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).