Antitrypanosomal Effect of Hydromethanolic Extract of Solanum anguivi Lam on Field Isolates of Trypanosoma congolense Infected Mice

Introduction. Trypanosomiasis is one of the world’s most serious infectious diseases caused by Trypanosoma parasites. Concern about resistance to conventional antitrypanosomal drugs, mosquito vector resistance to existing insecticide side effects of existing antitrypanosomal drugs justifies the urgent need for more effective, tolerable, and affordable drugs. Objective. The present study is aimed at determining the in vivo antitrypanosomal effect of the hydromethanolic extracts of Solanum anguivi fruit extracts against the field isolates of T. congolense. Methods. The 80% methanol extracts of S. anguivi fruits were prepared by cold maceration technique. In vivo curative tests were done to check the effect of plant extract against T. congolense in Swiss albino mice. Plant extracts were administered at doses of 100, 200, and 400 mg/kg/body weight. Acute toxicity of the extracts at 2000 mg/kg was performed according to OECD guidelines. Data obtained from the experiment were analyzed using one-way ANOVA followed by Tukey test. Results. This study indicated that extract did not exhibit any sign of acute toxicity up to 2000 mg/kg/body weight. In curative test, extracts reduced parasitemia, preventing the drop in packed cell volume and body weight significantly ( p < 0.05 ), compared to control. Groups provided with the extract before infection got prolonged incubation period with chemoprophylactic effect at the doses of 100, 200, and 400 mg/kg. Phytochemical analysis showed presence of flavonoids, steroids, triterpens, saponins, glycosides, tannins, and alkaloids. Conclusion. The extract showed promising curative. Further effort is required to isolate and purify specific compounds responsible for antitrypanosomal activity of studied plant.

Synergism in Antiplasmodial Activities of Artemether and Lumefantrine in Combination with Securidaca longipedunculata Fresen (Polygalaceae)

Malaria is the most lethal parasitic disease in the world. The frequent emergence of resistance by malaria parasites to any drug is the hallmark of sustained malaria burden. Since the deployment of artemisinin-based combination therapies (ACTs) it is clear that for a sustained fight against malaria, drug combination is one of the strategies toward malaria elimination. In Sub-Saharan Africa where malaria prevalence is the highest, the identification of plants with a novel mechanism of action that is devoid of cross-resistance is a feasible strategy in drug combination therapy. Thus, artemether and lumefantrine were separately combined and tested with extracts of Securidaca longipedunculata, a plant widely used to treat malaria, at fixed extract–drug ratios of 4:1, 3:1, 1:1, 1:2, 1:3, and 1:4. These combinations were tested for antiplasmodial activity against three strains of Plasmodium falciparum (W2, D6, and DD2), and seven field isolates that were characterized for molecular and ex vivo drug resistance profiles. The mean sum of fifty-percent fractional inhibition concentration (FIC50) of each combination and singly was determined. Synergism was observed across all fixed doses when roots extracts were combined with artemether against D6 strain (FIC50 0.403 ± 0.068) and stems extract combined with lumefantrine against DD2 strain (FIC50 0.376 ± 0.096) as well as field isolates (FIC50 0.656 ± 0.067). Similarly, synergism was observed in all ratios when leaves extract were combined with lumefantrine against W2 strain (FIC50 0.456 ± 0.165). Synergism was observed in most combinations indicating the potential use of S. longipedunculata in combination with artemether and lumefantrine in combating resistance.

Protein Biomarker Identification for the Discrimination of Brucella melitensis Field Isolates From the Brucella melitensis Rev.1 Vaccine Strain by MALDI-TOF MS

Brucella melitensis Rev.1 is a live attenuated vaccine strain that is widely used to control brucellosis in small ruminants. For successful surveillance and control programs, rapid identification and characterization of Brucella isolates and reliable differentiation of vaccinated and naturally infected animals are essential prerequisites. Although MALDI-TOF MS is increasingly applied in clinical microbiology laboratories for the diagnosis of brucellosis, species or even strain differentiation by this method remains a challenge. To detect biomarkers, which enable to distinguish the B. melitensis Rev.1 vaccine strain from B. melitensis field isolates, we initially searched for unique marker proteins by in silico comparison of the B. melitensis Rev.1 and 16M proteomes. We found 113 protein sequences of B. melitensis 16M that revealed a homologous sequence in the B. melitensis Rev.1 annotation and 17 of these sequences yielded potential biomarker pairs. MALDI-TOF MS spectra of 18 B. melitensis Rev.1 vaccine and 183 Israeli B. melitensis field isolates were subsequently analyzed to validate the identified marker candidates. This approach detected two genus-wide unique biomarkers with properties most similar to the ribosomal proteins L24 and S12. These two proteins clearly discriminated B. melitensis Rev.1 from the closely related B. melitensis 16M and the Israeli B. melitensis field isolates. In addition, we verified their discriminatory power using a set of B. melitensis strains from various origins and of different MLVA types. Based on our results, we propose MALDI-TOF MS profiling as a rapid, cost-effective alternative to the traditional, time-consuming approach to differentiate certain B. melitensis isolates on strain level.

Genetic Diversity of Plasmodium vivax Cysteine-Rich Protective Antigen (PvCyRPA) in Field Isolates from Five Different Areas of the Brazilian Amazon

The Plasmodium vivax Cysteine-Rich Protective Antigen (PvCyRPA) has an important role in erythrocyte invasion and has been considered a target for vivax malaria vaccine development. Nonetheless, its genetic diversity remains uncharted in Brazilian malaria-endemic areas. Therefore, we investigated the pvcyrpa genetic polymorphism in 98 field isolates from the Brazilian Amazon and its impact on the antigenicity of predicted B-cell epitopes. Genetic diversity parameters, population genetic analysis, neutrality test and the median-joining network were analyzed, and the potential amino acid polymorphism participation in B-cell epitopes was investigated. One synonymous and 26 non-synonymous substitutions defined fifty haplotypes. The nucleotide diversity and Tajima’s D values varied across the coding gene. The exon-1 sequence had greater diversity than those of exon-2. Concerning the prediction analysis, seven sequences were predicted as linear B cell epitopes, the majority contained in conformational epitopes. Moreover, important amino acid polymorphism was detected in regions predicted to contain residues participating in B-cell epitopes. Our data suggest that the pvcyrpa gene presents a moderate polymorphism in the studied isolates and such polymorphisms alter amino acid sequences contained in potential B cell epitopes, an important observation considering the antigen potentiality as a vaccine candidate to cover distinct P. vivax endemic areas worldwide.

Investigation of the sequence profile of the Plasmodium falciparum 18SrRNA diagnostic target in isolates from naturally infected children with uncomplicated malaria

The gold standard for malaria diagnosis is evidence of parasitological confirmation but the traditional method by light microscopy and the routinely used rapid diagnostic tests (RDTs) have limitations. Molecular assays are known to have higher sensitivity and specificity but there are indications that they may also be compromised by genetic variability of the target sequence. The aim of this study therefore, was to evaluate the DNA sequence profile of the diagnostic target of the P. falciparum 18S rRNA PCR assay in field isolates from North-Central Nigeria. Blood samples were collected from 324 children presenting with acute febrile illness suspected to be uncomplicated malaria. Light microscopy and 18S rRNA PCR assay were employed to determine the presence of P. falciparum parasites. Sequence profile of the diagnostic target was evaluated by Sanger sequencing of the PCR products on ABI PRISM® 3100 DNA sequencer (PE Applied Biosystems). Of the 324 children enrolled into this study, 134 (41.4%) were positive for P. falciparum by microscopy while 218 (67.3%) were positive by PCR. The sensitivity of microscopy was 61.47%(95% CI= 57.88% - 69.64%) using the PCR assay as reference standard. The degree of agreement between microscopy and PCR as measured by Cohen's kappa was moderate (κ = 0.502, 95% CI = 0.463 - 0.715).Sequence analysis showed that the DNA target of the P. falciparum 18S rRNA PCR from the field isolates were highly conserved. Only one A>T single nucleotide polymorphism was found within the target sequence among the isolates in this study. This study showed that the DNA target sequence of the18S rRNA PCR assay is highly conserved in field isolates in the study region suggesting little or no impact of selective pressure acting on the locus and has implications for the enhanced sensitivity of the molecular assay.

Genomic pattern analysis of Burkholderia mallei field isolates by pulsed-field gel electrophoresis (PFGE) discriminatory typing

Background and Objectives: Glanders is a serious zoonotic disease caused by Burkholderia mallei. Prevention, control, and treatment strategies of glanders are prerequisites for microbial source tracking. The present study was aimed to analyze the genomic pattern of B. mallei Iranian field isolates by pulsed-field gel electrophoresis (PFGE) typing. Materials and Methods: B. mallei isolates were aerobically cultured in nutrient broth/agar supplemented with glycerol 4% for 48 h at 37°C. API 20NE identification system was used for the biochemical characterization. Genomic DNA of bacterial isolates was extracted using OIE-recommended protocol. Molecular identification of bacterial isolates was done based on amplification of BimA and IS407-flip genes. PFGE was applied to prepare the genomic pattern of B. mallei isolates. The guinea pig was used as a suitable model for studying the histopathological characterization of B. mallei. Results: In both enzymatic digestion patterns by using Af1II and VspI, we found three different clonal types; І) PFGE type of B. mallei Razi 325 strain, ІІ) PFGE type of Tiger, Kordan, and Oshnavieh strains, and ІІІ) PFGE type of Semirom strain. B. mallei Razi 325 was categorized as unrelated strain which was belonged to the different cluster differing more than four bands. Conclusion: PFGE showed more discriminatory power and considerable reproducibility for molecular typing of B. mallei strains in our study. It is standardized the approaches for outbreak detection, pathogen phylogeny, molecular epidemiology, and population studies.

Foot-and-mouth Disease Virus type (O) Overhangs Livestock Industry of both Young and Adult Cattle and Buffalo in Egypt

Egyptian farms is still attacked by several outbreaks of FMDV, a highly contagious viral disease that threatens the livestock industry worldwide. Many of these outbreaks are unreported. Hence, the epidemiological situation and molecular characterization of the currently responsible strains of recent FMD outbreaks are missed. To identify FMDV strains currently circulating in cattle herds and catch the recent update in the morphophonological changes due to that virus, we carried out this study on the FMD outbreak from 2016–2017 in Egypt. Clinical samples (post mortem tissues samples, oral swabs, epithelial suspensions from ruptured vesicles, and blood) were collected from clinically examined animals suspected to be of being FMD virus = 229 cattle and 138 buffalo. Of 175, 96 (54.8%) samples and 85 (44.3%) from 192 samples were positive in 2016 and 2017, respectively. Pathological examination revealed the classical lesions of FMDV as vesicular and erosive lesions on cornified epithelial tissues. Multi-focal areas of lympho-plasmocytic myocarditis were seen in the heart of all calves. Similar lesions were also observed in the heart of adult cattle that died in that outbreaks. Detailed pathological alterations in adult and young animals were described. Polymerase chain reaction (PCR) screening of tissue specimens using specific primers for FMDV was done. Serotype O was the only serotype that was detected in our study and submitted in the Genebank. The four field isolates of serotype O that have been submitted in the Genebank had been found to belong to the EA-3 topotype. Nucleotide sequencing of the VP1 region and phylogenetic analysis has been used to determine the relationships between our field isolates, other recently isolated Egyptian isolates, and other isolates from some African countries and vaccinal strain used for serotype O in Egypt. The presence of strains of increased virulence among cattle and buffalo in Egypt could be attributed to the introduction of new viral strains through uncontrolled transboundary movements of animals during the last public revolution in Egypt in 2011.

Genetic Diversity of Microneme Protein 2 and Surface Antigen 1 of Eimeria tenella

Avian coccidiosis is a disease caused by members of the genus Eimeria. Huge economic losses incurred by the global poultry industry due to coccidiosis have increased the need for cost-effective and easily available recombinant vaccines. Microneme protein 2 (MIC2) and surface antigen 1 (SAG1) of E. tenella have been recognised as potential vaccine candidates. However, the genetic diversity of the antigens in field isolates, which affects vaccine efficacy, has yet to be largely investigated. Here, we analysed genetic diversity and natural selection of etmic2 and etsag1 in Korean E. tenella isolates. Both genes exhibited low levels of genetic diversity in Korean isolates. However, the two genes showed different patterns of nucleotide diversity and amino acid polymorphism involving the E. tenella isolates obtained from different countries including China and India. These results underscore the need to investigate the genetic diversity of the vaccine candidate antigens and warrant monitoring of genetic heterogeneity and evolutionary aspects of the genes in larger numbers of E. tenella field isolates from different geographical areas to design effective coccidial vaccines.

Comparison of Biological Activity of Field Isolates of Steinernema feltiae with a Commercial S. feltiae Biopesticide Product

Insect trap studies were carried out to determine the presence of entomopathogenic nematodes (EPN) from the family Steinernematidae in the soils of Poland and to compare the biological activities of field nematode isolates with nematodes from commercial biopesticide. The fauna of these organisms in central Poland is poorly studied in both taxonomic and biological terms. Tilled soils representative of this region were sampled from cultivated fields. EPN were isolated from soil samples under laboratory conditions and identified using a key for species identification and molecular analysis. Basic morphometric parameters of infective juveniles and adult males of the first generation were determined. The research showed that males and infective juveniles Steinernema feltiae from Łoniów were the largest. The smallest infective juveniles were found in the isolate from Oblasy, and the smallest males in the isolate from Danków. In Poland, new field isolates showed close genetic similarity to other S. feltiae isolates. The research showed that the field isolates from Poland had greater infectivity and rate of reproduction compared with nematodes from the commercial biopesticide. The findings indicate the potential use of field S. feltiae isolates from Poland (iso1Lon, iso1Dan and iso1Obl) to develop new biopesticide products.

Porcine Circovirus (PCV) Genotype 2d-Based Virus-like Particles (VLPs) Induced Broad Cross-Neutralizing Antibodies against Diverse Genotypes and Provided Protection in Dual-Challenge Infection of a PCV2d Virus and a Type 1 Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)

As PCV2d infection has been continuously reported in swine farms in which pigs were vaccinated with PCV2a- or 2d-based vaccines, we attempted to develop a novel vaccine using a PCV2d-based capsid to enhance its protective efficacy. In this study, recombinant virus-like particles (VLPs) of rPCV2a, rPCV2b and rPCV2d were synthesized from the capsid proteins of PCV2a, PCV2b and PCV2d field isolates, respectively. A cross-neutralization assay between the VLPs induced antisera and the field isolates demonstrated the broad cross-neutralizing activities of the rPCV2d-induced antisera. Then, the protective efficacy of rPCV2d as a vaccine candidate was investigated in commercial pigs by rPCV2d vaccination and a single- or dual-challenge infection using a PCV2d strain and a type 1 PRRSV strain. High levels of anti-PCV2d IgG and neutralizing antibodies were induced 3 weeks after vaccination. After the challenge infection, the average ADWG values of the vaccinated group were higher than those of the unvaccinated group. None or a significantly low amount of (p < 0.05) reduced PCV2 genomic DNA was found in the blood, saliva and tissues of the vaccinated pigs, when compared to the unvaccinated group. Moreover, macroscopic and microscopic lesions in the tissues were significantly (p < 0.05) reduced in the vaccinated groups. This study therefore suggests that rPCV2d may be highly useful for the control of diverse field genotypes.