Burhan Çetinkaya

Identification of Corynebacterium pseudotuberculosis isolates from sheep and goats by PCR

The present study was carried out to estimate the prevalence of caseous lymphadenitis (CL) in sheep and goats slaughtered at the local abattoir in Elazig province located in the east of Turkey, between September and December 2000. A total of 2046 sheep and 2262 goat carcasses were examined during the study period and 118 abscessed lymph nodes, 89 from sheep and 29 from goats, were collected. Corynebacterium spp. strains were isolated from 81.4% of the abscesses, giving an overall prevalence of 2.2%. The prevalence was 3.5 and 1.1% in sheep and goats, respectively. PCR on DNA extracted from 96 suspicious isolates, using a pair of Corynebacterium pseudotuberculosis-specific primers, was positive for 93. Although cross-reaction with C. ulcerans, a human/bovine species, was observed, the PCR assay used in this study may successfully be applied for the diagnosis of CL in goats and sheep as an alternative to conventional methods, owing to its advantages of specificity and speed.

Seroprevalence of coxiellosis in cattle, sheep and people in the east of Turkey.

Serum samples collected randomly from 416 cattle in 48 herds, and 411 sheep in 47 flocks, in eight different locations in the east of Turkey between June and December 1998, were examined by indirect fluorescent antibody test (IFAT) to determine the prevalence of Q fever. The age, sex, breed, tick control and abortion history of the animals were also recorded. In addition, 102 serum samples were collected from apparently healthy people who were at risk of contracting the disease, such as farmers, veterinarians, abattoir and laboratory workers, and veterinary students. Seropositivity was observed in 5.8 per cent (24/416) of the cattle in 17 (35.4 per cent) of the herds and in 10.5 per cent (43/411) of the sheep in 21 (44.7 per cent) of the flocks. Eight of the 102 people were seropositive, with the highest prevalence (12.0 per cent) in farmers and abattoir workers. All the seropositive farmers had seropositive animals. None of the laboratory workers or veterinary students was seropositive.

Genotyping of broiler-originated Campylobacter jejuni and Campylobacter coli isolates using fla typing and random amplified polymorphic DNA methods

Liver and intestine samples taken from 200 broilers at 20 flocks were inoculated onto Preston Enrichment broth and agar for selective isolation of Campylobacter jejuni and Campylobacter coli. The isolates were identified by both conventional and polymerase chain reaction (PCR) methods. Campylobacter spp. were identified in 102 of 400 samples (200 liver and 200 intestine), 57 (14.25%) of which were identified as C. jejuni and 45 (11.25%) as C. coli. PCR-restriction fragment length polymorphism (RFLP) of the flagellin gene (flaA) and random amplified polymorphic DNA (RAPD) typing were used to describe the heterogeneity among amplified DNA products of C. jejuni and C. coli isolates. Flagellin gene analysis by RFLP of the isolates produced seven different band profiles. On the other hand, five distinct band profiles were obtained in the examination of the isolates with RAPD assay using a random primer (OPA-11). The results of this study demonstrated that a relatively low heterogeneity existed among C. jejuni and C. coli strains isolated from the commercial broiler flocks in eastern Turkey. In the comparison of both typing methods, fla typing provided more discrimination than the RAPD assay used.

Investigation of toxin genes by polymerase chain reaction in Staphylococcus aureus strains isolated from bovine mastitis in Turkey.

Staphylococcus aureus causes a number of diseases in humans and animals, and it is the most common etiological agent of contagious bovine mastitis. The agent produces several virulence factors such as coagulase (coa), clumping factor, protein A, exfoliative toxins, staphylococcal enterotoxins (SEs), and toxic shock syndrome toxin-1. The aim of the present study was to characterize coa-positive S. aureus strains (n = 92) isolated from bovine subclinical mastitis in Turkey by polymerase chain reaction (PCR) amplification of exfoliative toxin (eta and etb) and toxic shock syndrome toxin-1 (tsst) genes. In addition, a multiplex PCR was employed to investigate the presence of SE genes sea, seb, sec, sed, see, seg, seh, sej, and sei. By PCR amplification, while eta and etb were not detected, only three isolates (3.3%) were positive for tsst. Twenty-seven (29.3%) isolates harbored one or more SE genes, and sei was the most common pattern by multiplex PCR. None of the isolates harbored the genes encoding sea, see, and seh. The application of this multiplex PCR assay could enable more samples to be rapidly characterized for enterotoxin production of S. aureus isolates from milk for epidemiological studies.

Detection of Brucella species DNA in the stomach content of aborted sheep fetuses by PCR

B. (4eednkaya, DVM, PhD, H. Ongor, DVM, A. Muz, DVM, PhD, H. B. Ertas, DVM, PhD, FO Veteriner Fakiutesi, Mikrobiyoloji Anabilim Dali, 23119, Elazig, Turkey H. Kalender, DVM, PhD, Veteriner Kontrol ve Arastirma Enstitiisu, 23119, Elazig, Turkey H. M. Erdogan, DVM, PhD, University of Liverpool, Department of Veterinary Clinical Science and Animal Husbandry, Leahurst, Neston, South Wirral L64 7TE THE ultimate aim of veterinary and medical disciplines is to control and eradicate disease in populations in order to improve health and productivity. Rapid and accurate diagnosis is essential if this aim is to be achieved. Brucellosis is an economically important infectious disease of livestock worldwide, characterised by abortion and infertility. Infertility, abortion and mastitis caused by brucella are the major reasons for culling animals. Six species of brucella are currently recognised (Alton and others 1988). Brucella melitensis is responsible for the most ofthe losses attributable to brucellosis in sheep in Turkey. Studies carried out in different regions of Turkey have shown that B melitensis is responsible for approximately 20 per cent of abortion cases in sheep (Arda and others 1987, Karaman and others 1993, Muz and others 1999). Considered together with high prevalence figures, the economic losses due to brucellosis are high. The disease can also affect humans through the consumption of meat, milk and milk products from infected animals (Young 1983, Wallach and others 1994). The diagnosis of brucellosis is usually made by bacteriological and immunological tests. Although bacteriology is the most reliable technique, it is time consuming and the cultures need to be handled with care because of the zoonotic potential. Immunological tests such as the complement fixation (CF) test and ELISA have frequently been used, but they lack either sensitivity or specificity and give false positive results due to cross-reactions with other bacteria including Yersinia enterocolitica 0:9, Vibrio cholera, Campylobacter fetus, Bordetella bronchiseptica and Salmonella species (Corbel 1985,Alton and others 1988). Another drawback of these tests is the inability to distinguish vaccinated animals from infected animals. The development of the polymerase chain reaction (PCR) has facilitated studies of microorganisms that are either difficult or time-consuming to grow, such as Mycobacterium avium subspecies paratuberculosis (Collins and others 1993, (etinkaya and others 1996) or are impossible to culture, such asM leprae (Rafi and others 1995, Wichitwechkarn and others 1995). This technique has recently found application in brucellosis and genusand species-specific PCR assays have been used in its diagnosis. Herman and de Ridder (1992) reported a PCR using a pair of primers derived from the 16S rRNA sequence ofB abortus and found that these primers could detect all six species in this genus. This PCR did not cross-react with closely related bacteria including Agrobacterium tumefaciens with which B abortus has 85 per cent homology in the 16S rRNA sequence. In another study, a primer cocktail, con1 2 R A K

Investigation of virulence and cytolethal distending toxin genes in Campylobacter spp. isolated from sheep in Turkey.

The presence of virulence and cytolethal distending toxin (Cdt) genes was investigated in isolates of Campylobacter jejuni, C. coli, C. lanienae, and C. lari that originated from intestinal contents and gallbladders of clinically healthy sheep. These genes have important roles in the pathogenicity of campylobacters. A total of 363 Campylobacter isolates (221 C. jejuni, 135 C. coli, five C. lanienae, and two C. lari) were used in this study. The frequency of racR, dnaJ, ciaB, pldA, flaA, and cadF virulence genes in all the isolates were determined to be 34.4%, 30%, 24.8%, 30.9%, 95%, and 81.3%, respectively, while the virB11 virulence gene could not be detected in any isolates. CdtA, cdtB, and cdtC genes were detected in 54.5%, 55.9%, and 52.3% of the isolates, respectively. None of the virulence and toxin genes examined here were detected in a total of 19 Campylobacter isolates consisting of 10 C. jejuni and nine C. coli. This is the first study investigating the presence of virulence and toxin genes in a large number of Campylobacter species isolated from clinically healthy sheep by scanning a large area. In addition, this is the first report investigating the presence of virulence and toxin genes in sheep-originated C. lanienae and C. lari isolates.

Isolation of Mycoplasma bovis from broiler chickens in Turkey

Mycoplasma bovis normally affects cattle, in which it causes pneumonia in calves, mastitis, arthritis and other diseases. In the present article we report the isolation of this bovine pathogen from the tracheas of broiler chickens with no clinical signs. The most probable source of infection was the cattle herd sharing the farm with the chickens.

Detection of avian metapneumovirus subtypes in turkeys using RT-PCR.

This study investigated the prevalence of avian metapneumovirus (aMPV) and the detection of molecular subtypes of field strains of the virus using RT-PCR in clinically healthy turkeys and those showing signs of respiratory disease. In the RT-PCR examination of 624 tracheal tissue samples collected from a local turkey abattoir, 2.9 per cent (18/624) of samples tested positive. In the examination of tracheal swab samples collected from flocks with respiratory problems, 18 of 20 samples tested positive. When the results were assessed at flock level, aMPV infection was detected in only one of the 23 clinically healthy turkey flocks, whereas all four flocks with respiratory problems were infected. Molecular typing using primers specific to the attachment glycoprotein (G) gene showed that all 36 positive samples belonged to subtype B. Partial sequence analysis of DNA samples showed 95 per cent homology between the field types and the reference strain aMPV subtype B. Whereas clinically healthy turkeys had been vaccinated with a subtype A virus vaccine, the flocks with respiratory problems had been vaccinated with a subtype B virus vaccine. Despite four blind passages of RT-PCRpositive samples on Vero and chicken embryo fibroblast cells, no cytopathic effect was detected by microscopic examination.

In vitro activity of Brucella melitensis isolates to various antimicrobials in Turkey

Abstract Background: Brucellosis is a systemic infectious disease caused by Brucella bacteria. A successful treatment requires antibiotics that can penetrate into the cell at high concentrations. The aim of this study was to assess the biotype and in vitro activity of 80 Brucella isolates obtained from blood against various antimicrobials for human brucellosis in Turkey. Methods: Identification of the types of the species designated Brucella species was made using the polymerase chain reaction (PCR), with type-specific primers. Serotyping was performed using mono-specific A and M antisera. The minimum inhibitory concentrations (MICs) of antibiotics known to have good intracellular penetration (doxycycline, rifampicin, ofloxacin, levofloxacin, moxifloxacin, clarithromycin, and azithromycin) were determined by the agar dilution method. Results: All of the 80 Brucella isolates were determined to be Brucella melitensis: 75 B. melitensis biotype 3 (93.7%) and 5 B. melitensis biotype 1 (6.3%). Doxycycline was the most effective among the tested antibiotics against Brucella species (MIC50–MIC90, 0.25–0.5 μg/ml), and it was followed by levofloxacin (MIC50–MIC90, 0.5–1 μg/ml), moxifloxacin (MIC50–MIC90, 1–1 μg/ml), ofloxacin (MIC50–MIC90, 1–1 μg/ml), rifampicin (MIC50–MIC90, 2–4 μg/ml), azithromycin (MIC50–MIC90, 4–8 μg/ml), and clarithromycin (MIC50–MIC90, 8–32 μg/ml), respectively. Conclusions: The in vitro activity of doxycycline and rifampicin, which are used in the classic treatment of brucellosis, was found to be very good. Quinolones were found to have in vitro activity against Brucella isolates. Among the macrolides, azithromycin had a higher level of activity compared with clarithromycin. A combination of quinolones and azithromycin could be an alternative to doxycycline and rifampicin in the treatment of brucellosis.

Isolation and molecular characterization of Escherichia coli O157 from broiler and human samples.

There is a lack of information about the role of poultry, specifically chicken, in transmission of Escherichia coli (E. coli) O157 and subsequent human illnesses. This study was therefore aimed at investigating the presence of E. coli O157 and its virulence genes in various samples collected from broiler chickens and humans in Eastern Turkey by culture, immunomagnetic separation (IMS), and polymerase chain reaction (PCR). The genetic relationship between broiler and human isolates was also examined by pulsed-field gel electrophoresis (PFGE). In the PCR analysis of sorbitol-negative isolates, E. coli O157 was identified in 0.1% (1/1000) and 0.4% (4/1000) of the liver and cecum samples of broiler chickens, respectively. On the other hand, none of the carcass samples were determined to be positive for E. coli O157. Overall, the results indicated that 12% (3/25) of the flocks were positive for E. coli O157. The differences between the flocks in terms of the positivity were determined to be statistically significant (p<0.001). Ten (2.7%) of 367 human stool samples were also positive for E. coli O157 in the PCR examination. None of the broiler and human E. coli O157 isolates possessed H7, shigatoxins 1-2, or enterohemolysin genes, whereas all the broiler isolates and one of the human isolates were positive for intimin gene. In the PFGE analysis, a total of eight different profiles (four from broiler and four from human isolates) were observed. However, there were no genetic relationships between broiler and human E. coli O157 isolates. It can be concluded that more detailed studies are needed in poultry to better understand the role of these species in the epidemiology of E. coli 0157 infections in humans.