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.

An analysis of factors affecting production performance in broiler flocks on Japanese commercial farms.

1. The present study determined descriptive values of the main production measurements of flocks and assessed the relationship between these measurements and related management factors in Japanese commercial broiler farms. 2. The data set included 5060 flock records from 183 farms. The production index was calculated as follows: liveability × average daily gain/feed conversion ratio × 10. Management factors included in the analysis were broiler breeder age, the time interval between successive flocks, the season of placement and stocking density. 3. The mean (±SD) production index was 283.9 ± 28.83. Management factors significantly associated with a decreased production index were low broiler breeder age, flocks placed in summer and high stocking density (P < 0.05). 4. In regard to an interaction for the production index, flocks with high stocking density had a lower production index than those with low stocking density in flocks with a low broiler breeder age (P < 0.05). In summer, flocks with a short time interval between successive flocks had a lower production index than those with an intermediate or long time interval (P < 0.05). 5. The present study identified factors related to flock performance. The knowledge obtained from this analysis will contribute to improve flock performance by optimising management.

Bovine Endocarditis Associated with Mycoplasma bovis.

Mycoplasma bovis is a microorganism associated with pneumonia, mastitis, arthritis and otitis media of cattle; however, there are no reports of this organism causing bovine endocarditis. Five adult cattle with endocarditis characterized by caseated lesions (diameter 5-12 cm) of the endocardial surface of the left atrium, but without lesions in heart valves or affecting the right side of the heart, were identified in slaughterhouses in Japan. M. bovis was successfully isolated from the lesions and M. bovis antigen was detected immunohistochemically within the lesions. The results suggest that the lesions may have been associated with M. bovis alone. To our knowledge, this is the first demonstration of bovine endocarditis associated with M. bovis.

Temporal distribution and genetic fingerprinting of Salmonella in broiler flocks from southern Japan.

During the 1998 to 2003 period, cecal contents of 4,024 broiler chickens from 252 flocks raised in 63 holdings were examined for Salmonella. The aims were to establish the actual status of the infection, its temporal distribution, prevalent serotype, and common genotype among broiler flocks brought at the slaughterhouse. Collected samples were preenriched in Hajna tetrathionate broth, and after 24 h of incubation, 10 microL of the broth was streaked on selective Rambach agar plate. Suspected scarlet color colonies of Salmonella were cloned on nutrient agar, confirmed through biochemical tests and sero-typed using O and H antigens. Pulsed field gel electrophoresis technique generated DNA fragments banding patterns and established their clonal relatedness. Salmonella was isolated from 563 (14%) samples in 179 (71%) flocks. The flock situation varied from Salmonella-negative holdings (n = 9), positive-flocks from persistently infected holdings (n = 21), and holdings (n = 19) that showed fluctuations with alternating negative and positive flocks for variable time periods. Fourteen holdings (negative, n = 5 and positive, n = 9) were sampled once throughout the study period. Seasonality component was not observed, and salmonellae were found colonizing broiler ceca in warm and cold months. Predominant serovar was Salmonella Infantis (93.3%; n = 525). Macrorestriction fingerprints of Salmonella Infantis using XbaI presumed the isolates to be derived from a common parent. Enhanced discrimination by BlnI digestion produced 3 banding patterns that were closely related genetically and hence epidemiologically related. Such epidemiological information may enable producers to formulate effective control action plan tailored for individual holdings with special emphasis on biosecurity, hygiene, and pest control.

Appearance of US-like porcine epidemic diarrhoea virus (PEDV) strains before US outbreaks and genetic heterogeneity of PEDVs collected in Northern Vietnam during 2012-2015.

Porcine epidemic diarrhoea virus (PEDV) is the aetiologic agent of porcine epidemic diarrhoea (PED), a highly contagious enteric disease that is threatening the swine industry globally. Since PED was first reported in Southern Vietnam in 2009, the disease has spread throughout the country and caused substantial economic losses. To identify PEDVs responsible for the recent outbreaks, the full-length spike (S) gene of 25 field PEDV strains collected from seven northern provinces of Vietnam was sequenced and analysed. The sequence analysis revealed that the S genes of Vietnamese PEDVs were heterogeneous and classified into four genotypes, namely North America and Asian non-S INDEL, Asian non-S INDEL, new S INDEL and classical S INDEL. This study reported the pre-existence of US-like PEDV strains in Vietnam. Thirteen Vietnamese variants had a truncated S protein that was 261 amino acids shorter than the normal protein. We also detected one novel variant with an 8-amino acid insertion located in the receptor-binding region for porcine aminopeptidase N. Compared to the commercial vaccine strains, the emerging Vietnamese strains were genetically distant and had various amino acid differences in epitope regions and N-glycosylation sites in the S protein. The development of novel vaccines based on the emerging Vietnamese strains may be contributive to the control of the current PED outbreaks.

To what extent do human-altered landscapes retain population connectivity? Historical changes in gene flow of wetland fish Pungitius pungitius.

Understanding how human-altered landscapes affect population connectivity is valuable for conservation planning. Natural connectivity among wetlands, which is maintained by floods, is disappearing owing to farmland expansion. Using genetic data, we assessed historical changes in the population connectivity of the ninespine stickleback within a human-altered wetland system. We predicted that: (i) the contemporary gene flow maintained by the artificial watercourse network may be restricted to a smaller spatial scale compared with the gene flow preceding alteration, and (ii) the contemporary gene flow is dominated by the downstream direction owing to the construction of low-head barriers. We evaluated the potential source population in both timescales. Seventeen studied populations were grouped into four genetically different clusters, and we estimated the migration rates among these clusters. Contemporary migration was restricted to between neighbouring clusters, although a directional change was not detected. Furthermore, we consistently found the same potential source cluster, from past to present, characterized by large amounts of remnant habitats connected by artificial watercourses. These findings highlight that: (i) artificial connectivity can sustain the short-distance connectivity of the ninespine stickleback, which contributes to maintaining the potential source populations; however, (ii) population connectivity throughout the landscape has been prevented by agricultural developments.

Radiopacity and physical properties of titanium-polymethacrylate composite.

Titanium-polymethacrylate composite was prepared as an x-ray-opaque filler for composite core materials. The base monomers of the composite were 2,2-bis [4-(3-methacryloyloxy-2-hydroxypropoxy) phenyl] propane (Bis-GMA) and triethyleneglycol dimethacrylate (TEGDMA). Pure titanium powder was used as a radiopaque element. Two functional methacrylates--isopropyl dimethacryloyl isostearoyl titanate (KR-7) and 4-methacryloyloxyethyl trimellitate anhydride (4-META)--were adopted as couplers of titanium powder. The radiopacity of composites was determined in terms of their equivalent thickness of aluminum per unit thickness of material. Compressive and transverse strengths of the composites were measured by use of coupled and untreated titanium fillers. The composites that contained 70% and 80% titanium by weight showed more radiopacity than enamel and less than amalgam. Radiopacity was controlled by the content of titanium filler in the composite. The decrease in compressive and transverse strengths of the composites was 0.7 to 15.9% and 43.7 to 52.9%, respectively, after one year's immersion in water. The decrease in transverse strength was remarkable; however, this deterioration was diminished by use of both KR-7 and 4-META as couplers for titanium. Thus, prepared titanium-polymethacrylate composite may be used as a radiopaque composite filler for self-curing composite core materials.

Isolation of porcine epidemic diarrhea virus in porcine cell cultures and experimental infection of pigs of different ages.

This paper describes the isolation of porcine epidemic diarrhea (PED) virus in Vero and porcine cell cultures, and the influence of age on disease in experimental infection. PED virus was isolated from the small intestine of piglets inoculated with PED samples and cultured in Vero, porcine bladder and kidney cells propagated in collagen-coated tissue culture plates in maintenance medium (MM) containing trypsin. In porcine bladder and kidney cell cultures inoculated with isolated PED virus, cytopathic effects (CPE) including cell fusion were detected. Specific brilliant fluorescence was observed in the cytoplasm of these cells. Two- and 7-day old, and 2-, 4-, 8- and 12-week old specific pathogen-free (SPF) pigs were orally inoculated with PED virus isolated from an outbreak. All 2- and 7-day old pigs inoculated developed severe watery diarrhea from post-inoculation day (PID) 1 and died between PID 3 and 4. Although three of five 2-week old pigs developed diarrhea on PID 1-4, they eventually recovered. In the 4-week old group, three of five pigs had mild diarrhea for 1-2 days. None of the 8- and 12-week old pigs showed any clinical signs. Antibodies against PED virus were detected in all surviving pigs by virus neutralization (VN) test and immunofluorescence assay (IFA). Therefore, there is an age-dependent resistance to pathogenic PED virus infection in pigs.

Comparative studies of the persistence of animal mycoplasmas under different environmental conditions.

A comparison of the persistence of mycoplasmas in animals was carried out. When inoculated into liquid media, strains of Mycoplasma bovis, M. arginini, Acholeplasma laidlawii, and A. axanthum persisted for 59-185 days post-inoculation. The survival periods were not significantly influenced by temperature (4, 30, 37 degrees C, and room temperature). The survival periods for M. bovigenitalium, M. gallisepticum, M. bovirhinis, and M. gateae ranged from <7 to 185 days depending on medium components and temperature. Further, it was determined that strains of M. bovigenitalium, M. bovis, M. bovirhinis, M. arginini, and A. laidlawii persisted in a dry paper disc for at most 28, 126, 154, 56 and over >168 days at 4 degrees C, respectively. At 4 degrees C, strains of M. gallisepticum, M. columborale, M. edwardii, M. felis, and M. gateae survived for at most 28, 21, 42, 28, 28 and 70 days, respectively. At 30 degrees C, strains of M. bovis, M. bovirhinis, M. arginini, A. laidlawii, and M. gallisepticum persisted for at most 28, 84, 56, >168 and 14 days, respectively, but strains of M. gallisepticum, M. columborale, M. edwardii, M. felis, M. gateae, and U. diversum did not survive for more than 14 days. In an outdoor environment, strains of M. bovirhinis and A. laidlawii survived for at most 28 and 14 days, respectively. Finally, it was found that 14 isolates of M. gallisepticum persisted for periods similar to those of the reference strains. The results under dry conditions at a variety of temperatures presented contribute to understanding the epizootiology of mycoplasmal infections in the field.

Detection of Akabane viral antigens in spontaneous lymphohistiocytic encephalomyelitis in cattle.

A 5-month-old Japanese black bull calf and twenty-seven 1-27-day-old calves exhibiting neurological signs between August and October 1998 were examined. The bull calf exhibited rapid breathing, fever, hypersensitivity, and ataxia and was euthanized 4 days after the onset of symptoms. The 27 calves primarily exhibited ataxia, and 15 had arthrogryposis. Histological examination of the bull calf revealed perivascular infiltraction by mononuclear cells, diffuse to multifocal gliosis, and neuronal necrosis in the brain and spinal cord. Multiple malacic foci were found in the midbrain in 5 cases. In contrast, in the 15 calves necropsied in October, there were fewer inflammatory changes, but there was neuronal cell loss in the ventral horn and a decrease in myelinated axons in the lateral and ventral funiculi. Immunohistochemical examination using a rabbit antiserum against Akabane virus strain OBE-1 revealed a large amount of viral antigen in the degenerating neurons and glial cells of the bull calf, mainly in the spinal gray matter. Small amounts of viral antigen in swollen axons and a few glial cells were found in 5 of 27 calves. Thirteen of the 27 calves had high neutralization antibody titers against the Akabane virus, whereas there was no significant antibody titer in most of the calves necropsied during August. The present study revealed that viral antigen detection was very useful for the diagnosis of Akabane diseases in the 5-month-old bull calf that was suspected to be infected postnatally, while it had limited usefulness in the other young calves.

Sarcoid granuloma-like lesions in the lungs of broiler chicks.

Sarcoid granuloma-like lesions were observed in the lungs of two broiler chicks showing depressed growth. Multinucleated giant cells in the lesions often contained foreign bodies comprising amorphous and laminated material. These were morphologically similar to Schaumann bodies found in human sarcoidosis. This is the first report of sarcoid granuloma-like lesions and Schaumann body-like material in chickens.

Natural infection with attaching and effacing Escherichia coli (O 103:H-) in chicks.

The existence of natural infection with attaching and effacing Escherichia coli (AEEC) in chicks was reported. Numerous Gram-negative bacilli were attached to the enterocyte in association with lesions characterized on a wavy appearance of the mucosal surface in the intestines of six chicks. Immunohistochemically, these bacteria reacted positively with antiserum to Escherichia coli (O 103). By electron microscopy, numerous colibacilli were seen to be closely attached to the surface membranes of the enterocytes. In regions of bacterial attachment, almost all cell microvilli were effaced, and some of the remaining ones were elongated and/or disoriented. Part of the cell membrane formed a cup invagination and pedestal-like protrusion associated with the attached bacteria. A concentration of electron-dense material was seen beneath the adherent organisms. Bacteriologically, numerous E. coli (O 103:H-) were isolated from the jejunal contents of two chicks and the E. coli did not produce verotoxin or enterotoxin. The characteristic lesions could also be induced in the cecal mucosa of young chicks experimentally inoculated with the isolated E. coli. This is the first report of natural infection in chicks with AEEC.

A chick model for the study of "attaching and effacing Escherichia coli" infection.

Young chicks were experimentally infected with 6 strains of AEEC isolated from calves, pigs, chicks, and humans. AEEC colonized the cecum of chicks and induced the AE lesions on the mucosal surface. In the early stages of the AE lesions, AEEC attached to the enterocyte were enfolded with the microvilli. In the advanced stages, microvilli and cytoskeletons of the enterocytes were disrupted, and cytoplasmic cups and pedestal-like protrusions were formed on the cell surface. The AE lesions interconnected with the adjacent lesions, and it formed the network on the mucosal surface. Leukocytes infiltrated in the mucosa associated with AE lesions, and lymphatic nodules also developed. The results of these studies support the conclusion that chicks can be used as a model for the study of the lesions caused by AEEC.

An immunohistochemical investigation of porcine epidemic diarrhoea.

A sudden outbreak of epidemic diarrhoea of piglets occurred in Japan, the principal features being watery diarrhoea, dehydration and high mortality in newborn animals. The microscopical lesions were villous atrophy in the small intestine, the villous enterocytes being vacuolated and cuboidal in shape. The villus-crypt ratio was severely reduced, varying from 1:1 to 3:1. Transmission electron microscopy showed numerous coronaviruses within the cytoplasm of enterocytes and among microvilli. Specific antigens of porcine epidemic diarrhoea (PED) virus were detected in the cytoplasm of enterocytes by the streptavidin-biotin (SAB) technique. Infected cells, which were most abundant in the villous epithelia of the jejunum and ileum, were present in small numbers in the large intestine, the crypt epithelia, the lamina propria and Peyer's patches. The study suggests that the SAB technique is useful for the diagnosis of PED.

A novel developmental process of intestinal epithelial lesions in a calf infected with attaching and effacing Escherichia coli.

A comparative study on the adhesion of attaching and effacing Escherichia coli (AEEC) to the enterocytes between the colon of a calf and the jejunum of a piglet showed differences in the developmental process of attaching and effacing (AE) lesions. In the calf, pedestals consisted of fused microvilli, while in the piglet they developed from the apical epithelial cell membranes after effacing microvilli. Microvilli adjacent to the AEEC attachment site were atrophic in the calf, whereas they were elongated in the piglet. The production of AE lesions in the calf may be indicative of a novel developmental process with AEEC infection.

A new adherent form of an attaching and effacing Escherichia coli (eaeA+, bfp-) to the intestinal epithelial cells of chicks.

The adherent site of "attaching and effacing Escherichia coli" (AEEC; O103: H-, SK-1 strain) on the intestinal epithelial cells of chicks infected naturally and experimentally was ultrastructurally investigated. The eaeA gene was detected by polymerase chain reaction in the SK-1 strain of E. coli isolated from the intestinal content of a chick infected naturally, however, the bundle-forming pilus (bfp) gene could not be detected. The SK-1 strain (bfp-) of AEEC could attach to the intestinal epithelial cell and induce attaching-effacing lesions in the intestine of chicks. Transmission electron microscopy revealed numerous pilus-like microfilaments in the space between colibacilli and the membranes of the intestinal epithelial cells. The present study suggests that SK-1 strain (eaeA+, bfp-) may attach closely to the intestinal epithelial cells by a novel adhesion different from bfp.

Intestinal spirochetosis in wild sika deer (Cervus nippon yesoensis) infected with Brachyspira species.

Seven adult free-ranging sika deer (Cervus nippon yesoensis) were examined by histology, immunohistochemistry and electron microscopy for intestinal spirochetal infection. Histologically epithelial and goblet cell hyperplasia and edema of the lamina propria mucosa with macrophage and lymphocyte infiltration were observed in the cecum and colon in 6 of the 7 deer. Numerous argyrophilic spirochetes were present in the crypts and some had invaded epithelial and goblet cells and caused degeneration. Immunohistochemically the organisms stained positively with polyclonal antisera against Brachyspira (Serpulina) hyodysenteriae and B. pilosicoli. Ultrastructurally they were 6-14 microm long, 0.2-0.3 microm wide and had 4-6 coils and 13 axial filaments per cell; such features were closely similar to those in the Brachyspira species. These results showed that the spirochetes were capable of inducing enteritis in deer and this intestinal spirochete infection might already be prevalent among wild sika deer in Japan. There is a possibility that this spirochetal colitis is a new syndrome in sika deer and that the same and/or similar spirochetes have infected ruminants, including sika deer and cattle.

Radiopacity of dental cements.

The radiopacity of zinc phosphate, polycarboxylate and glass ionomer dental luting materials was determined and the values were compared with those of tooth substrates. The radiographs of the 2 mm thick specimens were taken together with tooth slices and pure aluminum step wedges. The radiographic density of the specimens was measured using a densitometer and expressed in terms of the equivalent thickness of aluminum per unit thickness of material. All the zinc phosphate and polycarboxylate cements showed radiopacity values greater than that of enamel (3.5), while one glass ionomer material showed a radiopacity value lower than that of dentin (2.0). A radiopacity value greater than 3.5 may be desirable for the detection of luting materials or for their distinction from decayed tooth structure on the dental radiograph.

[Estimation of probiotics by infection model of infant rabbit with enterohemorrhagic Escherichia coli O157:H7].

We examined the preventative effect against enterohemorrhagic Escherichia coli O157:H7 (EHEC) infection in rabbits on administration of probiotics contained in Streptococcus faecalis, Clostridium butyricum and Bacillus mesentericus. The probiotics were administered to 5 days old Japanese white rabbits throughout the experiment. Inoculation of 10(6) colony forming units (CFUs) of EHEC per rabbit was tried 8 days after birth. Three days after EHEC inoculation, in the control group (not administered probiotics), diarrhea was observed in approximately 80% of the rabbits, on the other hand, in the group administered probiotics, rabbits showed diarrhea in 15%. In necropsy, the number of EHEC in the contents of the cecum and the number of attaching and effacing (AE) lesions were fewer in the probiotics group than in the control group. These results suggest that infant rabbits are useful as a colitis model for EHEC, and the probiotics are effective for the prevention of the growth of EHEC in the intestine and for diarrhea.