The detection and differentiation of canine respiratory pathogens using oligonucleotide microarrays

Abstract

Canine respiratory diseases are commonly seen in dogs along with co-infections with multiple respiratory pathogens, including viruses and bacteria. Virus infections in even vaccinated dogs were also reported. The clinical signs caused by different respiratory etiological agents are similar, which makes differential diagnosis imperative. An oligonucleotide microarray system was developed in this study. The wild type and vaccine strains of canine distemper virus (CDV), influenza virus, canine herpesvirus (CHV), Bordetella bronchiseptica and Mycoplasma cynos were detected and differentiated simultaneously on a microarray chip. The detection limit is 10, 10, 100, 50 and 50 copy numbers for CDV, influenza virus, CHV, B. bronchiseptica and M. cynos, respectively. The clinical test results of nasal swab samples showed that the microarray had remarkably better efficacy than the multiplex PCR-agarose gel method. The positive detection rate of microarray and agarose gel was 59.0% (n=33) and 41.1% (n=23) among the 56 samples, respectively. CDV vaccine strain and pathogen co-infections were further demonstrated by the microarray but not by the multiplex PCR-agarose gel. The oligonucleotide microarray provides a highly efficient diagnosis alternative that could be applied to clinical usage, greatly assisting in disease therapy and control.

Keywords: Bordetella bronchiseptica; Canine distemper virus; Canine herpesvirus; Influenza virus; Mycoplasma cynos; Oligonucleotide microarray.

Fig. 1

Multiplex PCR result of dog respiratory pathogens on an agarose gel. M: 100 bp ladder marker; 1: Influenza virus (245 bp); 2: B. bronchiseptica (291 bp); 3: CHV (427 bp); 4: M. cynos (449 bp); 5: CDV (531 bp); 6: CAV-2; 7: CPIV; 8: CRCoV; 9: Mixture of all pathogens containing 1–5. 10: Negative control.

Fig. 2

Detection and differentiation of dog respiratory pathogens using oligonucleotide microarrays. (A) Microarray map. The meaning of each probe and its detecting strains are shown in Table 2. P: positive control. (B) The microarray detection results.

Fig. 3

Detection limit comparison on agarose gel and microarray. (A) CDV (wild type NTU311 strain). (B) Influenza virus. (C) CHV. (D) B. bronchiseptic. (E) M. cyno. M: 100 bp ladder marker; 1: 10⁴ copies; 2: 10³ copies; 3: 10² copies; 4: 50 copies; 5: 10 copies; 6: 1 copy.