Isolation and molecular characterization of Mycoplasma spp. in sheep and goats in Egypt

Abstract

Background and aim: Different species of Mycoplasma are associated with many pathological problems in small ruminants including respiratory manifestation, this problem results in significant losses, especially in African countries. This study aimed to (I) study some epidemiological aspects of Mycoplasma species infections in Egyptian sheep and goats at Giza Governorate, (II) diagnosis of Mycoplasma species affections using bacterial isolation and identification, (III) apply the polymerase chain reaction (PCR) for typing of different Mycoplasma species, and (IV) illustrate the phylogenetic tree for the isolated Mycoplasma species and other species from GenBank using the purified PCR product.

Materials and methods: A total of 335 samples were collected from sheep and goats from Giza Governorate in Egypt as 142 nasal swabs from clinically affected animals, 167 pneumonic lungs, 18 samples from tracheal bifurcation, and 8 samples by bronchial wash were cultured on pleuropneumonia-like organisms (PPLOs) media for cultivation of Mycoplasma species. PCR and sequencing and phylogenetic analysis were adopted to identify and classify the isolated Mycoplasma species.

Results: A total of 24 Mycoplasma isolates were isolated on PPLO media, identified by biochemical tests, and confirmed and typed by PCR using specific primers. 10 isolates were confirmed as Mycoplasma arginini, four isolates as Mycoplasma ovipneumoniae by PCR, and 10 isolates as undifferentiated Mycoplasma species. A purified isolate of M. arginini and M. ovipneumoniae was sequenced and phylogenetic analysis was illustrated.

Conclusion: M. arginini and M. ovipneumoniae are prevalent in Egyptian sheep and goats. Further studies on M. arginini are required due to its high frequency of isolation from pneumonic sheep and goats and also from animals suffer from different respiratory manifestations.

Keywords: Mycoplasma; goats; polymerase chain reaction; sequencing and phylogenetic analysis; sheep.

Figure-1

(a) Buck suffers from different respiratory manifestations with nasal and ocular discharges. (b) Goats suffer from severe respiratory distress with nasal and ocular discharge (corneal opacity). (c) Pneumonic sheep lung with areas of red hepatization, especially at the cranial lobe. (d) Colonies of Mycoplasma appear as a fried egg under the microscope.

Figure-2

(a) Agarose gel of conventional polymerase chain reaction for detection of 16S gene using Mycoplasma genus-specific primer at amplicon size 278 bp. Lane M (Molecular weight marker, 100-1000 bp); Lanes 1-14, positive samples except for lane 3 negative control. (b) Agarose gel of conventional polymerase chain reaction for detection of Mycoplasma arginini species at amplicon size 525 bp. Lane M (Molecular weight marker, 100-1000 bp); Lanes 1-10 positive. (c) Agarose gel of conventional polymerase chain reaction for detection of Mycoplasma ovipneumoniae at amplicon size 418 bp. Lane M, (Molecular weight marker, 100-1000 bp); lanes 1-4 positive samples.

Figure-3

(a) Phylogenetic analysis of Mycoplasma arginini isolates based on 16S gene sequences, MH685445 isolate under study, KP972459 and KP972459 strains isolated from goats in Egypt, HQ661830 and HQ661829 strains from sheep in South Africa, LC158832 from sheep in Japan, and KX2304770 from goat in China. (b) Phylogenetic analysis of M. ovipneumoniae isolates based on 16S gene sequence, KU870650 and KU870647 strains isolated from goats in China, JN257120, EF687778, DQ0000588, and JN257121 from sheep in China, NR_025989 from wild sheep (Ovis aries) in the USA and EU265779 from bighorn sheep in the USA and KJ433280 from Norwegian Muskov in Norway.