were obtained by next generation sequencing from samples collected from both experimental groups. Subsequently, genomic, and evolutionary analyses were carried out to detect both, genomic reassortments and single nucleotide variants (SNV). Regarding the segments found per sample, the simultaneous presence of segments from both subtypes was much lower in vaccinated animals, indicating that the vaccine reduced the likelihood of genomic reassortment events. In relation to swine IAV intra-host diversity, a total of 239 and 74 SNV were detected within H1N1 and H3N2 subtypes, respectively. Different proportions of synonymous and nonsynonymous substitutions were found, indicating that vaccine may be influencing the main mechanism that shape swine IAV evolution, detecting natural, neutral, and purifying selection in the different analyzed scenarios. SNV were detected along the whole swine IAV genome with important nonsynonymous substitutions on polymerases, surface glycoproteins and nonstructural proteins, which may have an impact on virus replication, immune system escaping and virulence of virus, respectively. The present study further emphasized the vast evolutionary capacity of swine IAV, under natural infection and vaccination pressure scenarios.

Hepatic and Renal Histotripsy in an Anticoagulated Porcine Model

PurposeTo determine the risk of mechanical vessel wall damage resulting in hemorrhage during and after hepatic and renal histotripsy in an anticoagulated in vivo porcine model. Materials and MethodsNon–tumor-bearing pigs (n = 8; mean weight, 52.5 kg) were anticoagulated with warfarin (initial dose, 0.08 mg/kg) to a target prothrombin time (PT) of 30%–50% above baseline. A total of 15 histotripsy procedures were performed (kidney: n = 8, 2.0-cm sphere; liver: n = 7, 2.5-cm sphere). Treatments were immediately followed by computed tomography (CT) imaging. Animals were observed for 7 days while continuing anticoagulation, followed by repeat CT and necropsy. ResultsAll animals survived to complete the entire protocol with no signs of disability or distress. Three animals had hematuria (pink urine without clots). Baseline PT values (mean, 16.0 seconds) were elevated to 22.0 seconds (37.5% above baseline, P = .003) on the day of treatment and to 28.8 seconds (77.8% above baseline, P < .001) on the day of necropsy. At the time of treatment, 5 of 8 (63%) animals were at a therapeutic anticoagulation level, and all 8 animals (100%) reached therapeutic levels by the time of necropsy. There were no cases of intraparenchymal, peritoneal, or retroperitoneal hemorrhage associated with any treatments despite 5 of 7 (71%) liver and all 8 (100%) kidney treatments extending to the organ surface. ConclusionsLiver and kidney histotripsy seems safe with no elevated bleeding risk in this anticoagulated animal model, supporting the possibility of histotripsy treatments in patients on anticoagulation.

A Critical Assessment of the Requirement for a Single Time Point Vaginal Cytology on the Day of Necropsy in Rats.

For toxicology testing of (agro)chemicals, different study types are being performed with general and/or reproductive toxicity endpoints (see Organisation for Economic Co-operation and Development guidelines). In most of these rat studies, vaginal cytology is performed on serial samples (collected by lavage) for evaluation of cycle regularity and evidence of mating, and/or on a single sample collected on the day of necropsy for information on the estrous cycle stage and allowing correlation with histopathology. In the latter case, the utility of vaginal cytology can be argued. In this article, estrous cycle stages based on vaginal cytology of samples taken on the day of necropsy and histopathology of ovaries, uterus, and vagina (gold standard for estrous cycle stage assessment) were compared. The agreement was generally low. Disagreement between the two methods could be explained by time differences between lavage and necropsy, by manipulation of vaginal epithelium during lavage which may impact epithelial morphology on histology, and by misinterpretation of vaginal cytology during or shortly after lactation. Based on the results of estrous staging within different study types, we strongly discourage vaginal cytology from samples collected on the day of necropsy since there is no added value, vaginal manipulation can be stressful and may complicate the histologic diagnosis.

Effects of the Hydnophytum formicarum plant extract on collagen density, angiogenesis, wound length, and re-epithelialization in wound healing: Experimental study on rats.

The formation of scar tissue in the wound healing process is associated with fibroblasts that are produced during the proliferation phase (3-14 days after surgery/injury). One of the strategies to suppress the formation of excessive scar tissue is to use wound care material. The use of herbal extracts is currently being investigated by researchers, as it allows avoiding the side effects of synthetic drugs. The Hydnophytum formicarum extract has antioxidant and anti-inflammatory potential. The aim of the study was to analyze the effects of the Hydnophytum formicarum plant extract on collagen density, angiogenesis, wound length, and re-epithelialization in wound healing. Twenty-four Sprague-Dawley rats were divided into 2 groups: the control group; and the treatment group. Skin wounds were made on the dorsum of the rats, using the biopsy punch technique. Four rats from each group were sacrificed on days 4, 7 and 14 after injury. Collagen density, angiogenesis, wound length, and re-epithelialization were analyzed using hematoxylin and eosin (H&E) staining and Masson's trichrome staining. There were significant differences in the results of the angiogenesis analysis, wound length and re-epithelialization between the treatment and control groups. When considering angiogenesis, there were fewer vessels in the treatment group, but they were more mature as compared to the control group. There was also a meaningful interaction between the application of the Hydnophytum formicarum extract and the necropsy day with regard to collagen density and the re-epithelialization rate. No secondary infection was found in either group. The topical use of the Hydnophytum formicarum extract affected the formation of scar tissue, as indicated by the positive area of collagen, the extent of angiogenesis, wound length, and the re-epithelialization rate in the early, middle and final granulation phases. The inhibition of angiogenesis through the application of Hydnophytum formicarum was probably related to the formation of scar tissue in the wound.

Influence of lincomycin-spectinomycin treatment on the outcome of Enterococcus cecorum infection and on the cecal microbiota in broilers

BackgroundEnterococcus cecorum (EC) is one of the main reasons for skeletal disease in meat type chickens. Intervention strategies are still rare and focus mainly on early antibiotic treatment of the disease, although there are no data available concerning the effectivity of this procedure. The present study aimed to investigate the effectivity of early lincomycin-spectinomycin treatment during the first week of life after EC-infection. Furthermore, the impact of lincomycin-spectinomycin treatment and EC infection on the development of cecal microbiota was investigated.MethodsA total of 383 day-old broiler chicks were randomly assigned to four groups (non-infected and non-treated, non-infected and treated, EC-infected and non-treated, and EC-infected and treated). The EC-infected groups were inoculated orally with an EC suspension at the day of arrival and at study day 3. The treatment groups were treated with lincomycin-spectinomycin via the drinking water for six consecutive days, starting two hours after the first inoculation. Necropsy of 20 chickens per group was performed at study days 7, 14, 21, and 42. Bacteriological examination via culture and real-time PCR was performed to detect EC in different extraintestinal organs. Cecal samples of nine chickens per group and necropsy day were analyzed to characterize the composition of the cecal microbiota.ResultsNo clinical signs or pathologic lesions were found at necropsy, and EC was not detected in extraintestinal organs of the EC-infected and treated birds. Lincomycin-spectinomycin promoted the growth of the bacterial genus Escherichia/Shigella and reduced the amount of potentially beneficial Lactobacillus spp. in the ceca regardless of EC-infection. Unexpectedly, the highest abundances of the genus Enterococcus were found directly after ending antibiotic treatment in both treatment groups, suggesting the growth of resistant enterococcal species. EC was not detected among the most abundant members of the genus Enterococcus. Oral EC-infection at the first day of life did not influence the development of cecal microbiota in the present study.ConclusionsLincomycin-spectinomycin treatment during the first week of life can prevent the EC-associated disease in broiler type chickens and has a direct impact on the development of the cecal microbiota. The low abundance of EC in the ceca of infected chickens underlines the pathogenic nature of the disease-causing EC strains. Further research on alternative prevention and intervention strategies is needed with regard to current efforts on reducing the use of antibiotics in livestock animals.

Dimethyl Fumarate, an Approved Multiple Sclerosis Treatment, Reduces Brain Oxidative Stress in SIV-Infected Rhesus Macaques: Potential Therapeutic Repurposing for HIV Neuroprotection.

Dimethyl fumarate (DMF), an antioxidant/anti-inflammatory drug approved for the treatment of multiple sclerosis, induces antioxidant enzymes, in part through transcriptional upregulation. We hypothesized that DMF administration to simian immunodeficiency virus (SIV)-infected rhesus macaques would induce antioxidant enzyme expression and reduce oxidative injury and inflammation throughout the brain. Nine SIV-infected, CD8+-T-lymphocyte-depleted rhesus macaques were studied. Five received oral DMF prior to the SIV infection and through to the necropsy day. Protein expression was analyzed in 11 brain regions, as well as the thymus, liver, and spleen, using Western blot and immunohistochemistry for antioxidant, inflammatory, and neuronal proteins. Additionally, oxidative stress was determined in brain sections using immunohistochemistry (8-OHdG, 3NT) and optical redox imaging of oxidized flavoproteins containing flavin adenine dinucleotide (Fp) and reduced nicotinamide adenine dinucleotide (NADH). The DMF treatment was associated with no changes in virus replication; higher expressions of the antioxidant enzymes NQO1, GPX1, and HO-1 in the brain and PRDX1 and HO-2 in the spleen; lower levels of 8-OHdG and 3NT; a lower optical redox ratio. The DMF treatment was also associated with increased expressions of cell-adhesion molecules (VCAM-1, ICAM-1) and no changes in HLA-DR, CD68, GFAP, NFL, or synaptic proteins. The concordantly increased brain antioxidant enzyme expressions and reduced oxidative stress in DMF-treated SIV-infected macaques suggest that DMF could limit oxidative stress throughout the brain through effective induction of the endogenous antioxidant response. We propose that DMF could potentially induce neuroprotective brain responses in persons living with HIV.

Role of JAK-STAT Pathway in Broiler Chicks Fed with Chestnut Tannins.

Simple SummaryCurrent bans on the use of antibiotics in livestock feed have led to increasing demand for alternatives to antibiotics (ATA). One popular alternative is chestnut tannins (ChT). While there is growing evidence of the immune benefits of using phytobiotics, such as ChT, there is currently minimal information on the effect of tannins on the immune pathway of the host. A previous study discovered a large upregulation of IL-6 in broiler chicks at day 6 when provided with 1% ChT from hatch. In regard to potential immune modulation, ChT appear to influence host immunity via an IL-6 mediated response, which could be beneficial in host defenses against pathogens at the early stages of broiler growth and development. A critical pathway identified in the regulation of the immune system is the JAK-STAT signaling pathway. The role of JAK-STAT pathway is altered by the addition of ChT in the diet. By demonstrating the changes in the kinome of the broiler model, the information in this study will provide further insights into potential ATA to improve poultry health.The objective of this study was to identify the phosphorylation events associated with host immunity with the inclusion of chestnut tannins (ChT) in the diet. A total of 200 male day-of-hatch Cobb 500 chicks were randomly assigned to two treatment groups, totaling 50 chicks per pen per experiment (this study was repeated two times). The treatments were as follows: (1) control feed—normal starter feed (n = 50), and (2) 1% ChT inclusion feed (n = 50). The ceca were collected on each necropsy day for analysis via (1) a peptide array to provide tissue immunometabolism information from the host, and (2) quantitative PCR for mRNA expression. Of the top three immune pathways, the data identified the T-cell receptor signaling pathway, the chemokine signaling pathway, and the JAK-STAT signaling pathway. The results showed significantly altered phosphorylation of JAK and STAT peptides within the JAK-STAT pathway. These results support the mRNA expression data with the upregulated IL-6 response, due to the significant phosphorylation of IL6ST, JAK, and STAT peptides. In regard to immune modulation, ChT appear to influence host immunity via an IL-6 mediated response which could be beneficial in host defenses against pathogens at the early stages of broiler growth and development. Therefore, it is suggested that the role of the JAK-STAT pathway is altered by including ChT in the diet.

Safety assessment of fermented jackfruit (Artocarpus heterophyllus) pulp and leaves in Sprague-Dawley rats.

Fermented jackfruit (Artocarpus heterophyllus) extracts were produced using pure symbiotic culture of bacteria and yeast (SCOBY) under controlled fermentation process. Both female and male Sprague‐Dawley rats were orally administrated with 4,000 mg/kg of fermented jackfruit pulp and leaves extracts for 28 consecutive days. Body weight of rats was recorded at 1‐week interval until necropsy day. There was no mortality reported along the experiment with no significant differences (p > .05) record among organ histopathology and blood biochemical parameters in treated groups when compared to control group. Interestingly, there were significant differences (p < .05) in the lower body weight gained of treated rats groups as opposed to control group, indicating the potential anti‐obesity effect of fermented jackfruit extracts. In conclusion, no toxicity symptoms were observed in 28 days oral administration toxicity study of fermented jackfruit pulp and leaves extracts in Sprague‐Dawley rats for both sexes.

Evaluation of heat-treating heartworm-positive canine serum samples during treatment with Advantage Multi\xae for Dogs and doxycycline

BackgroundThe use of heat-treatment in canine and feline serum has been hypothesized to break the formation of antigen-antibody complexes, thereby freeing the heartworm antigen allowing for detection by commercially available heartworm antigen kits. While studies have analyzed the effect of heat-treating serum and plasma samples in the detection of heartworm antigen, these studies have not utilized necropsy verified results for validation. This study evaluated the use of heat-treating serum samples in experimentally infected dogs during adulticidal treatment in comparison with necropsy adult heartworm recovery.MethodsAs part of a primary study, a total of 16 dogs were experimentally infected with 16 sexually mature adult heartworms using surgical transplantation, allocating 8 dogs in both the control and treated group. Treated dogs received 10 months of topical administration of Advantage Multi® for Dogs (10% Imidacloprid + 2.5% Moxidectin) every 4 weeks and 30 days of 10 mg/kg doxycycline BID. Blood samples were collected from all study animals prior to surgical transplantation of adult heartworms, on study days 0, 1, 3, 7, 14, 21, 28, and every 4 weeks thereafter for the duration of this study. Concentration of heartworm antigen was tested using the DiroCHEK® heartworm antigen test kit using serum samples both pre- and post-heat-treatment. Serum samples were heat-treated at 103 °C in a dry heat block for 10 min and centrifuging at 1818× g for 20 min.ResultsThere were a total of 4 instances (days 56, 140, 224 and 252) in 3 treated dogs in which a serum sample converted from negative for the detection of heartworm antigen prior to heat-treatment to positive for the detection of heartworm antigen post-heat-treatment. At necropsy, these dogs had no adult heartworms recovered and were all negative on antigen testing prior to and after heat treatment. There was 100% accuracy in the detection of either no infection, or 1–2 adult heartworm infections using the DiroCHEK in serum samples with and without heat-treatment at the time of necropsy.ConclusionsThe DiroCHEK accurately diagnosed all dogs with live adults recovered at necropsy as heartworm antigen positive and all those dogs with no live adults recovered at necropsy as heartworm antigen negative without the use of heat-treatment for samples taken on the day of necropsy. Therefore, these results indicate that the use of heat-treating serum samples did not provide data of any additional value in the diagnosis of heartworm-positive dogs receiving treatment in this study. Additionally, these results may indicate that the conversion of serum samples from negative to positive for the presence of heartworm antigen with heat-treatment may not always accurately diagnose live adult heartworm infections since no adult heartworms were recovered at necropsy for those dogs in which a conversion event occurred. These conversion events may be detecting residual antigen leftover after all adult worms have died or may even be detecting off- target antigens, which have been denatured during heat-treatment. While a necropsy was not performed at the time of the conversion events, no live adult worms were recovered from any of the dogs in which a conversion event occurred earlier in treatment.

Novel Observations of Peroxiredoxin-2 Profile and Protein Oxidation in Skeletal Muscle from Pigs that Differ In Residual Feed Intake and Health Status

ObjectivesEfficiency of pork production is critical as demand for animal protein rises with increases in global populations and income. Feed efficiency and response to disease challenge are affected by stressors that increase reactive oxygen species in muscle. Reactive oxygen species are detrimental to growth, as they require the expenditure of energy for tissue repair that could be used for weight gain. Peroxiredoxin-2 (Prdx-2) is an antioxidant enzyme that converts hydrogen peroxide to water using reactive cysteines, thus mitigating oxidation. Prdx-2 can exist in multiple oxidation states (reduced, oxidized, and hyperoxidized) and quantenary structures (dimer and decamer). Prdx-2 concentration is greater in more color stable muscles and may be an indicator of tenderness. The objective of this study was to determine differences in Prdx-2 profile and protein oxidation between pigs that differed in feed efficiency and health status. Materials and MethodsPigs selected for differing feed efficiency based on residual feed intake (RFI) were used in a 2 × 2 factorial design for this study. At 50 ± 7 kg in body weight, high RFI (less efficient) and low RFI (more efficient) barrows were distributed between 2 rooms in the same building. One room was inoculated with a dual respiratory/enteric bacterial health challenge. At 21 d post-infection (projected peak of illness), pigs were necropsied (total = 24, n = 6 per group) and longissimus muscle samples were collected. Reduced and nonreduced sarcoplasmic protein samples (β-mercaptoethanol) were used for western blot analyses. Reduced samples were used to quantify total Prdx-2, while nonreduced samples were used to evaluate hyperoxidized peroxiredoxin, Prdx-2 decamer, and Prdx-2 profile in nonreducing gels (2 distinct bands analyzed). Carbonyl content and diagonal gel electrophoresis were performed on sarcoplasmic protein samples to determine protein oxidation. Statistical analysis was performed using SAS v. 9.4 (SAS Inst. Inc., Cary, NC) with fixed effects of RFI line, infection status, and RFI line × infection status interaction. Gel repetition and necropsy day were included as random effects. ResultsInfection status was significant for the Prdx-2 band comparison in nonreducing gels (P = 0.014) indicating potential differences in posttranslational modifications. Compared to low RFI pigs, high RFI pigs had greater total Prdx-2 (P = 0.035), greater Prdx-2 decamer (P = 0.0007), greater Prdx-2 in the second band of nonreducing gels (P = 0.0006), and less hyperoxidized peroxiredoxin relative to the total immunoreactive protein (P = 0.028). An interaction existed between RFI line and infection status for the Prdx-2 band comparison (P = 0.02), with high RFI challenged pigs having greater ratios, and high RFI control pigs having lesser ratios relative to low RFI pigs. Visual analysis of diagonal gels showed more proteins with intermolecular disulfide formation in low RFI muscle samples. No significant difference was seen for carbonyl content. ConclusionDifferences exist in Prdx-2 profile based on RFI line and infection status. High and low RFI pigs may respond differently to health challenges at the molecular level based on differences in antioxidant protein response. By defining Prdx-2 in livestock muscle, we may be better able to manage animals to meet demand for meat without increasing inputs.

Pre-clinical toxicology considerations for vaccine development.

Vaccine development requires pre-clinical toxicology studies, following good laboratory practice (GLP), before first in human (phase I) use. Many factors are critical in the final outcome of any pre-clinical toxicology study. The study design is one of these critical factors and should be carefully planned to avoid any false negative and/or false positive results. Preparation is another most critical factor in a successful study. Major changes in any procedure during the course of study should be avoided by all means. For example, if the protocol specified the tail as the site of blood collection and this procedure was used for the control group at the day of necropsy, this collection site should never be replaced by another site (e.g. foot, eye, or heart) in all other treatment groups. Food restrictions and acute restraint stress affect clinical pathology data and should be avoided in rodents. Institutional Animal Care and Use Committee (IACUC) guidelines for frequent blood collections (weekly, monthly, or at necropsy) in any animal species should be strictly followed. Clinical pathology data will be profoundly affected by any diversion from the recommended volumes. If CO2 is specified in the protocol for anesthesia and/or euthanasia, ensuring enough quantity to use for all groups at necropsy is a very important factor. Using two different anesthetics in any study (e.g. CO2 vs. pentobarbital) may result in false positive or false negative results in clinical chemistry parameters. Quality assurance elements (SOPs, instrument validation, lab certification etc.) affect the data interpretation and the final outcome of any toxicology study. SOPs should be up to date and written clearly. All lab instruments should be validated and all laboratories should be certified.

Salmonella Persistence within the Peripheral Lymph Nodes of Cattle following Experimental Inoculation

Utilizing a transdermal method of inoculation developed in our laboratory, the duration of infection of Salmonella in the peripheral lymph nodes of steers was examined. Thirty-six Holstein steers (mean body weight of 137 kg) were inoculated with Salmonella Montevideo (day 0) on each lower leg and both sides of the back and abdomen. Calves were euthanized beginning at 6 h and subsequently on each of days 1, 2, 4, 7, 9, 11, 14, and 21 postinoculation (four animals each time). The subiliac, popliteal, and superficial cervical (prescapular) lymph nodes were collected and cultured (quantitatively and qualitatively) for the challenge strain of Salmonella. The challenge strain was detected via direct culture within the lymph nodes at 6 h postinoculation and on each subsequent necropsy date. Salmonella levels in lymph node were 0.8 to 1.8 log CFU/g. Lymph nodes were generally positive after enrichment culture throughout the experiment. Salmonella elimination appeared to begin approximately 14 days postinoculation. However, elimination was not completed by day 21; therefore, a second experiment was conducted identical to the first except that the time from inoculation to necropsy was extended. Salmonella was recovered via direct culture on each of the necropsy days, and results in general were similar to those of experiment I, except that on days 20, 24, and 28 isolates from serogroups C2 and E1 were identified in addition to the inoculation strain C1 in multiple animals. The data from both experiments indicate that after a single inoculation event, Salmonella would be completely cleared by approximately 28 days. Further research with expanded times between inoculation and necropsy is required for verification.

Comparison of innate and Th1-type host immune responses in Oesophagostomum dentatum and Trichuris suis infections in pigs.

This study investigated the details of the innate and Th1/Treg-type-associated host immune responses in Trichuris suis and Oesophagostomum dentatum mono- and co-infected pigs and invitro in stimulated porcine dendritic cell cultures. Forty-eight pigs were allocated into a 2-factorial design with two groups trickle-inoculated with 10 T.suis eggs/kg/day (Group T) or 20 O.dentatum L3/kg/day (O). Another group (OT) was infected with both parasites. Group C remained uninfected. Expression of innate and Th1/Treg-cell-associated genes in gut mucosa and associated lymph nodes was determined by qPCR at necropsy day 35 and 71. Gene expression showed suppressed/inhibited Th1 and Treg-type immune reactions, in accordance with previous findings of a predominant Th2-type immune response to both nematodes. The invitro part examined the production of TNF-α in porcine dendritic cells (DC) exposed to T.suis and/or O.dentatum excretory/secretory (E/S) products. Further, binding capacity and structure of E/S products were characterized. Glycan and lectin-binding capacity were generally lower in O.dentatum E/S products compared to T.suis which may explain the earlier found weaker Th2 response to the former. Surprisingly, O.dentatum E/S products induced a significant (P<0·0001) increase in TNF-α DC production, potentially indicating a new mode of helminth-host immune response interaction.

Comparative efficacy of experimental inactivated and live-attenuated chimeric porcine circovirus (PCV) 1-2b vaccines derived from PCV1 and PCV2b isolates originated in China

BackgroundPorcine circovirus type-2b (PCV2b) is recognized as the etiological agent of the various clinical manifestations of porcine circovirus-associated disease (PCVAD). Previous studies have demonstrated effectiveness of chimeric PCV1-2 vaccines against PCV2b challenge. In this study, the efficacy of inactivated and live-attenuated (2 × 103.5 or 2 × 104.0 50 % tissue culture infective dose [TCID50] dose) chimeric PCV1-2b vaccines was compared side-by-side in conventional pigs.MethodsTwenty-seven non-PCV2 viremic pigs without PCV2-specific antibody were randomly divided into six groups, including four vaccinated and challenged groups, a nonvaccinated challenged group, and a mock group. All pigs except those in the mock group were challenged at 28 days post vaccination (DPV) using PCV2b.ResultsBoth inactivated and live-attenuated chimeric PCV1-2b vaccines induced a robust antibody responses, and significantly decreased microscopic lesion and lower viral loads in serum or superficial inguinal lymph nodes (SILN) compared with that in the nonvaccinated challenged group. PCV2 antibody titers decreased after 7 days post challenge (DPC) in pigs administered the inactivated PCV1-2b vaccine and they were lower than those in pigs inoculated with live-attenuated PCV1-2b on the day of necropsy. Moreover, no viremia was present in pigs inoculated with live-attenuated PCV1-2b vaccine at 21 DPC regardless of the dose difference.ConclusionsThe results demonstrated that both inactivated and live-attenuated chimeric PCV1-2b vaccines were effective to induce protective immunity against PCV2b infection.

Neutrophil gelatinase-associated lipocalin, a sensitive urinary biomarker of acute kidney injury in dogs receiving gentamicin

A sensitive urinary biomarker for acute kidney injury (AKI) was investigated in beagle dogs with nephrotoxicity induced by gentamicin. Gentamicin sulphate at 25 or 50 mg/kg was injected (s.c.) for 9 days, and conventional urinalysis, ELISA assay of neutrophil gelatinase-associated lipocal (NGAL) in urine, blood chemistry, and pathological examinations were performed. The dog given gentamicin at 25 mg/kg only showed slight deposition of lysosomal granules in the proximal tubular epithelium of the kidneys without any other significant changes even though urinary NGAL was elevated on Day 10 (day of necropsy). In the dog receiving gentamicin at 50 mg/kg, increases in urinary NGAL were observed on Days 3 and 5, and absence of urination, marked increases in serum urea nitrogen and creatinine, enlargement and discoloration of the kidneys with marked necrosis, and swelling of proximal epithelium were observed. In conclusion, urinary NGAL is considered to be a candidate as a sensitive predictable biomarker of AKI in the gentamicin-induced nephrotoxicity model in dogs.

Increased viral load and prevalence of Torque teno sus virus 2 (TTSuV2) in pigs experimentally infected with classical swine fever virus (CSFV)

Torque teno sus viruses (TTSuVs) are considered non-pathogenic viruses, although lately they have been linked to porcine circovirus diseases, mainly with post weaning multisystemic wasting syndrome (PMWS). These associations point out a possible pathogenic role of TTSuVs or, alternatively, that TTSuV replication is up-regulated under disease conditions. In order to further explore the association of TTSuVs with disease occurrence, TTSuVs prevalence and viral load were assessed before and after an experimental infection with a highly pathogenic classical swine fever (CSF) virus (CSFV) isolate. Serum samples from 56 animals were analyzed by means of a real time quantitative PCR (qPCR) for TTSuV1 and TTSuV2 before and after (between 6 and 13 days post-inoculation) the CSFV challenge. Based on the post-infection clinical evolution and immune response against CSFV, the animals were divided into two groups: group I, with protecting immunity against CSFV and no clinical signs at the day of necropsy, and group II, with no detectable immune response against CSFV and moderate to severe clinical signs. TTSuVs qPCR results indicated that TTSuV2 and not TTSuV1 load in serum increased significantly after challenge with CSFV in the group of pigs with clinical signs, specifically in those with a moderate course of the disease. Therefore, this study emphasizes the different behaviour of both TTSuVs, as already found in the PMWS background, and further supports the association of TTSuV2 with disease occurrence.