Infection survey, molecular, pathogenicity, and morphological characteristics of Sarcocystis species naturally infected water buffaloes (Bubalus bubalis) in Egypt

Abstract

Background: Sarcocystosis is a parasitic disease found worldwide, resulting from various Sarcocystis species. The current research was carried out in three significant economic areas in Egypt: Greater Cairo, the Nile Delta, and Upper Egypt. It aimed to investigate the occurrence of Sarcocystis spp. in locally bred water buffaloes Bubalus bubalis.

Methods: To achieve this objective, 317 buffalos were slaughtered in different slaughterhouses in various regions of Egypt. Samples of heart, skeletal muscle, esophagus, and tongue were assessed using macroscopic and microscopic tests. Examination methods included direct optical observation of tissues as well as digestion and examination of the sediment obtained from the tissues. Additionally, ultrastructural features were analyzed using scanning and transmission electron microscopy. Molecular characterization was conducted through PCR, followed by nucleotide sequencing and phylogenetic analysis.

Results: A total of 317 slaughtered buffaloes were examined for Sarcocystis during the period from September 2021 to October 2023. The prevalence of infection was recorded with 229 out of 317 (72.2%) infected with Sarcocystis spp. The results also showed that the prevalence of Sarcocystis species in females was higher than males. Based on the age of carcasses, adults (> 2 years) had a higher infection rate compared to young ones (< 2 years). Regarding seasonal variation, the highest prevalence of infection was recorded during the summer followed by spring, and then autumn, while winter had the lowest prevalence of infection. Additionally, the skeletal muscle was the most susceptible organ to sarcocystosis (87.3%) followed by the esophageal muscle (8.3%), the tongue (4.4%), and no infection in the heart muscle. The use of scanning and transmission electron microscopy allowed the identification of S. fusiformis and S. cruzi in buffaloes in Egypt. Furthermore, the Sarcocystis 18 S rRNA genes from skeletal tissue samples were cloned and sequenced under accession numbers OQ507387, OQ507388, and OQ507389 for S. fusiforms, and one OQ507391 for S. cruzi.

Conclusion: The findings revealed a notably high prevalence of Sarcocystis infection (72.2%) in buffaloes from Egypt, with skeletal muscle identified as the organ most susceptible to the parasite. Two Sarcocystis species were detected: S. fusiformis and S. cruzi.

Keywords: Sarcocystis cruzi; Sarcocystis fusiformis; Buffaloes; Molecular; Pathogenicity; Sarcocystosis; Ultrastructural.

Fig. 1

The map of Egypt shows sampling sites

Fig. 2

Photomicrograph of esophageal muscle showing cysts of Sarcocystis sp. embedded in the muscle fibers of the host, (a) showing an elongated fusiform cyst of Sarcocystis fusiformis (black star). (b) showing the rounded ends of the cyst of Sarcocystis cruzi (black stars)

Fig. 3

A photomicrograph from the skeletal muscles of the infected cattle shows heavy sarcocystosis infection with the presence of both macro and microcysts (black arrows) (a, b & c). The cyst is enclosed by a cyst wall (CW) (b) that is underlined by a layer of ground substance. This ground substance extends into the inner side of the cyst, forming septa (S) containing bradyzoites (Br) (d, e, & f). A thin fibroblastic tissue capsule can be seen surrounding large cystic lesions (black aster) (c). Mild vascular dilatation (green arrows) (a & c) edema (red aster) (d) and hyaline degeneration (red arrows) (b& c) are seen. Note that finger-like villar protrusions (vp) are clearly visible on the striated cyst wall

Fig. 4

Scanning electron microscope (SEM) photos, a & b: showing fusiform cyst of Sarcocystis fusiformis, c-f: showing the cyst of Sarcocystis cruzi. c: showing radially striated protrusions folded over the surface. d: showing both convex and concave sides of the cyst. e & f: showing both rounded ends of the cyst

Fig. 5

TEM of Sarcosystis sp. showing (a) the cyst is divided into many chambers separated from each other by the septa (S). (b) higher magnification of chambers with large numbers of merozoites (Me) and degenerated merozoites (DeM). (c) higher magnification of merozoites with micronemes (Mn), clear nucleus (N) with a nucleolus (Ne) in addition to amylopectin granules (Am). (d) clear banana-shaped merozoites (Me) with micronemes (Mn), rhoptries (Rh), and conoid (C), also figure showed degenerated (DeM) and developed (DM) merozoites. (e) well-developed merozoite (Me) with micronemes (Mn), and rhoptries (Rh). (f) the division of merozoites (Me)

Fig. 6

PCR-based assays targeted the 18S rDNA gene for Sarcocystis. L, L lanes: 100 bp DNA ladder. Lanes 1, 2, 4, 6, 8, 9, and 10 correspond to positive results for Sarcocystis. Lanes P and N represent positive and negative controls, respectively.

Fig. 7

The percentage of identity for our Sarcocystis fusiformis and Sarcocystis cruzi isolates based on the 18S rRNA gene. Their accession numbers are followed by (AZ-DRC)

Fig. 8

The phylogenetic relationship of the 18S rDNA gene for submitted isolates of Sarcocystis fusiformis and Sarcocystis cruzi