B. Sareyyüpoğlu

A Case of Aspergillosis in a Broiler Breeder Flock

SUMMARY. A case of aspergillosis in a broiler breeder flock having respiratory and nervous system problems caused by Aspergillus fumigatus and Aspergillus niger is documented. Dyspnea, hyperpnea, blindness, torticollis, lack of equilibrium, and stunting were observed clinically. On postmortem examination of the affected birds, white to yellow caseous nodules were observed on lungs, thoracic air sacs, eyes, and cerebellum. Histopathologic examination of lungs and cerebellum revealed classic granulomatous inflammation and cerebellar lesions, necrotic meningoencephalitis, respectively. No lesions were noted in the cerebrum histopathologically. Aspergillus hyphae were observed in stained sections prepared from lesioned organs. Fungal spores and branched septate hyphae were observed in direct microscopy. Aspergillus fumigatus and A. niger were isolated from the inoculations prepared from the suspensions of organs showing lesions.

Q fever abortions in ruminants and associated on-farm risk factors in northern Cyprus

Background Q fever is caused by the obligate intracellular bacterium, Coxiella burnetii [1,2]. This disease is regarded as endemic worldwide, with the exception of New Zealand [3-6]. Cattle, sheep and goats are considered to be the primary source of transmission for humans [7,8]. Humans are infected mainly by inhalation of contaminated aerosols or by the ingestion of infected milk and/or fresh dairy products. In animals, Q fever is mainly subclinical but has especially been associated with reproductive disorders such as late abortions, stillbirths, weak off springs, metritis and infertility in ruminants [8-11]. Abortions during Q fever epizootics have been described in goats and sheep, but rarely documented in dairy cows [7,12]. Domestic pets, such as cats, dogs and wild-domestic birds such as rock doves (Columba livia) and geese (Anser anser) are known to be an additional source of infection [8,12-14]. Previous studies have reported occurrences of C. burnetii in migratory wild birds, rodents and ticks in southern Cyprus [15-17]. More than 40 species of ticks are naturally infected with C. burnetii. However, besides the aerosol route, the significance of ticks in transmitting the disease in ruminants and humans has previously been documented [8,9]. On the other hand, recent studies showed that ticks seem to play a major role in the circulation of C. burnetii in cycles of nature especially in wild life cycles. Ticks are also believed to probably play another crucial role in the transmission of the agent from infected wild vertebrates to domestic animals [5,18,19]. In humans, Q fever is mostly asymptomatic, the acute disease form is mainly limited flu-like illness, pneumonia or hepatitis while the chronic disease manifests with chronic fatigue syndrome or endocarditis [4,5,20]. On the reproductive health point of view, C. burnetii infections are known to cause abortions, stillbirth and premature deliveries in pregnant women. In the past, a series of Q fever outbreaks in both human and animal populations resulting in abortions on the island of Cyprus have been reported [10,21]. Studies done on the islands as far back as the 1970’s showed that Q fever has been an ongoing public health problem. Recently, the prevalence of IgG antibodies against C. burnetii phase II antigens was estimated to be at 52.7% for humans, 48.2% for goats, 18.9% for sheep, and 24% for cows. In this context, control of C. burnetii infection in ruminants is a vital component of public health [19]. There is no known record of humans contracting Q fever in northern Cyprus, which might be linked with lack of routine screenings and/or insufficient diagnostic units for C. burnetii [Northern Cyprus Ministry of Health, 2008]. The diagnostic enigma is that C. burnetii is difficult to culture, and detection in Cyprus was first done by complement fixation of antibodies [22,23]. Lately detection and diagnosis of C. burnetii, has been more effectively done by PCR based techniques, targeting the isocitrate dehydrogenase, superoxide dismutase gene and a transposon-like repetitive region [15,24-28]. The current technological advancement in these techniques has made them the most useful diagnostic tools for detection of C. burnetii in bovine aborted foetuses and ovine genital swabs [29-31]. Since the division of Cyprus in 1974, there has not been any research work on this disease in the northern region. Therefore, the aim of this study was to determine the occurrence of Q fever abortion using a PCR based method on DNA isolated from aborted foetal abomasal contents and placental tissues from ruminants in northern Cyprus. In addition, to determine the on-farm risk factors associated with the disease.

Chlamydophila psittaci DNA Detection in the Faeces of Cage Birds

In this study, we investigated the shedding of Chlamydophila psittaci in faecal samples from cage birds using PCR testing. A total of 47 faeces samples were collected from four different aviaries. Main symptoms determined after clinical investigation and owner histories of the birds showed that the birds had respiratory system problems changing from mild to severe. They also showed conjunctivitis, diarrhoea or no symptoms at all. DNA extractions from faeces were performed with the QIAamp DNA Stool Mini Kit. Following PCR with Cp. psittaci specific primers, 43 (91.5%) samples were determined to harbour‐specific DNA. Only one bird from each aviary was found to be negative by PCR. As all the samples from birds showing clinical signs were PCR positive, these signs could be correlated to psittacosis in these birds. Cp. psittaci shedding in faeces was detected in all the aviaries. After restriction analysis of PCR amplicons with AluI enzyme, all the isolates showed the same RFLP (Restriction Fragment Length Polymorphism) patterns with the control Cp. psittaci DNA. PCR following QIAamp DNA stool mini kit extraction of faecal samples was found to be a rapid, specific, sensitive, reproducible test, which did not need additional nested PCR of samples.

Polymerase Chain Reaction Detection of Salmonella spp. in Fecal Samples of Pet Birds

Abstract The aims of this study were 1) to determine the prevalence of Salmonella in clinically ill birds in aviaries in Ankara, Turkey, and 2) to compare conventional culture and polymerase chain reaction (PCR) for detection of Salmonella in feces from clinically ill pet birds. In the study, 185 fecal samples (feces and/or swabs) collected from the pet birds kept in the seven different aviaries in the city of Ankara were investigated for the existence of Salmonella spp. by bacterial isolation and PCR. The conventional isolation and identification methods were performed for Salmonella isolation from fecal cultures. Suspected colonies were confirmed with the Salmonella polyvalent O antiserum and serogrouped with Salmonella group-specific antiserum. PCR was performed after the fecal swabs were incubated for 18 hr in 10 ml of tetrathionate broth. Three (1.63%) out of 185 fecal samples were found to harbor Salmonella spp. by conventional identification tests and were found to belong to serogroup B. Five (2.7%) swab samples were found to harbor Salmonella DNA by PCR tests. As a conclusion, PCR following incubation of clinical samples in pre-enrichment broth seemed to be a fast and practicable method for Salmonella spp. diagnosis when compared to protracted labor-intensive conventional culture techniques.

Detection of methicillin and mupirocin resistance in staphylococcal hospital isolates with a touchdown multiplex polymerase chain reaction

Staphylococcal hospital isolates (n = 166) were tested in a touchdown multiplex-polymerase chain reaction assay for the identification of methicillin and mupirocin resistance and discrimination of S. aureus (femA gene) from coagulase negative staphylococci and other bacteria. All isolates harbored the 16SrDNA (Staphylococcus genus specific internal control) gene, and 130 (78 %) the mecA (methicillin resistance) gene. Fifty-seven (44 %) of these were determined as methicillin-resistant S. aureus, while the remaining 73 (56 %) were methicillin-resistant coagulase-negative staphylococci. Seventy-five (45 %) isolates harbored the ileS-2 (high-level mupirocin resistance) gene and were determined as mupirocin-resistant. This assay represents a simple, rapid, reliable approach for the detection and discrimination of methicillin-and mupirocin-resistant staphylococci.

PCR Detection of Shiga Toxins, Enterohaemolysin and Intimin Virulence Genes of Escherichia coli O157:H7 Strains Isolated from Faeces of Anatolian Water Buffaloes in Turkey

The aim of this study was to detect Shiga toxins (stx1 and stx2), enterohaemolysin (EhlyA) and intimin (eaeA) virulence genes of 11 Escherichia coli O157:H7 strains isolated from faecal samples of 300 clinically healthy Anatolian water buffaloes by PCR. Multiplex PCR was used for the detection of stx1 and stx2, and singleplex PCRs were used for the detection of EhlyA and eaeA virulence genes respectively. A total of three (27.3%) strains were determined to harbour both of the stx1 and stx2 genes, of these, one (9.1%) only harboured these two genes alone, one (9.1%) also contained the EhlyA gene and one (9.1%) additionally contained the EhlyA and the eaeA genes. EhlyA gene was obtained from eight (72.7%) strains, six (54.5%) of these were alone. eaeA gene was positive in only one (9.1%) strain. Only one (9.1%) of the 11 E. coli O157:H7 strains harboured all the four virulence genes. Two (18.2%) of the isolates had none of the virulence genes. Enterohaemolysin was found to be the most common virulence factor. In conclusion, the virulence factors of E. coli O157:H7 strains isolated from the faeces of Anatolian water buffaloes were investigated and detected for the first time in Turkey.

Evaluation of PCR methods for detection of Brucella strains from culture and tissues

The genus Brucella causes significant economic losses due to infertility, abortion, stillbirth or weak calves, and neonatal mortality in livestock. Brucellosis is still a zoonosis of public health importance worldwide. The study was aimed to optimize and evaluate PCR assays used for the diagnosis of Brucella infections. For this aim, several primers and PCR protocols were performed and compared with Brucella cultures and biological material inoculated with Brucella. In PCR assays, genus- or species-specific oligonucleotide primers derived from 16S rRNA sequences (F4/R2, Ba148/928, IS711, BruP6-P7) and OMPs (JPF/JPR, 31ter/sd) of Brucella were used. All primers except for BruP6-P7 detected the DNA from reference Brucella strains and field isolates. In spiked blood, milk, and semen samples, F4-R2 primer-oriented PCR assays detected minimal numbers of Brucella. In spiked serum and fetal stomach content, Ba148/928 primer-oriented PCR assays detected minimal numbers of Brucella. Field samples collected from sheep and cattle were examined by bacteriological methods and optimized PCR assays. Overall, sensitivity of PCR assays was found superior to conventional bacteriological isolation. Brucella DNA was detected in 35.1, 1.1, 24.8, 5.0, and 8.0% of aborted fetus, blood, milk, semen, and serum samples by PCR assays, respectively. In conclusion, PCR assay in optimized conditions was found to be valuable in sensitive and specific detection of Brucella infections of animals.

RT-PCR/RFLP Typing of Infectious Bursal Disease Virus Field Strains in Turkey

The results of this study indicate the existence of field isolates with new molecular patterns different from those previously published, that may well be unique and specific to geographical regions. This result, that new molecular patterns of IBDV strains were detected in Turkey, was determined by examining 80 bursa samples from 56 commercially-reared chicken flocks in Turkey with clinical symptoms of IBD for IBDVs. The samples were examined using the reverse transcription (RT)-polymerase chain reaction (PCR)/ restriction fragment length polymorphism (RFLP) assay. Two new and unique molecular patterns (MP1 and MP2), which may well be typical of the region, were observed in this study. Observations indicated:

Restriction Fragment Length Polymorphism Typing of Infectious Bursal Disease Virus Field Strains in Turkey

Abstract Infectious bursal disease (IBD), also known as Gumboro disease, is a highly contagious, immunosuppressive disease of immature chickens. It is caused by IBD virus (IBDV) and is responsible for major economic losses in the poultry industry worldwide. In this study, 280 bursa samples from 56 commercially reared chicken flocks in Turkey with clinical symptoms of IBD were examined for IBDVs using the reverse transcription (RT)–polymerase chain reaction (PCR)/restriction fragment length polymorphism (RFLP) assay. The assay was conducted on a 743-bp fragment of the VP2 gene with the restriction enzymes BstNI, MboI, and SspI. The results indicate the existence of field isolates with new molecular patterns different from those previously published that may well be unique and specific to geographical regions.