At least three different EV-71 subgenotypes were identified from an outbreak in Malaysia in 1998. The subgenotypes C2 and B4 were associated with the severe and fatal infections, whereas the B3 virus was associated with mild to subclinical infections. The B3 virus genome sequences had >= 85% similarity at the 3' end to CV-A16. This offers opportunities to examine if there are characteristic similarities and differences in virulence between CV-A16, EV-71 B3 and EV-71 B4 and to determine if the presence of the CV-A16-liked genes in EV-71 B3 would also confer the virus with a CV-A16-liked neurovirulence in mice model infection.
Rotaviral infections in cynomolgus monkeys (Macaca fasicularis) were studied to ascertain its suitability as a model of infection and diarrhea caused by group A human rotaviruses. Formula-fed monkeys were used as they could be observed closely. Experimental rotaviral infection of cynomolgus monkeys was age-dependent; only young monkeys were readily infected. Formula-fed newborns were readily infected with cell-culture-adapted human (WA) and simian (SA11) viruses and with a rotavirus from a human fecal specimen. However, diarrhea was detected only in very young animals. A number of rotaviral shedding patterns as a function of time were observed. Although there was no typical viral shedding pattern which represented exclusive association of viral infection with diarrhea, the initial level of viral excretion and the maximum level of viral shedding attained were much higher in animals with diarrhea. Seroconversion occurred in less than half of the inoculated animals. The presence of maternal rotaviral antibodies did not prevent infection or diarrhea.
In order to attempt isolate the protozoan parasite Neospora caninum, an N. caninum seropositive pregnant Sahiwal Friesian cross heifer from a large-scale dairy farm in Malaysia was kept for observation until parturition at the Veterinary Research Institute, Ipoh. The heifer gave birth to a female calf that was weak, underweight and unable to rise. Precolostral serum from the calf had an N. caninum indirect fluorescent antibody test titre of 1:3200. It died 12 h after birth and necropsy was performed. Brain homogenate from the calf was inoculated into 10 BALB/c mice that were kept for 3 months after which brain tissue from the mice was inoculated onto 24 h fresh monolayer Vero cell lines. The cell cultures were examined daily until growth of intracellular protozoa was observed. DNA of the organisms from the cell cultures was analyzed by PCR and DNA sequencing. DNA fragments of the expected size were amplified from the isolate using N. caninum-specific primers, and sequence analysis of ITS1 clearly identified the isolate as N. caninum. This is the first successful isolation of N. caninum from a bovine in Malaysia, and the isolate is designated Nc-MalB1.
Fetal alcohol syndrome (FAS), a condition occurring in some children of mothers who have consumed alcohol during pregnancy, is characterized by craniofacial malformations, and physical and mental retardation. It is significant that even children with history of gestational ethanol exposure but relatively unaffected overall IQ performance, often exhibit learning difficulties and behavioral problems, suggestive of impaired memory formation. Hence, the specific aim of this study was to examine memory formation in chicks exposed to ethanol during early gestation toward the understanding of neurobehavioral disturbances in FAS. Chicks were exposed to alcohol on gestational days 1-3 by injection of ethanol into the airspace of freshly fertilized eggs. The effects of prenatal ethanol on physical growth and development, and memory formation were studied. The one-trial passive avoidance learning paradigm in 1-day-old chicks was used to study memory formation in these chicks. It was observed that chick embryos exposed to 10% ethanol on gestational days 1-3 had significant reduction in all body parameters when compared with appropriate controls. Further, ethanol-exposed chick embryos had significantly impaired (P
Adeno-associated virus (AAV)-based gene therapy is gaining popularity owing to its excellent safety profile and effective therapeutic outcomes in a number of diseases. Intravenous (IV) injection of AAV into the tail vein, facial vein and retro-orbital (RO) venous sinus have all been useful strategies to infuse the viral vector systemically. However, tail vein injection is technically challenging in juvenile mice, and injection at young ages (≤ postnatal day-(P)21) is essentially impossible. The temporal or facial vein is localized anterior to the ear bud and is markedly visible in the first couple of days postnatally. However, this method is age-dependent and requires a dissecting microscope. Retro-orbital injection (ROI), on the other hand, is suitable for all murine ages, including newborn and older mice, and is relatively less stressful to animals compared to tail vein injection. Although many reports have shown ROI as an effective route of AAV delivery, herein we aim to highlight and summarize the methods and benefits of ROI. To capture the full spectrum of transduction efficiency mediated by ROI, we transduced the editing-dependent reporter mice (Ai9 Cre reporter mice) with the AAV9 vector, which targets a wide range of peripheral tissues with exceptional brain tropism. We also provide a comprehensive description of the ROI technique to facilitate viral vector administration without complications.
Twelve goats were inoculated with 40,000 third-stage Haemonchus contortus larvae and two were killed on each of Days 4, 7, 11, 14, 18 and 21 after inoculation (DAI). The number of worms that established, and the site of development were recorded. More worms established in the fundic, than in the middle or pyloric thirds of the abomasum. Early development occurred within the mucosa; emergence into the lumen started between 7 and 11 days after infection. By 4 DAI, all worms had completed the third moult to the L4 stage. At 11 DAI the majority of the worms were adults. A mean of 13.2% of the female worms had eggs in their uteri at 18 DAI; by 21 DAI more than half of the female worms had eggs in their uteri. The development of H. contortus was essentially similar to that described in sheep.
Twelve goats were inoculated with 20,000 infective larvae of Trichostrongylus colubriformis and two were killed on each of Days 4, 7, 11, 14, 18 and 21 after inoculation (DAI). The number of worms that established, and the site of development were recorded. Most of the worms established within the first 3 m of the small intestine. There was little relocation or loss of nematodes after establishment. The worms started to migrate from the mucosa to the lumen 11 days after infection. At 4 DAI, 63% of the worms were still at the late L3 stage; the remainder of the worm population had completed the third moult to the L4 stage. The population at 11 DAI comprised largely young adults. When 21 DAI was reached, about 57% of the female worms had eggs in their uteri.
The objective of this study was to study the possible reproductive adverse effects of the diazinon on rat offspring exposed in utero and during lactation. Dams were gavaged daily (10, 15, and 30 mg/kg) before mating, during mating, and during pregnancy and lactation in separate groups. Reproductive outcome data of dams were examined. Body weight, testis weight, testicular marker enzyme activities (alkaline phosphatase, acid phosphatase, lactate dehydrogenase, and glucose-6-phosphate dehydrogenase), qualitative and quantitative testicular and epididymal histology, and immunohistochemisty for 3-β-hydroxysteroid dehydrogenase (HSD) were examined in male offspring at puberty and adulthood. The 30-mg/kg dose induced significant adverse effects at both puberty and adulthood in offspring. At puberty the male offspring showed a decrease in testicular weight, degenerative changes, and 3-β-HSD. Moreover, an increase in activity of alkaline and acid phosphatase also was observed. At adulthood, there was a decrease in testicular weight and 3-β-HSD with an increase in the levels of testicular marker enzyme. There was evidence of some adverse reproductive effects in male offspring at the 15-mg/kg dose. Most of the adverse effects were irreversible and were evident at both puberty and adulthood in offspring, although a few parameters reverted back to the normal growth pattern. Hence, diazinon is a reproductive toxicant in male offspring, which caused significant damage to the testes when exposed during prenatal and postnatal life.
Cytokines are extremely potent biomolecules that regulate cellular functions and play multiple roles in initiation and inhibition of disease. These highly specialised macromolecules are actively involved in control of cellular proliferation, apoptosis, cell migration and adhesion. This work, investigates the effect of transforming growth factor-beta2 (TGF-β2) on the biological regulation of chondrocyte and the repair of a created model wound on a multilayer culture system. Also the effect of this cytokine on cell length, proliferation, and cell adhesion has been investigated. Chondrocytes isolated from knee joint of rats and cultured at 4 layers. Each layer consisted of 2 × 105 cells/ml with and without TGF-β2. The expression of mRNA and protein levels of TGF-β receptors and Smad1, 3, 4, and 7 have been analysed by RT-PCR and western blot analysis. The effect of different supplementations in chondrocyte cell proliferation, cell length, adhesion, and wound repair was statistically analysed by One-way ANOVA test. Our results showed that the TGFβ2 regulates mRNA levels of its own receptors, and of Smad3 and Smad7. Also the TGF-β2 caused an increase in chondrocyte cell length, but decreased its proliferation rate and the wound healing process. TGF-β2 also decreased cell adhesion ability to the surface of the culture flask. Since, TGF-β2 increased the cell size, but showed negative effect on cell proliferation and adhesion of CHC, the effect of manipulated TGF-β2 with other growth factors and/or proteins needs to be investigated to finalize the utilization of this growth factor and design of scaffolding in treatment of different types of arthritis.
Introduction: Doppler mode ultrasound is widely used in prenatal scanning and known to produce a higher acoustic
output which later leads to higher heat energy conversion compared to other ultrasound modes. It has been reported
that the use of Doppler imaging might increase the temperature of tissues, thus, when Doppler is used in combination with 2D ultrasound, the risks of bioeffects tend to increase more. It is also known that prolonged exposure to
ultrasound during pregnancy can cause irreversible biological destructions to the fetus. Despite the benefits of using
Doppler ultrasound, its potential adverse effects have received scant attention in the research literature. Therefore,
this study aimed to examine a correlation between gestational stages (GS) and newborn rabbit’s body weight at different prenatal Doppler ultrasound exposure durations. Methods: Twelve pregnant New Zealand white rabbits (NZWR)
were exposed once using three different Doppler ultrasound exposure durations (30, 60, 90 minutes exposure) at
three different GSs (1st, 2nd, and 3rd GS). After delivery, the mean weights of the 62 newborns were statistically analysed. Results: Strong negative and positive correlation between newborn’s body weight at different GSs and Doppler
ultrasound exposure durations with a significant result found in 60 minutes exposure (p =
It has long been known that spatial memory and the ability to navigate through space are sexually dimorphic traits among mammals, and numerous studies have shown that these traits can be altered by means of sex hormone manipulation. Hippocampus, the main organ involved in this kind of memory, has specific signature genes with high expression level compared to other regions of the brain. Based on their expression levels and the role that products of these genes can play in processes like signal transduction, mediation of hormone effects and long term potentiation, these genes can be considered as genes necessary for routine tasks of hippocampus. Male and female rat pups were injected with estradiol and testosterone respectively. at early stage of their lives to examine the effect of sex hormone manipulation on mRNA expression of Slc9a4, Nr3c2, Htr5b and Mas1 using comparative quantitative real-time polymerase chain reaction. The results showed that expressions of these genes are strongly influenced by sex hormones in both the frontal cortex and hippocampus, especially in male hippocampus, in which expression of all genes were up-regulated. Htr5b was the only gene that was affected only in the males. Expression of Mas1 was contrary to expectations, showed stronger changes in its expression in cortex than in hippocampus. Nr3c2 was down regulated in all samples but up regulated in male hippocampus, and Slc9a4 also showed a huge up-regulation in male hippocampus compared to other samples.
The aim of this study was to investigate the changes in parathyroid hormone (PTH) level of rabbit foetal bodies exposed to ultrasound at different gestational stages. A total of 9 pregnant rabbits (Oryctolagus cuniculus) were insonated for 60 minutes at the middle of 1(st), 2(nd) and 3(rd) gestational stages for group A (n=14 newborns), group B (n=7 newborns) and group C (n=24 newborns) respectively. Seven pregnant rabbits with 41 newborns severed as negative control group. Blood samples were withdrawn from each newborn rabbits for Parathyroid Hormone-Intact (PTH-I) test. Results of the independent samples t-test implied statistically significant differences (P<0.05) between the control group and the 1(st) stage (P=0.001), the 2(nd) stage (P<0.001) and the 3(rd) stage group (P<0.001). This in-vivo study revealed diagnostic ultrasound heating has the potential of affecting foetal PTH level. This study observed significantly low PTH level for all the treated groups. A further study should be instituted to determine whether this finding in rabbit may also occur in human by means of clinical trials.
Ectopic implantation of donor testis cell aggregates in recipient mice results in de novo formation or regeneration of testis tissue and, as such, provides a unique invivo model for the study of testis development. However, currently the results are inconsistent and the efficiency of the model remains low. This study was designed to: (1) examine several factors that can potentially improve the consistency and efficiency of this model and (2) explore the use of ultrasound biomicroscopy (UBM) for the non-invasive invivo evaluation of implants. Testis cell aggregates, containing ~40% gonocytes, from 1-week-old donor piglets were implanted under the back skin of immunodeficient mice through skin incisions using gel matrices or through subcutaneous injection without using gel matrices. The addition of gel matrices led to inconsistent tissue development; gelatin had the greatest development, followed by collagen, whereas agarose resulted in poor development. The results also depended on the implanted cell numbers since implants with 100×106 cells were larger than those with 50×106 cells. The injection approach for cell implantation was less invasive and resulted in more consistent and efficient testis tissue development. UBM provided promising results as a means of non-invasive monitoring of implants.
MicroRNAs are small non-coding RNAs that play crucial roles in the regulation of gene expression and protein synthesis during brain development. MiR-3099 is highly expressed throughout embryogenesis, especially in the developing central nervous system. Moreover, miR-3099 is also expressed at a higher level in differentiating neurons in vitro, suggesting that it is a potential regulator during neuronal cell development. This study aimed to predict the target genes of miR-3099 via in-silico analysis using four independent prediction algorithms (miRDB, miRanda, TargetScan, and DIANA-micro-T-CDS) with emphasis on target genes related to brain development and function. Based on the analysis, a total of 3,174 miR-3099 target genes were predicted. Those predicted by at least three algorithms (324 genes) were subjected to DAVID bioinformatics analysis to understand their overall functional themes and representation. The analysis revealed that nearly 70% of the target genes were expressed in the nervous system and a significant proportion were associated with transcriptional regulation and protein ubiquitination mechanisms. Comparison of in situ hybridization (ISH) expression patterns of miR-3099 in both published and in-house-generated ISH sections with the ISH sections of target genes from the Allen Brain Atlas identified 7 target genes (Dnmt3a, Gabpa, Gfap, Itga4, Lxn, Smad7, and Tbx18) having expression patterns complementary to miR-3099 in the developing and adult mouse brain samples. Of these, we validated Gfap as a direct downstream target of miR-3099 using the luciferase reporter gene system. In conclusion, we report the successful prediction and validation of Gfap as an miR-3099 target gene using a combination of bioinformatics resources with enrichment of annotations based on functional ontologies and a spatio-temporal expression dataset.
Dermal exposure to organophosphate pesticide is important because of its popular use. This study planned to compare the changes in serum acetylcholinesterase, paraoxonase and neuronal density of hippocampus and iso-cortex between two age groups of Swiss albino mice (18-day-old and 150-day-old) after dermal application of (1/2) LD50 of chlorpyrifos for 14 days. Statistically significant reduction was observed in serum acetylcholinesterase (Mann-Whitney test, p<0.05) and neuronal density (Independent samples t-test, p<0.05) in exposed groups compared to the control. The reduction in serum AChE and neuronal density was more pronounced in exposed adult mice than in exposed neonatal mice. The paraoxonase level was insignificant in control neonatal mice, whereas it was 890-fold more in exposed neonatal mice. Upregulated paraoxonase levels may be extrapolated to produce relatively lower reduction of cholinesterase and neuronal density in neonatal mice.
The aim of this research was to determine the effects of Momordica charantia (MC) fruit aqueous extract on pancreatic histopathological changes in neonatal STZ-induced type-II diabetic rats. Diabetes mellitus was induced in one day Sprague-Dawley neonatal rats using a single intrapretoneal injection of streptozotocin (STZ) (85 mg/kg body weight) and monitored for 12 weeks thereafter. The diabetic rats were separated into three groups, as follows: the diabetic control group (i.e. nSTZ), the diabetic group (i.e. nSTZ/M) - which was orally given 20 mg/kg of MC fruit extract, and the diabetic group (i.e. nSTZ/G) - that was treated with glibenclamide, 0.1 mg/kg for a period of four weeks. At the end of treatment, the animals were sacrificed and blood samples were collected from the saphenous vein to measure the blood glucose and serum insulin level. The pancreatic specimens were removed and processed for light microscopy, electron microscopy examination and immunohistochemical study. The results of this study showed that MC fruit aqueous extract reduced the blood glucose level as well as glibenclamide and increased the serum insulin level in the treated diabetic rats (P<0.05). The fruit extract of MC alleviated pancreatic damage and increased the number of β-cells in the diabetic treated rats (P<0.05). Our results suggest that oral feeding of MC fruit extract may have a significant role in the renewal of pancreatic β-cells in the nSTZ rats.
Immunohistochemistry is a histological technique that allows detection of one or more proteins of interest within a cell using specific antibody binding, followed by microscopic visualization of a chromogenic substrate catalyzed by peroxidase and/or alkaline phosphatase. Here, we describe a method to localize Chikungunya virus (CHIKV) antigens in formalin-fixed and paraffin-embedded infected mouse brain.