A short review on behavioural assessment methods in rodents.

The rodent behavioural examination techniques are used to assess various psychological, neurological models and neurotoxicity studies. Therefore, it is of interest to document the various behavioural assessment methods used in rodent model to study the motor, sensory, cognitive functions and emotional behaviour.

Potency of Veterinary Rabies Vaccines Marketed in Sri Lanka.

Seven brands of veterinary rabies vaccines are commercially available in Sri Lanka, but there is no established procedure to test the potency of the vaccines at the local level, especially prior to their release. The aim of this study was to test the potency of these vaccines using a mouse challenge test in collaboration with the EU/WOAH/WHO Reference Laboratory for Rabies, ANSES-Nancy, France. Based on the European Pharmacopoeia, the inactivated rabies vaccines complied with the mouse potency test if the estimated potency is ≥1.0 IU in the smallest prescribed dose. Among the eight tested vaccines, four single-dose preparations (Rabisin™, Raksharab™, Nobivac™ RL, and Nobivac™ Rabies) were compliant, with potencies of 12 IU/dose, 7.2 IU/dose, 4.4 IU/dose, and 3.4 IU/dose, respectively. Three of the single-dose preparations (Canvac™ R, Defensor™ 3, and Rabies killed vaccine) were not compliant, with potency values <1.0 IU/dose. One multidose preparation (Raksharab™ multidose) had a potency of 1.3 IU/dose, even though the test was not validated. Based on these results, it appears that some rabies vaccine batches that are currently available in the local market do not comply with the mouse potency test. Testing the vaccines' potency before registration and release to the market appears to be an important step to allow good immunization to animals during pre-exposure vaccination programs.

Detection of immunity in sheep following anti-rabies vaccination.

Purpose: Rabies is a fatal but preventable disease with proper pre-exposure anti-rabies vaccination (ARV). Dogs, as household pets and strays, are the reservoir and vector of the disease, and dog bites have been associated with human rabies cases in Sri Lanka over the past few years. However, other susceptible species having frequent contact with humans may be a source of infection. One such species is sheep and immunity following ARV has never been tested in sheep reared in Sri Lanka. Materials and Methods: We have tested serum samples from sheep reared in the Animal Centre, Medical Research Institute of Sri Lanka for the presence of anti-rabies antibodies following ARV. Sheep serum samples were tested with Bio-Pro Rabies enzyme-linked immunosorbent assay (ELISA) antibody kits used for the first time in Sri Lanka and our results were verified by a seroneutralization method on cells (fluorescent antibody virus neutralization, FAVN test) currently recommended by World Organization for Animal Health and World Health Organization. Results: Sheep received annual ARV and maintained high neutralizing antibody titers in their serum. No maternal antibodies were detected in lamb around 6 months of age. Agreement between the ELISA and FAVN test, i.e., coefficient concordance was 83.87%. Conclusion: Annual vaccination in sheep has an effect on maintaining adequate protection against rabies by measurements of anti-rabies antibody response. Lambs need to be vaccinated earlier than 6 months of age to achieve protective levels of neutralizing antibodies in their serum. Introducing this ELISA in Sri Lanka will be a good opportunity to determine the level of anti-rabies antibodies in animal serum samples.

The Capability Maturity Model as a Measure of Culture of Care in Laboratory Animal Science.

Culture of care in Laboratory Animal Science (LAS) refers to a commitment toward improving animal welfare, scientific quality, staff wellbeing, and transparency for all stakeholders, ensuring that the animals and personnel involved are treated with compassion and respect. A strong culture of care can be established by the proactive implementation of the Three Rs, sharing best practices, caring for and respecting animals and colleagues, empowering staff, taking responsibility for our actions, and having a caring leadership. Culture of care, when established, should be evaluated continuously, in order to foster its progress and persistence. Even though several tools for assessing the culture of care within an institution have been proposed, an ultimate standard for measuring the concept is lacking. Here, we review the culture of care concept and propose the 'Capability Maturity Model' as a means of quantifying culture of care in the laboratory animal setting.

Severe acute kidney injury following Sri Lankan Hypnale spp. envenoming is associated with thrombotic microangiopathy.

CONTEXT: Acute kidney injury (AKI) is the most serious clinical manifestation of the Sri Lankan hump-nosed pit viper (Hypnale spp.) bites. Thrombotic microangiopathy (TMA) is increasingly recognized in association with AKI in cases of Hypnale spp envenomation. We investigated AKI in a cohort of cases of Hypnale envenomation, its association with TMA and the early diagnostic value of common biomarkers for AKI occurring. MATERIALS AND METHODS: We conducted a prospective observational study of suspected viper bites and included 103 confirmed cases of Hypnale envenomation, based on venom specific enzyme immunoassay of blood. AKI was defined using the Kidney Disease: Improving Global Outcomes (KDIGO) criteria. Thrombotic microangiopathy was diagnosed based on thrombocytopenia (platelet count < 150,000 × 103/µL) and microangiopathic haemolytic anaemia (MAHA). We investigated the diagnostic performance of creatinine, platelet count and INR for AKI within 4 h and 8 h post-bite by area under the receiver operator characteristic curve (AUC-ROC). RESULTS: Ten patients developed AKI: seven AKI stage 1 and three AKI stage 3. Ten patients (10%) developed thrombocytopaenia while 11 (11%) had MAHA. All three AKI stage 3 had thrombocytopaenia and MAHA fulfilling the criteria for TMA. Two of them presented with oliguria/anuria and all three required haemodialysis. Serum creatinine within 4 h post-bite was the best predictor of AKI with AUC-ROC of 0.83 (95% CI: 0.67-0.99) and was no better within 8 h of the bite. CONCLUSIONS: We found that AKI is uncommon in Hypnale spp. envenomation, but an important serious complication. Severe AKI was associated with TMA. A creatinine within 4 h post-bite was the best predictor of AKI.

Thrombotic microangiopathy and acute kidney injury following Sri Lankan Daboia russelii and Hypnale species envenoming.

Context: Thrombotic microangiopathy (TMA) resulting in acute kidney injury (AKI) is an important complication of venomous snakebites. We aimed to describe TMA secondary to Russell's viper (Daboia russelli) and hump-nosed viper (Hypnale spp.) bites and assess the effect of different treatments.Materials and methods: We undertook a prospective observational study of patients with AKI secondary to snakebite over a two-year period. Data recorded included: demographic details, clinical and laboratory features, treatment, complications and outcomes, until hospital discharge and at three months post-discharge. TMA was defined as the development of microangiopathic hemolytic anemia and thrombocytopenia along with AKI. Treatment with therapeutic plasma exchange (TPE; also known as plasmapheresis) and/or fresh frozen plasma (FFP) was determined by the treating clinician. Antivenom was given to all patients with evidence of systemic envenoming following Russell's viper bites.Results: Fifty-nine patients were included in the analysis. Thirty-three (56%) were males and median age was 56 years. Forty-five (76%) developed TMA while a further 11 and two developed isolated thrombocytopenia and microangiopathic hemolytic anemia, respectively. Presence of TMA was associated with increased dialysis requirements (5 vs. 3) and longer hospital stay (18 vs. 12 days). Of the patients with TMA, nine received TPE with or without FFP infusions. The use of TPE was not associated with improved outcomes in patients with TMA based on requirement for blood transfusion, recovery of thrombocytopenia, requirement of dialysis and duration of hospital stay. Patients who did not receive TPE had better renal function at three months compared to patients who received this treatment.Conclusion: Presence of TMA in patients with Daboia and Hypnale bites was associated with a more prolonged course of AKI. Patients with TMA who were treated with TPE did not have improved early or late outcomes compared to patients who were not treated with TPE.

Health in rural Sri Lanka: A cross-sectional survey of three rural districts

Introduction: Sri Lanka has a predominantly rural population. However, there is a dearth of research on health and socioeconomic issues in this group. Objective: To describe basic socioeconomic characteristics and health profile in a rural population. Methods: A descriptive cross-sectional household survey was conducted in 1950 households in three rural districts, selected by a three-stage stratified cluster sampling method. Results: The population pyramid showed an ageing population (dependency ratio of 50%). Only 39% had completed GCE (ordinary level). Unemployment rates were high (25% males, 76% females). Agriculture and related work were main occupations. Most lacked amenities (e.g. 61% households lacked a refrigerator) and practiced inappropriate methods of waste disposal (e.g. open burning by 72%). Household illnesses were frequent: episodes of acute illness within two weeks, injuries within past year and chronic illness were reported from 35.9%, 14.9% and 48.3% households. The prevalence of chronic diseases in adults >20 years were high: diabetes 13.5%, hypertension 16.7% and overweight/obesity 28.2%. Of the males, 22.1% smoked and 12.3% took alcohol. Almost 25% adults chewed betel. Reports of snake bite, dog bites and suicide/attempted suicide were seen in 15.5%, 9.7% and 3.0% households respectively. Conclusions: This study shows a unique clustering of health-related problems in rural Sri Lanka. This was characterized by demographic transition, burden from snake bites, chronic diseases and acute illnesses. There were resource limitations and low levels of education. Cohort studies and comparisons with urban areas will enable further elucidation of determinants of health and other issues in rural Sri Lanka.

Determination of the sub-lethal nephrotoxic dose of Russell's viper (Daboia russelii) venom in Wistar rats.

Wistar rats were administered increasing doses of Russell's viper venom (RVV; 0.025-0.4 mg/kg) intraperitoneally to investigate acute kidney injury (AKI) by measuring creatinine (1.5-fold increase in serum creatinine above baseline) and examining kidney histology. Approximately 50% of rats receiving 0.25-0.4 mg/kg venom died within 72 h. An increase in serum creatinine only occurred at a venom dose of 0.4 mg/kg, except in two rats. Acute tubular necrosis, glomerular necrosis, cortical necrosis and interstitial inflammation were observed at venom doses of ≥0.25 mg/kg in 12/36 rats. However, of those 12 rats only four survived to 48 h compared to the 24 rats not developing nephrotoxicity, in which 18 were alive at 48 h. There was poor correlation between histological nephrotoxicity and AKI based on creatinine measurement. The early death in rats with AKI makes this a poor model for studying RVV-induced AKI.

Report on first certificate course on applied physiology of pain, analgesia, anesthesia, and euthanasia for laboratory animals held in Sri Lanka.

Similar to human beings, pain is an unpleasant sensation experienced by animals as well. There is no exception when the animals are subjected to experimental procedures. Our duty as researchers/scientists is to prevent or minimize the pain in animals so as to lessen their suffering and distress during experimental procedures. The basics of the physiology of pain and pain perception, analgesia, anesthesia, and euthanasia of laboratory animals were included to complete the program, before the practical part was attempted and before advanced topics, such as comparison of anesthetic combinations, were discussed. Therefore, this course was organized in Sri Lanka for the first time in collaboration with the Comparative Biology Centre of Newcastle University, UK. During this course, we were able to demonstrate how an anesthesia machine could be used in laboratory animal anesthesia for the first time in the country. None of the animal houses in the country were equipped with an anesthesia machine at the time of conducting the course.

History and development of laboratory animal science in Sri Lanka.

The Faculty of Medicine, University of Colombo (UCFM), is the forerunner in the laboratory animal science (LAS) field in Sri Lanka. Here laboratory animals such as rats, frogs and rabbits were used in medical education to demonstrate physiological principles in the early 1950s. The establishment of the first animal house in 1969 at UCFM better facilitated the use of laboratory animals in medical education and research. By 2006, lack of ethical guide lines was an issue and a set of ethical guidelines was developed and published in 2009, which today are followed by researchers and reviewers in many universities and research institutions in the country. With this development, there was a need for an organized system of education for the LAS discipline. This was fulfilled by conducting the first certificate course on LAS in the Asian region in 2011 in collaboration with the Utrecht University, Netherlands. The Sri Lanka Association for LAS has continued LAS education since its establishment in 2012 at UCFM by conducting workshops and short courses.

Preliminary survey on animal research statistics in Sri Lanka from 2010 onwards.

BACKGROUND: Animal research is conducted in many countries across the world. However, concepts of recording and reporting the numbers of animals used vary. In the context of animal studies, inadequate reporting raises both ethical and scientific concerns. Sri Lanka has yet to publish data on animal research statistics. METHODS: The objective of this survey was to present the number and types of vertebrate animals used for research and establish the species used for different types of research studies. All facilities/personnel engaged in animal research were invited to participate and a web-based survey was performed. RESULTS: Based on the data collected, rats and mice were the most widely used animals in Sri Lankan laboratories. Of these, the most reported model were rats of the Wistar strain (42%) and mice of the BALB/c strain (15%). The highest number of animals used in laboratory research occurred in 2011, with a 12% decrease observed in 2012.

Reference values for selected hematological, biochemical and physiological parameters of Sprague-Dawley rats at the Animal House, Faculty of Medicine, University of Colombo, Sri Lanka.

BACKGROUND: Lack of available reference values in a research setting under local conditions can be a drawback for beginners, as the accuracy of data from control samples cannot be checked at the beginning of a research project. This affects comparisons with data from test samples. To avoid these complications in their research projects, beginners tend to have a greater number of animals in the control group compared to test groups in order to have control group measurements within 2 SDs of the mean. METHODS: As non-availability of reference values was a long-felt need, the described project was conducted in order to establish a reference database for selected haematological, biochemical and physiological parameters using apparently healthy Sprague-Dawley rats bred in the Animal House of Faculty of Medicine, University of Colombo (UCFM). RESULTS: Differences in mean values of packed cell volume (PCV), mean corpuscular volume (MCV) and mean corpuscular hemoglobin concentration (MCHC), serum creatinine and blood glucose levels between the two genders were statistically significant. Lipid profile measurements did not differ significantly between genders, but mean and median values of triglycerides (TG) between male and female rats showed a difference of more than 10 mg/dL. The liver enzymes alkaline phosphatase (AP) and aspartate aminotransferase (AST) were also statistically significantly different between sexes. Despite wide variation in mean alanine aminotransferase (ALT) between sexes, the difference was not statistically significant. CONCLUSION: The findings of this project should support to a certain extent the "Reduction" aspect of the 3Rs concept of Russell and Burch by reducing the number of Sprague-Dawley rats used in future research projects at UCFM.

Aerva lanata (Polpala): Its effects on the structure and function of the urinary tract.

CONTEXT: Aerva lanata is not prescribed for long-term use in Ayurveda as it is believed to produce structural changes in the urinary tract of the patients leading to renal failure. AIM: To investigate the toxic effects of Aerva lanata on the structure and function of urinary tract of a rat model (Sprague-Dawley rats). SETTINGS AND DESIGN: An experimental study. Thirty male and thirty female healthy rats were randomly assigned to three groups (test groups 1, 2 and control) and administered dried infusion of A. lanata 25g/200ml (low dose), 100g/200ml (high dose) and distilled water respectively, for 30 days. MATERIALS AND METHODS: Blood and urine were collected and creatinine was measured. Creatinine clearance (Ccr) and urine flow rate (UFR) of rats were determined to assess renal function. Kidneys, ureters, and bladders of rats were harvested for light microscopic (LM) studies. Electron microscopic (EM) studies were done on kidney tissues. STATISTICAL ANALYSIS USED: Difference in mean values of Ccr and UFR between test groups and the control group were compared statistically using independent T test. LM and EM findings of the two treated groups (T1 and T2) were statistically compared with the control group (C) using standard normal distribution. RESULTS: Ccr and UFR of test groups were not significantly different from that of the control group. LM studies did not show any histological changes suggesting toxicity. EM, however showed significant ultra structural changes in proximal convoluted tubular epithelial cells of rats in the two test groups. CONCLUSION: Administration of dried Aerva lanata for a period of one month did not produce significant effects on renal function of rats. However administration for the same period caused significant ultra structural changes in the proximal convoluted tubular epithelial cells.

Effects of Cinnamomum zeylanicum (Ceylon cinnamon) on blood glucose and lipids in a diabetic and healthy rat model.

OBJECTIVES: To evaluate short- and long-term effects of Cinnamomum zeylanicum on food consumption, body weight, glycemic control, and lipids in healthy and diabetes-induced rats. MATERIALS AND METHODS: The study was conducted in two phases (Phase I and Phase II), using Sprague-Dawley rats in four groups. Phase I evaluated acute effects on fasting blood glucose (FBG) (Groups 1 and 2) and on post-oral glucose (Groups 3 and 4) blood glucose. Groups 1 and 3 received distilled-water and Groups 2 and 4 received cinnamon-extracts. Phase II evaluated effects on food consumption, body weight, blood glucose, and lipids over 1 month. Group A (n = 8, distilled-water) and Group B (n = 8, cinnamon-extracts) were healthy rats, while Group C (n = 5, distilled-water) and Group D (n = 5, cinnamon-extracts) were diabetes-induced rats. Serum lipid profile and HbA1c were measured on D-0 and D-30. FBG, 2-h post-prandial blood glucose, body weight, and food consumption were measured on every fifth day. PHASE I: There was no significant difference in serial blood glucose values in cinnamon-treated group from time 0 (P > 0.05). Following oral glucose, the cinnamon group demonstrated a faster decline in blood glucose compared to controls (P < 0.05). Phase II: Between D0 and D30, the difference in food consumption was shown only in diabetes-induced rats (P < 0.001). Similarly, the significant difference following cinnamon-extracts in FBG and 2-h post-prandial blood glucose from D0 to D30 was shown only in diabetes-induced rats. In cinnamon-extracts administered groups, total and LDL cholesterol levels were lower on D30 in both healthy and diabetes-induced animals (P < 0.001). CONCLUSIONS: C. zeylanicum lowered blood glucose, reduced food intake, and improved lipid parameters in diabetes-induced rats.

Prolonged breastfeeding reduces risk of breast cancer in Sri Lankan women: a case-control study.

GOAL: To assess the association between duration of breastfeeding and the risk of breast cancer in Sri Lankan women. METHODS: We conducted a case-control study in women aged 30-64 years in selected health care facilities in the Western province. A total of 100 recent cases of breast cancer (histologically confirmed) and 203 controls (age and parity matched) were included. Detailed information regarding breastfeeding, menstruation, reproductive factors, passive smoking and other confounders was collected using a structured questionnaire. Adjusted odds ratios and 95% confidence intervals were calculated using multiple logistic regressions. PRINCIPLE RESULTS: Multivariate analysis found that those women who breastfed for > or =24 months during lifetime had significantly lower risk of breast cancer than those who breastfed for less than 24 months (OR=0.40; 95%CI=0.22, 0.73). Compared to 0-11 months of lifetime breastfeeding, there was a 66.3% reduction in breast cancer risk in women who breastfed for 12-23 months, 87.4% reduction in 24-35 months and 94% reduction in 36-47 months categories. The mean duration of breastfeeding per child for > or =12 months was also associated with reduced risk of breast cancer (OR=0.52; 95%CI=0.28, 0.94). The significant factors associated with increased risk of breast cancer were: post-menopausal women (OR=1.74; 95%CI=1.01, 3.01); having an abortion in the past (OR=3.42; 95%CI=1.75, 6.66) and exposure to passive smoking (OR=2.96, 95%CI=1.53, 5.75). MAJOR CONCLUSIONS: Prolonged breastfeeding significantly reduces the risk of breast cancer and this protective effect was supported by a dose-response relationship. Risk due to passive smoking should be emphasized in anti-smoking programmes.