The Clostridium perfringens epidemic threatens biosecurity and causes significant economic losses. C. perfringens infections are linked to more than one hundred million cases of food poisoning annually, and 8-60% of susceptible animals are vulnerable to infection, resulting in an economic loss of more than 6 hundred million USD. The enzymes and toxins (>20 species) produced by C. perfringens play a role in intestinal colonization, immunological evasion, intestinal micro-ecosystem imbalance, and intestinal mucosal disruption, all influencing host health. In recent decades, there has been an increase in drug resistance in C. perfringens due to antibiotic misuse and bacterial evolution. At the same time, traditional control interventions have proven ineffective, highlighting the urgent need to develop and implement new strategies and approaches to improve intervention targeting. Therefore, an in-depth understanding of the spatial and temporal evolutionary characteristics, transmission routes, colonization dynamics, and pathogenic mechanisms of C. perfringens will aid in the development of optimal therapeutic strategies and vaccines for C. perfringens management. Here, we review the global epidemiology of C. perfringens, as well as the molecular features and roles of various virulence factors in C. perfringens pathogenicity. In addition, we emphasize measures to prevent and control this zoonotic disease to reduce the transmission and infection of C. perfringens.

OBJECTIVE: Cryptosporidium spp. and Giardia duodenalis are two important foodborne human and animal parasites that can be disseminated through both food and water, leading to diarrheal disease. Nevertheless, available information on the circumstances of Cryptosporidium and Giardia duodenalis from Ningxia is limited.
METHODS: A total of 208 stool samples of dairy calves derived from large-scale farms (> 1000 heads) of five cities randomly in Ningxia were gathered randomly, were amplified and analyzed by nested PCR based on the three target genes (18S rRNA, gp60 and tpi)and phylogenetic systematics.
RESULTS: The prevalence of cryptosporidiosis and giardiasis in dairy calves in Ningxia were 13.0% (27/208 samples, 95% CI 9.1-18.2%) and 1.9% (4/208, 95% CI 0.8-4.9%) respectively. Three Cryptosporidium species appeared in this study which are Cryptosporidium parvum (C. parvum), Cryptosporidium andersoni (C. andersoni) and Cryptosporidium ryanae (C. ryanae) based on the 18S rRNA gene sequence. IIdA15G1 and IIdA13G1 belonging to the subtypes of Cryptosporidium were detected by the gp60 PCR. The genotypes of Giardia duodenalis were only assemblage E through the amplification of the triosephosphate-isomerase gene (tpi gene).
CONCLUSIONS: There is a risk of transmission to humans in Ningxia because of zoonotic genotypes (C. parvum, C. andersoni, assemblage E) and subtypes (IId) of Cryptosporidium spp. and G. duodenalis in dairy calves, and it is necessary to pay attention to the disease to prevent a widespread epidemic of the disease with the purpose to protect human and livestock health.

Pentatrichomonas hominis is a trichomonad protozoan that infects the cecum and colon of humans and other mammals. It is a zoonotic pathogen that causes diarrhea in both animals and humans. As companion animals, dogs infected with P. hominis pose a risk of transmitting it to humans. Current methods, such as direct smears and polymerase chain reaction (PCR), used for P. hominis detection have limitations, including low detection rates and the need for specialized equipment. Therefore, there is an urgent need to develop rapid, sensitive, and simple detection methods for clinical application. Recombinase polymerase amplification (RPA) has emerged as a technology for rapid pathogen detection. In this study, we developed a lateral flow dipstick (LFD)-RPA method based on the highly conserved SPO11-1 gene for detecting P. hominis infection by optimizing the primers, probes, and reaction conditions, and evaluating cross-reactivity with genomes of Giardia duodenalis and other parasites. The LFD-RPA method was then used to test 128 dog fecal samples collected from Changchun. The results confirmed the high specificity of the method with no cross-reactivity with the five other parasites. The lowest detection limit of the method was 102 copies/µL, and its sensitivity was 100 times higher than that of the conventional PCR method. Consistent with the positivity rate observed using nested PCR, 12 samples (out of 128) tested positive using this method (positivity rate, 9.38%). In conclusion, the LFD-RPA method developed in this study represents a simple and sensitive assay that allows for the rapid detection of P. hominis infection in dogs, especially in this field.

Streptococcus pasteurianus is a zoonotic pathogen causing meningitis and bacteremia in animals and humans. A lack of accurate and convenient detection methods hinders preventing and controlling diseases caused by S. pasteurianus. Additionally, there is limited knowledge about its pathogenicity and antimicrobial resistance characteristics, as there are only three complete genome sequences available. In this study, we established a multiplex PCR assay for the detection of S. pasteurianus, which was applied to six fecal samples from cattle with diarrhea and 285 samples from healthy pigs. Out of the samples tested, 24 were positive, including 5 from pig tonsils, 18 from pig hilar lymph nodes, and 1 from cattle feces. Two strains were isolated from positive samples, and their complete genomes were sequenced. The two strains were non-virulent in mice and multidrug-resistant by the antimicrobial susceptibility test. We first found the presence of genes tet(O/W/32/O) and lsa(E) in S. pasteurianus, leading to resistance to lincosamides and tetracyclines. The convenient and specific multiplex PCR assay provides essential technical support for epidemiological research, and the complete genome sequence of two non-virulent strains contributes to understanding this zoonotic bacterium\'s genomic characteristics and pathogenesis.

Pasteurella multocida (P. multocida) is an important zoonotic pathogen. In addition to lung lesions, necropsies have revealed macroscopic lesions in the heart in clinical cases. However, most previous studies focused on lung lesions while ignoring heart lesions. Therefore, to investigate the immune response of the P. multocida-infected heart, two murine infection models were established by using P. multocida serotype A (Pm HN02) and D (Pm HN01) strains. Histopathological examination revealed heterogeneous inflammatory responses, including immune cell infiltration in the epicardial and myocardial areas of the heart. Transcriptome sequencing was performed on infected cardiac tissues. To explore the traits of immune responses, we performed the functional enrichment analysis of differentially expressed genes, gene set enrichment analysis and gene set variation analysis. The results showed that the innate immune pathways were significantly regulated in both groups, including the NOD-like receptor signaling pathway, the complement and coagulation cascade and cytokine-cytokine receptor interaction. The Toll-like receptor signaling pathway was only significantly activated in the Pm HN02 group. For the Pm HN02 group, immunohistochemistry analysis further verified the significant upregulation of the hub component MyD88 at the protein level. In conclusion, this study reveals critical pathways for host heart recognition and defense against P. multocida serotypes A and D. Moreover, MyD88 was upregulated by P. multocida serotype A in the heart, providing a theoretical basis for future prevention, diagnosis and treatment research.

The emergence and rapid spread of the acute respiratory syndrome coronavirus-2 have confirmed that animal coronaviruses represent a potential zoonotic source. Porcine deltacoronavirus is a worldwide evolving enteropathogen of swine, detected first in Hong Kong, China, before its global identification. Following the recent detection of PDCoV in humans, we attempted in this report to re-examine the status of PDCoV phylogenetic classification and evolutionary characteristics. A dataset of 166 complete PDCoV genomes was analyzed using the Maximum Likelihood method in IQ-TREE with the best-fitting model GTR + F + I + G4, revealing two major genogroups (GI and GII), with further seven and two sub-genogroups, (GI a-g) and (GII a-b), respectively. PDCoV strains collected in China exhibited the broadest genetic diversity, distributed in all subgenotypes. Thirty-one potential natural recombination events were identified, 19 of which occurred between China strains, and seven involved at least one China strain as a parental sequence. Importantly, we identified a human Haiti PDCoV strain as recombinant, alarming a possible future spillover that could become a critical threat to human health. The similarity and recombination analysis showed that PDCoV spike ORF is highly variable compared to ORFs encoding other structural proteins. Prediction of linear B cell epitopes of the spike glycoprotein and the 3D structural mapping of amino acid variations of two representative strains of GI and GII showed that the receptor-binding domain (RBD) of spike glycoprotein underwent a significant antigenic drift, suggesting its contribution in the genetic diversity and the wider spread of PDCoV.

Arcobacter spp. is a globally emerging zoonotic and foodborne pathogen. However, little is known about its prevalence and antimicrobial resistance in China. To investigate the prevalence of Arcobacter spp. isolated from various sources, 396 samples were collected from human feces, chicken cecum, and food specimens including chicken meat, beef, pork, lettuce, and seafood. Arcobacter spp. was isolated by the membrane filtration method. For 92 strains, the agar dilution method and next-generation sequencing were used to investigate their antimicrobial resistance and to obtain whole genome data, respectively. The virulence factor database (VFDB) was queried to identify virulence genes. ResFinder and the Comprehensive Antibiotic Resistance Database (CARD) were used to predict resistance genes. A phylogenetic tree was constructed using the maximum likelihood (ML) method with core single-nucleotide polymorphisms (SNPs). We found that 27.5% of the samples (n = 109) were positive for Arcobacter spp., comprising Arcobacter butzleri (53.0%), Arcobacter cryaerophilus (39.6%), and Arcobacter skirrowii (7.4%). Chicken meat had the highest prevalence (81.2%), followed by seafood (51.9%), pork (43.3%), beef (36.7%), lettuce (35.5%), chicken cecum (8%), and human fecal samples (0%, 0/159). Antimicrobial susceptibility tests revealed that 51 A. butzleri and 40 A. cryaerophilus strains were resistant to streptomycin (98.1, 70%), clindamycin (94.1, 90%), tetracycline (64.7, 52.5%), azithromycin (43.1%, 15%), nalidixic acid (33.4, 35%), and ciprofloxacin (31.3, 35%) but were susceptible to erythromycin, gentamicin, chloramphenicol, telithromycin, and clindamycin (≤10%). A. skirrowii was sensitive to all experimental antibiotics. The virulence factors tlyA, mviN, cj1349, ciaB, and pldA were carried by all Arcobacter spp. strains at 100%, and the following percentages were cadF (95.7%), iroE (23.9%), hecB (2.2%), hecA, and irgA (1.1%). Only one A. butzleri strain (F061-2G) carried a macrolide resistance gene (ereA). One A. butzleri and one A. cryaerophilus harbored resistance island gene clusters, which were isolated from pork and chicken. Phylogenetic tree analysis revealed that A. butzleri, A. cryaerophilus, and A. skirrowii were separated from each other. To our knowledge, this is the first report of the isolation of Arcobacter spp. from vegetables and seafood in China. The resistance island gene cluster found in pork and chicken meat and the presence of virulence factors could be a potential risk to human health.

Hepatitis E virus (HEV) is an emerging zoonotic pathogen with multiple species and genotypes, which may be classified into human, animal, and zoonotic HEV. Codon usage bias of HEV remained unclear. This study aims to characterize the codon usage of HEV and elucidate the main drivers influencing the codon usage bias. A total of seven HEV genotypes, HEV-1 (human HEV), HEV-3 and HEV-4 (zoonotic HEV), HEV-8, HEV-B, HEV-C1, and HEV-C2 (emerging animal HEV), were included in the study. Complete coding sequences, ORF1, ORF2, and ORF3, were accordingly obtained in the GenBank. Except for HEV-8, the other six genotypes tended to use codons ending in G/C. Based on the analysis of relatively synonymous codon usage (RSCU) and principal component analysis (PCA), codon usage bias was determined for HEV genotypes. Codon usage bias differed widely across human, zoonotic, and animal HEV genotypes; furthermore, it varied within certain genotypes such as HEV-4, HEV-8, and HEV-C1. In addition, dinucleotide abundance revealed that HEV was affected by translation selection to form a unique dinucleotide usage pattern. Moreover, parity rule 2 analysis (PR2), effective codon number (ENC)-plot, and neutrality analysis were jointly performed. Natural selection played a leading role in forming HEV codon usage bias, which was predominant in HEV-1, HEV-3, HEV-B and HEV-C1, while affected HEV-4, HEV-8, and HEV-C2 in combination with mutation pressure. Our findings may provide insights into HEV evolution and codon usage bias.

The dissemination of antibiotic resistance genes (ARGs) in pathogens is becoming a pervasive global health threat, to which the importance of the environment attracts more and more attention. However, how natural minerals affect ARGs transfer in pathogens is still unclear. In this study, the concentration and size effects of hematite on the ARGs conjugative transfer to a common zoonotic pathogen Escherichia coli O157:H7 and underlying mechanisms were explored. Results revealed that bulk hematite reduced the conjugation of resistant plasmids by inhibiting cell growth at any concentration (1-100 mg/L), different from nano-hematite. Low concentrations of nano-hematite (≤ 25 mg/L) induced significant increases in conjugative transfer frequency of 1.83-4.49 folds, while its high concentrations (50 and 100 mg/L) showed no impact, compared with the control group. This low-concentration effect was likely attributed to the increased intracellular ROS level, the reduced intercellular repulsion by increasing the extracellular polymeric substances production and cell surface hydrophobicity, the formation of transfer channels and the increased membrane permeability evidenced by significant changes in gene expression level, and the increased proton motive force by increasing the transmembrane potential of recipients. These findings shed light on potential health risks caused by nano minerals-mediated ARGs dissemination in pathogens in the environment.

Anaplasma capra, a species of the family Anaplasmataceae, is zoonotic tick-borne obligate intracellular bacteria. There have been no reports of human infection with this pathogen since 2015. Therefore, the zoonotic characteristics of A. capra need to be further studied. To verify the ability of A. capra to infect human cells, A. capra were inoculated in human erythrocytes, HL-60, and TF-1 cell lines in vitro. Cell smears were taken after inoculation, using Giemsa staining, transmission electron microscope (TEM), chromogenic in situ hybridization and immunocytochemistry for detection. In the Giemsa staining, many dark colored corpuscles or purple granules were seen in the inoculated erythrocytes, HL-60, and TF-1 cells. The results of chromogenic in situ hybridization show that there were brown precipitates on the surface of most erythrocytes. Immunocytochemistry results show many dark brown vacuolar structures or corpuscles in the cytoplasm of erythrocytes, HL-60, and TF-1 cell lines. The A. capra morulae were seen in the cytoplasm of both HL-60 and TF-1 in TEM, and their diameter was about 295-518 nm. Both dense-cored (DC) and reticulate cell (RC) form morulae could be seen. This study confirmed the ability of A. capra to infect human erythrocytes, HL-60, and TF-1. This study is of profound significance in further verifying the zoonotic characteristics of the pathogen and for establishing an in vitro cultivation model.