title: Optimized protocol for multiplex nested polymerase chain reaction to detect and differentiate Haemophilus parasuis, Streptococcus suis, and Mycoplasma hyorhinis in formalin-fixed, paraffin-embedded tissues from pigs with polyserositis.
abstract: An optimized protocol was developed for the simultaneous detection and differentiation of Haemophilus parasuis, Streptococcus suis, and Mycoplasma hyorhinis in formalin-fixed, paraffin-embedded (FFPE) tissues with multiplex nested polymerase chain reaction (PCR). This method also determines the prevalence of these bacteria in pigs with polyserositis. DNA extraction with a combination of a commercial reagent and proteinase K resulted in more frequent detection of the pathogens than DNA extraction with proteinase K alone. Among FFPE tissue samples from 312 cases of polyserositis in which at least 1 bacterial species was detected, multiplex nested PCR detected H. parasuis in 239 (77%), S. suis in 124 (40%), and M. hyorhinis in 40 (13%). The disease was caused by a single pathogen in 224 (72%) of the cases and multiple pathogens in 88 (28%). Among the pigs positive for H. parasuis, S. suis, and M. hyorhinis by multiplex nested PCR, the pathogen was isolated from only 11%, 35%, and 28%, respectively. Therefore, the PCR protocol developed in this study is a useful diagnostic method when samples are negative after isolation methods and even for samples in which only 1 pathogen was isolated.
authors.affiliation: Seoul National University, College of Veterinary Medicine, Department of Veterinary Pathology, 599 Gwanak-ro, Gwanak-gu, 151-742, Seoul, Republic of Korea.
authors.forename: Ikjae
authors.initials: I
authors.lastname: Kang
authors.forename: Duyeol
authors.initials: D
authors.lastname: Kim
authors.forename: Kiwon
authors.initials: K
authors.lastname: Han
authors.forename: Hwi Won
authors.initials: HW
authors.lastname: Seo
authors.forename: Yeonsu
authors.initials: Y
authors.lastname: Oh
authors.forename: Changhoon
authors.initials: C
authors.lastname: Park
authors.forename: Jeehoon
authors.initials: J
authors.lastname: Lee
authors.forename: Marcelo
authors.initials: M
authors.lastname: Gottschalk
authors.forename: Chanhee
authors.initials: C
authors.lastname: Chae
mesh_terms: D000818:Animals; D005557:Formaldehyde; D006192:Haemophilus Infections / Q000382:microbiology / Q000662:veterinary; D044137:Haemophilus parasuis / Q000302:isolation & purification*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D009175:Mycoplasma Infections / Q000382:microbiology / Q000662:veterinary; D045790:Mycoplasma hyorhinis / Q000302:isolation & purification*; D016612:Paraffin Embedding; D012680:Sensitivity and Specificity; D012700:Serositis / Q000382:microbiology / Q000662:veterinary*; D013290:Streptococcal Infections / Q000382:microbiology / Q000662:veterinary; D017011:Streptococcus suis / Q000302:isolation & purification*; D013552:Swine; D013553:Swine Diseases / Q000175:diagnosis / Q000382:microbiology*; D016707:Tissue Fixation
chemical_list: D005557:Formaldehyde