Serap Savasan

Detection of methicillin resistance and slime factor production of Staphylococcus aureus in bovine mastitis

This study aimed to detect methicillin resistant and slime producing Staphylococcus aureus in cases of bovine mastitis. A triplex PCR was optimized targetting 16S rRNA, nuc and mecA genes for detection of Staphylococcus species, S. aureus and methicillin resistance, respectively. Furthermore, for detection of slime producing strains, a PCR assay targetting icaA and icaD genes was performed. In this study, 59 strains were detected as S. aureus by both conventional tests and PCR, and 13 of them were found to be methicillin resistant and 4 (30.7%) were positive for mecA gene. Although 22 of 59 (37.2%) S. aureus isolates were slime-producing in Congo Red Agar, in PCR analysis only 15 were positive for both icaA and icaD genes. Sixteen and 38 out of 59 strains were positive for icaA and icaD gene, respectively. Only 2 of 59 strains were positive for both methicillin resistance and slime producing, phenotypically, suggesting lack of correlation between methicillin resistance and slime production in these isolates. In conclusion, the optimized triplex PCR in this study was useful for rapid and reliable detection of methicillin resistant S. aureus. Furthermore, only PCR targetting icaA and icaD may not sufficient to detect slime production and further studies targetting other ica genes should be conducted for accurate evaluation of slime production characters of S. aureus strains.

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.

Detection of Candida species by nested PCR method in one-humped camels (Camelus dromedarius)

Systemic fungal diseases are the infections caused by false treatment protocols and generally are not taken into consideration especially in the veterinary field. One-humped camels are found in the western side of the Aegean region of our country and bred for wrestling. The aim of this study is the application of diagnosing systemic fungi infection from camel blood samples by the PCR method. In this study, specific primers for DNA topoisomerase II gene sequences were used. As a result, a systemic fungal infection was detected by the nested PCR method from 10 (20%) out of 50 DNA samples taken from camels located on the western side of the Aegean region. In this study, 3 (30%) samples were identified as Candida albicans, 3 (30%) samples were identified as C. glabrata, and 4 (40%) samples were identified as C. parapsilosis. In conclusion, the 20% positive systemic fungal infection rate in one-humped camels observed in the present study showed that the systemic fungal infections are not taken into considerations in veterinary medicine. Further studies are suggested in order to obtain and to maintain extensive data for systemic fungal diseases in our country for one-humped camels.

Molecular identification of Corynebacterium pseudotuberculosis in sheep

Caseous lymphadenitis is still a serious zoonotic problem in Turkey. Sheep suffer from the disease with yield loss in wool and meat production. Moreover, with inexperienced laboratory staff, biochemical identification may go unrevealed. The scope of this study was to demonstrate the presence of Corynebacterium pseudotuberculosis in sheep by PCR. The sampling was conducted via collecting lymph fluids from the lymph node internal pouch wall of 100 sheep that were examined for the presence of Corynebacterium pseudotuberculosis. Molecular identification of the Corynebacterium pseudotuberculosis isolates was carried out by establishing the presence of the proline iminopeptidase gene. All isolates were confirmed to be Corynebacterium pseudotuberculosis by polymerase chain reaction. The polymerase chain reaction procedure conducted in this research was observed to be reliable and fast, and could be utilized for confirmation of caseous lymphadenitis in sheep as an optional technique to timeconsuming biochemical identification methods. Pleomorphic bacteria, caseous lymphadenitis, proline iminopeptidase, PCR Caseous lymphadenitis (CL) is a bacterial disease that causes considerable economic loss in sheep and goat industries (loss of skin and carcass value, loss of weight, loss of reproductive activity, and reduced milk yield) in many countries around the world (Guimaraes et al. 2011). Corynebacterium pseudotuberculosis (C. pseudotuberculosis) is a gram-positive, facultative intracellular actinomycete that causes chronic bacterial disease in sheep, goats, and other warm-blooded animals with caseous lymphadenitis (CLA) (Dorella et al. 2006). Pleomorphism in fresh C. pseudotuberculosis cultures is like that of Corynebacterium diphtheriae, and other bacterial species, in terms of microscopic morphology. However, metachromatic granules are better detected using the staining methods of Neisser and Albert (Ilhan 2001). Colonies are easily broken down and dispersed on agar, but are less dispersed in liquid medium (Paracikoglu and Aydin 2006). Laboratory diagnosis is usually achieved through confirmation using bacterial cultures and by biochemical, serological and nucleic acid-based detection methods (Baird and Fontaine 2007; Guimaraes et al. 2011). The microorganism continues to multiply in host cells after being taken up by macrophages, which are disrupted and the microorganism is released thereafter. Released microorganisms are subsequently taken up by other circulating phagocytic cells and the cycle is repeated. This repeated phagocytosis cycle has been reported to cause recurrent lesions in C. pseudotuberculosis infections in sheep (Yeruham et al. 1997). In this study, the presence of C. pseudotuberculosis was identified in suspected cases of caseous lymphadenitis in sheep, using bacteriological cultures and polymerase chain reaction (PCR), in the Aydin Province of Turkey. ACTA VET. BRNO 2018, 87: 3-8; https://doi.org/10.2754/avb201887010003 Address for correspondence: Dr.Ugur Parin Department of Microbiology Faculty of Veterinary Medicine University of Adnan Menderes, 09016 Turkey Phone:+90 533 418 40 78 E-mail: uparin@adu.edu.tr http://actavet.vfu.cz/ Materials and Methods