Infected Swine Research Articles

NADH oxidase of Mycoplasma hyopneumoniae functions as a potential mediator of virulence

BackgroundMycoplasma hyopneumoniae (M. hyopneumoniae) is the etiological agent of enzootic pneumonia, a highly infectious swine respiratory disease that distributed worldwide. The pathogenesis and virulence factors of M. hyopneumoniae are not fully clarified. As an important virulence factor of bacteria, nicotinamide adenine dinucleotide (NADH) oxidase (NOX) participates in host-pathogen interaction, however, the function of NOX involved in the pathogenesis of M. hyopneumoniae is not clear.ResultsIn this study, significant differences in NOX transcription expression levels among different strains of M. hyopneumoniae differed in virulence were identified, suggesting that NOX may be correlated with M. hyopneumoniae virulence. The nox gene of M. hyopneumoniae was cloned and expressed in Escherichia coli, and polyclonal antibodies against recombinant NOX (rNOX) were prepared. We confirmed the enzymatic activity of rNOX based on its capacity to oxidize NADH to NAD+. Flow cytometry analysis demonstrated the surface localization of NOX, and subcellular localization analysis further demonstrated that NOX exists in both the cytoplasm and cell membrane. rNOX was depicted to mediate adhesion to immortalized porcine bronchial epithelial cells (hTERT-PBECs). Pre-neutralizing M. hyopneumoniae with anti-rNOX antibody resulted in a more than 55% reduction in the adhesion rate of high- and low-virulence M. hyopneumoniae strains to hTERT-PBECs. Moreover, a significant difference appeared in the decline in CCU50 titer between virulent (168) and virulence-attenuated (168L) strains. NOX not only recognized and interacted with host fibronectin but also induced cellular oxidative stress and apoptosis in hTERT-PBECs. The release of lactate dehydrogenase by NOX in hTERT-PBECs was positively correlated with the virulence of M. hyopneumoniae strains.ConclusionsNOX is considered to be a potential virulence factor of M. hyopneumoniae and may play a significant role in mediating its pathogenesis.

Clostridioides (Clostridium) difficile en porcinos Caracterización, consideraciones epidemiológicas y resistencia a los antimicrobianos

The aim of this review is to synthesize, gather and summarize some aspects related to Clostridioides difficile and its role as a microbiota and pathogen in pigs. C. difficile is an anaerobic, sporulated bacterium and one of the main etiological agents of diarrhea associated with the use of antibiotics. The main virulence factors are two toxins (A and B) that cause damage to the colon epithelium when a dysbiosis occurs, usually it is caused for the antibiotics. It has been determined that this bacterium has a high prevalence in pigs, mainly in the first months of the animal's life and also many of these animals colonized with non-toxigenic strains of C. difficile. However, there are reports of gastroenteritis caused by this bacterium and possibly related to the use and abuse of antibiotics, although the causes of these infections in swine are not yet completely clear and require further investigation. The RT028 is one of the most frequently isolated C. difficile genotypes in pigs, which has also been reported causing epidemic outbreaks in human hospitals; therefore a possible zoonosis has been postulated, although this hypothesis has not been fully demonstrated. Additionally, a high antimicrobial resistance of C. difficile isolates from pigs and multi-resistant phenotypes has been reported, thus this bacterium could be functioning as an ecological reservoir of antimicrobial resistance genes. Finally, it is necessary to establish measures for the rational use of antibiotics in pig farms and a surveillance system on circulating genotypes and resistance of C. difficile in pigs.

Long-Term Expansion of Porcine Intestinal Organoids Serves as an in vitro Model for Swine Enteric Coronavirus Infection

A reliable and reproducible model in vitro for swine enteric coronaviruses infection would be intestinal models that support virus replication and can be long-term cultured and manipulated experimentally. Here, we designed a robust long-term culture system for porcine intestinal organoids from the intestinal crypt or single LGR5+ stem cell by combining previously defined insights into the growth requirements of the intestinal epithelium of humans. We showed that long-term cultured swine intestinal organoids were expanded in vitro for more than 6 months and maintained the potential to differentiate into different types of cells. These organoids were successfully infected with porcine enteric coronavirus, including porcine epidemic diarrhea virus (PEDV) and transmissible gastroenteritis virus (TGEV), and were capable of supporting virus replication and progeny release. RNA-seq analysis showed robust induction of transcripts associated with antiviral signaling in response to enteric coronavirus infection, including hundreds of interferon-stimulated genes and cytokines. Moreover, gene set enrichment analysis indicated that PEDV infection could suppress the immune response in organoids. This 3D intestinal organoid model offers a long-term, renewable resource for investigating porcine intestinal infections with various pathogens.

Validation of a single liquid chromatography-tandem mass spectrometry approach for oxytetracycline determination in bull plasma, seminal plasma and urine.

Oxytetracycline is a broad‐spectrum antibiotic, which inhibits protein synthesis and is generally used for the treatment of pneumonia, shipping fever, leptospirosis and wound infections in cattle and swine. The present work proposes a novel liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for oxytetracycline quantification in bull plasma, seminal plasma and urine, requiring limited sample treatment before analysis. Extraction with trichloroacetic acid followed by dilution of the supernatant in mobile phase proved to be effective in all three matrices, allowing to rapidly process large batches of samples. Sharp and symmetrical peak shape was obtained using a BEH C18 reversed‐phase column in a chromatographic run of just 3.5 min. The mass spectrometer operated in positive electrospray ionization mode and monitored specific transitions for oxytetracycline (461.1 → 425.8) and the internal standard demeclocycline (465.0 → 447.6). The method was validated over concentration ranges suitable for field concentrations of oxytetracycline found in each matrix, showing good linearity during each day of testing (R 2 always >0.99), as also confirmed by analysis of variance (ANOVA) and lack‐of‐fit tests. Excellent accuracy and precision were demonstrated by calculated bias always within ±15% and CV% below 10% at all quality control (QC) levels in the three matrices. Matrix effect and recovery were investigated for both analytes, which showed consistent and comparable behaviour in each matrix. To our knowledge, this is the first validated approach for mass spectrometric determination of oxytetracycline in seminal plasma and urine. The method was successfully applied to samples collected during a pharmacokinetic study in bulls, allowing to assess the oxytetracycline concentration–time profile in plasma, seminal plasma and urine.

Development and Large-Scale Testing of a Novel One-Step Triplex RT-qPCR Assay for Simultaneous Detection of "Neurotropic" Porcine Sapeloviruses, Teschoviruses (Picornaviridae) and Type 3 Porcine Astroviruses (Astroviridae) in Various Samples including Nasal Swabs.

Porcine sapeloviruses, teschoviruses of family Picornaviridae and type 3 porcine astroviruses of family Astroviridae are (re-)emerging enteric pathogens that could be associated with severe, disseminated infections in swine, affecting multiple organs including the central nervous system (CNS). Furthermore, small-scale pioneer studies indicate the presence of these viruses in porcine nasal samples to various extents. The laboratory diagnostics are predominantly based on the detection of the viral RNA from faecal and tissue samples using different nucleic-acid-based techniques such as RT-qPCR. In this study, a novel highly sensitive one-step triplex RT-qPCR assay was introduced which can detect all known types of neurotropic sapelo-, tescho- and type 3 astroviruses in multiple types of samples of swine. The assay was evaluated using in vitro synthesized RNA standards and a total of 142 archived RNA samples including known sapelo-, tescho- and type 3 astrovirus positive and negative CNS, enteric and nasal specimens. The results of a large-scale epidemiological investigation of these viruses on n = 473 nasal swab samples from n = 28 industrial-type swine farms in Hungary indicate that all three neurotropic viruses, especially type 3 astroviruses, are widespread and endemically present on most of the investigated farms.

Comparison of PK/PD Targets and Cutoff Values for Danofloxacin Against Pasteurella multocida and Haemophilus parasuis in Piglets.

Danofloxacin is a synthetic fluoroquinolone with broad-spectrum activity developed for use in veterinary medicine. The aim of this study was to evaluate the pharmacokinetic/pharmacodynamic (PK/PD) targets, PK/PD cutoff values and the optimum doses of danofloxacin against P. multocida and H. parasuis in piglets. Single dose serum pharmacokinetics was determined in piglets after intravenous and intramuscular administration of 2.5 mg/kg. Danofloxacin was well absorbed and fully bioavailable (95.2%) after intramuscular administration of 2.5 mg/kg. The epidemiological cutoff (ECOFF) values of danofloxacin from 931 P. multocida isolates and 263 H. parasuis isolates were 0.03 and 4 mg/L, respectively. Danofloxacin MICs determined in porcine serum were markedly lower than those measured in artificial broth, with a broth/serum ratio of 4.33 for H. parasuis. Compared to P. multocida, danofloxacin exhibited significantly longer post-antibiotic effects (3.18–6.60 h) and post-antibiotic sub-MIC effects (7.02–9.94 h) against H. parasuis. The mean area under the concentration-time curve/MIC (AUC24h/MIC) targets of danofloxacin in serum associated with the static and bactericidal effects were 32 and 49.8, respectively, for P. multocida, whereas they were 14.6 and 37.8, respectively, for H. parasuis. Danofloxacin AUC24h/MIC targets for the same endpoints for P. multocida were higher than those for H. parasuis. At the current dose of 2.5 mg/kg, the PK/PD cutoff (COPD) values of danofloxacin against P. multocida and H. parasuis were calculated to be 0.125 and 0.5 mg/L, respectively, based on Monte Carlo simulations. The predicted optimum doses of danofloxacin for a probability of target attainment (PTA) of > 90% to cover the overall MIC population distributions of P. multocida and H. parasuis in this study were 2.38 and 13.36 mg/kg, respectively. These PK/PD-based results have potential relevance for the clinical dose optimization and evaluation of susceptibility breakpoints for danofloxacin in the treatment of swine respiratory tract infections involving these pathogens.

Expression and immunogenicity of nsp10 protein of porcine epidemic diarrhea virus

Porcine epidemic diarrhea virus (PEDV), a swine enteropathogenic coronavirus, causes lethal watery diarrhea to the piglets, which poses significant economic losses and public health concerns. The nsp10 protein of PEDV is essential regulatory subunits that are critical for virus replication. Since PEDV nsp10 is a crucial regulator of viral RNA synthesis, it is promising that nsp10 might become anti-virus drugs target or candidate for rapid diagnosis of PEDV infection. In this study, the PEDV nsp10 was inserted into pMAL-c2x-MBP / pET-28a vector, efficiently and stably expressed in E.coli system. Then the purified nsp10 protein was found to mediate potent antibody responses in immunized mice. The antibodies of immunized mice and PEDV infection swine strongly recognized purified nsp10 protein from cell lysates. Furthermore, cytokines test revealed that the expression of IL-2, IL-4, IL-10, TNF-α, IFN-γ were significantly higher than those in control group, indicated that purified nsp10 protein induce the cellular immune response mechanism in mice. Using modified seroneutralization test, we also demonstrated that sera from nsp10-immunized mice inhibited PEDV replication to some extent. These findings suggest that nsp10 has a high immunogenicity. This study may have implications for future development of PEDV detection or anti-virus drugs for swine.

Detection and molecular characterisation of Mycoplasma spp. from respiratory tract infections in pigs

Achieving food security involves the maintenance of intensive production systems and large animal populations where infectious diseases are the most important challenge that need to be addressed. Respiratory infections in pigs are a formidable threat to swine farmers worldwide. It is multifactorial and is caused by the interplay between bacterial pathogens, viral agents and environmental factors. The present study aimed to identify mycoplasmal agents causing respiratory infections in pigs. A total of 43 samples, comprising of nasal swabs, and necropsy samples with pneumonic lesions were collected. The DNA extracted from the samples were subjected to Mycoplasma genus-specific, followed by species-specific PCR. Twenty five samples were positive for Mycoplasma spp., but further molecular detection performed through species-specific primers, revealed that 12 samples were positive for M. hyorhinis. None of the samples were positive for M. hyopneumoniae. The identity of the amplicons were confirmed by nucleic acid sequencing and BLAST analysis. The evolutionary relationship between the detected organisms was also studied by phylogenetic analysis. This study hints at the significance of M. hyorhinis in causing respiratory infections in swine and also suggests that M. hyopneumoniae may not be a significant health hazard for swine populations of north Kerala.

Streptococcus gallolyticus and Bacterial Endocarditis in Swine, United States, 2015-2020.

To evaluate trends in bacterial causes of valvular endocarditis in swine, we retrospectively analyzed 321 cases diagnosed at Iowa State University Veterinary Diagnostic Laboratory (Ames, IA, USA) during May 2015–­April 2020. Streptococcus gallolyticus was the causative agent for 7.59% of cases. This emerging infection in swine could aid study of endocarditis in humans.

Transboundary Swine Infections in India: An Overview

In the recent past, India has seen a number of outbreaks and epidemics of transboundary infectious virus diseases associated with swine. Classical swine fever (CSF), African swine fever (ASF), Porcine reproductive and respiratory syndrome (PRRS), Porcine circovirus 2 (PCV2) infections, Porcine parvovirus, Swine Influenza and Japanese Encephalitis (JE) infections are the major transboundary viral diseases prevalent in Indian swine populations. Understanding the impact of factors that provoke the emergence of viral infections is the major challenge faced in the control and prevention of emerging transboundary viral diseases. In-depth knowledge on disease status is crucial in successfully implementing disease control programs. The movement of animals influences the spread of pathogens in swine populations. This article gives insight into major emerging transboundary emerging viral infections of swine and their current status in India.

A serological survey of porcine reproductive and respiratory syndrome virus in wild boar in Gifu Prefecture, Japan.

Porcine reproductive and respiratory syndrome (PRRS) is an infectious swine disease caused by the PRRS virus (PRRSV) that results in economic loss to the pig-rearing industry. To study PRRSV infection in wild boars and pigs, we conducted a serological survey in Gifu Prefecture, Japan, from 2020 to 2021. Three out of 453 (0.7%) wild boar sera were positive for PRRSV antibodies in a commercial ELISA. However, given that PRRSV RNA was not detected in these three wild boars and the specificity and sensitivity of the test kit, these are considered as false positives. Although seropositive pigs were found in multiple pig farms in the study area, the role of wild boars as a source of PRRS to pig farms appeared to be minimal.

Morphological and immunophenotypic characteristics of the liver of swine naturally infected with hepatitis E virus.

Hepatitis E virus (HEV), the zoonotic agent of infectious hepatitis, is present in swine farms in different geographical areas. Little is known about the mechanism of liver damage and type of local immune response by HEV in swine. Therefore, the aim of this study was to determine the morphological and immunophenotypic characteristics of hepatic lesions caused by hepatitis E virus in naturally infected swine. In this study, liver samples of 12 slaughtered 10 weeks old pigs which were RT-PCR positive for HEV RNA in rectal swab samples have been used. Livers were macroscopically examined and samples were taken for histopathological, immunohistochemical (CD3, CD79α and TGF-β1), semiquantitative, morphometric analysis, RT-nested-PCR, PCR and bacteriological analysis. Microscopically, mild and moderate multifocal lymphoplasmacytic hepatitis was observed. Apoptotic bodies were observed as areas of focal eosinophilic condensation in the cytoplasm of 33.33% liver samples, while in 16.67% liver samples portal fibrosis was detected. Immunohistochemically, portal and lobular lymphocytes in the mononuclear liver infiltrate were predominantly CD3+ T cells (234.80 ± 79.98). An intense TGF-β1 positive reaction was observed within the mononuclear cell infiltrate as well as polymorphonuclear cells in liver samples with apoptosis of hepatocytes. In all 12 tested liver samples HEV RNA was detected by RT-nested-PCR. HEV is noncytopathic, and this finding provides further evidence for an immune mediated pathogenesis in hepatitis E virus infection in swine. Also, the role of CD3+ cells in hepatocyte damage is clearly demonstrated.

Genomic Characterization of Streptococcus suis Serotype 24 Clonal Complex 221/234 From Human Patients.

Streptococcus suis is a zoonotic pathogen that causes invasive infections in humans and pigs. Although S. suis serotype 2 is prevalent among patient and swine infections, other serotypes are occasionally detected in humans. Of these, serotype 24 clonal complex (CC) 221/234 are recognized as emerging clones of human infection. Genomic exploration of three S. suis serotype 24 CC221/234 strains revealed antimicrobial resistance genes, pathotyping, virulence-associated gene (VAG) profiles, minimum core genome (MCG) typing, and comparison of the genomes. Based on these analyzes, all three serotype 24 strains were MCG7-3 and should be classified in the intermediate/weakly virulent (I/WV) group. All selected serotype 24 strains were susceptible to several antibiotics including β-lactam, fluoroquinolone, and chloramphenicol. Resistance to tetracycline, macrolide, and clindamycin was observed and attributed to the genes tet(O) and erm(B). Genomic comparison revealed the strains S12X, LSS66, LS0L, LS0E, 92–4,172, and IMT40201 that had phylogenetic affinity with serotype 24 CC221/234. Analysis of 80 virulence-associated genes (VAG) showed that all three serotype 24 strains lacked 24 genes consisting of adhesin P, epf, hyl, ihk, irr, mrp, nadR, neuB, NisK/R, ofs, permease (SSU0835), rgg, revS, salK/R, sao, sly, spyM3_0908, srtBCD, srtF, srtG, SSU05_0473, virA, virB4, and virD4. Eleven specific sequences were identified in the 3 serotype 24 genomes that differed from the genomes of the representative strains of epidemic (E; SC84), highly virulent (HV; P1/7), I/WV (89–1,591), and avirulent (T15 and 05HAS68).

Evaluation of an ASFV RNA Helicase Gene A859L for Virus Replication and Swine Virulence.

African swine fever virus (ASFV) is producing a devastating pandemic that, since 2007, has spread to a contiguous geographical area from central Europe to Asia. In July 2021, ASFV was detected in the Dominican Republic, the first report of the disease in the Americas in more than 40 years. ASFV is a large, highly complex virus harboring a large dsDNA genome that encodes for more than 150 genes. The majority of these genes have not been functionally characterized. Bioinformatics analysis predicts that ASFV gene A859L encodes for an RNA helicase, although its function has not yet been experimentally assessed. Here, we evaluated the role of the A859L gene during virus replication in cell cultures and during infection in swine. For that purpose, a recombinant virus (ASFV-G-∆A859L) harboring a deletion of the A859L gene was developed using the highly virulent ASFV Georgia (ASFV-G) isolate as a template. Recombinant ASFV-G-∆A859L replicates in swine macrophage cultures as efficiently as the parental virus ASFV-G, demonstrating that the A859L gene is non-essential for ASFV replication. Experimental infection of domestic pigs demonstrated that ASFV-G-∆A859L replicates as efficiently and induces a clinical disease indistinguishable from that caused by the parental ASFV-G. These studies conclude that the predicted RNA helicase gene A859L is not involved in the processes of virus replication or disease production in swine.

The Valproic Acid Derivative Valpromide Inhibits Pseudorabies Virus Infection in Swine Epithelial and Mouse Neuroblastoma Cell Lines.

Pseudorabies virus (PRV) infection of swine can produce Aujeszky’s disease, which causes neurological, respiratory, and reproductive symptoms, leading to significant economic losses in the swine industry. Although humans are not the natural hosts of PRV, cases of human encephalitis and endophthalmitis caused by PRV infection have been reported between animals and workers. Currently, a lack of specific treatments and the emergence of new PRV strains against which existing vaccines do not protect makes the search for effective antiviral drugs essential. As an alternative to traditional nucleoside analogues such as acyclovir (ACV), we studied the antiviral effect of valpromide (VPD), a compound derived from valproic acid, against PRV infection in the PK15 swine cell line and the neuroblastoma cell line Neuro-2a. First, the cytotoxicity of ACV and VPD in cells was compared, demonstrating that neither compound was cytotoxic at a specific concentration range after 24 h exposure. Furthermore, the lack of direct virucidal effect of VPD outside of an infected cell environment was demonstrated. Finally, VPD was shown to have an antiviral effect on the viral production of two strains of pseudorabies virus (wild type NIA-3 and recombinant PRV-XGF) at the concentrations ranging from 0.5 to 1.5 mM, suggesting that VPD could be a suitable alternative to nucleoside analogues as an antiherpetic drug against Aujeszky’s disease.

Evaluation of homeopathic treatment of Escherichia coli infected swine with indetification of virulence factors involved: Pilot study

Aim: To evaluate the efficacy of prophylactic homeopathic treatment of swine diarrhea to achieve a satisfactory balance between weight gain and cost. Methods: Escherichia coli (E. coli) was isolated from 4 piglets with diarrhea, their clinical signs were used to select the adequate homeopathic medicine China officinalis (Chin). Newborn piglets were divided in 4 groups (n=11 or 12): control group treated with the routinely used antimicrobial agent (ceftiofur 4 mg 1 ml/piglet for 3 days); one group with homeopathic medicine Chin 30 cH; one group treated with biotherapy prepared from locally isolated E. coli; and one group was treated with association of Chin and biotherapy. All isolated samples of E. coli were subjected to polymerase chain reaction (PCR) to identify virulence factors in each group. Results:There was no difference in the number of animals with diarrhea between groups treated with homeopathic medicine and control; weight gain was greater in groups treated with homeopathic medicine alone and associated with biotherapy compared to control (p

A PCR assay with high sensitivity and specificity for the detection of swine toxoplasmosis based on the GRA14 gene

Toxoplasma gondii, an intracellular apicomplexan protozoan parasite, can infect all warm-blooded animals. Infected swine are considered one of the most important sources of T. gondii infection in humans. Rapidly and effectively diagnosing T. gondii infection in swine is essential. PCR-based diagnostic tests have been fully developed, and very sensitive and specific PCR is crucial for the diagnosis of swine toxoplasmosis. In this study, we used the T. gondii dense granule protein 14 (GRA14) gene as a target to design specific primers and established a high-specificity and high-sensitivity PCR detection method for swine toxoplasmosis. Notably, this PCR method could detect T. gondii tachyzoite DNA in the acute infection phase. The GRA14 gene PCR assay detected a minimum of 2.35 tachyzoites of T. gondii and can be used for T. gondii detection in blood, tissue, semen, urine and waste feed specimens. A total of 5462 blood specimens collected from pigs in 5 provinces and autonomous regions in southern China during 2016–2017 were assessed by the newly established GRA14 gene PCR method. The overall T. gondii infection rate was 18.9 % (1033/5462). According to the statistical analysis of different regions in China, the positive rates of swine toxoplasmosis from 2016 to 2017 were highest in the Shaanxi, Fujian and Guangdong areas, at 31.7 % (44/139), 21.9 % (86/391) and 18.8 % (874/4645), respectively. Specimens collected in 2017 had a higher positive rate (19.1 %) than those collected in 2016 (16.1 %). In addition, specimens collected in autumn (39.4 %), spring (22.8 %) and winter (18.2 %) had higher positive rates than those collected in summer (3.8 %). These results indicate that the new PCR method based on the T. gondii GRA14 gene has utility for the diagnosis of swine toxoplasmosis and can facilitate the diagnosis of toxoplasmosis in clinical laboratories.

Programmed death-ligand 1 expression in swine chronic infections and enhancement of interleukin-2 production via programmed death-1/programmed death-ligand 1 blockade.

IntroductionChronic infections lead to the functional exhaustion of T cells. Exhausted T cells are phenotypically differentiated by the surface expression of the immunoinhibitory receptor, such as programmed death‐1 (PD‐1). The inhibitory signal is produced by the interaction between PD‐1 and its PD‐ligand 1 (PD‐L1) and impairs the effector functions of T cells. However, the expression dynamics of PD‐L1 and the immunological functions of the PD‐1/PD‐L1 pathway in chronic diseases of pigs are still poorly understood. In this study, we first analyzed the expression of PD‐L1 in various chronic infections in pigs, and then evaluated the immune activation by the blocking assay targeting the swine PD‐1/PD‐L1 pathway.MethodsIn the initial experiments, anti‐bovine PD‐L1 monoclonal antibodies (mAbs) were tested for cross‐reactivity with swine PD‐L1. Subsequently, immunohistochemical analysis was conducted using the anti‐PD‐L1 mAb. Finally, we assessed the immune activation of swine peripheral blood mononuclear cells (PBMCs) by the blockade with anti‐PD‐L1 mAb.ResultsSeveral anti‐PD‐L1 mAbs tested recognized swine PD‐L1‐expressing cells. The binding of swine PD‐L1 protein to swine PD‐1 was inhibited by some of these cross‐reactive mAbs. In addition, immunohistochemical analysis revealed that PD‐L1 was expressed at the site of infection in chronic infections of pigs. The PD‐L1 blockade increased the production of interleukin‐2 from swine PBMCs.ConclusionsThese findings suggest that the PD‐1/PD‐L1 pathway could be also involved in immunosuppression in chronic infections in pigs. This study provides a new perspective on therapeutic strategies for chronic diseases in pigs by targeting immunosuppressive pathways.

Determination of Susceptibility Breakpoint for Cefquinome against Streptococcus suis in Pigs.

Streptococcus suis (S. suis), a zoonotic pathogen, causes severe diseases in both pigs and human beings. Cefquinome can display excellent antibacterial activity against gram-negative and gram-positive bacteria. The aim of this study was to derive an optimal dosage of cefquinome against S. suis with a pharmacokinetic/pharmacodynamic (PK/PD) integration model in the target infection site and to investigate the cutoffs monitoring the changes of resistance. The minimum inhibitory concentration (MIC) distribution of cefquinome against 342 S. suis strains was determined. MIC50 and MIC90 were 0.06 and 0.25 μg/mL, respectively. The wild-type cutoff was calculated as 1 μg/mL. A two-compartmental model was applied to calculate the main pharmacokinetic parameters after 2 mg/kg cefquinome administered intramuscularly. An optimized dosage regimen of 3.08 mg/kg for 2-log10 CFU reduction was proposed by ex vivo PK/PD model of infected swine. The pharmacokinetic-pharmacodynamic cutoff was calculated as 0.06 μg/mL based on PK/PD targets. Based on the clinical effectiveness study of pathogenic MIC isolates, the clinical cutoff was calculated as 0.5 μg/mL. A clinical breakpoint was proposed as 1 μg/mL. In conclusion, the results offer a reference for determining susceptibility breakpoint of cefquinome against S. suis and avoiding resistance emergence by following the optimal dosage regimen.

Development and Validation of Liquid Chromatography-Tandem Mass Spectrometry Methods for the Quantification of Cefquinome, Ceftiofur, and Desfuroylceftiofuracetamide in Porcine Feces with Emphasis on Analyte Stability.

Cefquinome and ceftiofur are β-lactam antibiotics used for the treatment of bacterial infections in swine. Although these antimicrobials are administered intramuscularly, the exposure of the gut microbiota to these cephalosporins is not well described. This exposure can contribute to the emergence and spread of antimicrobials in the environment and to the possible spread of antimicrobial resistance genes. To assess the impact of drug administration on the intestinal excretion of these antimicrobials it is essential to measure the amounts of native compound and metabolites in feces. Two (ultra)-high-performance liquid chromatography-tandem mass spectrometry ((U)HPLC–MS/MS) methods were developed and validated, one for the determination of cefquinome and ceftiofur and the other for the determination of ceftiofur residues, measured as desfuroylceftiofuracetamide, in porcine feces. The matrix-based calibration curve was linear from 5 ng g−1 to 1000 ng g−1 for cefquinome (correlation coefficient (r) = 0.9990 ± 0.0007; goodness of fit (gof) = 3.70 ± 1.43) and ceftiofur (r = 0.9979 ± 0.0009; gof = 5.51 ± 1.14) and quadratic from 30 ng g−1 to 2000 ng g−1 for desfuroylceftiofuracetamide (r = 0.9960 ± 0.0020; gof = 7.31 ± 1.76). The within-day and between-day precision and accuracy fell within the specified ranges. Since β-lactam antibiotics are known to be unstable in feces, additional experiments were conducted to adjust the sampling protocol in order to minimize the impact of the matrix constituents on the stability of the analytes. Immediately after sampling, 500 µL of an 8 µg mL−1 tazobactam solution in water was added to 0.5 g feces, to reduce the degradation in matrix.