Babesia duncani Pyruvate Kinase Inhibitor Screening and Identification of Key Active Amino Acid Residues.

Babesia duncani (B. duncani), a protozoan parasite prevalent in North America, is a significant threat for human health. Given the regulatory role of pyruvate kinase I (PyK I) in glycolytic metabolism flux and ATP generation, PyK I has been considered the target for drug intervention for a long time. In this study, B. duncani PyK I (BdPyK I) was successfully cloned, expressed, and purified. Polyclonal antibodies were confirmed to recognize the native BdPyK I protein (56 kDa) using Western blotting. AlphaFold software predicted the three-dimensional structure of BdPyK I, and molecular docking with small molecules was conducted to identify potential binding sites of inhibitor on BdPyK I. Moreover, inhibitory effects of six inhibitors (tannic acid, apigenin, shikonin, PKM2 inhibitor, rosiglitazone, and pioglitazone) on BdPyK I were examined under the optimal enzymatic conditions of 3 mM PEP and 3 mM ADP, and significant activity reduction was found. Enzyme kinetics and growth inhibition assays further confirmed the reliability of these inhibitors, with PKM2 inhibitor, tannic acid, and apigenin exhibiting the highest selectivity index as specific inhibitors for B. duncani. Subsequently, key amino acid residues were mutated in both BdPyK I and Homo sapiens pyruvate kinase I (HPyK I), and two differential amino acid residues (isoleucine and phenylalanine) were identified between HPyK I and BdPyK I through PyK activity detection experiments. These findings lay foundation for understanding the role of PyK I in the growth and development of B. duncani, providing insights for babesiosis prevention and drug development.

A novel easy-to-desorb eluant contributes to address environmental contamination of African swine fever virus.

African swine fever virus (ASFV) is a highly pathogenic and rapidly disseminated virus with strong viability in the environment, suggesting the importance of environmental detection for prevention and control in all the pig industry. However, the detection results of environmental swabs cannot always reflect the accurate status of viral pollution, leading to persistent ASFV environmental contamination. In this study, we developed an ASFV eluant with higher environmental ASFV detection efficiency relative to 0.85% saline solution, which obtains the patent certificate issued by the China Intellectual Property Office (patent number:202010976050.9). qPCR analysis showed that in the environmental swab samples, the number of viral copies was 100 times higher for the ASFV eluant treatment than the traditional eluant treatment (0.85% saline solution). And besides, the high sensitivity of the ASFV eluant had be verified in a slaughterhouse environmental sampling detection. In soil samples, the ASFV eluent showed the same extraction effect as the TIANamp Soil DNA Kit, in contrast to no extraction effect for 0.85% saline solution. Simultaneously, this eluent could protect ASFV from degradation and allow the transportation of samples at ambient temperature without refrigeration. In clinical practice, we monitored the environmental contamination condition of the ASFV in a large-scale pig farm. The results shown that the ASFV load decreased after every disinfection in environment. This study provides an effective solution for surveilling the potential threat of ASFV in environment.

Isolation, Identification, Genomic Diversity, and Antimicrobial Resistance Analysis of Streptococcus suis in Hubei Province of China from 2021 to 2023.

Streptococcus suis (S. suis) is a zoonotic pathogen capable of causing severe diseases in humans and pigs, including meningitis, sepsis, polyserositis, arthritis, and endocarditis. This study aimed to investigate the biological characteristics of 19 strains of S. suis isolated from diseased pigs in Hubei Province between 2021 and 2023. Through bioinformatics analysis, we investigated the serotype, MLST, pan-genome characteristics, SNP, AMR, and ICE of the 19 S. suis isolates. Among the 19 S. suis strains, ten serotypes were identified, and serotype 9 was the most prevalent (21.05%). Ten new alleles and nine new sequence types (STs) were discovered, with ST28 and ST243 emerging as the predominant STs. The results of the pan-genomic analysis of S. suis indicate that there are 943 core genes, 2259 shell genes, and 5663 cloud genes. Through SNP evolutionary analysis, we identified a strong genetic similarity between SS31 and the reference genome P1/7. The analysis of antibiotic resistance genes revealed widespread presence of erm(B) and tet(O) genes among 19 strains of S. suis. This association may be linked to the high resistance of S. suis to lincosamides, macrolides, and tetracyclines. Integrative and conjugative elements (ICEs) and integrative and mobilizable elements (IMEs) were identified in 16 strains, with a carriage rate of 84.21%, and resistance genes were identified within the ICE/IME elements of 8 strains. Antimicrobial susceptibility testing revealed that all strains showed sensitivity to vancomycin and lincomycin but resistance to tilmicosin, tiamulin, amoxicillin, and doxycycline. This study contributes to our understanding of the genomic diversity of S. suis in Hubei Province of China, providing essential data for the comprehensive prevention and control of S. suis infections in China.

Babesia microti Infection Inhibits Melanoma Growth by Activating Macrophages in Mice.

Babesia microti is an obligate intraerythrocytic protozoan transmitted by an Ixodes tick. Infections caused by protozoa, including Plasmodium yoelii and Toxoplasma gondii, are shown to inhibit tumor development by activating immune responses. Th1 immune response and macrophages not only are essential key factors in Babesia infection control but also play an important role in regulating tumor development. In this study, we investigated the effects of B. microti infection on melanoma in tumor-bearing mice. The results showed that B. microti infection could inhibit the growth of melanoma, significantly enlarge the spleen size (p ≤ 0.0001), and increase the survival period (over 7 days) of tumor-bearing mice. Mouse spleen immune cell analysis revealed that B. microti-infected tumor-bearing mice could increase the number of macrophages and CD4+ T cells, as well as the proportion of CD4+ T cells and M1 macrophages in the tumor. Immunohistochemical assays showed that B. microti infection could inhibit tumor angiogenesis (p ≤ 0.0032). Meanwhile, both B. microti-infected erythrocytes and culture supernatant were observed to significantly (p ≤ 0.0021) induce the mRNA expression of iNOS, IL-6, and TNF-α in macrophages. Moreover, B. microti culture supernatant could also repolarize IL-4-induced M2 macrophages to the M1 type. Overall, B. microti exerted antitumor effects by stimulating the immune system of tumor-bearing mice and inducing the polarization of immunosuppressive M2 macrophages to pro-inflammatory M1 macrophages.

Seroprevalence Investigation of Classic Swine Fever Virus Before, During, and After African Swine Fever Virus Outbreak in Some Provinces of China from 2017 to 2021.

Classic swine fever is a severe infectious and fatal disease in pigs caused by the classic swine fever virus (CSFV). Surveillance and investigation for CSFV seroprevalence contribute to knowing the immune efficiency of CSFV vaccines and reflect health status of swine herd, especially since the African swine fever virus (ASFV) outbreak in China in 2018. A total of 40,489 pig serum samples with related descriptive variables were obtained from 12 provinces and 2 cities of China from December 2017 to May 2021, covering before, during, and after three periods of ASFV outbreak. Pearson chi-square test and multivariable logistic regression analysis were used to identify impact factors related to variations in CSFV seroprevalence. Total CSFV seroprevalence was 60.40% (95% confidence interval: 59.92-60.88). Seroprevalence and antibody blocking rate mean of CSFV before outbreak of ASFV in China are higher and change gently compared with that after outbreak of ASFV. Serum collected from "summer and autumn," "north, southwest and northwest of China," "pig farm located in hill or mountain," " period before outbreak of ASFV," "PRRSV negative farm," and "replacement gilts, multiparous sows and boars" show high seroprevalence of CSFV. These results show trends in prevalence of CSFV antibody in recent years in China, especially when ASFV entered China. Identified impact factors provide references for improving immune efficiency of CSFV vaccine and benefit for prevention of CSFV.

Identification of a novel variant erythrocyte surface antigen-1 (VESA1) in Babesia orientalis.

Babesia orientalis, belonging to the phylum Apicomplexa, is mainly accountable for water buffalo babesiosis, which adversely affected the livestock industry in China. Variant erythrocyte surface antigen-1 (VESA1), an antigen that helps infected erythrocytes to escape from host immune responses, was first reported in Babesia bovis. Various VESA1 proteins have also been characterized in other Babesia species. Nevertheless, there is no research on the identification and characterization of VESA1 proteins in Babesia orientalis. In this study, the BoVESA1 gene was amplified from both gDNA and cDNA. The results revealed that it is an intronless gene with a full length of 753 bp, encoding a protein of 250 amino acids with a predicted molecular weight of 28 kDa. The coding sequence (CDS) was cloned into the pGEX-6p-1 vector using a homologous recombination kit and expressed as a glutathione-S-transferase (GST)-fusion protein with a molecular weight of 53 kDa. The tertiary structure of BoVESA1 was predicted using the I-TASSER software. The recombinant protein was subjected to western blotting; the immunogenicity of recombinant BoVESA1 (rBoVESA1) was identified by incubating it with B. orientalis-positive serum. The native BoVESA1 was identified using the lysates of B. orientalis-infected water buffalo erythrocytes incubated with the anti-rBoVESA1 mouse serum. The results showed a band of ~ 28 kDa, which is similar to the predicted size. Immunofluorescence assay (IFA) using anti-rBoVESA1 serum probed indicated a strong signal in the infected RBCs, while the negative control showed no signal. In conclusion, the VESA1 protein was first identified in B. orientalis. This study facilitated further investigation of B. orientalis, and the results indicated that BoVESA1 may serve as a potential candidate antigen for diagnosis and detection of B. orientalis infection.

Evaluation of immunoprotective effects of recombinant protein and DNA vaccine based on Eimeria tenella surface antigen 16 and 22 in vivo.

Coccidiosis triggered by Eimeria tenella is accompanied by haemorrhagic caecum and high morbidity. Vaccines are preferable choices to replace chemical drugs against coccidiosis. Surface antigens of apicomplexan parasites can adhere to host cells during the infection process. Therefore, truncated fragments coding E. tenella surface antigen 16 (EtSAG16) and 22 (EtSAG22) were cloned into pET-28a prokaryotic vector to express recombinant protein 16 (rEtSAG16) and 22 (rEtSAG22), respectively. Likewise, pEGFP-N1-EtSAG16 and pEGFP-N1-EtSAG22 plasmids were constructed using pEGFP-N1 eukaryotic vector. Further, pEGFP-N1-EtSAG4-16-22 multiple gene plasmid carrying EtSAG4, 16 and 22 were designed as cocktail vaccines to study integral immunoprotective effects. Western blot and RT-PCR (reverse transcription) assay were performed to verify expressions of EtSAG16 and 22 genes. Immunoprotective effects of recombinant protein or DNA vaccine were evaluated using different doses (50 or 100 µg) in vivo. All chickens in the vaccination group showed higher cytokine concentration (IFN-γ and IL-17), raised IgY antibody level, increased weight gain, lower caecum lesion score and reduced oocyst shedding compared with infection control groups (p < 0.05). The highest anticoccidial index (ACI) value 173.11 was from the pEGFP-N1-EtSAG4-16-22 plasmid (50 µg) group. In conclusion, EtSAG16 and 22 might be alternative candidate genes for generating vaccines against E. tenella infection.

Characterization of the variable merozoite surface antigen (VMSA) gene family of Babesia orientalis.

Due to its wide presence in apicomplexan parasites as well as high polymorphism and antigenic diversity, the variable merozoite surface antigen (VMSA) family in Babesia sp. has attracted increasing attention of researchers. Here, all the reported VMSA genes of Babesia spp. were obtained from GenBank, and multiple alignments were performed by using conserved regions to blast the Babesia orientalis genome database (unpublished data). Five MSA genes (named MSA-2a1, MSA-2a2, MSA-2c1, MSA-1, and MSA-2c2, respectively) were identified, sequenced, and cloned from B. orientalis, which were shown to encode proteins with open reading frames ranging in size from 266 (MSA-2c1) to 317 (MSA-1) amino acids. All the five proteins contain an MSA-2c superfamily conserved domain, with an identical signal peptide and glycosyl phosphatidyl inositol (GPI)-anchor for each of them. The five proteins were also predicted to contain B cell epitopes, with only three for BoMSA-2c1, the smallest protein in the BoVMSA family, while at least six for each of the others. Notably, BoMSA-2a1 has 2 identical copies, a specific phenomenon only present in B. orientalis. This research has determined the MSA genes of B. orientalis and provides a genetic basis for further research of functional genes in B. orientalis.

A novel 53 kDa protein (BoP53) in Babesia orientalis poses the immunoreactivity using the infection serum.

Babesia orientalis (B. orientalis) is responsible for water buffalo babesiosis, which caused serious economic losses in the south of China. Although the invasion process has been roughly described, there are still some unknown molecules that have not yet been identified. Recently, an invasion-related protein BOV57 has been identified in the Babesia bovis. However, there is no report available about the gene in B. orientalis. B. orientalis P53 (BoP53) sequence was obtained by blast BOV57 sequence in B. orientalis genome database, and the full length of the BoP53 gene is 1599 bp. BoP53 gene was cloned into a pGEX-6P-1 expression vector and expressed as a GST-tag fusion protein. The tertiary structure of BoP53 was predicted with the I-TASSER software. The native BoP53 was identified from of B. orientalis lysate incubation with mouse antiserum against rBoP53. BoP53 as a novel identified protein promotes the study of B. orientalis, the reaction of rBoP53 with the serum of B. orientalis-infected water buffalo but not with healthy buffalo serum indicated its good antigenicity. It may be a candidate antigen for the diagnosis of B. orientalis infection.