J Vet Sci.  2014 Sep;15(3):361-367. 10.4142/jvs.2014.15.3.361.

Detection of porcine reproductive and respiratory syndrome virus in oral fluid from naturally infected pigs in a breeding herd

• Trang NT
• Hirai T
• Yamamoto T
• Matsuda M
• Okumura N
• Giang NT
• Lan NT
• Yamaguchi

• KMID: 2155619
• DOI: http://doi.org/10.4142/jvs.2014.15.3.361

Abstract

The objectives of the present study were to evaluate the anatomic localization of porcine reproductive and respiratory syndrome virus (PRRSV) in naturally infected pigs and to determine whether oral fluid could be used to detect the virus in infected animals. Two sows, seven 2-month-old grower pigs, and 70 6-month-old gilts were included in this study. PRRSV in sera and oral fluid were identified by nested reverse transcription PCR (nRT-PCR) while lung, tonsil, and tissue associated with oral cavity were subjected to nRT-PCR, immunohistochemistry, and in situ hybridization. In sows, PRRSV was identified in oral fluid and tonsils. PRRSV was also detected in oral fluid, tonsils, salivary glands, oral mucosa, and lungs of all seven grower pigs. However, viremia was observed in only two grower pigs. Double staining revealed that PRRSV was distributed in macrophages within and adjacent to the tonsillar crypt epithelium. In gilts, the North American type PRRSV field strain was detected 3 to 8 weeks after introducing these animals onto the farm. These results confirm previous findings that PRRSV primarily replicates in tonsils and is then shed into oral fluid. Therefore, oral fluid sampling may be effective for the surveillance of PRRSV in breeding herds.

Keyword

acclimatization; localization; oral fluid; porcine reproductive and respiratory syndrome virus; tonsil

MeSH Terms

Animals
Female
In Situ Hybridization/veterinary
Lung/virology
Male
Palatine Tonsil/virology
Polymerase Chain Reaction/veterinary
Porcine Reproductive and Respiratory Syndrome/*virology
Porcine respiratory and reproductive syndrome virus/*physiology
Saliva/*virology
Salivary Glands/virology
Swine/virology
Virus Replication/physiology

Figure

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  Fig. 1 Tonsil tissue from piglet 1 naturally infected with porcine reproductive and respiratory syndrome virus (PRRSV). Tonsillar crypt with macrophages, neutrophils, lymphocytes, and cryptal epithelial cells in the lumen. (A) Hematoxylin and eosin staining. (B) Higher magnification. PRRSV nucleic acids were detected in the cytoplasm of cells resembling macrophages in the tonsillar crypts. (C) ISH and hematoxylin counterstaining. (D) Higher magnification. Scale bars = 10 µm.

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  Fig. 2 Tonsil section from piglet 1 naturally infected with PRRSV. PRRSV nucleic acids were stained brown and lysozymes were stained red. Double-positive cells showed mixed red and brown staining in the cytoplasm (arrow). Double labeling of the lysozymes with IHC and PRRSV RNA using ISH. Hematoxylin counterstaining. Scale bars = 10 µm.

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  Fig. 3 Tonsil section from piglet 7 naturally infected with PRRSV. Double immunofluorescence staining for PRRSV antigen (red) and lysozymes (green). Double-positive cells contained both red and green signals or orange fluorescence in the cytoplasm (arrows). Nuclei were counterstained with 4',6-diamidino-2-phenylindole (blue). Scale bars = 10 µm.

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  Fig. 4 Phylogenetic tree showing the relationships based on the North American-type PRRSV ORF 5 nucleotide sequences. The tree was constructed using the neighbor-joining method for eight isolates recovered in this study (Sow A, Sow B, Gilt 3, Gilt 6, Gilt 7, Grower 1, Grower 3, and Grower 6), the vaccine strain (Vaccine), and three previously obtained PRRSV isolates from the farm (Farm 10/2011, Farm 11/2011, and Farm 12/2011). A bootstrap analysis was performed with 1,000 replicates.

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