scholarly journals A Comparative Genomic Study of Attenuated and Virulent Strains of Babesia bigemina

Pathogens ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 318
Author(s):  
Bernardo Sachman-Ruiz ◽  
Luis Lozano ◽  
José J. Lira ◽  
Grecia Martínez ◽  
Carmen Rojas ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Virulence Genes ◽  
Laboratory Strain ◽  
Host Cells ◽  
Comparative Genomic ◽  
Local Alignment ◽  
Host Immune System ◽  
Babesia Bigemina ◽  
Genomic Study

Cattle babesiosis is a socio-economically important tick-borne disease caused by Apicomplexa protozoa of the genus Babesia that are obligate intraerythrocytic parasites. The pathogenicity of Babesia parasites for cattle is determined by the interaction with the host immune system and the presence of the parasite’s virulence genes. A Babesia bigemina strain that has been maintained under a microaerophilic stationary phase in in vitro culture conditions for several years in the laboratory lost virulence for the bovine host and the capacity for being transmitted by the tick vector. In this study, we compared the virulome of the in vitro culture attenuated Babesia bigemina strain (S) and the virulent tick transmitted parental Mexican B. bigemina strain (M). Preliminary results obtained by using the Basic Local Alignment Search Tool (BLAST) showed that out of 27 virulence genes described and analyzed in the B. bigemina virulent tick transmitted strain, only five were fully identified in the attenuated laboratory strain. In all cases, the identity and coverture of the identified genes of the wildtype strain were higher than those of the laboratory strain. This finding is putatively associated with the continuous partial loss of virulence genes in the laboratory strain after several passages of the parasite population under optimal in vitro growth conditions. The loss of virulence factors might be reflected in the absence of symptoms of the disease in cattle inoculated with the attenuated strain despite the presence of infection in the bovine host cells.

scholarly journals Association of Sequence types, Antimicrobial Resistance and Virulence Genes in India isolates of Klebsiella pneumoniae: A Comparative Genomics Study

2021 ◽  
Author(s):  
Abhirami Krishnamoorthy Sundaresan ◽  
Keerthana Vincent ◽  
Ganesh Babu Malli Mohan ◽  
Jayapradha Ramakrishnan
Keyword(s):  
Comparative Genomics ◽  
Klebsiella Pneumoniae ◽  
Virulence Genes ◽  
Respiratory Infections ◽  
Comparative Genomic ◽  
Clinical Strains ◽  
Genomic Study ◽  
Comparative Genomic Study ◽  
Phylogenetic Evolution ◽  
Tract Infections

Abstract Klebsiella pneumoniae is an important ESKAPE pathogen that causes sepsis, urinary tract infections, peritonitis, intraabdominal abscesses and upper respiratory infections. The strains exhibiting multidrug resistance and hypervirulence are priority pathogens for which immediate treatment and dissemination prevention strategies are required. The hypervirulent drug resistant K. pneumoniae is associated with high mortality rates. Numbers of environmental strains also have acquired virulence genes. Hence to gain a better understanding of the spread of antimicrobial resistant genes across the country over 10 years and to delineate environmental and clinical K. pneumoniae, a comparative genomics investigation was made. This is the first comparative genomic study using India isolates of K. pneumoniae, which includes publicly available WGS of 144 clinical and 9 environmental strains collected during 2010–2020. The blaCTX-M-15 was widely distributed in clinical isolates since 2013 and increased over time from 5 % to 30 %. The co-existence of blaNDM and blaOXA was observed in 22 % of clinical strains. Diverse serotypes were found among the 153 K. pneumoniae isolates, of which, K51 (28%) and K64 (21.56%) were majorly found. Most of the K51 isolates belong to ST231 (93.02 %). And more than 50% of KL51 strains were found to have both rmpA and magA. The number of associated virulence genes (rmpA, magA, entB, ybtS, iutA, alls,) appeared to be higher in ST231-KL51 and ST23-KL1 isolates. Of greatest concern, these virulence genes are observed in environmental strains aswell. More than 97% of clinical strains have yersinibactin (ybtS), aerobactin (iutA) genes. Importantly, 98% of ESBL and 62% of carbapenamasen isolates harboured ybtS, iutA and rmpA, magA respectively. The IncF conjugative plasmids are predominant in K. pneumoniae, which contribute to the spread of AMR, and virulence genes. The increasing trend in hypervirulent strains was observed from 2017. The phylogenetic analysis separates the environmental from clinical strains and is characterized by uncommon STs and serotypes. Thus, the study illustrates the K. pneumoniae genomic surveillance in India representing the phylogenetic evolution, STs, AMR, virulence, serotype to provide more attention for immediate treatment and preventing the dissemination of K. pneumoniae.

scholarly journals Multiparameter Optimization of Trypanocidal Cruzain Inhibitors With In Vivo Activity and Favorable Pharmacokinetics

2022 ◽  
Vol 12 ◽  
Author(s):  
Ivani Pauli ◽  
Celso de O. Rezende Jr. ◽  
Brian W. Slafer ◽  
Marco A. Dessoy ◽  
Mariana L. de Souza ◽  
...  
Keyword(s):  
Parasite Burden ◽  
Host Cells ◽  
Benzimidazole Derivatives ◽  
Pharmacokinetic Studies ◽  
Host Immune System ◽  
Novel Drugs ◽  
Main Metabolic Pathway ◽  
Multiparameter Optimization

Cruzain, the main cysteine protease of Trypanosoma cruzi, plays key roles in all stages of the parasite’s life cycle, including nutrition acquisition, differentiation, evasion of the host immune system, and invasion of host cells. Thus, inhibition of this validated target may lead to the development of novel drugs for the treatment of Chagas disease. In this study, a multiparameter optimization (MPO) approach, molecular modeling, and structure-activity relationships (SARs) were employed for the identification of new benzimidazole derivatives as potent competitive inhibitors of cruzain with trypanocidal activity and suitable pharmacokinetics. Extensive pharmacokinetic studies enabled the identification of metabolically stable and permeable compounds with high selectivity indices. CYP3A4 was found to be involved in the main metabolic pathway, and the identification of metabolic soft spots provided insights into molecular optimization. Compound 28, which showed a promising trade-off between pharmacodynamics and pharmacokinetics, caused no acute toxicity and reduced parasite burden both in vitro and in vivo.

scholarly journals Innovative Alternatives for Continuous In Vitro Culture of Babesia bigemina in Medium Free of Components of Animal Origin

Pathogens ◽  
2020 ◽  
Vol 9 (5) ◽  
pp. 343
Author(s):  
Jesús A. Álvarez Martínez ◽  
Julio V. Figueroa Millán ◽  
Massaro W. Ueti ◽  
Carmen Rojas-Martínez
Keyword(s):  
In Vitro Culture ◽  
Culture Media ◽  
Culture Method ◽  
Animal Origin ◽  
Acid Mixture ◽  
Babesia Bigemina ◽  
Lipid Mixture ◽  
Proliferation In Vitro ◽  
Animal Component

In this study, we report Babesia bigemina proliferation in culture medium free of components of animal origin supplemented with a lipid mixture. Babesia bigemina continuously proliferated in VP-SFM with a higher percent parasitized erythrocyte as compare to using other animal component-free culture media. Compared with Advanced DMEM/F12 (ADMEM/F12), VP-SFM had a similar percent parasitized erythrocyte (PPE). Supplementation of VP-SF with a lipid acid mixture improved B. bigemina proliferation in vitro culture, with a maximum PPE of 11.3%. Growth of B. bigemina in a perfusion bioreactor using VP-SFM medium supplemented with lipid mixture resulted in a PPE above 28%. In conclusion, we demonstrated that B. bigemina proliferated in an animal component-free medium supplemented with the fatty acid mixture. This innovation to B. bigemina in vitro culture method presented herein is an important source of biological material for live vaccine production and understanding the mechanisms and molecules involved in parasite attachment and invasion of bovine erythrocytes.

scholarly journals Clinical Candida albicans Vaginal Isolates and a Laboratory Strain Show Divergent Behaviors during Macrophage Interactions

mSphere ◽  
2020 ◽  
Vol 5 (4) ◽  
Author(s):  
Franziska Gerwien ◽  
Christine Dunker ◽  
Philipp Brandt ◽  
Enrico Garbe ◽  
Ilse D. Jacobsen ◽  
...  
Keyword(s):  
Candida Albicans ◽  
Fungal Infections ◽  
Laboratory Strain ◽  
Infection Process ◽  
Vulvovaginal Candidiasis ◽  
Host Immune System ◽  
Host Pathogen Interactions ◽  
Content Type ◽  
Host Pathogen

ABSTRACT Typically, established lab strains are widely used to study host-pathogen interactions. However, to better reflect the infection process, the experimental use of clinical isolates has come more into focus. Here, we analyzed the interaction of multiple vaginal isolates of the opportunistic fungal pathogen Candida albicans, the most common cause of vulvovaginal candidiasis in women, with key players of the host immune system: macrophages. We tested several strains isolated from asymptomatic or symptomatic women with acute and recurrent infections. While all clinical strains showed a response similar to the commonly used lab strain SC5314 in various in vitro assays, they displayed remarkable differences during interaction with macrophages. This coincided with significantly reduced β-glucan exposure on the cell surface, which appeared to be a shared property among the tested vaginal strains for yeast extract/peptone/dextrose-grown cells, which is partly lost when the isolates faced vaginal niche-like nutrient conditions. However, macrophage damage, survival of phagocytosis, and filamentation capacities were highly strain-specific. These results highlight the high heterogeneity of C. albicans strains in host-pathogen interactions, which have to be taken into account to bridge the gap between laboratory-gained data and disease-related outcomes in an actual patient. IMPORTANCE Vulvovaginal candidiasis is one of the most common fungal infections in humans with Candida albicans as the major causative agent. This study is the first to compare clinical vaginal isolates of defined patient groups in their interaction with macrophages, highlighting the vastly different outcomes in comparison to a laboratory strain using commonly applied virulence-determining assays.

scholarly journals What a Difference a Gene Makes: Identification of Virulence Factors of Cowpox Virus

2019 ◽  
Vol 94 (2) ◽  
Author(s):  
Aistė Tamošiūnaitė ◽  
Saskia Weber ◽  
Timo Schippers ◽  
Annika Franke ◽  
Zhiyong Xu ◽  
...  
Keyword(s):  
Wistar Rats ◽  
High Throughput Sequencing ◽  
Virulence Genes ◽  
Expression Profiles ◽  
Survival Rates ◽  
Laboratory Strain ◽  
General Interest ◽  
Infection Model ◽  
Cowpox Virus

ABSTRACT Cowpox virus (CPXV) is a zoonotic orthopoxvirus (OPV) that causes spillover infections from its animal hosts to humans. In 2009, several human CPXV cases occurred through transmission from pet rats. An isolate from a diseased rat, RatPox09, exhibited significantly increased virulence in Wistar rats and caused high mortality compared to that caused by the mildly virulent laboratory strain Brighton Red (BR). The RatPox09 genome encodes four genes which are absent in the BR genome. We hypothesized that their gene products could be major factors influencing the high virulence of RatPox09. To address this hypothesis, we employed several BR-RatPox09 chimeric viruses. Using Red-mediated mutagenesis, we generated BR-based knock-in mutants with single or multiple insertions of the respective RatPox09 genes. High-throughput sequencing was used to verify the genomic integrity of all recombinant viruses, and transcriptomic analyses confirmed that the expression profiles of the genes that were adjacent to the modified ones were unaltered. While the in vitro growth kinetics were comparable to those of BR and RatPox09, we discovered that a knock-in BR mutant containing the four RatPox09-specific genes was as virulent as the RatPox09 isolate, causing death in over 75% of infected Wistar rats. Unexpectedly, the insertion of gCPXV0030 (g7tGP) alone into the BR genome resulted in significantly higher clinical scores and lower survival rates matching the rate for rats infected with RatPox09. The insertion of gCPXV0284, encoding the BTB (broad-complex, tramtrack, and bric-à-brac) domain protein D7L, also increased the virulence of BR, while the other two open reading frames failed to rescue virulence independently. In summary, our results confirmed our hypothesis that a relatively small set of four genes can contribute significantly to CPXV virulence in the natural rat animal model. IMPORTANCE With the cessation of vaccination against smallpox and its assumed cross-protectivity against other OPV infections, waning immunity could open up new niches for related poxviruses. Therefore, the identification of virulence mechanisms in CPXV is of general interest. Here, we aimed to identify virulence markers in an experimental rodent CPXV infection model using bacterial artificial chromosome (BAC)-based virus recombineering. We focused our work on the recent zoonotic CPXV isolate RatPox09, which is highly pathogenic in Wistar rats, unlike the avirulent BR reference strain. In several animal studies, we were able to identify a novel set of CPXV virulence genes. Two of the identified virulence genes, encoding a putative BTB/POZ protein (CPXVD7L) and a B22R-family protein (CPXV7tGP), respectively, have not yet been described to be involved in CPXV virulence. Our results also show that single genes can significantly affect virulence, thus facilitating adaptation to other hosts.

Babesia bigemina : Advances in continuous in vitro culture using serum-free medium supplemented with insulin, transferrin, selenite, and putrescine

2018 ◽  
Vol 67 (3) ◽  
pp. 294-301 ◽  
Author(s):  
Carmen Rojas-Martínez ◽  
Roger I. Rodríguez-Vivas ◽  
Julio V. Figueroa Millán ◽  
Karla Y. Acosta Viana ◽  
Edwin J. Gutiérrez Ruíz ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Serum Free Medium ◽  
Free Medium ◽  
Babesia Bigemina ◽  
Serum Free

scholarly journals Temporal Expression of Enteropathogenic Escherichia coli Virulence Genes in an In Vitro Model of Infection

2005 ◽  
Vol 73 (2) ◽  
pp. 1034-1043 ◽  
Author(s):  
Laura Q. Leverton ◽  
James B. Kaper
Keyword(s):  
Escherichia Coli ◽  
In Vitro Model ◽  
Virulence Genes ◽  
Host Cells ◽  
Temporal Expression ◽  
Lesion Formation ◽  
Culture Models ◽  
Vitro Model ◽  
Enterocyte Effacement

ABSTRACT The hallmark of enteropathogenic Escherichia coli (EPEC) infection is the ability of EPEC to cause attaching and effacing (A/E) lesions on intestinal epithelium. This event is reproducible in in vitro tissue culture models of infection. We used real-time PCR to measure transcription from several locus of enterocyte effacement (LEE) operons (LEE1 to LEE5) and from bfp during a 5-h infection of HEp-2 cells with EPEC. We found that after the initial formation of A/E lesions, which occurs as early as 5 min postinfection, EPEC continues to increase transcription from LEE3 to LEE5 as well as from bfp. These levels are maximized by 3 h postinfection and remain constant throughout the course of infection. This increase in transcription from LEE3 to LEE5 occurs when LEE1 (ler) transcription is decreasing. EspA, EspB, intimin, Tir, and bundle-forming pilus expression is detectable during the entire 5-h infection. These results indicate that the EPEC genes involved in localized and intimate adherence are continually expressed after the initial stages of A/E lesion formation on host cells.

scholarly journals Exogenous nitric oxide stimulates early egress of Eimeria tenella sporozoites from primary chicken kidney cells in vitro

Parasite ◽  
2021 ◽  
Vol 28 ◽  
pp. 11
Author(s):  
Xinlei Yan ◽  
Wenying Han ◽  
Xianyong Liu ◽  
Xun Suo
Keyword(s):  
Nitric Oxide ◽  
Vital Role ◽  
Eimeria Tenella ◽  
Host Cells ◽  
Host Immune System ◽  
Kidney Cells ◽  
Apicomplexan Parasites ◽  
Immune Molecule ◽  
Chicken Kidney

Egress plays a vital role in the life cycle of apicomplexan parasites including Eimeria tenella, which has been attracting attention from various research groups. Many recent studies have focused on early egress induced by immune molecules to develop a new method of apicomplexan parasite elimination. In this study, we investigated whether nitric oxide (NO), an immune molecule produced by different types of cells in response to cytokine stimulation, could induce early egress of eimerian sporozoites in vitro. Eimeria tenella sporozoites were extracted and cultured in primary chicken kidney cells. The number of sporozoites egressed from infected cells was analyzed by flow cytometry after treatment with NO released by sodium nitroferricyanide (II) dihydrate. The results showed that exogenous NO stimulated the rapid egress of E. tenella sporozoites from primary chicken kidney cells before replication of the parasite. We also found that egress was dependent on intra-parasitic calcium ion (Ca2+) levels and no damage occurred to host cells after egress. The virulence of egressed sporozoites was significantly lower than that of fresh sporozoites. The results of this study contribute to a novel field examining the interactions between apicomplexan parasites and their host cells, as well as that of the clearance of intracellular pathogens by the host immune system.

scholarly journals Genome Survey Sequencing of In Vivo Mother Plant and In Vitro Plantlets of Mikania cordata

Plants ◽  
2020 ◽  
Vol 9 (12) ◽  
pp. 1665
Author(s):  
Yongfeng Hong ◽  
Xia Huang ◽  
Chunmei Li ◽  
Xiaoxian Ruan ◽  
Zhen Wang ◽  
...  
Keyword(s):  
In Vitro Culture ◽  
Genome Size ◽  
Culture Technique ◽  
Mother Plant ◽  
Comparative Genomic ◽  
Invasive Weed ◽  
In Vitro Plantlets ◽  
Long Read

Mikania cordata, the only native congener of the invasive weed Mikania micrantha in China, is an ideal species for comparative study to reveal the invasion mechanism. However, its genome resources are lagging far behind its congener, which limits the comparative genomic analysis. Our goal is to characterize the genome of M. cordata by next-generation sequencing and propose a scheme for long-read genome sequencing. Previous studies have shown that the genomic resources of the host plant would be affected by the endophytic microbial DNA. An aseptic sample of M. cordata will ensure the proper genome in downstream analysis. Because endophytes are ubiquitous in the greenhouse-grown M. cordata, the in vitro culture with cefotaxime or timentin treatment was undertaken to obtain the aseptic plantlets. The in vivo mother plant and in vitro plantlets were used to survey the genome. The microbial contamination in M. cordata was recognized by blast search and eliminated from the raw reads. The decontaminated sequencing reads were used to predict the genome size, heterozygosity, and repetitive rate. The in vivo plant was so contaminated that microbes occupied substantial sequencing resources and misled the scaffold assembly. Compared with cefotaxime, treatment with timentin performed better in cultivating robust in vitro plantlets. The survey result from the in vitro plantlets was more accurate due to low levels of contamination. The genome size was estimated to be 1.80 Gb with 0.50% heterozygosity and 78.35% repetitive rate. Additionally, 289,831 SSRs were identified in the genome. The genome is heavily contaminated and repetitive; therefore, the in vitro culture technique and long-read sequencing technology are recommended to generate a high-quality and highly contiguous genome.

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