Currently, most methods of AIV, NDV, IBV, IBDV and other avian viral agents detection are adapted to specific detection of one agent in a sample. Multiplex RT-PCR is successfully used for detection of AIV and its subtypes [19, 20] and for diagnosing double infections such as combination of NDV and AIV. Also methods with use of multiplex real-time RT-PCR for AIV, NDV and IBV subtypes differentiation have been developed [22–24]. At present development of a test based on microarray technology for simultaneous detection of AIV, NDV, IBV and IBDV in one sample is important for poultry industry in the Republic of Kazakhstan.

Use of microarray improves quality and shortens the analysis duration in molecular diagnosis of infectious diseases and therefore is employed as an independent method in screening for several genes of large numbers of pathology samples [25–27]. There are biochips for influenza diagnosis that allow screening not only for HA and NA, but for M and NP genes of influenza A virus [25, 28]. In identification of NDV molecular methods with use of oligonucleotides specific to conservative regions of NP-gene of NDV were used. Recently VP2 gene region of IBDV is successfully used in synthesis of oligonucleotide primers and probes from highly conservative regions for molecular diagnosis [30–33]. Molecular methods for IBV diagnosis are oriented at using more conservative sequences located in S1 and S2 genes of IBV [34, 35].

In the proposed microarray probes were developed on the basis of conservative regions of gene fragments encoding NP and M (AIV), NP (NDV), VP2 (IBDV), S1 (IBV) array proteins from Genbank Database. All viral gene fragments demonstrated high rate of conservatism and therefore the test is universal for detecting AIV, NDV, IBV and IBDV strains. So, high homology of nucleotide sequences of gene regions encoding AIV, NDV, IBV and IBDV array proteins compared to GenBank data confirms specificity of the developed microarray for rapid diagnosis of avian influenza, Newcastle disease, infectious bronchitis and infectious bursal disease.

Total analysis duration without time required for the viral RNA extraction is 5–6 h, and 16 specimens can be simultaneously assayed. Duration of the assay with use of the proposed microarray is not longer than in other molecular methods and simultaneous testing of samples for AIV, NDV, IBV and IBDV provides its advantage over other methods.

Various methods have been developed for the diagnosis of bird infection, such as virus isolation in cell culture, embryonated chicken eggs, or young specific-pathogen-free (SPF) chickens and localization of the virus in infected tissues by electron microscopy, fluorescence assay, agar immunodiffusion, antigene-capture enzyme-linked immunosorbent assay (ELISA), or immunohistochemistry. All these methods have disadvantages, such as being time consuming, labor intensive, expensive, or nonspecific. These methods lack the ability to detect low levels of antigens in tissues [36–40].

In the present study field samples (122 in total) were used to test effectiveness and reliability of the microarray. Nevertheless, positive result of using molecular and biological methods, being very important in emergency cases, should always be confirmed by the method of virus isolation.

The results of the study show that diagnostic sensitivity (99.16%) and diagnostic specificity (100%) of the DNA microarray are comparable with the same of the real-time RT-PCR (99.15 and 100%, respectively).

Diagnostic effectiveness as percentage ratio of true results to the total number of obtained results for the developed DNA microarray and real-time RT-PCR was 99.18%.

Analysis of the obtained data shows that the microarray test for rapid diagnosis of avian infections demonstrates the effectiveness comparable to that of the molecular method real-time RT-PCR and is more rapid and less resource-consuming owing to its ability to detect simultaneously AIV, NDV, IBV IBDV positive samples in the course of one experiment. Universality of the test makes it suitable for wide use in veterinary laboratories for prompt detection of avian infections.

Statistical analysis was performed using GraphPad Prism 6.0. Correlation of C. psittaci bacterial load and adenovirus viral load was determined using linear regression analysis. For statistical calculation, 50% of the lower detection limit (6,000 copies/ml for C. psittaci and 600 copies/ml for adenovirus) was assigned to specimens that tested negative. Log-transformed viral loads were used for statistical analysis.

Results from a conventional RT-PCR using the IBV N gene confirmed that the obtained RT-PCR products were 130 bp in length, and the PCR products were detectable at a concentration of 100 copies/µl (Fig. 6).

The patient was a 55-year-old male artisan working at the NTNAMC. He presented after four days of dyspnea and two days of hemoptysis. Chest radiograph showed bilateral patchy consolidation. Blood test showed neutrophilia and elevated liver enzymes with alanine transaminase of 51 U/L. He was put on non-invasive positive pressure ventilation for respiratory failure. Bronchoalveolar lavage tested positive for C. psittaci and rhinovirus by PCR and reverse transcriptase-PCR respectively. Direct fluorescent antigen detection and viral culture did not reveal other viral co-pathogens. Paired serology, collected 18 days apart (on days 2 and 20 after hospitalization), showed a rise of C. psittaci IgG titer from <32 to 128 by microimmunofluorescence assay, but there was no increase in adenovirus or other respiratory virus antibody titer. The patient recovered with oral doxycycline. He had contact with birds, monkeys, iguanas and snakes at the NTNAMC within one month of symptom onset.

The optimized methods of SYBR green I real-time RT-PCR and conventional RT-PCR were applied to clinical samples from broiler chickens showing clinical signs of IB, including coughing, sneezing, rales, and nasal discharge. From 34 tracheal swabs tested, all samples and positive controls were positive for IBV by SYBR green I real-time RT-PCR targeting the IBV N gene (data not shown and Fig. 8 for a subset of the total swabs tested). However, only 28 of 34 samples were positive for IBV by conventional RT-PCR (data not shown).

Influenza virus, an acute respiratory infectious agent, can rapidly propagate in the upper respiratory tract. It is capable of airborne transmission to other individuals. Prescribing anti-influenza drugs following rapid and accurate diagnosis of an infection is thus critical to mitigate viral spread in the early stage of an outbreak. Many primary care providers use RIDT to diagnose influenza infections because it is simple to use with relatively rapid results. However, the accuracy of this approach is lower than that of qRT-PCR. A highly accurate gene-based diagnostic assay would serve as a valuable local on-site tool if it is easy to use with rapid results. Although many CLIA-waived molecular tests satisfy these criteria due to recent advancement, most of these developed tests are limited to detect seasonal influenza without appropriate subtype discrimination. In this study, we developed a multiplex RT-LAMP method to distinctively diagnose human influenza (e.g., seasonal influenza type B, H1N1, H3N2) and avian influenza viruses infecting humans (e.g., H5N1, H5N6, and H7N9). Hence, this developed method would be essential in some countries where these viruses are co-circulating in humans. Moreover, the RT-LAMP method described here can be used to intuitively detect these viruses using a one-pot colorimetric visualization approach, making it a more feasible POCT.

Our multiplex RT-LAMP detection system was designed to be a more feasible RIDT with accuracy as high as RT-PCR methods for detecting most recent human influenza viruses and avian influenza viruses infecting humans. Although a variety of reliable and affordable RT-LAMP methods have been developed to detect human or avian influenza viruses, each can only detect an individual subtype [23–29, 34, 35] or limited subtypes of human influenza viruses and/or avian influenza viruses infecting humans [18, 30, 36, 37]. In a pandemic, it is critical to rapidly differentiate patients infected by seasonal flu from those infected by an emerging strain. Thus, reliable and affordable multiplex detection tools should be capable of identifying broad-spectrum influenza viruses infecting humans. Moreover, RT-LAMP method for most recent emerging HPAI H5N6 and H5N8 viruses which have high potential to infect humans has rarely been developed or evaluated for its detection efficacy. In addition to broad-spectrum detection of human influenza viruses using our multiplex RT-LAMP assay, we optimized commercially available colorimetric RT-LAMP enzyme. Previous studies have also shown that the colorimetric visualization system using dyes (e.g., SYBR) is detectable by naked eyes. However, these colorimetric methods require additional steps (e.g., adding dye to test for color changes after reaction or the use of a UV device for visualization) that can decrease their utility in resource-limited primary care settings.

RT-LAMP assay is one of promising diagnostics tools that can be utilized to empower disease detection in developing countries as it does not require sophisticated, expensive equipment or trained personnel. The complex design of each specific primer sets made to amplify target sequence with high degree of sensitivity and specificity is one of major recognized constraints of this assay. Recognizing its advantages when detecting RNA genome of viral pathogens, RT-LAMP assay still requires improvement, particularly for the RNA extraction step, to be an efficient and suitable field-based diagnostic tool. Current approaches for RNA extraction of nasopharyngeal swabs or aspirate specimens collected from influenza-suspected patients usually require laboratory processing equipment (e.g., use of centrifuge system and others laboratory devices), technical support, and electricity. Thus, the use of chaotropic salt extraction which does not require centrifugation would improve the feasibility of the developed multiplex RT-LAMP method as it utilizes a syringe with RNA-binding filter method. Furthermore, it has been reported that the RT-LAMP reaction could be performed without RNA extraction step for some RNA viruses. Although our multiplex RT-LAMP assay has not been optimized to directly detect influenza viral RNA without RNA sample preparation, it would significantly increase its feasibility as a diagnostic tool for POCT.

Although RT-LAMP assays designed to detect Victoria lineage Type-B viruses has 10 times less sensitivity compared to one-step RT-PCR and qRT-PCR approaches, RT-LAMP reactions for other subtypes exhibited similar or higher sensitivity without cross-reaction to various human infectious viruses and other subtypes of avian influenza viruses. Furthermore, our multiplex RT-LAMP assay was more sensitive than a conventional RT-PCR approach using clinical or spiked samples. Although the RT-LAMP detection method for H7N9 was optimized and evaluated, single spiked sample for clinical verification would be a limitation of the method. The specificity of multiplex RT-LAMP assay demonstrated in this current study might have limitations for use as a primary detection method based on local epidemiological setting. However, this assay’s rapid detection has a valuable contribution by providing diagnostics specifically in areas with prevalent avian influenza viruses infecting humans.

To further demonstrate assay sensitivity and specificity, 1,011 new specimens were tested in parallel using the multiplex real-time RT-PCR and the WHO-recommended real-time RT-PCR protocol. The results showed that, using the multiplex assay, 192 specimens tested positive for FluA, including 35 specimens that were positive for the H7N9 virus. No co-infections with viruses were found in the multiplex tests, 819 samples tested negative for FluA, and the Ct values of RP gene were all positive with 18.5 ~ 29.6 for 1,011 clinical specimens (Table 5). In comparison, the Ct values of RP gene were also all positive with 18.6 ~ 29.8 in 1,011 samples, 192 specimens tested positive for FluA using the WHO protocol. With respect to the H7 gene, both assays identified 35 positive samples. However, the multiplex assay also detected 35 positive samples with respect to the N9 gene, while the WHO protocol only detected 32 positives, and the Ct value of N9 gene in three patients was high with 36 ~ 38 indicating low titres of the H7N9 virus. In an addition, to demonstrate whether the three N9 samples that differed between the two assays were positive or negative, firstly, we amplified DNA products using N9-specific primers, and DNA sequencing of the cloned products revealed that these three samples were confirmed to be positive. Secondly, the sputum of the three cases was individually re-collected and re-detected again, and the Ct values of the three cases in N9 gene were all less than 34. In the three cases, the Ct values of RP gene were individually 27.5 ~ 28.5 (the early samples) and 26.8 ~ 28.5 (the late samples) in the twice samples. Moreover, 35 patients with confirmed novel H7N9 virus infections accepted hospital treatments and their clinical specimens (including throat swab [135 specimens], sputum or tracheal aspirates [160 specimens], etc.) were tested for the presence and distribution of the H7N9 virus by the multiplex and WHO-recommend real-time RT-PCR assays every day following admission, which contributed to the evaluation of therapeutic treatments received by the patients. Eight fecal samples from the 35 patients tested positive for the H7N9 virus using the multiplex assay, while only seven tested positive using the WHO-recommend assay. No positive results for the H7N9 virus were found in blood (35 specimens), urine (30 specimens), cerebrospinal fluid (one specimen), pleural fluid (one specimen) and bone marrow specimens (two specimens). The results of the throat swabs and sputum or tracheal aspirates specimens from 35 patients with H7N9 infections were shown in Figure 1. The results demonstrate that the H7N9 virus persisted in sputum or tracheal aspirate specimens for an average of 6.14 days, while the virus persisted in throat swab samples for 2.42 days.

A total of 73 influenza-positive (confirmed using a qRT-PCR method) clinical nasopharyngeal aspirate samples collected from patients who demonstrated flu-like symptoms at Chungbuk National University Hospital, Republic of Korea were used for clinical evaluation of the RT-LAMP diagnostic assay developed in this study. In addition, 18 spiked samples in which 104 TCID50/ml of viruses (i.e., H1N1, H5N6, H5N8, and H7N9) were diluted into flu-negative human nasopharyngeal aspirate samples were used for clinical evaluation. RNA was extracted from clinical/spiked samples and subjected to multiplex RT-LAMP and conventional RT-PCR for comparative detection of specific influenza viruses. To verify the specificity of the RT-LAMP assay using clinical samples, 44 RNA samples of human infectious viruses other than those targeting influenza viruses including human enterovirus (HEV), adenovirus (AdV), parainfluenza virus (PIV), human metapneumovirus (MPV), human bocavirus (HboV), human rhinovirus (HRV), human coronavirus 229E (229E), human coronavirus NL63 (NL63), human coronavirus OC43 (OC43), respiratory syncytial virus A (RSVA), respiratory syncytial virus B (RSVB), and Middle East Respiratory Syndrome coronavirus (MERS-CoV) were subjected to multiplex RT-LAMP assay. RNA samples of all other human infectious viruses except MERS-CoV were extracted from clinical swab or nasopharyngeal aspirate samples collected from patients and confirmed positive by qRT-PCR. RNA samples from MERS-CoV grown in cell culture were extracted and diluted in human nasopharyngeal aspirate as additional samples for spiked test.

One hundred and thirty throat swabs or sputum specimens were tested at the Center for Clinical Laboratory of the First Affiliated Hospital of the Medical School of Zhejiang University and the State Key Laboratory for Diagnosis and Treatment of Infectious Diseases. These specimens were determined to be positive for FluA and the H7N9 subtype specifically by tissue culture, and each sample was blindly tested and compared using the multiplex real-time RT-PCR and the WHO-recommended real-time RT-PCR assays. Moreover, tissue culture results were used as the true result for sensitivity and specificity calculations.

The developed microarray for rapid diagnosis of avian viral diseases can be used in mass analysis in the system of routine epidemiological surveillance owing to its ability to test one sample for simultaneous detection of AIV, NDV, IBV and IBDV in cases of single and mixed viral infections. At the same time duration of the analysis decreases many times versus classical methods and the proposed scheme of specimen preparation allows conducting assays immediately in small veterinary laboratories thus avoiding transportation of thermolabile RNA.

The study was conducted in years 2015–2016 under the grant research project (Ministry of Education and Science, Republic of Kazakhstan) “Development and testing of microarray for rapid diagnosis of avian viral diseases”, No. 0920/GF 4.

In conclusion, we have reported an efficient, specific and sensitive assay that has been evaluated on field specimens to be able to detect a wide variety of H5N1 influenza virus isolates. Accurate and sensitive detection of viral RNA is also strongly influenced by the sample type. The rapid one-step single tube reaction described here not only reduce the detection time but also lowers the risk of cross-contamination which has a higher probability in two-steps RT-PCR methods. This cost effective gel-based system has a lower limit of detection in the picogram range which is equivalent to 1 × 103 copies, and is designed to cater for use in the field in regions where real-time PCR platform and equipments may not be available. Clearly, the results would be further strengthened with the inclusion of more known H5N1 influenza in archived samples from humans, but the availability of human samples are difficult due to the low number of human infections at this point. However, observations from this study strongly suggest that the primers are specific for H5N1 which can be very useful for the early detection and monitoring of avian influenza outbreaks.

LFPN, IB, RSPT, ECR conceived the study, its design and coordination, and results analysis. TN, SMN, LVA, SG, HK, TLT and SSH carried out the experiments and analysis. LFPN and ECR drafted the manuscript.

The RPM-Flu assay panel simultaneously addresses 30 different categories of viruses and bacteria that are more or less likely to be encountered in specimens related to respiratory infections (Figure 1, Figure S1). Each assay simultaneously enables detection and identification of targeted pathogens from one or more of 188 different target gene resequencing detector tiles.

For comparison, the recently FDA-cleared Luminex® Respiratory Viral Panel (RVP) Multiplex Nucleic Acid Detection Assay Respiratory Virus Panel (510(k) K063765) is a twelve-plex RT-PCR panel for detection and differentiation of a few strains and types of respiratory viruses including adenovirus, influenza A virus (as A/H1 or A/H3), respiratory syncytial virus, parainfluenza virus, metapneumovirus and rhinovirus (no bacterial respiratory pathogens tested). The RVP assay yields no specimen-specific viral gene sequences that could be used for more detailed identification and differentiation of detected virus strains and variants of detected virus(es).

For human and avian type A influenza viruses in particular, the RPM-Flu provides direct detection and identification capability for any of the combinatorial 144 A/HN subtypes that may be present in a specimen, using an ensemble of 39 different influenza virus target gene resequencing detectors. The Luminex RVP multiplex RT-PCR panel uses three of its twelve primer-probe sets to detect type A influenza virus (M gene-specific primer pair) and distinguish A/H1 from A/H3 (HA1- and HA3-specific primer pairs). While this RT-PCR panel may infer influenza virus subtype, not even testing for the NA component, the assay result is only based on a pattern of signals from a small panel of RT-PCR primer-probes.

Some RT-PCR protocols require serial analysis with hierarchical panels of primer-probes in order to expand the practical multiplicity of pathogen targets or variants. This is the case with the benchmark PCR testing used in this report –a type A influenza virus-positive specimen may be identified in a first round RT-PCR panel, followed by testing with another panel to distinguish A/H1 from A/H3. Failure to establish subtype A/H1 or A/H3 in the follow-on test is a first tier screening results for possible identification of the 2009 Novel A/H1N1 influenza virus outbreak strain. According to the FACT SHEET FOR HEALTHCARE PROVIDERS: INTERPRETING CDC HUMAN INFLUENZA VIRUS REAL-TIME RT-PCR DETECTION AND CHARACTERIZATION PANEL FOR RESPIRATORY SPECIMENS (NPS, NS, TS, NPS/TS, NA1) AND VIRAL CULTURE TEST RESULTS (Authorized by FDA 02 May 2009),

It is not at all clear that such hierarchical RT-PCR protocols, attempting to increase analytical multiplicity through serial testing of the same specimen(s) at different laboratory venues over a period likely longer than a single day can be either efficient or cost-effective. Far more diagnostic intelligence about the patient and specimen would be available in first-day same-day results from a single RPM-Flu test of the original specimen.

Virus binding assays with a low pathogenic AIV (LPAIV) H5N2 (A/H5N2/chicken/Pennsylvania/7659/1985) and human pandemic H1N1 virus (A/H1N1/Virginia/2009) were performed as previously described14. Virus binding assays were performed following strict biosafety level-2 (BSL-2) practices. Briefly deparaffinised tissue sections were incubated with a 250 μl of LPAI H5N2 virus (106 TCID50/ml) or human H1N1 virus (106 TCID50/ml) for 2 h at RT. Tissues incubated with PBS served as mock treated controls. Sections were washed with TBS before blocking with inactivated goat serum for 30 min and immunostained with primary mouse monoclonal antibody to influenza hemagglutinin H5 (ab82455, Abcam) or influenza nucleoprotein (ab20343, Abcam). Following 40 min of incubation with the primary antibody, sections were washed and incubated with a secondary goat anti mouse IgG-Cy5 antibody (ab6563, Abcam). After 40 min of incubation with secondary antibody at RT, sections were washed with TBS and mounted in ProLongGold antifade reagent with DAPI. Following 24 h of curing, the sections were imaged using Olympus FluoView™ FV1000 confocal microscope.

Lectin histochemistry on paraffin embedded tissues was performed as previously described14. Briefly, 5 µm thick tissue sections were deparaffinized in xylene and rehydrated in ethanol. Following 10 min presoaking of the rehydrated sections in Tris-buffered saline (TBS), sections were incubated with biotinylated Maackia amurensis (MAAII) and FITC labelled Sambucus Nigra (SNA) lectins specific to SA α2,3-Gal and SA α2,6-Gal receptor respectively each at a concentration of 10 μg/ml, overnight at 4°C. Both lectins were purchased from Vector laboratories (Burlingame, CA, USA). Following three washes with TBS, sections were incubated with Streptavidin Alexafluor 594 conjugate for 2 h at 4°C. The sections were washed three times with TBS and mounted in ProLongGold antifade reagent with nuclear stain 4′,6-diamino-2-phenylindole, dihydro-chloride (DAPI). Negative controls were performed omitting the primary reagents. Following 24 h of curing at room temperature (RT), sections were imaged using Olympus FluoView™ FV1000 confocal microscope. Settings of the confocal microscope were determined using negative controls to avoid any background fluorescence and the same settings were used to scan all the other sections for consistency. To rule out nonspecific binding of the lectins and IAVs, control tissue sections were treated, prior to lectin staining or virus binding, with Sialidase A (N-acetylneuraminate glycohydrolase; Prozyme, San Leandro, CA), for 2 h at 37°C, which cleaves all non-reducing terminal sialic acid residues in the order α(2,6) > α(2,3) > α(2,8) > α(2,9). Sialidase treated and control sections were further subjected to lectin staining or virus binding.

Influenza infections do not necessarily preclude co-infection of the same individual by other viral and/or bacterial pathogens (including other strains of influenza virus). Such co-infections may confound a simple diagnosis of influenza, and may also compound morbidity and mortality for a co-infected patient. However it is not common practice that a positive result from a routine diagnostic influenza test would be followed by a call for one or more tests of possible secondary agents of infection. The single specimen single aliquot RPM-Flu assay simultaneously targets 30 different categories of viruses and bacteria, and these were each selected as a respiratory pathogens reported to cause “flu-like” symptoms at some stage of human infection.

Implementation of a highly multiplexed differential diagnostic assay as RPM-Flu would be of great advantage in anticipation of highly likely future influenza outbreaks, epidemics and pandemics. For many decades, leaders in the area of influenza epidemic and pandemic risk assessment and outbreak management have reviewed the pathology of influenza with respect to rapid deterioration and patient deaths attributable to secondary bacterial infections and pneumonia,.

It has taken almost twenty-five years from the first research reports of the polymerase chain reaction until recent regulatory clearances have enabled introduction specific PCR- and RT-PCR-based diagnostic assays and instrumentation into clinical practice. Such a traditional regulatory clearance timeline may assure safety and efficacy of diagnostic devices beyond imaginable limits of liability, but it also assures that best practice standards for clinical diagnostics are constrained to technologies that perform with nearly obsolete capabilities and specifications. Within the last decade resequencing microarrays have been introduced for research applications and clinical research,,. It would be disappointing to anticipate that another decade or more may pass before benefits from diagnostic implementations of RPM or other highly multiplexed sequencing-based platforms can be realized in routine clinical diagnostics.

The study protocol and informed consent documents were reviewed and approved by the Ethics Committee of the First Affiliated Hospital, College of Medicine, Zhejiang University (ZJU Hospital), the Shanghai Public Clinical Health Center (SPHCC), the Nanjing Municipal Centers for Disease Control and Prevention (Nanjing CDC), the Zhejiang Provincial Centers for Disease Control and Prevention (Zhejiang CDC), the Guangdong Provincial Centers for Disease Control and Prevention (Guangdong CDC), the Guangzhou Municipal Centers for Disease Control and Prevention (Guangzhou CDC) and the Jiangsu Provincial Centers for Disease Control and Prevention (Jiangsu CDC). Written informed consent for the research use of clinical samples was obtained from all of the patients involved in the study.

The results of the three clinical trials are shown in Table 5. Among 294 clinical samples that were determined to be positive using the WHO-CNIC assay, 291 were also found to be positive using the Liferiver assay, and all the known negative samples were correctly identified as negative. Thus, the positive and negative agreement values were 99% and 100%, respectively. For the DAAN assay, the positive and negative agreement values were 98.5% (403/409) and 99.9% (876/877), respectively, with respect to the results of the WHO-CNIC assay. The six positive samples with results that were discordant between the DAAN assay and the reference assay were all throat-swab samples; they were subjected to RT-PCR and sequencing for confirmation. Among these discordant samples, three showed negative results in the RT-PCR and sequencing analysis, indicating that there was an extremely small amount of target RNA in these samples. Both the positive and negative agreements were 100% for the Puruikang assay.

Five panels of sera were evaluated in this study and were all provided by the FAO-OIE and National Reference Laboratory for Newcastle disease and Avian influenza, Istituto Zooprofilattico delle Venezie. Panel 1 consisted of 10 sera positive for antibodies to the LPAI H5N2 vaccine strain (A/chicken/Mexico/232/94/CPA) obtained from chickens vaccinated at 21 days of age and boosted after 3 weeks with a commercially available inactivated vaccine, which has been used in previous studies [12, 15]. Panel 2 consisted of 10 sera positive for H7 collected from turkeys during an Italian outbreak caused by an LPAI virus H7N3. Panel 3 consisted of 10 sera positive for H5 with stratified incremental HI titers (ranging from 1 : 4 to 1 : 2048) collected from chickens vaccinated with an inactivated adjuvanted H5N2 vaccine and were used for comparative firefly luciferase and GFP-pseudotype neutralization assays. In order to test for influenza HA group-specific virus neutralization, panel 4, consisting of 16 reference hyperimmune sera produced against 16 influenza subtypes (from Group 1 and Group 2), was also provided. These antisera (H1N1, H2N3, H3N8, H4N8, H5N1, H6N2, H7N3, H8N4, H9N2, H10N1, H11N9, H12N5, H13N6, H14N5, H15N9, and H16N2) were produced in specific pathogen-free chickens by inoculation with viruses (inactivated by beta-propriolactone if HPAI viruses) as described previously. A panel of 41 negative sera (panel 5) confirmed by agar gel immunodiffusion assay (AGID) and Enzyme-linked immunosorbent assay (ELISA) was also employed.

Caprine arthritis encephalitis virus (CAEV) is a member of the lentivirus family (in small ruminants) leading to chronic disease of the joints and rarely encephalitis in goat kids under the age of six months. The virus is in close intimation with white blood cells. Thus, any kinds of body secretions containing blood cells are potential sources for virus spread to other animals in the herd [141, 142]. In goats,in order to detect caprine arthritis encephalitis virus (CAEV), serological tests or cell cultures are mainly used. Besides, PCR has also been developed for detection of CAEV sequences from peripheral blood mononuclear cells (PBMC), synovial fluid cells (SFC), and milk cells (MC) from the infected goats. This type of PCR assay especially provides a useful method to detect CAEV infection in goats [66–68]. A two-step TaqMan quantitative (q) PCR, which is specific as well as sensitive for the detection of infection due to CAEV by the use of a set of primers (specific), and a TaqMan probe that targets a region which is highly conserved within the gene that encodes the capsid protein of the virus have been developed. In the total deoxyribonucleotide (DNA) extracts, the proviral DNA can be detected successfully by this assay. The TaqMan qPCR assay provides a fast as well as specific and sensitive means for detection of proviral DNA of the virus and thereby proves to be useful for detection in large scale for eradication programs as well as epidemiological studies.

PCR techniques have been standardizedin several laboratories for the detection of proviral DNA. Other molecular techniques such as cloning and sequencing are also used to provide knowledge on a country or region's specific strain of CAEV. Phylogenetic analyses of the proviral DNAs of CAEV throughout the world have given the suggestion that in certain areas CAEV causes natural infection not only in goats but also in sheep. In order to track the transmission of the disease in near future, phylogenetic analyses may be used [66, 69, 70]. Molecular techniques such as cloning and sequencing are also used to provide knowledge on the prevalence of specific strain of CAEV in a country or a region which may have influence on serological assay as well as corresponding CAEV antigen [33, 71].

Parainfluenza is mainly characterized at necropsy by purulent bronchopneumonia (focal) along with moderate to severe pulmonary congestion. Histopathological analysis has revealed the presence of acute and severe as well as diffuse necrotizing and fibrinous or suppurative bronchopneumonia. There is also a presence of diffuse congestion as well as pulmonary edema. As a diagnostic method, comparison of enzyme immunoassay has been done with complement fixation test (CFT). The cross-reactivity of the viruses can be detected by the application of such tests. Parainfluenza is a viral infection of the lower respiratory tract causing an enormous burden of disease in small ruminants. Direct immunofluorescence technique along with cross-neutralization tests is required for antigenic analysis of the parainfluenza virus isolates. For detection of the virus associated with it, new diagnostic test like multiplex PCR has got enormous advantages mainly because of its specificity. Real-time PCR (RT-PCR) is a useful molecular tool for detection of parainfluenza virus type 3 (Pi3) from ribonucleic acid (RNA) samples from cells of the lungs from the slaughtered animals. This is followed by sequencing as well as restriction enzyme patterns of the fragment amplified of the F gene which confers confirmation of the distinctness of the isolates. Availability of suitable PCR primers allows detection of the ovine virus specifically. Phylogenetic analysis of the amino acid as well as the nucleotide sequences is also equally important. In some of the instances, it has been seen that the in-house RT-PCR methods cannot yield expected products for which the nucleotide sequence analysis has been initiated. Multiplex RT-PCR can help distinguish parainfluenza viruses from other respiratory virus like adenovirus. Nucleic acid sequence based amplification (NASBA) has been developed for which primers as well as probes have been selected from the haemagglutinin-neuraminidase (HN) gene as well as from the phosphoprotein (P) of the parainfluenza virus [61, 65].

Measurement of Neutralizing Antibodies Using GFP and Firefly Luciferase HPAI H5 Pseudotypes. In order to determine the reliability and the applicability of the pp-NT assay using different reporter systems, H5 A/Vietnam/1194/04 and A/chicken/Egypt 1709-1/2007 pseudotypes carrying the GFP were tested against a panel of sera positive by HI with incremental titers ranging from 1 : 8 to 1 : 2048. Three sera (3929-1, 3929-9, and 3929-6) were scored as 100% neutralization activity with pp titers >1 : 1280 (no GFP expression was observed) and sera 3930-19, 3931-26, and 3930-20 showed 50% neutralization activity at 1 : 80, 1 : 160, and 1 : 320 when tested against A/Vietnam/1194/04. IC50 values corresponding to titers around ≤1 : 40 were obtained for sera with HI titers lower than 1 : 32. For A/chicken/Egypt 1709-1/2007 pseudotypes, a similar pattern was observed: 4 sera (3929-1, 3929-9, 3929-6, and 3930-19) have shown complete neutralization; for 3 sera (3931-26, 3930-20, and 3933-41) 50% neutralization activity was scored between 1 : 640 and 1 : 1280. For 2 sera (3933-42 and 3933-50) percentage values of 50% lay between 1 : 80 and 1 : 320.

In order to support the quantitative results obtained using GFP-pseudotypes, the panel of sera was tested in parallel against firefly luciferase HA pseudotype. pp-NT results were found to correlate strongly with HI showing a similar neutralization profile; however, for sera with an HI titre lower than 1 : 32, it has not been possible to determine the respective pp-NT neutralization values when H5 A/Vietnam/1194/04 pseudotypes have been used (Table 2).

Viral-induced cytotoxicity was determined by measuring adenylate kinase activity in apical washes using a commercially available assay (Lonza, Inc.). Apical samples were centrifuged prior to freezing to remove any cellular contaminants present in the wash. Luminescence detected in samples from infected HAE were normalized to uninfected HAE and expressed as fold change over AK measured in uninfected (mock) HAE. Morphological assessment of cytotoxicity in HAE was performed with paraformaldehyde (PFA, 4%)-fixed histological sections (5 µm) stained with hematoxylin and eosin.

Haptoglobin concentrations were determined by a sandwich ELISA as described previously. The detection limit was 66 μg/ml.

A commercially available sandwich ELISA assay (Phase SAA assay, Tridelta Development Ltd., Kildare, Ireland) was used for determination of Serum Amyloid A (SAA) concentrations. This assay was based on anti-human monoclonal antibodies in a sandwich set-up as originally described by McDonald et al.[39]. The detection limit of the assay was 125 μg/ml (porcine SAA equivalents).

C – Reactive Protein (CRP) was analyzed by a sandwich type ELISA using dendrimer-coupled cytidine diphosphocholine (a CRP-binding ligand) in the coating layer as previously described employing polyclonal rabbit anti-human antibodies with cross-reactivity towards porcine CRP followed by peroxidase-conjugated goat anti rabbit antibody for detection (both antibodies from DAKO, Glostrup, Denmark). The cross-reactivity of the anti human CRP antibody with porcine CRP was demonstrated previously. The detection limit was 1416 ng/mL (human equivalents).

Blood was drawn from the metatarsal or brachial vein and aliquoted into additive-free, EDTA, and lithium heparin blood collection tubes (Becton Dickinson, Franklin Lakes, NJ). Samples were kept cool on ice for transport back to the University of California, Davis where they were aliquoted and stored at -80°C until shipment to the diagnostic laboratories. Serological testing for avian adenovirus, Chlamydophila psittaci, infectious bronchitis virus (IBV) (Arkansas (Ark), Connecticut (Conn) and Massachusetts (Mass) strains), Mycoplasma gallisepticum, Mycoplasma synoviae, avian paramyxovirus-1 (Newcastle Disease virus), avian paramyxovirus-2, avian paramyxovirus-3, and avian reovirus was performed at Texas Veterinary Medical Diagnostic Laboratory (College Station, TX) using assays optimized for poultry species. Because these assays have not been validated in condors, vultures, or eagles, we relied on cut-off titers established for poultry. Exposure to avian adenovirus was evaluated using an agar gel immunodiffusion (AGID) test. Chlamydophila psittaci exposure status was determined by a direct complement fixation (DCF) assay. Exposure status for the three strains of IBV was determined by hemagglutination inhibition (HI) assay. A titer of 1:16 or greater was considered positive for exposure to IBV. Mycoplasma gallisepticum exposure status was determined using serial tests. Samples were initially screened using a plate agglutination assay. Any samples that were positive on the first assay were then tested by HI. A titer of 1:80 or above on the HI assay was considered positive. A sample testing positive on both the plate agglutination test and HI assay was considered positive for exposure to M. gallisepticum. Mycoplasma synoviae exposure status was determined in the same manner as M. gallisepticum. Avian paramyxovirus 1, 2 and 3 exposure status was determined by HI. A titer of 1:16 or greater on the HI assay was considered positive for each of the three paramyxoviruses. Avian reovirus exposure status was determined using AGID. The number of individuals tested varied between pathogens due to limitations in sample volume.

Serological testing for Toxoplasma gondii was performed at University of California, Davis (Department of Pathology, Microbiology, and Immunology, University of California-Davis) using a T. gondii agglutination test kit (Eiken Chemical Co., LTD. Tokyo, Japan, distributed by Tanabe USA, Inc., San Diego, CA). Manufacturer’s recommendations were followed, and a titer of 1:32 or greater was considered positive. Testing for arboviruses common to California was performed at the Center for Vectorborne Diseases (University of California-Davis). West Nile virus titers were determined using an indirect enzyme immunoassay (EIA) as previously described [75–78]. West Nile Virus cross reacts with St. Louis encephalitis virus (SLEV) and Western equine encephalitis virus on EIA, so EIA positive samples were tested using end-point plaque neutralization (PRNT) assays using the NY99 strain of WNV and the Kern 217 strain of SLEV. The PRNT assays were performed using >75 plaque forming units of virus grown on Vero cell culture. To be considered positive, sera had to neutralize >90% of the virus in at least a 1:4 dilution. Sera from free-flying California condors were also evaluated for active WNV infection by real time-polymerase chain reaction (rt-PCR) using primers as previously described [79, 80]. Additionally, free-flying condors that had a high WNV titer (≥ 1:256) were also screened for active WNV infection by PCR on whole blood (n = 9).

Liver and lung tissue samples collected from 14 condors that died of various causes between 1997 and 2009 were also selected for detection of a subset of potential pathogens. These 14 condors had all been in captivity and in the wild at different points in their lives. Representative cases were selected for analysis based on availability of frozen tissues at San Diego Zoo Safari Park and degree of autolysis. Tissue samples were screened for presence of avian adenovirus, coronavirus (including infectious bronchitis virus), paramyxovirus and Mycoplasma spp. DNA from tissues was extracted using the DNeasy Blood and Tissue kit (Qiagen, Valencia, CA, USA) following the manufacturer’s protocol. RNA was also extracted from the lung tissue samples using the QIAamp cador Pathogen Mini kit (Qiagen, Valencia, CA, USA) following the manufacturer’s guidelines. PCR primers were synthesized by Integrated DNA Technologies (San Diego, CA, USA) and were utilized in nine assays using the following primer pairs: adenovirus primers AdenokissF/AdenokissR, coronavirus primers Corona8pF/Corona7mR, IN-2F/IN-4R, Cor-p-F2/ Cor-p-R1 and Cor-p-F3/ Cor-p-R1, paramyxovirus primers NCD-3/NCD-4[84], ParamyxoP1/ParamyxoPR and ParamyxoP2 /ParamyxoPR and Mycoplasma spp. primers MycogenusF/MycogenusR and MycaldP/CapaldM. Positive controls were available for adenovirus and Mycoplasma spp.