FRAX486, a PAK inhibitor, overcomes ABCB1-mediated multidrug resistance in breast cancer cells

The overexpression of P-glycoprotein (P-gp/ABCB1) is a leading cause of multidrug resistance (MDR). Hence, it is crucial to discover effective pharmaceuticals that counteract ABCB1-mediated multidrug resistance. FRAX486 is a p21-activated kinase (PAK) inhibitor. The objective of this study was to investigate whether FRAX486 can reverse ABCB1-mediated multidrug resistance, while also exploring its mechanism of action. The CCK8 assay demonstrated that FRAX486 significantly reversed ABCB1-mediated multidrug resistance. Furthermore, western blotting and immunofluorescence experiments revealed that FRAX486 had no impact on expression level and intracellular localization of ABCB1. Notably, FRAX486 was found to enhance intracellular drug accumulation and reduce efflux, resulting in the reversal of multidrug resistance. Docking analysis also indicated a strong affinity between FRAX486 and ABCB1. This study highlights the ability of FRAX486 to reverse ABCB1-mediated multidrug resistance and provides valuable insights for its clinical application.

Progress of Research on ABC Transporters in Tumor Multidrug Resistance / 肿瘤防治研究

The ATP-binding cassette (ABC) transporter superfamily comprises membrane proteins that efflux various substrates across extra- and intracellular membranes. Among them, ABCB1, ABCG2, and ABCC1 are directly linked to tumor multidrug resistance (MDR). This review provides an overview of the current understanding on the novel mechanisms and functions of ABCB1, ABCG2, and ABCC1 transporters in tumor MDR, discusses the latest strategies to target these transporters, and explores further opportunities to overcome MDR.

Progress in the studies on the molecular mechanisms associated with multidrug resistance in cancers

Chemotherapy is one of the important methods to treat cancer, and the emergence of multidrug resistance (MDR) is one major cause for the failure of cancer chemotherapy. Almost all anti-tumor drugs develop drug resistance over a period of time of application in cancer patients, reducing their effects on killing cancer cells. Chemoresistance can lead to a rapid recurrence of cancers and ultimately patient death. MDR may be induced by multiple mechanisms, which are associated with a complex process of multiple genes, factors, pathways, and multiple steps, and today the MDR-associated mechanisms are largely unknown. In this paper, from the aspects of protein-protein interactions, alternative splicing (AS) in pre-mRNA, non-coding RNA (ncRNA) mediation, genome mutations, variance in cell functions, and influence from the tumor microenvironment, we summarize the molecular mechanisms associated with MDR in cancers. In the end, prospects for the exploration of antitumor drugs that can reverse MDR are briefly discussed from the angle of drug systems with improved targeting properties, biocompatibility, availability, and other advantages.

Cryo-EM structures for the Mycobacterium tuberculosis iron-loaded siderophore transporter IrtAB

The adenosine 5'-triphosphate (ATP)-binding cassette (ABC) transporter, IrtAB, plays a vital role in the replication and viability of Mycobacterium tuberculosis (Mtb), where its function is to import iron-loaded siderophores. Unusually, it adopts the canonical type IV exporter fold. Herein, we report the structure of unliganded Mtb IrtAB and its structure in complex with ATP, ADP, or ATP analogue (AMP-PNP) at resolutions ranging from 2.8 to 3.5 Å. The structure of IrtAB bound ATP-Mg2+ shows a "head-to-tail" dimer of nucleotide-binding domains (NBDs), a closed amphipathic cavity within the transmembrane domains (TMDs), and a metal ion liganded to three histidine residues of IrtA in the cavity. Cryo-electron microscopy (Cryo-EM) structures and ATP hydrolysis assays show that the NBD of IrtA has a higher affinity for nucleotides and increased ATPase activity compared with IrtB. Moreover, the metal ion located in the TM region of IrtA is critical for the stabilization of the conformation of IrtAB during the transport cycle. This study provides a structural basis to explain the ATP-driven conformational changes that occur in IrtAB.

Research progress on the gene polymorphism of tacrolimus transporter in organ transplantation / 器官移植

Tacrolimus (Tac) is a commonly used immunosuppressant after organ transplantation, which has high immunosuppressive efficacy. However, the pharmacokinetics of Tac significantly differ among individuals, and gene polymorphism is the main influencing factor. In recent years, the gene polymorphism of drug transporter has become a novel research hotspot. Nevertheless, the effect of the gene polymorphism of transporter on Tac pharmacokinetics remains controversial. Consequently, the correlation between the gene polymorphism of transporter and Tac blood concentration plays a significant role in guiding Tac-based individualized immunosuppressive therapy. In this article, the research progresses on the gene polymorphism of adenosine triphosphate-binding cassette (ABC) transporter and solute carrier (SLC) transporter in organ transplantation was reviewed. The correlation between the gene polymorphism of transporter and Tac blood concentration was summarized, aiming to provide reference for Tac-based individualized therapy.

Traditional Chinese herbal medicine Astragalus Radix and its effects on intestinal absorption of aconite alkaloids in rats / 中草药·英文版

Objective: Astragalus Radix (AR, Huangqi in Chinese) has been widely used as a qi (energy) restoring herb that is thought to act through reinvigorating the spleen and lung. Aconite is used to rebalance the body temperature during illness and played an irreplaceable role in disease control since ancient times, but it is limited by its strong neuro and cardiotoxicity. Since the Song Dynasty (1227), the two herbs have been commonly used as herbal pairs including in the famous Qifu Decotion, from the “Wei's Family Prescription”. However, many ancient texts also record that they are not compatible using together, suggesting they can have negative outcomes when mixed. This study investigated whether Astragali Radix had either positive or negative effects on absorption of six different active alkaloids derived from aconite. Methods: Single intestinal perfusion model was used to study the effects of Astragali Radix on aconite alkaloids absorption. Response of ABC transporters and distribution of three tight junction proteins on the surface of intestinal enothelium were assessed by Reverse Transcription-Polymerase Chain Reaction (RT-PCR), Western blot and immunofluorescence microscopy, respectively. Results: The results showed that aconite alkaloids absorption could be inhibited, and different concentrations of Astragali Radix considerably increased the expression levels of the ABC transporters and tight junction proteins with Astragali Radix treatment. Conclusion: These results suggest that Astragali Radix can block absorption of aconite alkaloids through the upregulation expression of ATP-binding cassette transporters (ABC transporters) and tight junction proteins. It demonstrates that co-administration of Astragali Radix with other drugs might change the absorption profile of the second drug which is important to know in clinic therapy.

Rociletinib (CO-1686) enhanced the efficacy of chemotherapeutic agents in ABCG2-overexpressing cancer cells and o

Overexpression of adenosine triphosphate (ATP)-binding cassette subfamily G member 2 (ABCG2) in cancer cells is known to cause multidrug resistance (MDR), which severely limits the clinical efficacy of chemotherapy. Currently, there is no FDA-approved MDR modulator for clinical use. In this study, rociletinib (CO-1686), a mutant-selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), was found to significantly improve the efficacy of ABCG2 substrate chemotherapeutic agents in the transporter-overexpressing cancer cells and in MDR tumor xenografts in nude mice, without incurring additional toxicity. Mechanistic studies revealed that in ABCG2-overexpressing cancer cells, rociletinib inhibited ABCG2-mediated drug efflux and increased intracellular accumulation of ABCG2 probe substrates. Moreover, rociletinib, inhibited the ATPase activity, and competed with [I] iodoarylazidoprazosin (IAAP) photolabeling of ABCG2. However, ABCG2 expression at mRNA and protein levels was not altered in the ABCG2-overexpressing cells after treatment with rociletinib. In addition, rociletinib did not inhibit EGFR downstream signaling and phosphorylation of protein kinase B (AKT) and extracellular signal-regulated kinase (ERK). Our results collectively showed that rociletinib reversed ABCG2-mediated MDR by inhibiting ABCG2 efflux function, thus increasing the cellular accumulation of the transporter substrate anticancer drugs. The findings advocated the combination use of rociletinib and other chemotherapeutic drugs in cancer patients with ABCG2-overexpressing MDR tumors.

Minor temperature shifts do not affect chromosomal ploidy but cause transcriptomic changes in Leishmania braziliensis promastigotes in vitro

BACKGROUND The leishmaniases are complex neglected diseases caused by protozoan parasites of the genus Leishmania. Leishmania braziliensis is the main etiological agent of cutaneous leishmaniasis in the New World. In recent studies, genomic changes such as chromosome and gene copy number variations (CNVs), as well as transcriptomic changes have been highlighted as mechanisms used by Leishmania species to adapt to stress situations. OBJECTIVES The aim of this study was to determine the effect of short-term minor temperature shifts in the genomic and transcriptomic responses of L. braziliensis promastigotes in vitro. METHODS Growth curves, genome and transcriptome sequencing of L. braziliensis promastigotes were conducted from cultures exposed to three different temperatures (24ºC, 28ºC and 30ºC) compared with the control temperature (26ºC). FINDINGS Our results showed a decrease in L. braziliensis proliferation at 30ºC, with around 3% of the genes showing CNVs at each temperature, and transcriptomic changes in genes encoding amastin surface-like proteins, heat shock proteins and transport proteins, which may indicate a direct response to temperature stress. MAIN CONCLUSIONS This study provides evidence that L. braziliensis promastigotes exhibit a decrease in cell density, and noticeable changes in the transcriptomic profiles. However, there were not perceptible changes at chromosome CNVs and only ~3% of the genes changed their copies in each treatment.

A novel ready-to-use dry-reagent polymerase chain reaction for detection of Escherichia coli & Shigella species

Background & objectives: Polymerase chain reaction (PCR) has wide acceptance for rapid identification of pathogens and also for diagnosis of infectious conditions. However, because of economic and expertise constraints, a majority of small or peripheral laboratories do not use PCR. The objective of the present study was to develop a dry-reagent PCR assay as an alternative to conventional PCR to assess its applicability in routine laboratory practice using malB gene for identification of Escherichia coli as a model. Methods: A total of 184 isolates were selected for the study comprising clinical isolates of E. coli and non-E. coli including Shigella sp. and a few other control strains. The DNA was isolated from all the isolates. The isolated DNA as well as the overnight grown bacterial cultures were subjected to both conventional wet PCR and dry-reagent PCR. Results: The genomic DNA isolated from E. coli showed amplification of malB gene in both conventional wet and dry-reagent PCR and the band was observed at 491 bp. In dry-reagent PCR, the overnight grown E. coli cells also showed positive result. The non-E. coli strains other than Shigella sp. showed negative in both conventional wet and dry-reagent PCR. Shigella sp. showed positive in both conventional wet and dry-reagent PCR. Interpretation & conclusions: Considering the elimination of genomic DNA isolation step, and similar results with the conventional wet PCR, dry-reagent PCR may be a good alternative for the conventional wet PCR.

High degree of pharmacokinetic compatibility exists between the five-herb medicine XueBiJing and antibiotics comedicated in sepsis care

Managing the dysregulated host response to infection remains a major challenge in sepsis care. Chinese treatment guideline recommends adding XueBiJing, a five-herb medicine, to antibiotic-based sepsis care. Although adding XueBiJing further reduced 28-day mortality modulating the host response, pharmacokinetic herb-drug interaction is a widely recognized issue that needs to be studied. Building on our earlier systematic chemical and human pharmacokinetic investigations of XueBiJing, we evaluated the degree of pharmacokinetic compatibility for XueBiJing/antibiotic combination based on mechanistic evidence of interaction risk. Considering both XueBiJing‒antibiotic and antibiotic‒XueBiJing interaction potential, we integrated informatics-based approach with experimental approach and developed a compound pair-based method for data processing. To reflect clinical reality, we selected for study XueBiJing compounds bioavailable for drug interactions and 45 antibiotics commonly used in sepsis care in China. Based on the data of interacting with drug metabolizing enzymes and transporters, no XueBiJing compound could pair, as perpetrator, with the antibiotics. Although some antibiotics could, due to their inhibition of uridine 5'-diphosphoglucuronosyltransferase 2B15, organic anion transporters 1/2 and/or organic anion-transporting polypeptide 1B3, pair with senkyunolide I, tanshinol and salvianolic acid B, the potential interactions (resulting in increased exposure) are likely desirable due to these XueBiJing compounds' low baseline exposure levels. Inhibition of aldehyde dehydrogenase by 7 antibiotics probably results in undesirable reduction of exposure to protocatechuic acid from XueBiJing. Collectively, XueBiJing/antibiotic combination exhibited a high degree of pharmacokinetic compatibility at clinically relevant doses. The methodology developed can be applied to investigate other drug combinations.

Construction and pathogenicity of mutant strain ATP binding cassette transporter SSU05_0948 of Streptococcus suis type 2 / 中华传染病杂志

Objective@#To construct the mutant strain ATP binding cassette transporter SSU05_0948 of Streptococcus suis type 2 and comprehensively study its pathogenicity, and to provide useful insights for understanding the mechanism that Streptococcus suis avoid host innate immunity. @*Methods@#The mutant strain 05ZYH33Δ0948 was constructed through homologous recombination technology. The differences between the mutant strain and the wild type strain were evaluated through bacterial adhesion, whole blood killing, mice meningitis assay, mice and piglets virulence assay. Chi-square test and t test were used. @*Results@#Successfully constructed the mutant strain 05ZYH33Δ0948. The adhesion results showed that the adhesion rate (0.663±0.047)% of the wild strain to A549 cell was significantly higher than that of the mutant (0.246±0.074)%, the difference was statistically significant (χ2=5.267, P=0.014); the adhesion rate (16.540±2.320)% of the wild strain to Hep2 cell was significantly higher than that of the mutant (1.970±0.320)%, the difference was statistically significant (χ2=0.014, P<0.01); the adhesion rate (5.497±0.174)% of the wild strain to Hep2 cell was significantly higher than that of the mutant (1.950±0.335)%, the difference was statistically significant (χ2=0.016, P<0.01). The killing rate (32.970±3.589)% of the wild strain in whole blood is no difference with the mutant (29.560±3.737)% (χ2=1.200, P=0.133). Piglets competitive infection showed that, the competitive index at 12 h, 24 h and 36 h were 0.046±0.003, 0.107±0.003, 0.064±0.001, respectively. 12 h and 24 h was significant differences(t=15.490, P=0.041), 24 h and 36 h was significant differences(t=5.660, P=0.047), 12 h and 36 h was no differences(t=1.445, P=0.285). @*Conclusions@#Streptococcus suis type 2 ABC transporter SSU05_0948 is a new adhesion factor and virulence factor of Streptococcus suistype 2, and also a new meningitis factor, which plays important roles in Streptococcus suis against host innate immunity.

Olmutinib (HM61713) reversed multidrug resistance by inhibiting the activity of ATP-binding cassette subfamily G member 2 and

Overexpressing of ATP-binding cassette (ABC) transporters is the essential cause of multidrug resistance (MDR), which is a significant hurdle to the success of chemotherapy in many cancers. Therefore, inhibiting the activity of ABC transporters may be a logical approach to circumvent MDR. Olmutinib is an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI), which has been approved in South Korea for advanced EGFR T790M-positive non-small cell lung cancer (NSCLC). Here, we found that olmutinib significantly increased the sensitivity of chemotherapy drug in ABCG2-overexpressing cells. Furthermore, olmutinib could also increase the retention of doxorubicin (DOX) and rhodamine 123 (Rho 123) in ABC transporter subfamily G member 2 (ABCG2)-overexpressing cells. In addition, olmutinib was found to stimulate ATPase activity and inhibit photolabeling of ABCG2 with [I]-iodoarylazidoprazosin (IAAP). However, olmutinib neither altered ABCG2 expression at protein and mRNA levels nor blocked EGFR, Her-2 downstream signaling of AKT and ERK. Importantly, olmutinib enhanced the efficacy of topotecan on the inhibition of S1-MI-80 cell xenograft growth. All the results suggest that olmutinib reverses ABCG2-mediated MDR by binding to ATP bind site of ABCG2 and increasing intracellular chemotherapeutic drug accumulation. Our findings encouraged to further clinical investigation on combination therapy of olmutinib with conventional chemotherapeutic drugs in ABCG2-overexpressing cancer patients.

Ligand binding and conformational changes of SUR1 subunit in pancreatic ATP-sensitive potassium channels

ATP-sensitive potassium channels (K) are energy sensors on the plasma membrane. By sensing the intracellular ADP/ATP ratio of β-cells, pancreatic K channels control insulin release and regulate metabolism at the whole body level. They are implicated in many metabolic disorders and diseases and are therefore important drug targets. Here, we present three structures of pancreatic K channels solved by cryo-electron microscopy (cryo-EM), at resolutions ranging from 4.1 to 4.5 Å. These structures depict the binding site of the antidiabetic drug glibenclamide, indicate how Kir6.2 (inward-rectifying potassium channel 6.2) N-terminus participates in the coupling between the peripheral SUR1 (sulfonylurea receptor 1) subunit and the central Kir6.2 channel, reveal the binding mode of activating nucleotides, and suggest the mechanism of how Mg-ADP binding on nucleotide binding domains (NBDs) drives a conformational change of the SUR1 subunit.

Identification and differential expression analysis of ABC transporter gene from medicinal plant Dendrobium officinale / 药学学报

ABC transporters (ATP-binding cassette transporters) are a family of trans-membrane transport proteins, which are ubiquitous in prokaryotes and eukaryotes. They are functionallyinvolved in the transport and accumulation of plant secondary metabolites, phytohormone transport, lipid metabolism, exogenous toxins detoxification, plant disease and other aspects. Based on the genome and transcriptome data of Dendrobium officinale, 88ABC transporters were preliminarily identified in D. officinale and their functions and subcellular localization were predicted. These proteins are divided into seven subfamilies, ABCA-ABCI, including 4 ABCA, 19 ABCB, 12 ABCC, 3ABCD, 9 ABCF, 37ABCG and 4 ABCI, which are mainly located in plasma membrane, vacuole and Golgi apparatus. Comparative transcriptomic analysis of ABC protein expression profile between asymbiotic and symbiotic germination of D. officinale show that some members of ABCB and ABCG proteins are highly expressed during seed germination inoculated fungi. qPCR validated that 2 ABCB11 and 2 ABCG-PDR are significantly up-regulated in symbiotic assay compared to asymbiotic germination (fold change ≥ 10.0). These proteins are mainly involved in abscisic acid and auxin transport, suggesting that these proteins play an important role in the germination of D. officinale seed and in the interaction with microorganisms.

Comparative transcriptome profile of the leaf elongation zone of wild barley (Hordeum spontaneum) eibi1 mutant and its isogenic wild type

Abstract The naturally occurring wild barley mutant eibi1/hvabcg31 suffers from severe water loss due to the permeable leaf cuticle. Eibi1/HvABCG31 encodes a full ATP-binding cassette (ABC) transporter, HvABCG31, playing a role in cutin deposition in the elongation zone of growing barley leaves. The eibi1 allele has pleiotropic effects on the appearance of leaves, plant stature, fertility, spike and grain size, and rate of germination. Comparative transcriptome profile of the leaf elongation zone of the eibi1 mutant as well as its isogenic wild type showed that various pathogenesis-related genes were up-regulated in the eibi1 mutant. The known cuticle-related genes that we analyzed did not show significant expression difference between the mutant and wild type. These results suggest that the pleiotropic effects may be a compensatory consequence of the activation of defense genes in the eibi1 mutation. Furthermore, we were able to find the mutation of the eibi1/hvabcg31 allele by comparing transcript sequences, which indicated that the RNA-Seq is useful not only for researches on general molecular mechanism but also for the identification of possible mutant genes.

Functional analysis of ATP-binding cassette transporter of Streptomyces coelicolor.

ATP- binding cassette (ABC) transporters are characterized as multi drug resistant transporters utilizing ATP hydrolysis. The SCO5113 (GeneID: 1100554) encoding a multidrug resistance ABC transporter in Streptomyces coelicolor was expressed in E.coli BL21 (DE3) plysS, using pET 21 a(+). The expression of his – tag fused SCO5113 at 65 kDa under 0.1 M IPTG in 6 h was detected on SDS-PAGE after purification using affinity chromatography. The E. coli BL21 (DE3) plysS harboring SCO5113 was used to test for the antibiotic resistance, using disc diffussion method. The results showed the resistance of E. coli BL21 (DE3) plysS harboring SCO5113 to five kinds of tested antibiotics such as cephalothin (1mg/ml), kanamycin (1mg/ml), ampicillin (10mg/ml), erythromycin (10mg/ml), and chloramphenicol (1mg/ml). The study reported the function of the SCO5113 gene of Streptomyces coelicolor A3(2).

The pharmacological impact of ATP-binding cassette drug transporters on vemurafenib-based therapy

Melanoma is the most serious type of skin cancer and one of the most common cancers in the world. Advanced melanoma is often resistant to conventional therapies and has high potential for metastasis and low survival rates. Vemurafenib is a small molecule inhibitor of the BRAF serine-threonine kinase recently approved by the United States Food and Drug Administration to treat patients with metastatic and unresectable melanomas that carry an activating BRAF (V600E) mutation. Many clinical trials evaluating other therapeutic uses of vemurafenib are still ongoing. The ATP-binding cassette (ABC) transporters are membrane proteins with important physiological and pharmacological roles. Collectively, they transport and regulate levels of physiological substrates such as lipids, porphyrins and sterols. Some of them also remove xenobiotics and limit the oral bioavailability and distribution of many chemotherapeutics. The overexpression of three major ABC drug transporters is the most common mechanism for acquired resistance to anticancer drugs. In this review, we highlight some of the recent findings related to the effect of ABC drug transporters such as ABCB1 and ABCG2 on the oral bioavailability of vemurafenib, problems associated with treating melanoma brain metastases and the development of acquired resistance to vemurafenib in cancers harboring the BRAF (V600E) mutation.

Reversal effect of vatalanib on BCRP-mediated multidrug resistance / 中国药理学通报

Aim To investigate the reversal effect of vatalanib, a novel kinase inhibitor, on multidrug re-sistance in cancer cells and its mechanism. Methods The cytotoxicity and reversal effects of vatalanib were evaluated in both resistant and sensitive tumor cell lines by MTS or SRB assays. The intracellular accumu-lation of fluorescence substrates ( Rh-123 , MX and ADR for P-gp, BCRP, MRP1, respectively) were ana-lysed by flow cytometry. Western blot or qRT-PCR was used to determine the protein or mRNA expression lev-el of BCRP. The effect of vatalanib on ATPase activity of BCRP was determined using crude membranes pre-pared from HEK293/ABCG2 cells. Results Vata-lanib at the nontoxic dose ( 5 μmol · L-1 ) potentially reversed BCRP-mediated MDR in cancer cells, howev-er it had no effect on P-gp or MRP1 mediated MDR. Vatalanib did not alter the intracellular accumulation of MX in HEK 2 9 3 / ABCG 2 , and had no influence on the BCRP-mediated drug efflux. The ATPase assay indica-ted that vatalanib may serve as a substrate of BCRP. Furthermore, vatalanib dramatically suppressed levels of both the protein and mRNA expression of BCRP in concentration-and time-dependent manners. However, reversal concentration of vatalanib had no influence on the total and phosphorylated forms of AKT and ERK1/ 2 in resistant cancer cells. Conclusion Vatalanib could significantly reverse BCRP-mediated MDR with specificity, and its mechanism may correlate with the down-regulation levels of BCRP both mRNA and pro-tein in resistant cancer cells.

Neonatal Diabetes Caused by Activating Mutations in the Sulphonylurea Receptor

Adenosine triphosphate (ATP)-sensitive potassium (KATP) channels in pancreatic beta-cells play a crucial role in insulin secretion and glucose homeostasis. These channels are composed of two subunits: a pore-forming subunit (Kir6.2) and a regulatory subunit (sulphonylurea receptor-1). Recent studies identified large number of gain of function mutations in the regulatory subunit of the channel which cause neonatal diabetes. Majority of mutations cause neonatal diabetes alone, however some lead to a severe form of neonatal diabetes with associated neurological complications. This review focuses on the functional effects of these mutations as well as the implications for treatment.

Isolation and Cloning of an ABC Transporter-Like Gene of Haemophilus parasuis and Its Use in a New Diagnostic PCR

The aim of this study was to identify a new gene of Haemophilus parasuis that could be used to develop a polymerase chain reaction (PCR) test for this porcine pathogen. H. parasuis genomic DNA was cloned into a set of expression vectors, and transformants expressing His-tagged polypeptides were identified by colony blotting. An ABC transporter-like gene was isolated. The cloned DNA fragment is 1,105 base pair and shows 78% similarity at the nucleotide level with an ABC transporter gene of H. ducreyi. Based on this sequence, two PCR primers were designed to amplify the entire 1,105-bp fragment in the proposed diagnostic PCR test. PCR amplification was able to detect a minimum of 1 x 10(4) CFU/ml of H. parasuis organisms. Fifteen different H. parasuis serovars were positive using the PCR test. No amplification was observed when the test was done using DNA from 16 other bacterial species commonly isolated from swine.