Evaluation of field and laboratory protocols used to detect avian influenza viruses in wild aquatic birds

Abstract

Careful selection and observance of standard field and laboratory protocols are critical for successful detection and characterization of avian influenza viruses (AIV) from wild birds. Cloacal swabs were collected from hunter-killed or nesting waterfowl and shorebirds from wildlife refuges in Alabama, Georgia, and Florida during 2006 to 2008. Swab samples were inoculated into embryonated eggs followed by hemagglutination (HA) test to determine the presence of hemagglutinating agents. Antigen capture-ELISA (AC-ELISA) and real-time reverse transcription-PCR (RRT-PCR) were used to detect AIV from both allantoic fluids (AF) and swab specimens of HA-positive samples. Hemagglutination inhibition test was used to detect Newcastle disease virus, another hemagglutinating virus common in wild birds. The HA-positive AF were sent to the National Veterinary Services Laboratory for subtyping of the isolates. Out of 825 samples tested, 19 AIV and 3 avian paramyxovirus subtypes were identified by the National Veterinary Services Laboratory. Without egg passage, AC-ELISA did not detect virus, whereas matrix gene of 13 AIV were detected using RRT-PCR. When testing was done on AF, 14 were positive for influenza A by AC-ELISA and 20 by RRT-PCR. Antigen capture-ELISA did not detect influenza A when the HA titer was lower than 125, whereas RRT-PCR detected AIV from AF with HA titer as low as 4. The highest isolation rate was from Florida, where out of 109 samples analyzed, 14 AIV were detected by RRT-PCR from AF. Real-time reverse transcription-PCR was more sensitive, specific, and cost-effective than AC-ELISA. However, to avoid false-negative results, testing should be performed on AF and not directly from cloacal swabs. Our procedures to detect AIV directly from cloacal swabs need further optimization for improved sensitivity.

Comparison of Pooling 11 or 5 Oropharyngeal Swabbings for Detecting Avian Influenza Virus by Real-Time Reverse Transcription–PCR in Broiler Chickens

“…At 2 wk of age, broilers in the AIVinfected treatment group were inoculated with 10 5 50% embryo infectious dose per bird with the H7N2 low pathogenicity (LP) AIV strain, chicken/Maryland/Minh Ma/04, via the intranasal route. On days 2, 3,4,5,7,9,11, and 14 postinoculation (PI), O/P swabbings were collected from individual AIV-infected and uninfected control broilers. A goal of the research was to simulate situations of low viral loads in the pools, thus making detection more difficult.…”

“…Pollos de engorde de dos semanas de edad, fueron inoculados por vía intranasal con una cepa del virus de la influenza aviar de baja patogenicidad Pollo/Maryland/ Minh Ma/04 H7N2, o se mantuvieron como controles no inoculados. En los días 2, 3,4,5,7,9,11, y 14 después de la inoculación, se recolectaron hisopos orofaríngeos de las aves infectadas individualmente y se combinaron con cuatro o 10 hisopos de pollos de engorda no infectados para producir 10 replicas de 5 y 11 hisopos, respectivamente. El virus de la influenza aviar fue detectado con facilidad (en un 80% a un 100%) mediante RRT-PCR en las muestra agrupadas con cinco y once hisopos desde los dos a los cinco días después de la inoculación.…”

“…E-mail: bladman@udel.edu AVIAN DISEASES 56:227-229, 2012 capture immunoassay (ACIA). While both ACIA and RRT-PCR testing are used to carry out active (preslaughter) surveillance programs, RRT-PCR is considered to be essential because it is more sensitive than ACIA for detecting avian influenza virus (AIV) infection (3,4,5). The cost of RRT-PCR, which is approximately 2 to 3-fold greater than ACIA, has had the effect of limiting the number of RRT-PCR tests performed in surveillance programs.…”

Preliminary comparison of different immune and production components in local and imported Saanen goats reared under a sub-tropical environment

“…The hemagglutination titer of each collected goat-plasma sample against c-RBC was performed according to a previous documented procedure (AAAP 1997;Dormitorio et al 2009). Briefly, half the volume of each collected plasma sample was heat inactivated at 56°C for 30 min to inactivate the complement enzymatic activity.…”

Viral Coinfections

“…Beperet et al successfully isolated two different subtypes of alphabaculoviruses from coinfection samples by an endpoint dilution assay [162]. Dormitorio et al successfully detected avian influenza virus (AIV) from suspicious allantoine fluid samples using this method [169]. (iii) The Ab neutralization method is suitable for different serotype viruses or two viruses with a distant genetic relationship.…”