Prevalence and antimicrobial resistance profiles of mastitis causing bacteria isolated from dairy goats in Mukurweini Sub-County, Nyeri County, Kenya.

BACKGROUND: Ruminant mastitis continues to be a cause of economic losses in the dairy industry and remains a major public health hazard globally. OBJECTIVES: This cross-sectional study was carried out in Mukurweini Sub-County of Nyeri County, Kenya, to investigate the prevalence of bacteria causing mastitis, risk factors associated with goat mastitis and the antibiotic resistance profiles of bacteria isolated from the goat milk. METHODS: Farm level data on risk factors for mastitis was obtained from 56 farmers using a semi structured questionnaire. A total of 189 goat milk samples were collected. The goat's udder was observed for signs of clinical mastitis and the California Mastitis Test (CMT) used to test the milk for sub-clinical mastitis. All samples were then cultured for morphological identification of bacteria and strain typing by Matrix Assisted Laser Desorption/Ionization (MALDI)-Time of Flight (ToF) technique. Antimicrobial susceptibility of the isolated Staphylococcus aureus, coagulase-negative Staphylococcus (CoNS), Escherichia coli, Klebsiella oxytoca, Pseudomonas spp., Enterobacter spp., Proteus vulgaris and Escherichia vulneris to eight commonly used antibiotics was done by the disc diffusion method and validated by determining the presence of antibiotic resistance genes (mecA and blaTEM) using polymerase chain reaction method. RESULTS: The prevalence of clinical mastitis was 1.1% (2/189) while that of sub-clinical mastitis was 84.7% (160/189). Higher (p < 0.05) prevalence of mastitis was observed in goats whose houses were cleaned fortnightly and in cases where farmers used same towel to dry different does' udders during the milking process. Thirteen different bacterial species were isolated from the milk samples and identified by MALDI-ToF, and these included S. aureus (22.0%), CoNS (20.3%), E. coli (18.1%), Pseudomonas spp. (14.3%), Enterobacter spp. (10.4%), K. oxytoca (6.0%), E. vulneris (1.7%), P. vulgaris (1.7%), Raoutella ornithinolytica (1.7%), Stenotrophomonas maltophilia (1.1%), Pantoea agglomerans (1.1%), Serratia marcescens (1.1%) and Cedeceas spp. (0.6%). One hundred pathogenic bacterial isolates were randomly selected and tested for antibiotic sensitivity to eight antibiotics out of which S. aureus were 97.5% resistant to Oxacillin and 100% sensitive to Ciprofloxacin. The CoNSs were 100% resistant to Oxacillin and 100% sensitive to Ciprofloxacin. E. coli were 93.9% resistant to Oxacillin, 69.7% sensitive to Ciprofloxacin and 87.9% sensitive to both Amoxicillin/Clavulanic acid and Meropenem. The antimicrobial resistant genes detected in S. aureus and E. coli were mecA [66.7%, 0%], and blaTEM [20% and 78.3%], respectively. CONCLUSION: In conclusion, the study showed that most of the does were affected by subclinical mastitis with the main causative bacteria being Staphylococci spp. and coliforms. Farmers need to be trained on improved control of mastitis by adoption of good milking practices and use of CMT kit for early detection of mastitis. Occurrence of multidrug resistance by key mastitis causing pathogens was shown to be prevalent and therefore there is need for development of intervention strategies.

Sub-acute and sub-chronic toxicity assessment of the antimicrobial peptide Dermaseptin B2 on biochemical, haematological and histopathological parameters in BALB/c mice and Albino Wistar rats.

Background: Dermaseptins (Drs) are peptides found in the skin secretions of a variety of Hylid frogs, particularly those belonging to the Agalychnis and Phyllomedusa families. Dermaseptin B2 (Drs B2), an amphipathic, α-helical polypeptide was reported as the most active of the Dermaseptin B family. We have previously shown that Drs B2 has strong anti-proliferative activities against RD cells in vitro and thus required further evaluations for future medical applications. Aim: The aim the study was to evaluate the 14-day sub-acute and 90-day sub-chronic toxicities Drs B2 in vivo. Materials and Methods: BALB/c mice were treated with increasing concentrations of 5-25 mg/kg of Drs B2. Rats were treated with 2, 4 and 10-fold concentrations of the calculated LD50 of Drs B2 following OECD recommendations. At the end of the experimentation periods, the animals were sacrificed and dissected to collect blood and selected organs for analysis of any effects caused by Drs B2 treatment on the biochemical, haematological, and histological parameters. Results: The 14-day sub-acute toxicity tests did not cause significant alteration in the biochemical, hematological and histological parameters. The 90-day sub-chronic toxicity study showed lower ALT and AST than control at doses 1.9 mg/kg and 4.6 mg/kg, respectively. Their haematology results also showed higher platelet count than the controls but the differences were not statistically significant. Histological analysis showed increased megakaryocytes in the spleen for both the mice and the rats. Conclusion: The results of this study indicate that short term treatment of Drs B2 could be safe to the animals, however, long-term treatment can have mild effects on the liver parameters and cause an inflammatory response in the spleen.

Evaluation of lethality and cytotoxic effects induced by Naja ashei (large brown spitting cobra) venom and the envenomation-neutralizing efficacy of selected commercial antivenoms in Kenya.

Neutralization of lethality in mice model at the preclinical level has been established by the World Health Organization as the gold standard for the evaluation of antivenom efficacy. The assessment of the neutralization profiles of antivenoms helps to discern the efficacy or otherwise of these antivenoms at neutralizing the toxic effects induced by medically significant snake venoms. However, for many antivenoms, information on their preclinical efficacy remains limited. Therefore, to strengthen global efforts at reducing the impact of snakebite envenoming, the provision of information on the preclinical efficacy of antivenoms, especially in parts of the world where antivenom availability and accessibility is problematic, including sub-Saharan Africa is crucial. This study presents the lethal and toxic activities of N. ashei venom and the neutralizing capacity of two commonly used commercial antivenoms in Kenya; VINS™ and Inoserp™. Median lethal dose (LD50), minimum necrotizing dose (MND) and minimum edema-forming dose (MED) of N. ashei venom as well as the neutralization of these effects were evaluated in mice. The LD50 of N. ashei venom was found to be 4.67 (3.34-6.54) mg/kg while MND and MED were 11.00 µg and 0.80 µg respectively. Both VINS™ and Inoserp™ antivenoms demonstrated capacity to neutralize the lethal and toxic effects induced by Naja ashei venom albeit at varying efficacies. Our results thus confirm the toxic effects of N. ashei venom as previously observed with other Naja sp. venoms and also underscore the relevance of para-specific neutralizing capacity of antivenoms in the design of antivenoms.

Antimicrobial Resistance and Virulence Characteristics of Klebsiella pneumoniae Isolates in Kenya by Whole-Genome Sequencing.

Klebsiella pneumoniae is a globally significant opportunistic pathogen causing healthcare-associated and community-acquired infections. This study examined the epidemiology and the distribution of resistance and virulence genes in clinical K. pneumoniae strains in Kenya. A total of 89 K. pneumoniae isolates were collected over six years from five counties in Kenya and were analyzed using whole-genome sequencing and bioinformatics. These isolates were obtained from community-acquired (62/89) and healthcare-associated infections (21/89), and from the hospital environment (6/89). Genetic analysis revealed the presence of blaNDM-1 and blaOXA-181 carbapenemase genes and the armA and rmtF genes known to confer pan-aminoglycoside resistance. The most abundant extended-spectrum beta-lactamase genes identified were blaCTX-M-15 (36/89), blaTEM (35/89), and blaOXA (18/89). In addition, one isolate had a mobile colistin resistance gene (mcr-8). Fluoroquinolone resistance-conferring mutations in gyrA and parC genes were also observed. The most notable virulence factors were those associated with hyper-virulence (rmpA/A2 and magA), yersiniabactin (ybt), salmochelin (iro), and aerobactin (iuc and iutA). A total of 38 distinct sequence types were identified, including known global lineages ST14, ST15, ST147, and ST307, and a regional clone ST17 implicated in regional outbreaks. In addition, this study genetically characterized two potential hypervirulent isolates and two community-acquired ST147 high-risk clones that contained carbapenemase genes, yersiniabactin, and other multidrug resistance genes. These results demonstrate that the resistome and virulome of Kenyan clinical and hospital environmental K. pneumoniae isolates are diverse. The reservoir of high-risk clones capable of spreading resistance, and virulence factors have the potential to cause unmanageable infection outbreaks with high morbidity and mortality.

Development and Characterization of Anti-Naja ashei Three-Finger Toxins (3FTxs)-Specific Monoclonal Antibodies and Evaluation of Their In Vitro Inhibition Activity.

Antivenom immunotherapy is the mainstay of treatment for snakebite envenoming. Most parts of the world affected by snakebite envenoming depend on broad-spectrum polyspecific antivenoms that are known to contain a low content of case-specific efficacious immunoglobulins. Thus, advances in toxin-specific antibodies production hold much promise in future therapeutic strategies of snakebite envenoming. We report anti-3FTxs monoclonal antibodies developed against N. ashei venom in mice. All the three test mAbs (P4G6a, P6D9a, and P6D9b) were found to be IgG antibodies, isotyped as IgG1. SDS-PAGE analysis of the test mAbs showed two major bands at approximately 55 and 29 kDa, suggestive of immunoglobulin heavy and light chain composition, respectively. The immunoaffinity-purified test mAbs demonstrated higher binding efficacy to the target antigen compared to negative control. Similarly, a cocktail of the test mAbs was found to induce a significantly higher inhibition (p-value < 0.0001) compared to two leading commercial brands of antivenoms on the Kenyan market, implying a higher specificity for the target antigen. Both the test mAbs and 3FTxs polyclonal antibodies induced comparable inhibition (p-value = 0.9029). The inhibition induced by the 3FTxs polyclonal antibodies was significantly different from the two antivenoms (p-value < 0.0001). Our results demonstrate the prospects of developing toxin-specific monoclonal-based antivenoms for snakebite immunotherapy.

Increase in the Immune Response in Balb/c Mice after the Co-Administration of a Vector-Based COVID-19 Vaccine with Cytosine Phosphoguanine Oligodeoxynucleotide.

The effects of cytosine phosphoguanine oligodeoxynucleotides (CPG ODNs) on immune response have been demonstrated for different vaccines; however, such information is limited for the vector-based Coronavirus disease 2019 (COVID-19). This paper aims to demonstrate the potential effect of CPG ODNs on immunological response against the vector-based COVID-19 vaccine on Balb/c mice using a JNJ-78436735 Ad26.COV2-S recombinant as a model vaccine. A total of 18 BALB/c mice clustered into six groups were used. All groups were observed for 14- and 28-days post immunization. Qualitative determination of IgG was performed using indirect Enzyme-Linked Immunosorbent Assay (ELISA) and qPCR for cytokine profiling. A significant (p ≤ 0.001) rise in antibody response was observed for groups 3 and 4, who also showed increased expression levels of Tumor Necrosis Factor (TNF) and Interferon Gamma (IFN-γ). Immunological parameters for toxicity were normal in all treatment groups. We conclude that supplementing vector-based COVID-19 vaccines with CpG ODNs has the potential to boost the body's immune responses to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection.

Preliminary In Vivo Evidence of Oral Selenium Supplementation as a Potentiating Agent on a Vector-Based COVID-19 Vaccine in BALB/c Mice.

Evidence of efficacy and toxicity of oral selenium supplementation in vaccine administration against severe acute respiratory syndrome coronavirus 2 (SARS-COV-2) in mice models is scarce. In this study, 4 × 109 virus particles (40 µL) dose of Janssen COVID-19 intramuscular injection vaccine was supplemented with a commercial selenium supplement and sodium selenite orally in BALB/c mice (N = 18). Qualitative determination of anti-spike IgG antibody response using indirect Enzyme-Linked Immunosorbent Assay (ELISA) showed significant (p ≤ 0.001) increase in anti-spike IgG antibody response for mice groups immunized with vaccine and supplemented selenium. Furthermore, cytokine profiling using real-time quantitative polymerase chain reaction also showed an increase in IL-6 and IL-10 mRNA levels normalized using hypoxanthine phosphoribosyl transferase 1 (Hprt1) and glyceraldehyde 3-phosphate dehydrogenase (Gadph) housekeeping genes. There was no statistical significance (p < 0.465) among treated and untreated groups for alanine transaminase (ALT), aspartate transaminase (AST), urea, and creatinine parameters. The study presents preliminary findings and suggests that supplementing Janssen COVID-19 vaccines with selenium can generate more robust immune responses.