Molecular detection of Nigerian field isolates of Mycoplasma mycoides subsp. mycoides as causative agents of contagious bovine pleuropneumonia

Abstract

Contagious bovine pleuropneumonia (CBPP) is a highly contagious respiratory disease affecting cattle and is widely distributed in the sub-Saharan Africa. The objective of this study was to detect Mycoplasma mycoides subspecies mycoides (Mmm) the causative agent of CBPP from 90 cattle at slaughter using polymerase chain reaction-Restriction fragment length polymorphism. In this study, 450 samples suggestive of CBPP in Maiduguri, Yola and Gombe township abattoirs were processed according to standard protocols. The isolation rate was found to be 3.33% and percentage of identification with PCR-RFLP yielded 1.56%. Subsequently, QIAxcel revealed molecular size of 574 bp for Mycoplasma mycoides subcluster. Further analysis of PCR amplicons with restriction digestion, confirmed the presence of Mmm 16 S rRNA of CAP 21 genomic region with molecular sizes of 180 bp and 380 bp. Thus, the 380 bp fragments delineated Mmm from Mycoplasma mycoides subsp. capri. Three isolates (BL5, BL6 and AL1) were from lungs and four from pleural fluids (APF2, APF8A, APF8B and APF9) were isolated and identified, while a vaccine strain T1/44 was re-detected along with the field isolates. No sample from Gombe had Mmm. In conclusion, the findings of this study have detected the presence of Mmm as causative agent of CBPP. Measures such as surveillance, quarantine and vaccination are hereby recommended for the control of CBPP in Nigeria.

Keywords: CBPP; Cattle; Mycoplasma; PCR; RFLP.

Fig. 1

Distribution of sampling location in North-Eastern Nigeria.

Fig. 2

A typical CBPP infected cattle lung cut open to reveal the marbled appearance from Maiduguri abattoir, North-Eastern Nigeria. Pneumonia and pleurisy (a), hepatization (b), thickened interlobular septae (c).

Fig. 3

Mycoplasma isolated from Yola at ten days of incubation (25X) at IZS, Teramo, Italy.

Fig. 4

(a) Capillary electrophoresis of PCR amplicons for Mycoplasma mycoides subcluster using Q1Axcel. Lances O1A to O6B: Samples in duplicate (OIA and OIB as sample number one in concentration of 50 μg and 30 μg and so on) † = Alignment marker, bp = Base pair. (b) Capillary electrophoresis of PCR amplicons for Mycoplasma mycoides subcluster using Q1Axcel. Lances Q1Axcel Lances O8A to O8B: T1/44,Lances: 09A and O9B: Positive controls for Mmm and Mmc, Lane O10A: Negative control, † = Alignment marker, bp = Base pair.

Fig. 5

PCR-RFLP amplicons on 3% agarose gel for the identification of Mycoplasma mycoides subspecies mycoides Lanes 1 and 20: Molecular marklers, lane 2 to 17: Samples in duplicate (T1/44 inclusive), Lane 18 and 19: Mmm and Mmc positive controls.