Generation of chicken-based IgY polyclonal antibodies against Dendroaspis polylepis and preclinical evaluation of envenomation-neutralizing efficacy vis-à-vis selected commercial antivenoms.

The Black mamba, D. polylepis, is one of the many venomous snakes found in Kenya, and known to account for some snakebite incidents. The Kenyan Ministry of Health data reveals annual 15,000 snakebites occurrences. Also, 1 in 15 people in Kenya gets bitten by a snake, and tragically, 1 in 147 of these individuals die of snakebite yearly. Traditionally, antivenoms for treatment are produced from horse or sheep but have complicated and expensive production issues. Alternative production approaches, such as using IgY antibodies derived from chicken egg yolks, may overcome disadvantages with traditional antivenom manufacturing techniques. In this current study, D. polylepis specific IgY polyclonal antibodies were purified from the egg yolks of chickens immunized with D. polylepis venom. These antibodies were subsequently assessed for their in-vivo neutralizing capacity vis-à-vis commercial antivenoms, PANAF-Premium and VINS. The IgY antibodies were purified by ammonium sulfate precipitation and affinity-chromatography, with quality and specificity determined by SDS-PAGE and ELISA. The LD50 of D. polylepis was found to be 0.54 mg/kg in chicks, and 0.34 mg/kg in mice, respectively. Pool of extracted IgY yielded 2.8 mg/mL concentration. Purified IgY under non-reducing and reducing conditions on SDS-PAGE exhibited a single-protein band of about 183 kDa and two bands (67 kDa and 25 kDa), respectively. The minimum-edematogenic dose was 0.05 µg. Anti-D. polylepis IgY antibodies and two antivenoms demonstrated the capacity to neutralize the toxic activities of D. polylepis venom. This study confirms a successful IgY generation against Black mamba venom for the first time, and observed toxic effects of the venom as well as neutralizing capacity of antivenoms.

Preclinical efficacy testing of three antivenoms against Naja ashei venom-induced lethality.

This study aimed to determine the efficacy of Inoserp, Vins bioproducts, and South African Institute of Medical Research (SAIMR) polyvalent antivenoms in neutralizing Naja ashei venom-induced lethality in mice. The neutralization efficacy of the antivenoms were expressed as effective dose, median effective ratio, potency, normalized potency, volume, and the number of vials of antivenom required to neutralize 100 mg of Naja ashei venom (NAV).

Artemia salina as an animal model for the preliminary evaluation of snake venom-induced toxicity.

Lethality and cytotoxicity assays of snake venoms and their neutralization by antivenom require many mice for the experiments. Recent developments have prompted researchers to seek alternative strategies that minimize the use of mice in line with Russel and Burch's 3Rs philosophy (Replacement, Reduction, and Refinement). Artemia salina is an animal model widely used for toxicity screening. However, its use in snake venom toxinology is limited by a lack of data. The present study compared the toxicity of venoms from Bitis arietans, Naja ashei, and Naja subfulva using mice and Artemia salina. In the Artemia salina test at 24 h and the dermonecrotic test in mice, the toxicity of the venoms was in the order Naja ashei ~ Naja subfulva > Bitis arietans. In the lethality test in mice, the toxicity of the venoms was in the order Naja subfulva > Naja ashei > Bitis arietans. These findings suggest that the toxicity of the venoms in Artemia salina and the dermonecrotic bioassay in mice have a similar trend but differ from the lethality test in mice. Therefore, it may be relevant to further explore the Artemia salina bioassay as a potential surrogate test of dermonecrosis in mice. Studies with more venoms may be needed to establish the correlation between the Artemia salina bioassay and the dermonecrotic assay in mice.

Enzymatic activity and brine shrimp lethality of venom from the large brown spitting cobra (Naja ashei) and its neutralization by antivenom.

OBJECTIVE: Naja ashei is a snake of medical importance in Kenya, Ethiopia, Somalia, Uganda, and Tanzania. Little is known about the enzymatic (snake venom phospholipases A2; svPLA2's) and toxic (lethal) activities of N. ashei venom and crucially, the safety and capacity of available antivenom to neutralize these effects. This study aimed to determine the enzymatic and toxic activities of N. ashei venom and the capacity of Indian and Mexican manufactured antivenoms to neutralize these effects. The protein content of the venom and the test antivenoms were also evaluated. A 12-point log concentration-response curve (0.5-22.5 µg/mL) was generated on an agarose-egg yolk model to predict the svPLA2 activity of the venom. The toxicity profiles of the venom and antivenoms were evaluated in the brine shrimp lethality assay. Lowry's method was used for protein estimation. RESULTS: Low and intermediate concentrations of the venom exhibited similar svPLA2 activities. The same was true for concentrations > 15 µg/mL. Intermediate and high doses of the venom exhibited similar mortalities in brine shrimp and test antivenoms were generally non-toxic but poorly neutralized svPLA2 activity. Mexican manufactured antivenom had lower protein content but neutralized venom-induced brine shrimp lethality much more effectively than Indian manufactured antivenom.

Management and cost of snakebite injuries at a teaching and referral hospital in Western Kenya.

Background: Data on the cost of snakebite injuries may inform key pillars of universal health coverage including proper planning, allocation, and utility of resources. This study evaluated the injuries, management, and costs resulting from snakebites at Jaramogi Oginga Odinga Teaching and Referral Hospital (JOOTRH) in Kenya.  Methods: In total, medical records of 127 snakebite victims attending JOOTRH between January 2011 and December 2016 were purposely selected and data on the age, gender, type of residence (urban or rural), part of the body bitten, time of bite, injuries, pre-hospital first aid, time to hospital, length of stay, treatment, and costs were collected. Regression analysis was used to predict the total indirect cost of snakebite injuries and p≤ 0.05 was considered significant. Mortality and loss of income of hospitalized victims were considered as direct costs. Results: It was found that 43 victims were 13-24 years of age, 64 were female, 94 were from rural areas, 92 were bitten on the lower limbs, 49 were bitten between 6.00 pm and midnight, 43 attempted pre-hospital first aid, and the median time to hospital was 4.5 hours. Antivenom, supportive therapy, antibiotics, antihistamines, corticosteroids, analgesics, and non-steroidal anti-inflammatory drugs were used. Cellulitis, compartment syndrome, gangrenous foot, psychiatric disorder, and death were the main complications. Most victims spent 1-5 days in hospital and the median cost of treating a snakebite was 2652 KES (~$26). Drugs, ward charges, and nursing procedures were the highest contributors to the total indirect cost. Victims hospitalized for 6-10 days and >10 days incurred 32% and 62% more costs, respectively, compared to those hospitalized for 1-5 days.  Conclusions: The longer snakebite victims are hospitalized, the higher the cost incurred. Continuous medical education on the correct management of snakebites should be encouraged to minimize complications that may increase hospital stays and costs incurred.

Revered but Poorly Understood: A Case Report of Dendroaspis polylepis (Black Mamba) Envenomation in Watamu, Malindi Kenya, and a Review of the Literature.

The black mamba (Dendroaspis polylepis) ranks consistently as one of the most revered snakes in sub-Saharan Africa. It has potent neurotoxic venom, and envenomation results in rapid onset and severe clinical manifestations. This report describes the clinical course and reversal of effects of black mamba envenomation in a 13-year-old boy in the Jimba area of Malindi. The victim presented to Watamu Hospital, a low resource health facility with labored breathing, frothing at the mouth, severe ptosis and pupils non-responsive to light. His blood pressure was unrecordable, heart rate was 100 beats per minute but thready, his temperature was 35.5 °C, and oxygen saturation was 83%. Management involved suction to clear salivary secretions, several hours of mechanical ventilation via ambu-bagging, oxygen saturation monitoring, and the use of South African Vaccine Producers (SAVP) polyvalent antivenom. Subcutaneous adrenaline was used to stave off anaphylaxis. The victim went into cardiac arrest on two occasions and chest compressions lasting 3⁻5 min was used to complement artificial ventilation. Hemodynamic instability was corrected using IV infusion of ringers lactate and normal saline (three liters over 24 h). Adequate mechanical ventilation and the use of specific antivenom remain key in the management of black mamba envenomation.

Epidemiology of snake bites in selected areas of Kenya.

INTRODUCTION: Snake bites are a silent public health problem in Kenya. Previous studies on snake bites in the country have mainly focused on identifying offending snake species, assessing the severity of envenomation and testing the efficacy of antivenom. Factors associated with snake bites in the country are yet to be fully understood. The aim of this work was to determine pharmaco-epidemiological factors associated with snake bites in areas of Kenya where incidence, severity and species responsible for snake bites have been reported. METHODS: Kakamega provincial hospital, Kabarnet, Kapenguria and, Makueni district hospitals were selected as study sites based on previous findings on incidence, severity and species responsible for snake bites in catchment areas of these hospitals. Persistent newspaper reports of snake bites in these areas and distribution of snakes in Kenya were also considered. Cases of snake bites reported between 2007-2009 were retrospectively reviewed and data on incidence, age, site of the bites, time of bite and antivenom use was collected. RESULTS: 176 bites were captured, 91 of which occurred in 2009. Individual incidence was between 2.7/100,000/year and 6.7/100,000/year. Bites peaked in the 1-15 year age group while 132/176 bites were in the lower limb area and 49/176 victims received antivenom. Most bites occurred during the dry season, in the bush and in the evening. Overall mortality was 2.27%. CONCLUSION: There is a need to sensitize the Kenyan public and healthcare personnel on preventive measures, first aid and treatment of snake bites.

Antimicrobial residues and compositional quality of informally marketed raw cow milk, Lamu West Sub-County, Kenya, 2015.

INTRODUCTION: unadulterated milk, free of antimicrobial residues is important for industrial processing and consumers' health. Antimicrobial residues in foods of animal origin can cause adverse public health effects like drug resistance and hypersensitivity. Milk produced in Lamu West sub-county is sold raw directly to consumers. We estimated the compositional quality and prevalence of antimicrobial residues in informally marketed raw cow milk in Lamu West Sub-County, Kenya. METHODS: we randomly recruited 152 vendors and 207 farmers from four randomly selected urban centers in a cross-sectional study and interviewed them using a pretested standardized questionnaire. A100-ml raw milk sample was aseptically collected from each vendor and farm and tested for antimicrobial residues using Charm Blue Yellow II kit following the European Union Maximum Residue Limits (EU-MRLs) while an Ekomilk® Analyzer was used to measure compositional quality where samples with either solid not fat (SNF) < 8.5 or added water ≥ 0.01% or both were considered adulterated. We analyzed data using univariate analysis and unconditional logistic regression to calculate odds ratios (OR) and 95% confidence intervals (CI). RESULTS: thirty-two of the 207 (15.5%) samples from farmers and 28 (18.4%) of the 152 samples from vendors tested positive for antimicrobial residues. Thirty-six (17.4 %) samples from farmers and 38 (25.0%) from vendors were found to be adulterated with water. Farmers' awareness of the danger of consuming milk with antimicrobial residues and farmers having training on good milking practices were protective against selling milk with antimicrobial residues (adjusted OR and 95% CI 0.20, 0.07-0.55 and 0.33, 0.11-0.99, respectively). CONCLUSION: the antimicrobial residues above EU MRLs and adulteration of raw marketed cow milk observed in this study provide evidence for routine testing of marketed milk and educating farmers to observe antimicrobial withdrawal period.