Prevalence of Cryptosporidium spp. in Sheep and Goats in Jiangsu, China.

Sheep and goats serve as crucial hosts for Cryptosporidium spp. and are primarily responsible for its transmission via the fecal-oral route. This can result in symptoms such as lamb weight loss, diarrhea, and even fatalities, leading to significant economic losses. Currently, there is a lack of scholarly research investigating the prevalence of Cryptosporidium spp. infection in sheep and goats specifically within Jiangsu province. This study collected fecal samples from sheep and goats, extracted their DNA, amplified target bands using nested PCR, sequenced the DNA, constructed a phylogenetic tree, and identified the genetic genotype. In total, 3 positive samples were identified out of 398 samples. Furthermore, the gene sequences of these samples exhibited significant homology with C. xiaoi in GenBank. The phylogenetic analysis revealed that the Cryptosporidium spp. parasites under investigation are phylogenetically related to C. xiaoi. Conducting epidemiological investigations and accurately identifying the species of Cryptosporidium spp. is of utmost importance not only for the mutton sheep farming industry in Jiangsu but also for the proactive safeguarding of human health.

Prevalence and risk factors associated with gastrointestinal parasites in goats (Capra hircus) and sheep (Ovis aries) from three provinces of China.

Gastrointestinal (GI) parasites in small ruminants, especially goats and sheep, have caused significant socio-economic and public health challenges worldwide. The aim of the present study was to investigate the diversity and prevalence of GI parasites in goats and sheep in Jiangsu, Shaanxi and Hunan provinces of China, and to assess whether the age of animals, sampling season and feeding mode influence the distribution and infection of GI parasites. A total of 1,081 fecal samples collected from goats (n = 835) and sheep (n = 246) were detected by saturated saline flotation technique and nylon sifter elutriation and sieving method for eggs/oocysts, respectively. Based on the morphological observation of eggs and oocysts, one tapeworm, five nematodes, three trematodes and nineteen coccidia were identified, of which seven helminths belong to zoonotic parasites. The infection rate of parasites was 83.4% (902/1081) in total samples, 91.6% (765/835) in goats, and 55.7% (137/246) in sheep. The infection rate of coccidia was 71.0% (767/1081), and that of helminths was 56.2% (607/1081). The dominant species was E. alijeri (67.3%, 562/835) in goats, E. parva (30.1%, 74/246) in sheep. The highest prevalent helminths were Trichostrongylidae spp. in goats (58.3%, 487/835), and Moniezia spp. in sheep (22.76%, 56/246). Of 902 positive samples, 825 (91.5%, 825/902) contained multiple (2-10) parasites. The feeding mode, sampling season and regions were relevant risk factors which have significant influence on the occurrence of GI parasites in goats and sheep. The risk coefficient of parasite infection in autumn was 2.49 times higher than spring (Odds ratio = 2.49, 95% CI = 1.51-4.09, p < 0.001). Compared to raising on the high beds, the goats and sheep raising on the ground had the higher risk of parasite infection (OR = 3.91, 95% CI = 2.07-7.40, p < 0.001). The risk coefficient of parasite infection in Shaanxi and Hunan was 3.78 and 1.25 times higher than that in Jiangsu (OR = 3.78, 95% CI = 2.01-7.12, p < 0.001; OR = 1.25, 95% CI = 1.21-1.29, p < 0.001). These data are significant for the development of prevention strategies to minimise economic losses from small ruminant production and to reduce the risk of water and food infecting humans as vectors of zoonotic parasitic diseases.

An Attenuated Escherichia coli K88ac LT(S63K)ΔSTb Efficiently Provides Protection Against Enterotoxigenic Escherichia coli in the Mouse Model.

To develop an attenuated vaccine candidate against K88ac enterotoxigenic Escherichia coli (ETEC), a novel Escherichia coli (E. coli) K88ac LT(S63K)ΔSTb with LT(S63K) mutation and ST1 deletion was generated using site mutagenesis and λ-Red homologous recombination based on wild paternal ETEC strain C83902. E. coli K88ac LT(S63K)ΔSTb showed very similar fimbriae expression and growth kinetics to the wild strain C83902, but it was significantly attenuated according to the results of a rabbit ligated ileal loop assay and mouse infection study. Oral inoculation with E. coli K88ac LT(S63K)ΔSTb stimulated the mucosa immune response and induced the secretion of IgA to K88ac in the intestines in mice. A challenge experiment revealed that the attenuated strain provided efficient protection against C83902 in the following 7 days and at the 24th day post-inoculation, suggesting that the attenuated isolate could act as an ecological protectant and vaccine in preventing K88ac ETEC.

CRISPR/Cas9/sgRNA-mediated targeted gene modification confirms the cause-effect relationship between gyrA mutation and quinolone resistance in Escherichia coli.

Quinolones are broad-spectrum antibiotics that have been used for decades in treating bacterial infections in humans and animals, and subsequently bacterial resistance to these agents has increased. While studies indicated the relationship between gyrA mutations and bacterial resistance to quinolones, CRISPR/Cas9 was used in this study to investigate causal role of gyrA mutation in the quinolone resistance. In this study, 818 clinical Escherichia coli isolates were analyzed for gyrA mutations and their resistance to quinolones. The CRISPR/Cas9 system was used to generate gyrA mutations in quinolone-susceptible E. coli ATCC 25922, and quinolone-resistant clinical E. coli. The antimicrobial resistance prevalence rate in E. coli against nalidixic acid, ciprofloxacin and enrofloxacin was 77.1% (631/818), 51.1% (418/818) and 49.8% (407/818), respectively. The gyrA mutations were identified in nucleotide positions 248, 255, 259, 260, 261, 273 and 300, and mutations at positions 248 and 259 resulting in amino acid changes at positions 83 and 87 were associated with quinolone resistance. Double-site amino acid mutations increase resistance to quinolones. The gyrA mutations causing changes at amino acids 83 and 87 reversed the features of quinolone resistance in ATCC and clinical strains, verifying the causal role of gyrA mutation in the quinolone resistance of E. coli.

Antimicrobial resistance in clinical Escherichia coli isolates from poultry and livestock, China.

Poultry and livestock are the most important reservoirs for pathogenic Escherichia coli and use of antimicrobials in animal farming is considered the most important factor promoting the emergence, selection and dissemination of antimicrobial-resistant microorganisms. The aim of our study was to investigate antimicrobial resistance in E. coli isolated from food animals in Jiangsu, China. The disc diffusion method was used to determine susceptibility to 18 antimicrobial agents in 862 clinical isolates collected from chickens, ducks, pigs, and cows between 2004 and 2012. Overall, 94% of the isolates showed resistance to at least one drug with 83% being resistance to at least three different classes of antimicrobials. The isolates from the different species were most commonly resistant to tetracycline, nalidixic acid, sulfamethoxazole, trimethoprim/sulfamethoxazole and ampicillin, and showed increasing resistance to amikacin, aztreonam, ceftazidime, cefotaxime, chloramphenicol, ciprofloxacin. They were least resistant to amoxicillin/clavulanic acid (3.4%) and ertapenem (0.2%). MDR was most common in isolates from ducks (44/44, 100%), followed by chickens (568/644, 88.2%), pigs (93/113, 82.3%) and cows (13/61, 21.3%). Our finding that clinical E. coli isolates from poultry and livestock are commonly resistant to multiple antibiotics should alert public health and veterinary authorities to limit and rationalize antimicrobial use in China.

Prevalence and isoforms of the pathogenicity island ETT2 among Escherichia coli isolates from colibacillosis in pigs and mastitis in cows.

To study the prevalence and isoforms of the pathogenicity island ETT2 among pathogenic Escherichia coli, as well as to determine the relationship between the ETT2 locus and other virulence factors, PCR amplifications target to the 35 ETT2-associated genes were established and used to investigate the presence of the ETT2 locus in 168 E. coli isolates from weaned piglets with edema and/or diarrhea or dairy cows with mastitis. The results showed that the ETT2 locus could be identified in the pathogenic E. coli isolates from colibacillosis in pigs and in the ones from mastitis in cows, but the presence of ETT2 among the isolates of porcine origin were significantly higher (85.87%) than that (47.37%) of bovine origin. Furthermore, 11 ETT2 isoforms were found in this research, including an intact form and 10 deletion types. The intact ETT2 was the prevalent form among the pathogenic E. coli isolates of porcine origin, and highly associated with the presence of shigatoxin type 2e (Stx2e), while the great majority isolates of bovine origin just carried various deletion types, and no distinct association with other virulence factors, e.g., the presence/absence of LT1, ST2, Cnf2, Tra, HPI, Hly, and F17a fimbriae.

The immune enhancement of sodium lauryl sulfoacetate in chickens.

The purpose of this study is to investigate feasibility of sodium lauryl sulfoacetate (SLS) as an immunoadjuvant in chickens. After treating with 62.5, 125, 250, or 500 mug/mL SLS in vitro, lymphocyte proliferation assay of chicken peripheral blood mononuclear cells showed that the OD(570) values of all experimental groups, as well as Con A-stimulated group, were significantly higher than that of the untreated control group. After injection with 1.0, 2.0, or 4.0 mg/kg of SLS for 3 consecutive days, chickens were vaccinated with an attenuated vaccine against Newcastle disease virus (NDV), and the immunoadjuvant effects of SLS were evaluated on the basis of immune organ index, antibody response, and CD(4) (+)/CD(8) (+) T-cell ratio. The results confirmed that SLS could enhance NDV-specific antibody response and increase CD(4) (+)/CD(8) (+) T-cell ratio in vivo. Furthermore, SLS could improve NDV-specific antibody response in thiamphenicol-treated chickens. These data indicate that SLS not only can improve humoral immune response but also reverse the immunosuppressive effect of thiamphenicol in chickens.

[Prevalence of LEE and HPI pathogenicity islands of Escherichia coli isolates from weaned piglets in China].

To investigate the distribution of LEE pathogenicity island and HPI of Yersinia entercolitica in Escherichia coli isolates from weaned piglets, PCR based on intimin gene (eaeA) of LEE pathogenicity island and high molecular weight protein 2 (HMWP1) gene (irp2) of HPI was developed. A total of 240 isolates from 140 diarrheic, 76 edematous and 24 edematous/diarrheic weaned piglets from different farms were tested for the presence of the two genes. Sequence analysis of randomly selected PCR products showed that eaeA gene of 5 isolates was 100%, irp2 gene of 7 isolates was 98.2%, fyuA gene of 5 isolates was 98.3% and Asn-tRNA-intB of 5 isolates was 95.8% identical to the published sequences. Isolates with LEE + HPI gene were more frequently detected in diarrheic swine than in edematous swine and edematous/diarrheic swine, and isolates with LEE gene were more frequently detected in edematous/diarrheic piglets than in edematous and diarrheic piglets. Furthermore, isolates with LEE or HPI or LEE + HPI were more frequently detected in diarrheic swine. 72.5% of HPI + isolates were fyuA positive and linked to asn-tRNA.

PCR detection of virulence factor genes in Escherichia coli isolates from weaned piglets with edema disease and/or diarrhea in China.

Fimbriae, toxins and pathogenicity islands (PAIs) are main virulence factors of the pathogenic Escherichia coli strains. To investigate into their prevalence in clinical E. coli isolates associated with porcine postweaning diarrhea (PWD) and/or pig edema disease (ED), 240 isolates were obtained from diseased piglets (140 from PWD, 76 from ED and 24 from ED/PWD) and submitted to PCR detection for genes coding for fimbriae, enterotoxins, shiga toxins, intimin and high-molecular-weight protein 2 (HMWP2). Among the 240 isolates detected, detection rates of the genes for F18, F4, intimin, HMWP2, Stx2e, LTa, STa and STb were 26.25%, 3.75%, 28.33%, 16.67%, 35%, 10.83%, 14.58% and 9.17%, respectively, and 67.92% of the isolates could be assigned into 20 different virulence factor patterns. Further more, F18ab+ STEC are the prevalent pathogens of ED, and F18+ and/or intimin+ STEC/ETEC are the dominant pathogens of ED/PWD, while F18ab+, F4+ and/or intimin+ ETEC and HPI+ and/or LEE+ E. coli are more frequently associated with PWD.

Prevalence of fimbial colonization factors F18ab and F18ac in Escherichia coli isolates from weaned piglets with edema and/or diarrhea in China.

F18ab and F18ac are important fimbrial colonization factors of verotoxigenic Escherichia coli (VTEC) and/or enterotoxigenic E. coli (ETEC) in weaned piglets with edema disease and/or diarrhea. To further investigate their prevalence and correlation to pathogenic E. coli, a duplex PCR, using three primers derived from the nucleotide sequence of the F18 major fimbrial subunit gene (fedA), and a direct agglutination test, using a monoclonal antibody specific for the antigenic factor 'a' of F18, were performed. Among 60VTEC, 24VTEC/ETEC and 24 ETEC isolates tested from weaned piglets with edema disease and/or diarrhea in different pig farms in the Jiangsu Province of China, 52 isolates (48.15%) were positive in the direct agglutination test and 63 isolates (58.33%) were positive in the duplex PCR. Among 63 PCR-positive isolates, 53 isolates (49.07%) were F18ab-positive and 10 isolates (9.26%) were F18ac-positive. In addition, the F18ab gene was more frequently detected in VTEC (61.67%) or VTEC/ETEC (62.50%) than in ETEC (4.17% only), while the F18ac gene was more frequently detected in VTEC/ETEC (33.33%) than in ETEC (8.33%) or VTEC (0%). Furthermore, F18ab was more frequently associated with Shiga toxin 2e (Stx2e), whereas F18ac was more frequently associated with enterotoxin ST I. These results suggest that the duplex PCR performed in this experiment is a more reliable method for identification of F18+E. coli, and that F18 is a more important virulence factor of VTEC and VTEC/ETEC.