Pathological and molecular study of mycotic abortion in ewes

Abstract

The aims of the present study is to isolate the fungal species associated with ewe abortion and to examine the histopathological changes in the placenta associated with fungal isolates .In addition to study the genetic information of TNF alpha gene, intensity and score of TNF-alpha protein in aborted and normal placenta by PCR and IHC respectivly. Frozen placental tissue and paraffin embedded tissue belong to aborted and normal groups were used for assessment of TNF alpha gene by studying the genetic information of TNF-alpha gene in aborted and normal placental sample by PCR .immmunohistochemical study was carried out to determine TNF protein in the placental sample. The same frozen placenta tissue samples were also used for DNA extraction to detect TNF alpha gene in the two groups. A fragment of (238 bp ) of the TNF alpha gene was amplified by PCR using primers ,the sequence of sense and antisense primer for TNF alpha gene was: GAA TAC CTG GAC TAT GCC GA, CC TCA CTT CCC TAC ATC CCT (bp 238) Gene Bank: X56756. The study showed that 10 fungal species were isolated from aborted placenta with high percentage of these isolates was recorded in March ,.The fungal isolates were variable according to region of sample collecting ,high percentage in the AL-Manathera(36%),followed by Al-Kuzweenia (34%) and Al-Shabaka(30%). The fungal isolates include Coccidioides immitis (16%). Asperigllus.fumigitus (14%), Trichophyton, Rhodetella rubra, and Aspergillus.ochraus ,(10%) for each one . Candida kruzei,(8%). Aspergillus.flavus and Geotrichum ,6%) for each one . Prototheca..zopfi, Saccharomyces.cerevisia, Blast.capitatuss, Cryptococcus uniguttultus and Candida zeylanoides,(4%) for each one . This study revealed the presence of ~238bp band in addition to the ~300 ,400 and 600 bp desired fragment in PCR products that related to aborted samples compared to normal one .,this may be due to polymorphism within the TNF gene itself. We found a significant elevated TNF alpha protein in inflammatory cell (macrophage) of aborted sheep placenta (increasing of intensity and score), while no changes could be observed for pro-inflammatory molecules in the control sample (normal delivery) The current study expressed correlation between polymorphisms in TNF- alpha gene ,immunohistochimistry and the fungal isolated species. The histopathological examination showed acute suppurative placentitis, necrosis of chorion villi, congestion of blood vessels with fibrin deposition in the villus stroma and inter villus space, in addition to calcification in the villus stroma and sloughing and desquamation of cytotrophoblast .numerous yeast and spheriols were detected in the placental tissue by periodic acid Schiff reaction(PAS).