Campylobacter and Salmonella are prevalent in broiler farms in Kyushu, Japan: results of a 2-year distribution and circulation dynamics audit.

AIM: To elucidate the distribution and circulation dynamics of Campylobacter and Salmonella in Japanese chicken broiler flocks. METHODS AND RESULTS: A 2-year investigation of the distribution of Campylobacter and Salmonella was conducted in 25 broiler flocks at nine farms in Japan from 2013 to 2014. Campylobacter and Salmonella tested positive in 11 (44·0%) and 24 (96·0%) broiler flocks respectively. One hundred and ninety-five Campylobacter and 184 Salmonella isolates were characterized into 12 Campylobacter (including two novel genotypes) and three Salmonella MLST genotypes. Only Salmonella isolation between caecal and environmental samples were significantly correlated. Further, one litter sample tested positive for Salmonella before new chicks were introduced. The Campylobacter strains rapidly lost culturability within 2-18 days; in contrast, the Salmonella strains survived from 64-211 days in artificially inoculated water samples. CONCLUSION: No persistent circulation-mediated Campylobacter contamination was observed. In contrast, circulation of Salmonella in broiler houses was seen, apparently due to the litter excreted from broiler flocks, as well as Salmonella-contaminated water and feed. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper provides the distribution, genotypic data and circulation dynamics of Campylobacter and Salmonella as recently observed in Japanese chicken broiler farms.

Polypharmacy of medications and fall-related fractures in older people in Japan: a comparison between driving-prohibited and driving-cautioned medications.

WHAT IS KNOWN AND OBJECTIVE: Polypharmacy is a risk factor for fall-related fractures. However, it is unclear whether polypharmacy itself is a direct risk factor. The aim of this study was to assess the association between the risk of fall-related fractures and polypharmacy of driving-prohibited and driving-cautioned medications in older outpatients. METHODS: We conducted a cross-sectional study of outpatients aged ≥65 years receiving any medication, using two sampling data sets from the October 2011 and October 2012 national insurance claims in Japan. Using logistic regression models, we analysed the association between the numbers of driving-prohibited or driving-cautioned medications administered or dispensed to patients and the occurrence of fall-related fractures. RESULTS AND DISCUSSION: In both analysis populations (n = 303 311 and n = 326 219), the adjusted odds ratio of driving-prohibited medications for the occurrence of fall-related fractures significantly increased as the number of these medications per patient increased (95% confidence interval: 0, 1-2, 3-4, 5-6, 7-8 and ≥9 medications; reference, 0·95-1·24, 1·18-1·79, 1·47-2·96, 1·26-5·21 and 1·50-15·2 in October 2011 and reference, 1·11-1·42, 1·39-2·03, 1·33-2·72, 1·53-5·49 and 1·30-13·0 in October 2012). The association was maintained even for sensitivity analyses restricted to medications administered orally or orally and by injection. However, a similar association was not observed for driving-cautioned medications. WHAT IS NEW AND CONCLUSION: Medication class is a more important risk factor for fall-related fractures rather than polypharmacy alone with no regard to medication class.

Comparing patient dissatisfaction and rational judgment in intentional medication non-adherence versus unintentional non-adherence.

WHAT IS KNOWN AND OBJECTIVE: Patients' poor adherence to medications is reported to be related to the individual patients' beliefs and cognitions and their trust of the medical staff. However, the causes of the two forms of non-adherence, intentional and unintentional behaviours, have yet to be clarified. This study compared psychological latent factors associated with intentional and unintentional non-adherence to chronic medication regimens, focusing on the potential effects of (i) patients' dissatisfaction with treatment and their relationships with the medical staff and (ii) patients' subliminal rational thinking processes, which weighed the positive values such as their expectations of benefits from treatment against negative values such as their dissatisfaction. METHODS: Two cross-sectional surveys were undertaken of patients given medications for chronic diseases, using a questionnaire developed and validated in this study. One survey was undertaken in three hospitals and the other survey, online throughout Japan. We scored the individual latent factors using the questionnaire and calculated the differential score between two negatively correlated latent factors to quantify patients' subliminal rational thinking process. We compared the adjusted odds ratio (OR) of latent factors between intentional and unintentional non-adherence to medication in both surveys. RESULTS AND DISCUSSION: Of the eligible subjects, 149 hospitalized patients and 524 survey participants completed the questionnaire. Intentional non-adherence was associated with patient dissatisfaction with treatment including interpersonal relationships with medical staff in both hospitalized patients and online survey participants (95% confidence interval of adjusted OR for Dissatisfaction, 1·20-16·26 in the hospital-based survey and 1·33-3·45 in the online survey). In both surveys, intentional non-adherence was significantly associated with the differential score between two negatively correlated latent factors, Willingness and Dissatisfaction (P = 0·02 in the hospital-based survey and P < 0·001 in the online survey). However, these associations were not evident in unintentionally non-adherent patients. WHAT IS NEW AND CONCLUSIONS: Patients' dissatisfaction and their resulting rational judgments are unique, consistent determinants of intentional non-adherence to medications, but not of unintentional non-adherence.

Simultaneous evaluation of long-lasting knee synovitis in patients undergoing arthroplasty by power Doppler ultrasonography and contrast-enhanced MRI in comparison with histopathology.

OBJECTIVES: We simultaneously assessed ultrasonography (US) and magnetic resonance imaging (MRI) in comparison with histopathological changes in the knee joints of long-lasting arthritis patients. METHODS: We studied 15 patients with rheumatoid arthritis and 5 patients with osteoarthritis, who underwent total knee arthroplasty. On the day before surgery, the joints were examined by US and contrast-enhanced MRI. In US, synovitis was graded with 0-3 grey scale (GSUS) and power Doppler (PDUS). In MRI, synovitis was graded according to OMERACT-RAMRIS (grade 0-3). Synovial tissue samples were obtained during arthroplasty and evaluated on the basis of inflammatory cell infiltrates (grade 0-3), synovial lining layer thickness (grade 0-3) and vascularity (grade 0-3). RESULTS: Positive findings of PDUS and contrast-enhanced MRI were 45% and 85% of 20 operated joints, respectively. GSUS, PDUS and MRI synovitis were well correlated with overall histopathological grades of synovitis (Spearman correlation coefficients 0.48, 0.84 and 0.48, p<0.05, p<0.01 and p<0.05, respectively). Moreover, positive PDUS findings were closely associated with all pathological comportments of synovitis including inflammatory cell infiltrates, synovial lining layer thickness and vascularity. CONCLUSIONS: The present study revealed that positive PDUS findings more faithfully illustrated active synovitis than MRI, whereas contrast-enhanced MRI was more sensitive in detecting synovitis in patients with long-lasting arthritis. It is important to understand distinct features of the both modalities for clinical assessment of chronic joint diseases.

Role of phosphorylation of Alzheimer's amyloid precursor protein during neuronal differentiation.

Alzheimer's amyloid precursor protein (APP), the precursor of beta-amyloid (Abeta), is an integral membrane protein with a receptor-like structure. We recently demonstrated that the mature APP (mAPP; N- and O-glycosylated form) is phosphorylated at Thr668 (numbering for APP695 isoform), specifically in neurons. Phosphorylation of mAPP appears to occur during, and after, neuronal differentiation. Here we report that the phosphorylation of mAPP begins 48-72 hr after treatment of PC12 cells with NGF and that this correlates with the timing of neurite outgrowth. The phosphorylated form of APP is distributed in neurites and mostly in the growth cones of differentiating PC12 cells. PC12 cells stably expressing APP with Thr668Glu substitution showed remarkably reduced neurite extension after treatment with NGF. These observations suggest that the phosphorylated form of APP may play an important role in neurite outgrowth of differentiating neurons.

Potassium channels in synaptosomal membrane examined using patch-clamp techniques and reconstituted giant proteoliposomes.

Synaptosomes isolated from the rat cerebral cortex were mixed with sonicated phospholipid vesicles and subjected to freezing-thawing to acquire giant proteoliposomes. Membranes of these giant proteoliposomes could thus be studied using patch-clamp techniques. Single-channel currents were measured with the inside-out patch of the membrane, in KCl solutions. Three different potassium channels were detected and unit conductances were 15.1, 28.6 and 91.0 pS, respectively, in a symmetrical 150 mM KCl solution. All these channels are more permeable to potassium than to sodium ions, the permeability ratio being about 2:1. Tetraethylammonium ions blocked these channels. The gating of these potassium channels is independent of the membrane potential. Presumably, these channels play a role in the resting membrane potential of presynaptic nerve terminals.

Comparative electrophysiological study of reconstituted giant vesicle preparations of the rabbit skeletal muscle sarcoplasmic reticulum K+ channel.

Sarcoplasmic reticulum (SR) membranes isolated from rabbit skeletal muscle were reconstituted into two types of giant vesicles: (1) Giant proteoliposomes prepared by freeze-thawing of a mixture of SR vesicles and sonicated phospholipid vesicles without the use of detergent. (2) Giant SR vesicles prepared by fusion of SR vesicles using poly(ethylene glycol) (PEG) as a fusogen and without the addition of exogenous lipid. These giant vesicles were patch-clamped and properties of the single voltage-dependent potassium channel in the excised patch were studied. Single-channel conductance in a symmetrical solution of 0.1 M KCl and 1 mM CaCl2 was 140.0 +/- 10 pS (n = 5) for freeze-thawed vesicles and 136.4 +/- 15 pS (n = 7) for PEG vesicles. Both types of vesicles exhibited a sub-conductance state having 55% of the fully open state conductance. The voltage-dependence of open-channel probability could be expressed in terms of thermodynamic parameters of delta Gi = 0.95 kcal/mol and z = -0.77 for freeze-thawed vesicles and delta Gi = 0.92 kcal/mol and z = -0.87 for PEG vesicles. These values correlated well with previous data obtained by fusion of native SR vesicles with a planar lipid membrane. Channel orientation was found to be conserved in both types of vesicles used in the present study.

Novel radioactive phospholipid probes as a tool for measurement of phospholipid translocation across biomembranes.

In an attempt to develop a new method to measure transbilayer phospholipid translocation, with a higher sensitivity and higher temporal resolution, novel radioactive phospholipid probes (*C5-PC, *C5-PE, and *C5-PS) with a short acyl chain at the 2-position were synthesized. The *C5-PC probe was made by coupling lysophosphatidylcholine with [14C]pentanoic acid, using N,N-carbonyldiimidazole as a coupling agent (yield 37%), and *C5-PE and *C5-PS were synthesized by exchanging the choline moiety of *C5-PC for ethanolamine and L-serine, respectively, as catalyzed by phospholipase D. The usefulness of the probes was confirmed by measuring phospholipid translocation across the human erythrocyte plasma membrane, in which the presence of aminophospholipid translocase was revealed using EPR techniques (Zachowski, A., Farve, E., Cribier, S., Herve, P. and Devaux, P.F. (1986) Biochemistry 25, 2585-2590). Using the present probes, ATP-dependent and SH-reagent-inhibitable translocation of *C5-PS and *C5-PE from outer to inner leaflets, which is characteristic to the translocation mediated by aminophospholipid translocase, was detected with a higher sensitivity than seen with the EPR technique. These radioactive phospholipid probes will be useful to measure phospholipid translocation with a high sensitivity and have the potential for application in measurements of transbilayer lipid-translocation for a wide variety of membranes.

Rapid determination of internal volumes of membrane vesicles with electron spin resonance-stopped flow technique.

We have developed an electron spin resonance (ESR)-stopped flow technique and employed it for the simple and rapid determination of internal volumes of biomembrane vesicles and liposomes. A vesicle suspension containing a neutral and membrane-permeable spin label, 2,2,6,6-tetramethyl-4-oxopiperidine-1-oxyl (TEMPONE), was mixed in the stopped-flow apparatus with an isotonic solution of relatively impermeable line broadening agents, potassium tris(oxalato)chromate(III) or potassium ferricyanide, and an ESR spectrum was recorded. From the relative intensity of the sharp triplet signal due to TEMPONE in the aqueous space within vesicles, the determination of the internal aqueous volume was straightforward. Using this technique, it is possible to measure intravesicular volumes in 0.1 s. The internal volume of sonicated phospholipid vesicles was approximately 0.3 microliter/mg lipid. The light fraction of sarcoplasmic reticulum membrane vesicles isolated from rabbit skeletal muscle was estimated to have an internal volume of 2.2-2.6 microliter/mg protein in its resting state. Activation of Ca2+ pumps in the membrane upon addition of ATP and Ca2+ ions decreased the internal volume by about 10%. This finding supports the hypothesis that the Ca2+ pump is electrogenic and that the efflux of potassium ions compensates for the influx of positive charges. The present technique is widely applicable to the simple and rapid determination of the internal volumes of membrane vesicles.

Two mode ion channels induced by interaction of acidic amphipathic alpha-helical peptides with lipid bilayers.

In order to investigate the ion permeability and selectivity of ion channel formed by amphipathic alpha-helical peptides, we designed to synthesize an acidic peptide Ac(Leu-Ala-Glu-Leu)3NHCH3 (Glu-4(3)) and its channel property was compared with a basic peptides Arg-4(3) in which Glu in Glu-4(3) was replaced by Arg. Two modes of the conductance change were observed by the interaction of Glu-4(3) with planar lipid bilayers; a steady increase of the conductance with the elapse of time (mode 1) and a spike-like increase of the current (mode 2) appearing with a lag time and overlapping the model current increase. The application of negative membrane potential induced the mode 1 current and the lowering pH decreased it, suggesting that the mode 1 current is caused by slow insertion of Glu-4(3) into the lipid bilayer and then by forming certain unknown bundles like semichannels. Mode 2 was found to be consisted of channel type opened-close current with several different conductances and relatively short opening lifetimes. There was no ion selectivity in the mode 1 current, whereas the mode 2 current was cation selective. The peptide Arg-4(3) has formed a cation-selective ion channel but not shown such two mode current changes. The membrane potential formation experiment in liposomes using DiSC3(5) also showed the cation selectivity for Arg-4(3) and non-ion selectivity for Glu-4(3). The difference between Arg-4(3) and Glu-4(3) was also observed in conformational analysis by CD and in dye-release experiment from liposome. Such difference was discussed in terms of electrostatic interaction between peptides and lipid head groups.

A voltage-dependent chloride channel from Tetrahymena ciliary membrane incorporated into planar lipid bilayers.

Membrane vesicles from cilia of Tetrahymena thermophila were incorporated into a planar phospholipid bilayer membrane, and single-channel currents across the planar membrane were recorded under voltage-clamp conditions. A novel and reproducible chloride channel was observed when a mixture of phosphatidylethanolamine and phosphatidylcholine was used to form the planar lipid membrane but not when acidic phospholipid mixtures such as asolectin or a mixture containing phosphatidylserine. Using symmetrical 100 mM KCl solutions, the single-channel conductance of the fully open state (O1) was 73.1 pS, with sub-level (O2) conductance of 9.0 pS. The permeability ratio Pc1/Pk was calculated as 3.7, according to the Goldman-Hodgkin-Katz current equation. This channel exhibited characteristic voltage-dependent burst activities. With an increase in membrane potential, the lifetimes of both the burst and interburst states decreased. In the burst state, the frequency of transition between the O1 and O2 states was also voltage-dependent, mainly due to the decrease in the lifetime of the O1 state, with an increase in membrane potential. In addition, channel activity was inhibited by indanyloxyacetic acid-94 (IAA-94), an inhibitor of epithelial chloride channels.

Formation of ion channels in planar lipid bilayer membranes by synthetic basic peptides.

We made use of a planar lipid bilayer system to examine the action of synthetic basic peptides which model the prepiece moiety of mitochondrial protein precursors and have antibacterial activity against Gram-positive bacteria. The sequences of the peptides used were as follows: Ac-(Ala-Arg-Leu)3-NHCH3 (3(3], Ac-(Leu-Ala-Arg-Leu)2-NHCH3 (4(2], Ac-(Leu-Ala-Arg-Leu)3-NHCH3 (4(3], Ac-(Leu-Leu-Ala-Arg-Leu)2-NHCH3 (5(2]. These peptides interacted differently with planar lipid bilayer membranes and membrane conductance increased by the formation of ion channels. The effects of the peptides on the macroscopic current-increase and on the probability of channel formation, at the single channel level were in the order of 4(3) greater than 4(2) approximately 5(2) much greater than 3(3), a finding which correlates with the antibacterial activity of these peptides. The micromolar (microM) order concentration at which the channel was formed resembles that causing antibacterial activity. Thus, the peptide antibacterial activity may occur through an increase in ion permeability of the bacterial membrane. The single-channel properties were investigated in detail using 4(3), the peptide with the highest ion channel-forming activity. Many types of channels were observed with respect to conductance (2-750 pS) and voltage dependency of gating. However, the channels were all cation-selective. These results suggest that the ion channels formed by peptide 4(3) may be able to take on a variety of conformations and/or assembly.

Effect of salts on conformational change of basic amphipathic peptides from beta-structure to alpha-helix in the presence of phospholipid liposomes and their channel-forming ability.

A synthetic model peptide, H-(Leu-Al alpha-Arg-Leu)3-(Leu-Arg-Al alpha-Leu)3-OH (4(6)) can form ion channels in planar lipid bilayers by taking an amphipathic alpha-helix (Agawa, Y., Lee, S., Ono, S., Aoyagi, H., Ohno, M., Taniguchi, T., Anzai, K. and Kirino, Y. (1991) J. Biol. Chem. 266, 20218-20222). For further study of ion channels formed by this type of peptides, we planned to synthesize [Trp1]-4(6)(Ser) and [Trp12]-4(6)(Ser) in which a hydrophilic amino acid, Ser, was introduced in several positions of 4(6) instead of hydrophobic ones. This modification was expected to decrease the ability of membrane perturbation and to simplify various current levels of the channel observed for 4(6). Furthermore, additional Trp was introduced to the N-terminus or position 12 to monitor the lipid-peptide interaction. CD study showed that both peptides formed a random structure in buffer, but an alpha-helix in the presence of egg PC and a beta-structure in egg PC/egg PG (3:1). Moreover, addition of NaCl to the acidic liposomes induced the conformational transition in the peptide from beta-structure to alpha-helix. Salt-induced conformational transition in the presence of acidic liposomes was discussed in terms of membrane binding and ion-channel formation in planar lipid bilayer. Despite introduction of hydrophilic residues instead of hydrophobic residues in 4(6), the peptide showed nearly the same dye-release ability from egg PC- egg PG liposomes as 4(6). [Trp12]-4(6)(Ser) was able to form cation-selective ion channels with two levels of conductance (mainly 250 and occasionally 125 pS) in asolectin planar lipid bilayer, suggesting that appropriate orientation of hydrophobic and hydrophilic residues in amphipathic peptide can simplify channel current levels.

Assignment of ESR signals of Escherichia coli terminal oxidase complexes.

The ESR signals of all the major components of the aerobic respiratory chain of Escherichia coli were measured and assigned at liquid helium temperature. Cytochrome b-556 gives a weak high-spin signal at g = 6.0. The terminal oxidase cytochrome b-562 . o complex gives signals at g = 6.0, 3.0 and 2.26, and the terminal oxidase cytochrome b-558 . d complex gives signals at g = 6.0, 2.5 and 2.3. A signal derived from cupric ions in the purified cytochrome b-562 . o complex was observed near g = 2.0. It was shown by the effects of KCN or NaN3 on cytochromes under the air-oxidized conditions that cytochrome o has a high-spin heme and cytochrome d has a low-spin heme. The E'm values for cytochromes b-558 and d, respectively, determined by potentiometric titration of the ESR signals were 140 and 240 mV in the membrane preparation, and 30 and 240 mV in the purified preparation. The oxidized cytochrome d gave intense low-spin signals at g = 2.5 and 2.3, while cytochrome d under the air-oxidized conditions gave corresponding signals of only very low intensity. These results suggested that most of the cytochrome d under the air-oxidized conditions contains a diamagnetic iron atom with a bound dioxygen.

Solution structure of micelle-bound H5 peptide (427-452): a primary structure corresponding to the pore forming region of the voltage dependent potassium channel.

A 26-mer peptide with the sequence of the pore forming region (residues 427-452) of the Shaker K(+) channel (H5 region) was chemically synthesized. Analyses by CD and two-dimensional 1H NMR spectroscopy were used to investigate the structure of the peptide bound to SDS micelles in solution, which are commonly used in biophysical studies. The tertiary structure of the peptide as a monomer was composed of an alpha-helix (431-438), a turn (439-442), and random coils (427-430, 443-452), and was very similar to that of the pore forming region of the native K(+) channel from Streptomyces lividans determined by X-ray analysis. This result suggests that even an isolated peptide forms a native-like conformation for residues from 431 to 442, depending on its intrinsic amino acid sequence and the surrounding environment.

N-methyl-D-aspartate receptors play important roles in acquisition and expression of the eyeblink conditioned response in glutamate receptor subunit delta2 mutant mice.

Classical eyeblink conditioning has been known to depend critically on the cerebellum. Apparently consistent with this, glutamate receptor subunit delta2 null mutant mice, which have serious morphological and functional deficiencies in the cerebellar cortex, are severely impaired in delay paradigm. However, these mutant mice successfully learn in trace paradigm, even in '0-trace paradigm,' in which the unconditioned stimulus starts just after the conditioned stimulus terminates. Our previous studies revealed that the hippocampus and the muscarinic acetylcholine receptors play crucial roles in 0-trace paradigm in glutamate receptor subunit delta2 null mutant mice unlike in wild-type mice, suggesting a large contribution of the forebrain to 0-trace conditioning in this type of mutant mice. In the present study, we investigated the role of N-methyl-D-aspartate receptors in 0-trace eyeblink conditioning in glutamate receptor subunit delta2 null mutant mice. Mice were injected intraperitoneally with the noncompetitive N-methyl-d-aspartate receptor antagonist (+)MK-801 (0.1mg/kg) or saline, and conditioned with 350-ms tone conditioned stimulus followed by 100-ms periorbital shock unconditioned stimulus. Glutamate receptor subunit delta2 null mutant mice that received (+)MK-801 injection exhibited a severe impairment in acquisition of the conditioned response, compared with the saline-injected glutamate receptor subunit delta2 null mutant mice. In contrast, wild-type mice were not impaired in acquisition of 0-trace conditioned response by (+)MK-801 injection. After the injection solution was changed from (+)MK-801 to saline, glutamate receptor subunit delta2 null mutant mice showed a rapid and partial recovery of performance of the conditioned response. On the other hand, when the injection solution was changed from saline to (+)MK-801, glutamate receptor subunit delta2 null mutant mice showed a marked impairment in expression of the pre-acquired conditioned response, whereas impairment of the expression was small in wild-type mice. Injection of (+)MK-801 had no significant effects on spontaneous eyeblink frequency or startle eyeblink frequency to the tone conditioned stimulus in either glutamate receptor subunit delta2 null mutant mice or wild-type mice. These results suggest that N-methyl-D-aspartate receptors play critical roles both in acquisition and expression of the conditioned response in 0-trace eyeblink conditioning in glutamate receptor subunit delta2 null mutant mice.

Variable penetrance of a familial progressive necrotising encephalopathy due to a novel tRNA(Ile) homoplasmic mutation in the mitochondrial genome.

INTRODUCTION: We present a family comprising a clinically normal mother and two daughters, each with severe encephalopathy with onset in late childhood. A third daughter had died previously of an earlier onset but neuropathologically similar disease. METHODS: Sequence analysis of the entire mtDNA was carried out in muscle, fibroblasts, and lymphocytes of the affected daughters and unaffected mother. Biochemical analysis of individual respiratory chain enzymes was performed on the same tissues, and on several transmitochondrial cybrid clones containing the nucleus of a 143B.206 osteosarcoma cell line and the mutant mtDNA. RESULTS: Genetic analyses revealed in both daughters and mother the presence of a novel mutation in the tRNA(Ile) gene of mtDNA, which was homoplasmic in fibroblasts, lymphocytes, and skeletal muscle of the two patients. It was also homoplasmic in fibroblast and skeletal muscle samples of the mother, and approximately 97% heteroplasmic in her lymphocytes. Combined defects of complexes I and IV of the mitochondrial respiratory chain were found not only in fibroblasts of the two probands, but surprisingly also in those of their clinically unaffected mother. The respiratory chain defect segregated in transmitochondrial cybrids containing the nucleus of a 143B.206 osteosarcoma cell line and the mutant mtDNA, indicating that the latter was responsible for the biochemical phenotype. DISCUSSION: Our results support the concept that homoplasmic mutations in tRNA genes can be responsible for mitochondrial disorders characterised by extremely variable penetrance. Albeit still unexplained, this phenomenon has important consequences in the nosological characterisation, clinical management, and genetic counselling of mitochondrial disorders.

Increase of Clostridium perfringens in association with Eimeria in haemorrhagic enteritis in Japanese beef cattle.

A coprological survey with detailed clinical observation of naturally occurring haemorrhagic enteritis (HE) cases was conducted to understand the pathophysiology of HE by clarifying the infection status of Eimeria and enteropathogenic bacteria in cattle. Faecal samples from 55 cases of HE and 26 clinically normal animals were collected, and a quantitative examination of Eimeria and potential enteropathogenic bacteria was performed. The number of Eimeria species oocysts per gram of faeces (OPG) exceeded 10,000 in 69.1 per cent of HE cases with a maximum of 1,452,500 OPG and Eimeria zuernii was found to be overwhelmingly dominant. A significant increase in faecal coliform count was observed in HE cases compared with clinically normal animals. Among the animals shedding >10,000 OPG, 42.9 per cent showed a remarkable increase in Clostridium perfringens abundance (>104 CFU/g) in the faeces. In the cases with C. perfringens detected, its abundance was positively correlated with Eimeria OPG and high C. perfringens abundance was always accompanied by high Eimeria OPG. E. zuernii is likely to play a crucial role in massive multiplication of C. perfringens in HE in cattle.