Epidemiological investigation of porcine circovirus type 2 and its coinfection rate in Shandong province in China from 2015 to 2018.

BACKGROUND: Porcine circovirus type 2 (PCV2) is one of the crucial swine viral pathogens, caused porcine circovirus associated diseases (PCVAD). Shandong province is one of the most important pork producing areas and bears a considerable economic loss due to PCVAD. However, there is limited information on epidemiology and coinfection rate of PCV2 with other critical swine diseases in this area, such as porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), Pseudorabies virus (PRV), and porcine epidemic diarrhea virus (PEDV). RESULTS: Overall, 89.59% serum samples and 36.98% tissue samples were positive for PCV2 specified ELISA and PCR positive for PCV2, respectively. The coinfection rates of PCV2 with PRRSV, PRV, CSFV, and PEDV were 26.73%, 18.37%, 13.06%, and 3.47%, respectively. Moreover, genetic characteristic of PCV2 were analyzed based on the cap genes showing that PCV2d is the dominant sub-genotype circulating in the province. CONCLUSIONS: Our findings reveal that PCV2d, as the dominant strain, is prevailing in pig farms in Shandong province at high levels. There was a high frequency of coinfection of PCV2 and PRRSV.

Detection of p53 mutation and serum monitoring alert caused by Marek's disease virus in poultry.

BACKGROUND: Marek's disease (MD) is a chicken neoplastic disease, which brings huge economic losses to the global poultry industry. The wild type p53, a tumor suppressor gene, plays a key role in blocking cell cycle, promoting apoptosis, and maintaining the stability of the genome. However, the mutant p53 losses its tumor inhibitory role and become an oncogene when a mutation has happened. RESULTS: The mutation rate of p53 was 60% in the experimentally and naturally infected chickens. The mutations included point-mutations and deletions, and mostly located in the DNA-binding domain. The mutated p53 was expressed in various tumor tissues in an infected chicken. The mutant P53 proteins were notably accumulated in the cytoplasm due to the loss in the function of nuclear localization. Unlike the study on human cancer, the concentrations of P53 in the serums of MD infected chicken were significantly lower than the control group. CONCLUSIONS: The p53 mutations were apparent in the development of MD. P53 and P53 antibody level in serum could be a useful marker in the diagnosis and surveillance of MD.

Genotypic and pathotypic characterization of Newcastle disease virus isolated from racing pigeons in China.

A Newcastle disease virus (NDV) isolated from an outbreak in racing pigeons in China was characterized in this study. Complete gene of the NDV isolate was sequenced and phylogenetic analysis. Pathogenicity experiment was carried out in pigeons, chickens, and ducks. Phylogenetic analysis revealed that the strain clustered with the Class II viruses, has highly phylogenetically similar to NDV strains isolated from pigeons in China, but was distant from the viruses prevalence in chickens and vaccine strains used in China. The deduced amino acid sequence of the cleavage site of the fusion (F) protein confirmed that the isolate contained the virulent motif (112)RRQKRF(117) at the cleavage site, but it caused no appearance disease in chickens and ducks. However, the isolate had virulence in pigeons, resulting in severe nervous signs and highly mortality. Pigeons were considered as a potential source of NDV infection and disease for commercial poultry flocks. Therefore, new vaccines to prevent the NDV infection in the pigeon flocks should be developed as soon as possible, and strict biosecurity measures should be taken to reduce the risk of pigeon Newcastle disease outbreaks.

Production and evaluation of anti-BP26 monoclonal antibodies for the serological detection of animal brucellosis.

Brucella BP26 proves to be a highly immunogenic antigen with excellent specificity in brucellosis detection. In China, the authorized use of the Bp26-deleted vaccine M5ΔBP26 for preventing small ruminant brucellosis highlights the importance of developing accurate detection methods targeting BP26, particularly for the diagnosis of differentiation between infected and vaccinated animals (DIVA). Using the traditional mouse hybridoma technique, we successfully obtained 12 monoclonal antibodies (mAbs) targeting BP26. The efficacy of these mAbs in detecting various animal brucellosis cases using the competitive ELISA method was evaluated. Among them, only the E10 mAb exhibited significant efficiency, being inhibited by 100, 97.62, and 100% of brucellosis-positive sera from cattle, small ruminants, and canines, respectively. The E10-based competitive enzyme-linked immunosorbent assay (cELISA) outperformed the BP26-based indirect enzyme-linked immunosorbent assay (iELISA) in accuracy, particularly for cattle and small ruminant brucellosis, with cELISA sensitivity reaching 97.62% compared to 64.29% for iELISA for small ruminants. Although cELISA showed slightly lower specificity than iELISA, it still maintained high accuracy in canine brucellosis detection. The epitope of mAb E10 was identified in the amino acid sequence QPIYVYPDDKNNLKEPTITGY, suggesting its potential as a diagnostic antigen for brucellosis. In conclusion, the E10-based cELISA presents an effective means of detecting animal brucellosis, particularly significant for DIVA diagnosis in China, where the BP26-mutant vaccine is widely used.

Development and evaluation of a triplex droplet digital PCR method for differentiation of M. tuberculosis, M. bovis and BCG.

Introduction: Tuberculosis, caused by Mycobacterium tuberculosis complex (MTBC), remains a global health concern in both human and animals. However, the absence of rapid, accurate, and highly sensitive detection methods to differentiate the major pathogens of MTBC, including M. tuberculosis, M. bovis, and BCG, poses a potential challenge. Methods: In this study, we have established a triplex droplet digital polymerase chain reaction (ddPCR) method employing three types of probe fluorophores, with targets M. tuberculosis (targeting CFP-10-ESAT-6 gene of RD1 and Rv0222 genes of RD4), M. bovis (targeting CFP-10-ESATs-6 gene of RD1), and BCG (targeting Rv3871 and Rv3879c genes of ΔRD1), respectively. Results: Based on optimization of annealing temperature, sensitivity and repeatability, this method demonstrates a lower limit of detection (LOD) as 3.08 copies/reaction for M. tuberculosis, 4.47 copies/reaction for M. bovis and 3.59 copies/reaction for BCG, without cross-reaction to Mannheimia haemolytica, Mycoplasma bovis, Haemophilus parasuis, Escherichia coli, Pasteurella multocida, Ochrobactrum anthropi, Salmonella choleraesuis, Brucella melitensis, and Staphylococcus aureus, and showed repeatability with coefficients of variation (CV) lower than 10%. The method exhibits strong milk sample tolerance, the LOD of detecting in spike milk was 5 × 103 CFU/mL, which sensitivity is ten times higher than the triplex qPCR. 60 clinical DNA samples, including 20 milk, 20 tissue and 20 swab samples, were kept in China Animal Health and Epidemiology Center were tested by the triplex ddPCR and triplex qPCR. The triplex ddPCR presented a higher sensitivity (11.67%, 7/60) than that of the triplex qPCR method (8.33%, 5/60). The positive rates of M. tuberculosis, M. bovis, and BCG were 1.67, 10, and 0% by triplex ddPCR, and 1.67, 6.67, and 0% by triplex qPCR, with coincidence rates of 100, 96.7, and 100%, respectively. Discussion: Our data demonstrate that the established triplex ddPCR method is a sensitive, specific and rapid method for differentiation and identification of M. tuberculosis, M. bovis, and BCG.

Construction of triphase interface for catalytic ozonation of polluted water.

The utilization efficiency of ozone determines the cost of catalytic ozonation in water treatment. Herein, a triphase catalytic system was constructed by aerating ozone through a CeO2 loaded Al2O3 ceramic membrane (CeO2-CM) for disinfection and antibiotic degradation. Ozone aeration and a packed catalyst system (CeO2-Packing) were set as the controls. Results showed that CeO2-CM reduced the ozone escape by 34.6%-56.2%. The ozone utilization capacity of CeO2-CM for E. coli inactivation was 33.1% and 33.8% higher than those of CeO2-Packing and ozone aeration, respectively. The ozone utilization capacity of CeO2-CM for sulfamethoxazole degradation was 88.5% and 183.1% higher than those of CeO2-Packing and ozone aeration, respectively. CeO2-CM, with the lowest ozone escape and highest ozone utilization efficiency, significantly enhanced the performance of catalytic ozonation in disinfection and antibiotic degradation. This work proposes a feasible strategy for minimizing ozone consumption in water treatment.

Ecological risk under the dual threat of heavy metals and antibiotic resistant Escherichia coli in swine-farming wastewater in Shandong Province, China.

Mineral elements and antibiotic-resistant bacterial pollutants in livestock and poultry farms' wastewater are often sources of ecological and public health problems. To understand the heavy-metal pollution status and the characteristics of drug-resistant Escherichia coli (E. coli) in swine-farm wastewater in Shandong Province and to provide guidance for the rational use of mineral-element additives, common antibiotics, and quaternary ammonium compound disinfectants on swine farms, 10 mineral elements were measured and E. coli isolated from wastewater and its resistance to 29 commonly used antibiotics and resistance genes was determined. Finally, phylogenetic and multi-locus sequence typing (MLST) analyses was performed on E. coli. The results showed serious pollution from iron and zinc, with a comprehensive pollution index of 708.94 and 3.13, respectively. It is worth noting that average iron levels in 75% (12/16) of the districts exceed allowable limits. Multidrug-resistant E. coli were found in every city of the province. The E. coli isolated from swine-farm wastewater were mainly resistant to tetracyclines (95.3%), chloramphenicol (77.8%), and sulfonamides (62.2%), while antibiotic resistance genes for quinolones, tetracyclines, sulfonamides, aminoglycosides, and ß-lactams were all more than 60%. The clonal complex 10 (CC10) was prevalent, and ST10 and ST48 were dominant in E. coli isolates. Multidrug-resistant E. coli were widely distributed, with mainly A genotypes. However, the mechanism of the effect of iron on antibiotic resistance needs more study in this area. Thus, further strengthening the prevention and control of iron and zinc pollution and standardizing the use of antibiotics and mineral element additives in the swine industry are necessary.

Pathogenicity and epidemiological survey of fowl adenovirus in Shandong Province from 2021 to 2022.

In recent years, the poultry industry had been markedly affected by adenoviral diseases such as hydropericardium syndrome and inclusion body hepatitis caused by fowl adenovirus (FAdV), which have become increasingly prevalent in China. Shandong Province, China, is an important area for poultry breeding where various complex and diverse FAdV serotypes were isolated. However, the dominant strains and their pathogenic characteristics are not yet reported. Therefore, a pathogenicity and epidemiological survey of FAdV was conducted, showing that the local dominant serotypes of FAdV epidemics were FAdV-2, FAdV-4, FAdV-8b, and FAdV-11. Their mortality rates in the 17-day-old specific-pathogen-free (SPF) chicks ranged from 10 to 80%; clinical signs included mental depression, diarrhea, and wasting. The maximum duration of viral shedding was 14 days. The highest incidence in all infected groups was on days 5-9, and then gradual regression occurred thereafter. The most pronounced symptoms occurred in chicks infected with FAdV-4, including pericardial effusion and inclusion body hepatitis lesions. Our results add to the current epidemiological data on FAdV in poultry flocks in Shandong and elucidate the pathogenicity of dominant serotypes. This information may be important for FAdV vaccine development and comprehensive epidemic prevention and control.

The impact of Mycobacterium tuberculosis complex in the environment on one health approach.

Tuberculosis caused by the Mycobacterium tuberculosis complex (MTBC) has become one of the leading causes of death in humans and animals. Current research suggests that the transmission of MTBC in the environment indirectly transmit to humans and animals with subsequent impact on their wellbeing. Therefore, it is of great significance to take One Health approach for understanding the role of MTBC in not only the interfaces of humans and animals, but also environment, including soil, water, pasture, air, and dust, etc., in response to the MTBC infection. In this review, we present the evidence of MTBC transmission from environment, as well as detection and control strategies in this interface, seeking to provide academic leads for the global goal of End Tuberculosis Strategy under multidisciplinary and multisectoral collaborations.

Research Note: Molecular characterization of antimicrobial resistance and virulence gene analysis of Enterococcus faecalis in poultry in Tai'an, China.

Enterococcus faecalis (E. faecalis) is a zoonotic pathogen that causes severe economic losses in the poultry-breeding industry. In our study, cecal samples from broilers with cecal enlargement at slaughterhouses in Tai'an, China, were analyzed. The results revealed that the 61 E. faecalis strains had drug resistance rates ranging from 96.72 to 8.20% against 11 antibiotics in 5 classes, of which erythromycin (96.72%) and tetracycline (96.72%) had the highest rates and vancomycin (8.20%) the lowest. The highest detection rate of multiple drug-resistant strains in 61 isolates was 72.13%. The results of polymerase chain reaction showed that, of the 12 virulence genes, ccf had the highest detection rate (80.33%), followed by asal and cob (both 78.69%), whereas hyl had the lowest (6.56%). Among 15 drug resistance genes, ermB had the highest detection rate (95.08%), followed by tetM (91.80%) and tetL (90.16%), whereas tetK (0.00%) and vanB (0.00%) remained undetected. Of the 34 sequence types found with multilocus sequence typing, the most predominant were ST631 (13.11%, 8/61) and ST634 (8.2%, 5/61). Our results provide a theoretical basis for guiding the rational use of antibiotics and preventing the spread of drug-resistant bacteria, along with epidemiological data for the risk analysis of food-borne bacteria and antimicrobial resistance in poultry farms in Shandong Province.

Prevalence and characteristics of multidrug-resistant Proteus mirabilis from broiler farms in Shandong Province, China.

Animal-derived Proteus mirabilis (P. mirabilis) is an important food-borne zoonotic bacillus and widely exists in the broiler-breeding industry. The present study was designed to explore the P. mirabilis prevalence and antimicrobial resistance characteristics in 6 conventional broiler-fattening farms in Shandong Province, China. The overall isolation rate of P. mirabilis was 7.07% (50/707). Antimicrobial resistance was very common in the P. mirabilis isolated from these farms and varied for different antibacterial drugs, with chloramphenicol, ciprofloxacin, and trimethoprim-sulfamethoxazole having the highest resistance rate (98%) and aztreonam the lowest (0%). Multidrug resistance was as high as 100%. The majority of the MDR isolates were resistant to between 9 and 12 of the antibiotics, with these accounting for 76% (38/50) of multidrug resistant strains. These P. mirabilis isolates carried 24 drug-resistance genes in 6 types, with stcM having the highest rate (96%) and cmlA, blaTEM, and qnrC the lowest (2%). Superdrug resistance gene blaNDM-1 was found in 10% (5/50) of isolates from poultry farms in Shandong. All the P. mirabilis isolates carried at least 6 virulence genes, with 100% detection rates of the ireA and hpmA genes. Our study revealed that the P. mirabilis strains isolated in the Shandong area all showed the MDR phenotype and the poultry-derived carbapenem-resistant MDR P. mirabilis strains may pose a potential risk to humans. Surveillance findings presented herein will be conducive to our understanding of the prevalence and characteristics of carbapenem-resistant P. mirabilis strains in Shandong, China.

Prevalence and antimicrobial susceptibility profiles of ESBL-producing Klebsiella Pneumoniae from broiler chicken farms in Shandong Province, China.

Klebsiella pneumoniae (K. pneumoniae) is a conditionally pathogenic bacterium present in the intestinal or the respiratory tract of animals, and it is a common factor in acquired infections and a major threat to public health. Increased production of extended-spectrum ß-lactamases (ESBLs) has become a serious issue in the treatment of K. pneumoniae infections. In this study, we examined the serotypes and antibiotic resistance profiles of K. pneumoniae isolated from broiler chickens on farms in Shandong Province, China. The K. pneumoniae isolation rate was 4.67% (33/707), and the serotype Capsular K54 (42.42%, 14/33) was the most prevalent serotype in broilers in Shandong. The antimicrobial susceptibility assay revealed that the 33 isolates were resistant to 28 antimicrobial drugs to varying degrees; among these, the highest resistance rate was observed for tetracyclines (90.91%), and the lowest rate of resistance was observed for moxifloxacin and fosfomycin (0%). The multidrug resistance (MDR) rate was 87.88% (29/33). The carrying rate of ß-lactam-resistance genes was as high as 100%, with blaSHV having the highest rate (93.94%). It is worth noting that one carbapenem-resistant K. pneumoniae (CRKP) isolate carrying blaNDM-1 and one colistin-resistant K. pneumoniae (COLR-KP) isolate carrying mcr-3 were found in broiler chickens. This study indicates that ESBL-producing CRKP isolates and COLR-KP isolates have emerged on poultry farms in Shandong and could be a potential threat to food safety and public health.

Molecular epidemiological survey of porcine epidemic diarrhea in some areas of Shandong and genetic evolutionary analysis of S gene.

Responsible for the acute infectious disease porcine epidemic diarrhea (PED), PED virus (PEDV) induces severe diarrhea and high mortality in infected piglets and thus severely harms the productivity and economic efficiency of pig farms. In our study, we aimed to investigate and analyze the recent status and incidence pattern of PEDV infection in some areas of Shandong Province, China. We collected 176 clinical samples of PED from pig farms in different regions of Shandong Province during 2019-2021. PEDV, TGEV, and PORV were detected using RT-PCR. The full-length sequences of positive PEDV S genes were amplified, the sequences were analyzed with MEGA X and DNAStar, and a histopathological examination of typical PEDV-positive cases was performed. RT-PCR revealed positivity rates of 37.5% (66/176) for PEDV, 6.82% (12/176) for transmissible gastroenteritis virus, and 3.98% (7/176) for pig rotavirus. The test results for the years 2019, 2020, and 2021 were counted separately, PEDV positivity rates for the years were 34.88% (15/43), 39.33% (35/89), and 36.36% (16/44), respectively. Histopathological examination revealed atrophied, broken, and detached duodenal and jejunal intestinal villi, as typical of PED, and severe congestion of the intestinal submucosa. Moreover, the results of our study clearly indicate that the G2 subtype is prevalent as the dominant strain of PEDV in Shandong Province, where its rates of morbidity and mortality continue to be high. Based on a systematic investigation and analysis of PEDV's molecular epidemiology across Shandong Province, our results enrich current epidemiological data regarding PEDV and provide some scientific basis for preventing and controlling the disease.

Research Note: The prevalence and vertical transmission of avian hepatitis E virus novel genotypes in Tai'an city, China.

To investigate the prevalence of avian hepatitis E virus (HEV) in chickens and gather evidence of viral vertical transmission, we collected 288 cloacal swabs and 288 yolks samples from 12 farms with clinically healthy chickens in 4 different areas in Tai'an City, Shandong Province, China (i.e., Daiyue District, Xintai City, Feicheng City, and Ningyang County). We also collected 240 samples from 2 breeder farms (from each of which 30 chicks, 30 dead embryos, 30 live embryos, and 30 hatching eggs were taken). PCR detection revealed that the positive rates of cloacal swabs and yolks were 6.25% (18/288) and 4.51% (13/288), respectively. Besides, avian HEV was detected with higher positive rates in the chicks (11.67%), hatching eggs (10.00%), live embryos (13.33%), and dead embryos (26.67%) from 2 breeder farms. Sequence and genetic evolution analyses revealed that the nucleotide homology of the isolated strains was 76.4to 83.9% compared with 4 reported genotypes, but the isolated strains were located in a separate branch, indicating they were potential novel genotypes. In conclusion, those results indicate that the latent infection of avian HEV novel genotypes has been widespread in chicken farms in Tai'an City, and provide reliable evidence of the possible vertical transmission of avian HEV.

Epidemiological survey of PRRS and genetic variation analysis of the ORF5 gene in Shandong Province, 2020-2021.

Since the rise of porcine reproductive and respiratory syndrome virus (PRRSV) in China, mutations have occurred regularly. In particular, the emergence of HP-PRRSV has significantly improved the pathogenicity of PRRSV. It has brought huge economic losses to the Chinese pig farming industry. To understand the current prevalence and evolution of PRRSV in Shandong Province, 1,344 samples suspected of having PRRSV were collected from local hog farms of different sizes. Genetic variation in the isolated PRRSV ORF5 gene was analyzed using the RT-PCR method. The results showed that the detection rate of PRRSV in the collected samples was 25.44%. The predominant strain of PRRSV in Shandong Province is still NADC30-like. However, it cannot be ignored that NADC34-like is also starting to become a prevalent strain. Mutations in ORF5 amino acids 13, 151 and neutralizing epitope (aa36-aa52) in some isolates can cause changes in virulence and ability to escape immunity. This study enriches the epidemiological data on PRRSV in Shandong Province, China. It provides an important reference for the development of new vaccines and for the prevention and control of PRRSV.

Comparison of BP26, Omp25 and Omp31 and a Multiepitope-Based Fusion Protein in the Serological Detection of Canine Brucellosis.

Background: Brucellosis is one of the most important zoonotic diseases in the world. Canine brucellosis, caused mainly by Brucella canis, is seriously neglected, and there is a lack of accurate diagnostic tools. Methods: In this study, to compare BP26, Omp25, Omp31 and a multiepitope-based fusion protein in the serological detection of canine brucellosis, using 34 brucellosis-positive dog sera and 62 negative control sera, the Brucella outer membrane proteins Omp31, BP26, Omp25 and a multiepitope-based fusion protein were evaluated by iELISA for their potential use as antigens in the serological diagnosis of canine brucellosis. Results: The results showed that the multiepitope-based fusion protein performed best in distinguishing brucellosis-positive and brucellosis-negative dog sera, with a positive predictive value (PPV) of 100% and a negative predictive value (NPV) of 98.41%. BP26 and Omp31 showed excellent sensitivity in detecting brucellosis-positive dog sera, but their cross reaction to sera infected with Vibrio parahaemolyticus and Listeria monocytogenes may hinder their application as diagnostic reagents. Omp25 lacked sufficient sensitivity and showed limited ability in distinguishing positive and negative dog sera. Conclusion: The multiepitope-based fusion protein can be used as an ideal antigen for serologically diagnosing canine brucellosis currently prevalent worldwide.

HoBi-like pestivirus infection leads to bovine death and severe respiratory disease in China.

HoBi-like pestivirus is an emerging atypical pestivirus in cattle and small ruminants, causing clinical signs similar to those observed in bovine viral diarrhoea virus infections. Natural infection of HoBi-like pestivirus has been reported in cattle herds and small ruminants in multiple countries in South America, Europe and Asia. However, HoBi-like pestiviruses were only identified from contaminated bovine serum and small ruminants in China. So far, no clinical cases induced by HoBi-like pestivirus infection were reported in Chinese cattle herds. Here, for the first time, we reported natural infection of HoBi-like pestivirus in a cattle herd in China. Sick cattle with severe respiratory and diarrhoea and high fatality rate were found in a beef cattle herd in Shandong province in November 2017. RT-PCR, viral isolation, sequencing and phylogenetic analysis showed that the primary causative agent was HoBi-like pestivirus. The isolated HoBi-like pestivirus strain, SDJN-China-2019, shared 94.1%-97.5% homology with the LV168-20_16RN strain from Brazil in nucleotide of 5'UTR, Npro and E2 while it shared only 88.5%-92.1% homology with Asian HoBi-like virus strain Th/04-Khonkaen. Multiple unique mutations of amino acid were observed in Npro and E2 proteins of SDJN-China-2019, which were different from that of other reference strains. In summary, this study provides the first evidence of HoBi-like pestivirus infection in Chinese cattle herds, raising potential threat to the cattle industry in China.

Analysis of In Vivo Transcriptome of Intracellular Bacterial Pathogen Salmonella enterica serovar Typhmurium Isolated from Mouse Spleen.

Salmonella enterica serovar Typhimurium (S. Typhimurium) is an important intracellular pathogen that poses a health threat to humans. This study tries to clarify the mechanism of Salmonella survival and reproduction in the host. In this study, high-throughput sequencing analysis was performed on RNA extracted from the strains isolated from infected mouse spleens and an S. Typhimurium reference strain (ATCC 14028) based on the BGISEQ-500 platform. A total of 1340 significant differentially expressed genes (DEGs) were screened. Functional annotation revealed DEGs associated with regulation, metabolism, transport and binding, pathogenesis, and motility. Through data mining and literature retrieval, 26 of the 58 upregulated DEGs (FPKM > 10) were not reported to be related to the adaptation to intracellular survival and were classified as candidate key genes (CKGs) for survival and proliferation in vivo. Our data contribute to our understanding of the mechanisms used by Salmonella to regulate virulence gene expression whilst replicating inside mammalian cells.

Excessive copper in feed not merely undermines animal health but affects food safety.

BACKGROUND: Blackened intestines in slaughtered pigs have been commonly observed in China in recent years. However, no cause has been reported. OBJECTIVES: We attempted to determine whether the blackening of the pig intestine was related to an excess of copper (Cu) in their feed. METHODS: In this study, we observed and collected porcine intestines in small- and large-scale pig slaughterhouses in Shandong province from May to October 2018. Twelve types of metal ions were detected in the black intestinal samples. RESULTS: The Cu level in the intestine samples was mostly higher than the Chinese national limit for food. Further study showed that Cu supplementation in most commercial porcine feed also exceeded the national standard. An animal model (mouse) that could mimic the intestinal blackening in pigs was established. Compared to control mice, Cu accumulated in the liver and intestines of mice fed an excessive Cu level, confirming the excessive Cu in the feed may be considered the major cause of blackened porcine intestines. Microscopic examination revealed that black intestines had many particles containing Cu in the lamina propria of the intestinal mucosa, and the intestinal mucosal epithelial cells showed degeneration and necrosis. CONCLUSIONS: In conclusion, overuse of Cu in animal feed can lead to animal poisoning and Cu accumulation in animal products. Such overuse not only harms the health of livestock but can also affect public health.

Longitudinal monitoring of multidrug resistance in Escherichia coli on broiler chicken fattening farms in Shandong, China.

The extensive use of antibiotics has, in recent years, caused antimicrobial resistance and multidrug resistance in Escherichia coli to gradually develop into a worldwide problem. These resistant E. coli could be transmitted to humans through animal products and animal feces in the environment, thereby creating a problem for bacterial treatment for humans and animals and resulting in a public health issue. Monitoring the resistance of E. coli throughout the broiler fattening period is therefore of great significance for both the poultry industry and public health. In this longitudinal study, samples were taken from 6 conventional broiler fattening farms in Shandong Province, China, at 3 different times within 1 fattening period. The overall isolation rate of E. coli was 53.04% (375/707). Antibiotic resistance was very common in the E. coli isolated from these farms, and differed for different antibiotics, with ampicillin having the highest rate (92.86%) and cefoxitin the lowest (10.12%). Multidrug resistance was as high as 91.07%. More importantly, both the resistance rate of E. coli to the different drugs and the detection rate of drug resistance genes increased over time. The mobile colistin resistance (mcr-1) gene was detected in 24.40% of the strains, and these strains often carried other drug resistance genes, such as those conferring aminoglycoside, ß-lactamase, tetracycline, and sulfonamide resistance. Antimicrobial resistance and drug resistance genes in E. coli were least common in the early fattening stage. The individual detection rates of sul1, sul3, aacC4, aphA3, and mcr-1 were significantly lower (P < 0.05) for the early fattening stage than for the middle and late stages. The rational use of antibiotics, in conjunction with the improvement of the breeding environment during the entire broiler fattening cycle, will be helpful in the development of the poultry industry and the protection of public health.