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Acta Veterinaria Hungarica
Authors:
Zsolt Becskei
,
Sanja Aleksić-Kovačević
,
Miklós Rusvai
,
Gyula Balka
,
Csaba Jakab
,
Tamaš Petrović
, and
Milijana Knežević

The lymphatic organs of 50 pigs from a total of eight farms located at different sites in the epizootiological region of North Bačka County were studied to obtain data on the prevalence of circoviral infections in Serbia. All of the pigs examined had clinical signs suggestive of postweaning multisystemic wasting syndrome (PMWS). All pigs underwent necropsy and tissue samples were taken for histopathological, immunohistochemical (IHC) and PCR analysis. The presence of porcine circovirus 2 (PCV2) was established by PCR analysis in the organs of the pigs tested. The most frequent histopathological lesions of lymphoid tissue linked with the presence of positive immunostaining for PCV2 Cap antigen confirmed the existence of PMWS in all farms tested in North Bačka County. Using PCR, histopathological and IHC techniques, the presence of PMWS was proved in the Republic of Serbia. During necropsy, generalised enlargement of the lymph nodes was evident. The most common histopathological finding was lymphocyte depletion in the follicular and perifollicular areas of lymph nodes. Infiltration by macrophages was also recorded. By IHC analysis, the cytoplasm of macrophages was shown to contain a large amount of the ORF2-coded Cap antigen of PCV2. Lymphocyte depletion and large numbers of macrophages were recorded in the tonsils, spleen, intestinal lymphatic tissue, Peyer’s patches and ileocaecal valve. The presence of typical granulomatous lesions with multinuclear giant cells (MGCs) was also recorded in the lymphatic tissue. Cap antigen was shown to be present in macrophages and less often in lymphocytes.

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Acta Veterinaria Hungarica
Authors:
Ching-Yang Cheng
,
Jing-Ruei Chi
,
Sin-Rong Lin
,
Chi-Chiang Chou
, and
Chin-Cheng Huang

The objective of this study was to use a 5′-nuclease (TaqMan) real-time PCR method with primers and probe specific to the spaQ gene as a rapid approach to quantitatively determine Salmonella Typhimurium. The result showed that the correlation coefficient between real-time PCR estimates and bovine serum albumin (BSA) plate counts of S . Typhimurium was 0.99, independently of 10 5 -fold numbers of bystander Escherichia coli O157:H7 or total viable counts. The sensitivity of the real-time quantitative PCR assay was 10 CFU/mL for pure S . Typhimurium culture without enrichment. A known number of S . Typhimurium target cells were inoculated to dumpling fillings and chicken nuggets and DNA was extracted for real-time PCR analysis. The sensitivity was 60 CFU/g for S . Typhimurium inoculated to the food samples without any preceding procedure of enrichment. The duration of the entire experiment from DNA isolation and purification to PCR amplification was less than 12 h. This study demonstrated that realtime PCR is a rapid and reliable technique for quantifying S . Typhimurium possessing the spaQ gene in pure culture and in meat products.

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Up to now, only a single adenovirus (AdV) isolate seemingly specific for pigeons, hence named pigeon AdV-1 (PiAdV-1), has been characterised at DNA sequence level. In the present work, the prevalence and diversity of AdVs occurring in domestic pigeon were examined by a survey performed on randomly collected samples using a very efficient, consensus nested PCR targeting the viral DNA polymerase gene. The newly detected viruses were characterised by sequencing and phylogeny analysis. Amplification of additional genome fragments was attempted by the use of several other PCR methods aiming at the hexon gene. During a 4-year survey, samples from dead or live, healthy pigeons originating from 27 lofts were examined in Hungary. Almost 50% of the samples (48 out of 97) proved to be positive for AdV. Sequence analysis revealed the presence of four hitherto unknown pigeon AdV types. PiAdV-1 was also identified in one sample. Two novel viruses named PiAdV-2 and -3 were found to belong to the genus Aviadenovirus, and two other novel types (PiAdV-4 and -5) to the genus Siadenovirus. This is the first report on the occurrence of siadenoviruses in birds belonging to the order Columbiformes. Approximately two-thirds of the PiAdV-2 genome was sequenced and analysed.

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Acta Microbiologica et Immunologica Hungarica
Authors:
Mojtaba Moosavian
,
Sakineh Seyed-Mohammadi
,
Ahmad Farajzadeh Sheikh
,
Saeed Khoshnood
,
Aram Asarehzadegan Dezfuli
,
Morteza Saki
,
Gholamreza Ghaderian
,
Fatemeh Shahi
,
Mahtab Abdi
, and
Fariba Abbasi

Shigella spp. are a major cause of bacillary dysentery, particularly among children in developing countries such as Iran. This study aimed to investigate the presence of two important Shigella enterotoxins (ShET-1 and ShET-2), encoded by the set and sen genes, respectively, by polymerase chain reaction (PCR) assay among Shigella species isolated from children affected by shigellosis in Ahvaz, southwest of Iran. In this cross-sectional study, from June 2016 to April 2017, altogether 117 Shigella isolates were collected from fecal specimens of children aged <15 years with diarrhea in Ahvaz, southwest Iran. All isolates were identified by standard microbiological and molecular methods. The presence of enterotoxin genes was determined by PCR. The most prevalent isolate was Shigella flexneri (47.9%), followed by Shigella sonnei (41%) and Shigella boydii (11.1%), respectively. Shigella dysenteriae was not detected in patients’ samples. The frequencies of set1A, set1B, and sen genes were 5.1% (6/117), 15.4% (18/117), and 76.9% (90/117), respectively. This study provides initial background on the prevalence and distribution of the Shigella enterotoxin genes in Shigella isolates in southwest of Iran. In addition, this study revealed a high prevalence of sen enterotoxin gene in Shigella species.

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A total of 146 Pasteurella multocida strains isolated from swine in Hungary in the last 20 years were examined. Biochemical characterisation and PCR-based techniques were used to determine species, subspecies, biovar, capsule type and presence of the toxA gene. Eighty-seven percent of the isolates belonged to P. multocida ssp. multocida , and 98% of these had biovar 3 or were trehalose-or lactose-fermenting or ornithine decarboxylase negative variants of that. Ten percent of the strains were P. multocida ssp. septica , and within this group 80% of the strains showed sorbitol-negative biovars (5, 6 and 7). The rest of the strains (20%) were lactose positive. Only 3% of the porcine isolates were P. multocida ssp. gallicida and 3 out of the 4 strains belonged to the dulcitol-fermenting biovar 8. Using a capsule-specific multiplex PCR, 60% of the strains belonged to capsule type D, 38% to capsule type A, and only 1 isolate had capsule type F. In contrast with data published in the literature, only 3% of capsule type D isolates carried the toxA gene, while this ratio was 41% for the type A strains. A remarkable regional distribution of toxA gene positive strains was observed. All but two isolates were found in swine herds located in the Transdanubian region, separated from other parts of Hungary by the river Danube.

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A real-time PCR assay was evaluated for the rapid detection (10 h) of Salmonella in meats using molecular beacon probes available as a commercial kit (iQ-Check, Bio-Rad laboratories). Raw (chicken, pork) and ready-to-eat (RTE) meats were artificially contaminated with Salmonella enterica serovar Typhimurium at the estimated level of 2 to 4 cells per 25 g. After 8 h of pre-enrichment in buffered peptone water, a molecular beacon-based PCR assay was performed to detect contamination in raw and RTE meats. The sensitivity and accuracy of the assay were compared with the conventional USDA microbiological procedure. Comparative evaluation of the USDA procedure with the rapid PCR assay for meat samples (n = 63) revealed 1 false negative pork sample with the PCR assay. All uninoculated controls (n = 34) but one sample were negative by both the 10-h PCR assay and the USDA procedure. Developing rapid pathogen detection methods with shorter pre-enrichment times (8-h) and real-time data monitoring capabilities will benefit the industry in preventing recall of contaminated meats by stopping the contaminated products from being introduced into the marketplace.

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Simple real-time PCR assay with one set of primer and probe for rapid, sensitive qualitative and quantitative detection of Entamoeba histolytica has been used. Consensus sequences were used to amplify a species-specific region of the 16S rRNA gene, and fluorescence resonance energy transfer hybridization probes were used for detection in a LightCycler platform (Roche). The anchor probe sequence was designed to be a perfect match for the 16S rRNA gene of Entamoeba species, while the acceptor probe sequence was designed for Entamoeba histolytica, which allowed differentiation. The performed characteristics of the real-time PCR assay were compared with ELISA antigen and microscopical detection from 77 samples of individuals with suspected clinical diagnosis of imported E. histolytica infection. Stool and liver abscess pus samples were examined with analytical sensitivity of 5 parasites per PCR reaction. The melting curve means Tms (standard deviation) in clinical isolates were 54°C. The real-time assay was 100% sensitive and specific for differentiation of Entamoeba histolytica, compared with conventional ELISA or microscopy. This real-time PCR assay with melting curve analysis is rapid, and specific for the detection and differentiation of Entamoeba histolytica. The suitability for routine use of this assay in clinical diagnostic laboratories is discussed.

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Sixty-one avian strains of Pasteurella multocida were characterised and compared by biochemical tests, capsular PCR typing and ERIC-PCR. The strains were recovered from various avian species (goose, duck, Muscovy duck, turkey, chicken and pheasant) and represented different geographic locations in Hungary. Forty-two strains (69%) were identified as P. multocida subsp. multocida and 19 strains (31%) as P. multocida subsp. septica . The strains were grouped into 7 different biovars (1, 2, 3, 4, 5, 6 and 7). The most prevalent biovars were 1 (25%), 3 (21%) and 6 (21%). Most of the duck isolates (90%) belonged to biovar 1 or 6. The most frequent capsular type was A (93.5%). Type F represented only a small number (6.5%) of the strains. Other capsular types were not identified. From the 61 isolates 24 different fingerprint patterns were generated by ERIC-PCR assay. Based on cluster analysis the strains could be grouped into four larger and four mini-clusters that showed considerable correlation with the geographical origin and the host species. The results indicate that ERIC-PCR may be a suitable technique for studying the host adaptation of P. multocida and the epidemiology of fowl cholera.

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Water is necessary to life so when supplied as drinking water to consumers, a satisfactory quality must be maintained. In Egypt, infectious intestinal diseases are the major cause of hospitalization in almost all regions. The purpose of this study was to evaluate the microbiological quality of treated and untreated water samples from urban and rural communities. Thirty-five samples of treated (chlorinated) water from taps, 25 samples of bottled water and 15 samples of hand pump (untreated) water collected from different cities alongside the River Nile during the winter of 2007 were bacteriologically tested for safety as drinking water. This study indicated good quality of tap water and bottled water. The untreated water samples (hand pumps) were, however, slightly contaminated by faecal coliforms, faecal enterococci, Clostridium perfringens, Salmonella and Shigella . Consequently, the consumers in the villages receiving water through hand pumps are often exposed to the risk of water-borne diseases due to inadequate treatment of the raw water. Therefore, there are guidelines necessary to protect groundwater quality. Moreover, PCR-amplified by some functional gene fragments such as dctA, dcuB, frdA, dcuS and dcuR genes of the E. coli was adapted for use as a non-cultivation-based molecular approach for detection of E. coli populations from water samples without the need for pure and identified cultures.

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Pasteurella multocida, Mannheimia haemolytica and Trueperella pyogenes are three bacterial pathogens closely associated with the bovine respiratory disease complex (BRDC). In the current study, a multiplex PCR for the simultaneous detection of these three bacteria in cultures was established. After serial optimisation, the detection limit of the method for the genomic DNA of the three bacteria was 40 pg/μl. The method could detect the genomic DNA of these three bacteria but not the genomic DNA of seven other bacterial strains. Together with the bacterial enrichment technology, the multiplex PCR could be used for detecting the three bacteria in animal tissues. This method might be valuable for speeding up laboratory diagnosis and directing the treatment of BRDC to these three bacterial pathogens.

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