ABSTRACT
ABSTRACT
Chicken infectious anaemia virus (CIAV) is a widely distributed immunosuppressive agent. SPF flocks and eggs used for vaccine production and diagnostics must be CIAV-free. Detection of CIAV infection in SPF flocks involves primarily serology or other invasive methods. In order to evaluate different types of samples for rapid detection of CIAV infection, a trial was conducted in serologically negative broiler breeder pullets vaccinated with a commercial live-attenuated CIAV vaccine. Controls and vaccinated groups were sampled before and after vaccination. Invasive and non-invasive samples were used for CIAV DNA detection by real-time PCR. Seroconversion occurred at 14 days post-inoculation (DPI) in the vaccinated group, whereas CIAV genome was detected by qPCR at 7 DPI in both invasive and non-invasive samples. Only invasive samples remained qPCR positive for CIAV DNA by 21 DPI despite seroconversion of the chickens.
Introduction
Chicken infectious anaemia virus (CIAV) is a relatively small, non-enveloped, icosahedral virus that contains a single-stranded, circular 2.3 kb DNA genome and belongs to the gyrovirus genus, recently reassigned to the Anelloviridae family (Rosario et al., 2017). CIAV was first recognized in Japan and is the causative agent of chicken infectious anaemia. The virus is highly stable and resistant to physical and chemical agents and is considered ubiquitous worldwide (Yuasa et al., 1979; Schat & van Santen, 2008). The disease primarily affects young birds and is characterized by aplastic anaemia resulting from infection of haemocytoblasts, which may result in depletion of erythrocyte, granulocyte and thrombocyte progenitor cells (Taniguchi et al., 1983). CIAV-induced immunosuppression may develop from infection of T lymphocyte precursor cells, which in turn results in depletion of functional T cells (Adair, 2000). The virus also infects non-lymphoid cells, which is relevant for persistence in the reproductive tract and for vertical transmission (Cardona et al., 2000b).
CIAV infection in commercial chickens is frequently complicated by secondary or opportunistic viral, bacterial or fungal infections (Goryo et al., 1987). The economic impact of subclinical CIAV infection on broiler flock performance represents a major threat for the poultry industry mainly due to decreased body weight and increased feed-conversion at processing (McIlroy et al., 1992).
The ability of the virus to spread vertically was suggested in 1983 (Yuasa & Yoshida, 1983) and reported in 1992 (Hoop, 1992). Vertical transmission occurs when hens are infected during the laying period and thus may produce infected chicks without CIAV maternal antibody. Maternal antibodies are protective for chicks against infection, and thus a common practice is to vaccinate pullets with live-attenuated vaccines before the onset of egg production (Yuasa et al., 1980; Otaki et al., 1992; Fussell, 1998). However, it has also been reported that CIAV can persist in the reproductive tract of pullets long after seroconversion (even in the absence of virus in spleen) and the antibodies against CIAV do not prevent vertical transmission (Cardona et al., 2000b; Brentano et al., 2005).
Establishing and maintaining CIAV seronegative source flocks used for the production of veterinary biologics is extremely challenging due to the ubiquitous nature of CIAV even under high biosecurity filtered-air and positive-pressure houses (Cardona et al., 2000a). SPF flocks are not required to be serologically monitored for CIAV according to the Veterinary Service Memorandum (VSM) No. 800.65 (https://www.aphis.usda.gov/animal_health/vet_biologics/publications/memo_800_65.pdf). Although CIAV is not listed in the Agents of Concern, SPF source flocks are required to report CIAV outbreaks following procedures described in VSM No. 800.65. Regulatory agencies in the United States and Europe require screening of avian vaccines for the detection of extraneous viral agents including CIAV (United State Department of Agriculture, 2008; European Pharmacopoeia 7.0, 2010). Very possibly, the most common reasons for SPF source flocks being disqualified include accidental infection with CIAV, avian orthoreovirus or fowl adenovirus.
Following regulations, SPF source flocks that have seroconverted to CIAV are discarded to use for the production of human vaccines and for poultry vaccines intended for applications in birds younger than 3 weeks of age (Miller & Schat, 2004). CIAV infection and subsequent seroconversion have been reported after the onset of sexual maturity in SPF flocks in spite of the adoption of strict biosecurity guidelines (McNulty, 1991; Cardona et al., 2000b). It has been proposed that a well-adapted relationship between the host and pathogen exists when conditions of low-stress, low challenge dose of virus and flocks with chronic infection are met. Under these conditions, CIAV may evade immune system detection until hormonal activity, related with the onset of laying, possibly allows for reactivation of virus replication thus permitting transmission of virus to the progeny (Miller & Schat, 2004).
Furthermore, virus isolation in cell culture or in embryonating eggs is considered the gold standard procedure for CIAV detection and has been used routinely, but it is tedious, expensive and time-consuming (Yuasa et al., 1979; McNulty et al., 1989). In addition, numerous methods for the detection of CIAV infection have been reported including immunofluorescence and immunoperoxidase staining in situ hybridization and ELISA, but the time required for sample processing may be extensive (Hoop & Reece, 1991; McNeilly et al., 1991; Sander et al., 1997; Toro et al., 2006). In addition, specificity, sensitivity and cost may limit the use of these methods. PCR techniques provide a relatively inexpensive and fast alternative with high sensitivity and specificity, including nested and conventional PCR, strain-specific real-time PCR for quantitation and real-time quantitative PCR-based serum neutralization (Imai et al., 1998; Cardona et al., 2000b; Markowski-Grimsrud et al., 2002; van Santen et al., 2004b; Haitham et al., 2011). The latter has been developed recently for the detection and quantification of CIAV genomes (van Santen et al., 2004b). PCR techniques have been used to detect CIAV DNA in different types of tissue samples including the thymus, caecal tonsils, Harderian gland, reproductive tissues, spleen and infected cell lines (Cardona et al., 2000b; Markowski-Gimsrud et al., 2002; van Santen et al., 2004a; Joiner et al., 2005). Because most types of clinical samples used conventionally for the detection of CIAV infections are invasive and require direct contact with birds for sampling, they pose a biosecurity risk for SPF flocks.
In this study, an experimental infection in broiler breeder pullets was performed in order to analyse the CIAV infection dynamic around the time when hens mature sexually. The study consisted of measuring the viral load in different types of samples at three time points. The aim was not only to provide information on viral pathogenesis but also to obtain data that can be useful for the election of samples that might be utilized in a rapid detection method, particularly for monitoring SPF flocks.
Materials and methods
Experimental inoculation, and sample collection
Twenty-three-week-old Cobb-500 FF broiler breeders were obtained from Dr. Jeanna Wilson, Department of Poultry Science, the University of Georgia after having tested negative for the presence of CIAV-induced antibodies using a commercially available enzyme-linked immunosorbent assay (IDEXX CIAV Ab Test, IDEXX Laboratories, Westbrook, ME, USA). The chickens were separated into two groups consisting of 40 hens and eight males each. Both groups were maintained in isolated rooms at the Poultry Diagnostic and Research Center with food and water provided ad libitum throughout an experimental period that lasted four weeks post-CIAV infection. Group 1 served as uninfected controls. All the birds in group 2 were inoculated at 23 and 24 weeks of age by the intramuscular (IM) route (0.5 ml) and oral route (1 ml), respectively, each time with a full dose of a commercial modified live CIAV vaccine (Circomune® CEVA-Biomune, Lenexa, Kansas Serial No: 333-047, Del-Ros strain). Immediately before vaccination and on days 7, 14 and 21 after vaccination, blood, cloacal swabs and organ/tissue samples were collected from 10 birds from each group. Environmental (boot swabs) samples were taken during each sampling period from both groups. Males were kept commingled with the females until the last sampling date. Samples were collected and identified individually by bird to check any inconsistency in the ELISA antibody titres and virus detection results in the various organs. The cloacal swabs were taken before euthanasia and placed in 1 ml sterile phosphate-buffered saline solution containing 2% antibiotic–antimycotic 100× (Gibco, Grand Island, NY, USA) and 2% new-born calf serum (Gibco, Grand Island, NY, USA). Tissue samples were collected aseptically and stored at −80°C until further use.
Serology
CIAV antibodies were determined using a commercially available CIAV antibody ELISA kit according to the manufacturer's recommendations (IDEXX CAV Ab Test, IDEXX Laboratories, Westbrook, ME, USA). The CIAV 100 dilution method was conducted according to the manufacturer's guidelines in order to obtain a quantitative result for CIAV antibodies.
Nucleic acid extractions
Thymus, liver, spleen, bone marrow, feather pulp, blood, ovary/testis, cloacal and boot swab samples were stored at −80°C for subsequent CIAV DNA isolation, nucleic acid extraction and qPCR. The frozen organ samples were thawed immediately before homogenization and total DNA was extracted using the MagaZorb® DNA extraction mini-prep 96-well kit (Cortex Biochem™, San Leandro, CA). Briefly, 50 µl of sample was incubated with 5 µl of Proteinase K and 50 µl of Lysis Buffer at 56°C for 10 min in a 96-well plate. Ten microlitres of MagaZorb Reagent (beads) was added along with 125 µl of binding buffer to each well and incubated for 10 min at room temperature. The supernatants were separated from the beads (with the DNA on their surfaces) by means of a magnetic frame, and the beads were washed twice with washing buffer. Finally, the extracted DNA was eluted in 100 µl of elution buffer and stored at −80°C until further use. Additionally, total DNA was extracted from cloacal and boot swabs using the High Pure PCR Template Preparation Kit from Roche (Roche Diagnostics®. Indianapolis, IN) following the instructions of the manufacturer.
Real-time quantitative PCR (qPCR)
The CIAV DNA was amplified using a qPCR procedure. Briefly, CIAV real-time PCR was performed in a duplex assay along with an internal control (host DNA). Primers and probe used for the detection of CIAV (Table 1) were as described elsewhere (van Santen et al., 2004b). The primers amplify a 181 bp fragment of the CIAV VP2 open reading frame, including the 3′ end of the overlapping VP3 open reading frame and extending 11 bp into the overlapping VP1 open reading frame. As an internal control (housekeeping gene), a 76 bp region of the chicken (Gallus gallus domesticus) α2-collagen gene was amplified using primers and probe (Table 2), as previously described (Islam et al., 2004). The amplification of the housekeeping gene along with the viral DNA (target) allowed us to control the DNA extraction procedure as well as the amplification reaction (Livak & Smchmittgen, 2001). The amplification of both the target and the internal control was performed in a duplex reaction, which was set up to a final volume of 20 µl as follows: 12.5 of 2× master mix (Quanti Tect multiplex PCR non-ROX kit; Qiagen, Valencia, CA); all primers were diluted to a final concentration of 12.5 µM; CIAV and collagen probes to a final concentration of 2.5 and 1 µM, respectively; 0.05 U/µL of Uracil (Epicentre, Madison, WI); and 5 µl of DNA template. The thermal cycling profile used was 50°C for 2 min, 95°C for 2.5 min, 45 cycles of 94°C (denaturation) for 15 s and 55°C (annealing/extension) for 45 s.
Table 1. Real-time qPCR primers and probes as reported in van Santen et al. (2004b).
Table 2. α2-collagenª (Gallus gallus domesticus) primers and probes.
In order to quantify the viral load of each sample, a standard curve was made using purified DNA from CIAV vaccine cloned into the pCR-XL-TOPO vector (Invitrogen, Carlsbad, CA) following the manufacturer's directions. The solution of purified plasmid with the inserted amplicon (verified by DNA sequencing) was measured in a spectrophotometer NanoDrop 2000 (Thermo Scientific, Wilmington, DE, USA), and dilutions with a known number of molecules of plasmids (copy number/μl) were prepared. Then, the dilutions were run (as indicated above) in triplicate and the results were used to plot a standard curve. From the standard curve a linear equation (y = −3.29x + 50.97 with R2 = 0.98) was obtained, which was used to quantify the viral target in each sample (data shown in the supplementary material). The efficiency of the reaction (E = 10(−1/slope) − 1) was calculated as 1.01. The ability of the qPCR to detect low amounts of template dropped dramatically over a Ct of 41. The analytical sensitivity (as the minimum amount of template detected) was 2 femtograms (fg) per µl, which is equivalent to 5.1 × 102 molecules (supplemental material). The amount of DNA in each sample was measured by spectrophotometer (NanoDrop 2000, Thermo Scientific, Wilmington, DE, USA), and it was used for the quantitation of viral load, which was expressed as copy number per microgram of template DNA.
Statistical analysis
Differences in viral DNA detection and antibody titres were examined statistically by One Way Analysis of Variance (ANOVA). The Tukey’s multiple comparison test was used to compare groups with significant differences (P < 0.05) using the Minitab® Statistical Software (www.minitab.com). When two groups were analysed, the unpaired t tests were performed.
Ethical statement
All bird experiments conducted in this study (Project # 10-31-DM188-002) were performed under the Animal Use Proposal A2008 10-229-A1 approved by the Animal Care and Use Committee (IACUC) in accordance with regulations of the Office of the Vice President for Research at the University of Georgia.
Results
Serology
All broiler breeder chickens (80 females and 16 males) were seronegative for CIAV-specific antibodies one week before being moved to isolated rooms at the Poultry Diagnostic and Research Center. The negative serology was confirmed two days before inoculation with the CIAV vaccine in the isolated units (Figure 1). As expected, ELISA antibodies against CIAV were detected in all the samples taken at 14 days post-inoculation (DPI) with the CIAV vaccine in the inoculated group, whereas the non-infected control group remained serologically negative throughout the experimental period (Figure 1).
Published online:
25 July 2018Figure 1. CIAV antibody titres of broiler breeder pullets, before and after inoculation at 23 weeks of age with a commercial modified live CIAV vaccine by IM route, detected using a commercial ELISA. Values under 0.2 and above 0.59 of s/n ratio must be considered positive (protected) and negative (un-protected), respectively (according to the manufacturer’s indications).
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/cavp20/2018/cavp20.v047.i05/03079457.2018.1492089/20180921/images/medium/cavp_a_1492089_f0001_ob.jpg"})
Figure 1. CIAV antibody titres of broiler breeder pullets, before and after inoculation at 23 weeks of age with a commercial modified live CIAV vaccine by IM route, detected using a commercial ELISA. Values under 0.2 and above 0.59 of s/n ratio must be considered positive (protected) and negative (un-protected), respectively (according to the manufacturer’s indications).
Published online:
25 July 2018Figure 2. Viral load (measured as log10 of copy number per µg of template) in organ tissues. (A) CIAV vaccine virus loads at 7 DPI. (B) CIAV vaccine virus loads at 14 DPI. (C) CIAV vaccine virus loads at 21 DPI. The dotted line in the graphs indicates the negative cut-off (102 copies), which is related to the analytical sensitivity of the test. Superscripts on the boxes indicate whether the differences were statistically significant. The repetition of symbols (*) indicates the degree of significance of the amount of CIAV quantified in the tissue. The analysis was performed with Tukey’s multiple comparison test (P < 0.05).
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/cavp20/2018/cavp20.v047.i05/03079457.2018.1492089/20180921/images/medium/cavp_a_1492089_f0002_ob.jpg"})
Figure 2. Viral load (measured as log10 of copy number per µg of template) in organ tissues. (A) CIAV vaccine virus loads at 7 DPI. (B) CIAV vaccine virus loads at 14 DPI. (C) CIAV vaccine virus loads at 21 DPI. The dotted line in the graphs indicates the negative cut-off (102 copies), which is related to the analytical sensitivity of the test. Superscripts on the boxes indicate whether the differences were statistically significant. The repetition of symbols (*) indicates the degree of significance of the amount of CIAV quantified in the tissue. The analysis was performed with Tukey’s multiple comparison test (P < 0.05).
Virus detection and quantification by qPCR
Results for CIAV genome detection were included in the analysis only when the housekeeping gene (α2-collagen) DNA was successfully amplified. This ensured successful DNA extraction and amplification for CIAV. Viral genome was detected in the majority of the invasive samples (thymus, liver, spleen and bone marrow), and in feather pulp (which was considered a non-invasive type of sample), throughout an experimental period of 21 days by qPCR. The CIAV genome distribution in hens from both groups is shown in Table 3. As seen in this table, at 7 DPI CIAV genomes were detected by qPCR in 10/10 (100%), 6/9 (67%), 9/9 (100%), 9/9 (100%) and 8/10 (80%) of the thymus, liver, spleen, bone marrow and feather pulp samples respectively, while seroconversion was not detected at this time point (Figure 1). CIAV genomes were not detected in other types of samples like plasma, cloacal swabs or environmental samples from any of the hens tested at 7 DPI (Table 3). At 14 DPI, CIAV genomes were detected in 10/10 (100%), 6/9 (67%), 4/8 (50%), 9/10 (90%) and 7/10 (70%) of the thymus, liver, spleen, bone marrow and feather pulp samples, respectively, of vaccinated hens (Table 3). Seroconversion against CIAV was detected in all (100%) of the hens at 14 DPI with the live CIAV vaccine. CIAV genome was not detected in plasma, cloacal swabs or environmental samples from hens tested at 14 DPI when the MagaZorb® DNA extraction mini-prep 96-well kit (Cortex Biochem™, San Leandro, CA) was used. However, when the DNA from cloacal swab samples was obtained using the High Pure PCR Template Preparation Kit from Roche (Roche Diagnostics®, Indianapolis, IN), a significantly higher viral load was observed in the vaccinated group when compared with the non-vaccinated group at 7 DPI (Figure 3(A)) and at 14 DPI (Figure 3(B)). At 21 DPI, CIAV genome was detected only in the thymus and spleen, 9/10 (90%) and 5/9 (56%), respectively, in the vaccinated group which was strongly seropositive (Figure 1). None of the non-invasive samples were positive for CIAV DNA at 21 DPI. Samples of ovaries and testicles were obtained only at 21 DPI, whereas plasma, cloacal swabs and environmental samples were taken throughout the experimental time. CIAV DNA was not detectable in either the ovaries or testicles when sampled at 21 DPI, or in plasma or environmental swabs sampled throughout the experimental period.
Published online:
25 July 2018Figure 3. Viral load (as log10 of copy number) in cloacal swabs. (A) CIAV vaccine virus loads at 7 DPI from non-vaccinated (NVx) and vaccinated (Vx) groups. The means between groups, analysed by unpaired t test, were significantly different (P = 0.0039). (B) CIAV vaccine virus loads at 14 DPI from non-vaccinated (NVx) and vaccinated (Vx) groups. NVx group was significantly different from the Vx group (P = 0.0002). Superscript (*) indicates whether the difference between groups was statistically significant (P < 0.05). Statistical analysis was performed with unpaired t test.
![]({"type":"image","src":"/na101/home/literatum/publisher/tandf/journals/content/cavp20/2018/cavp20.v047.i05/03079457.2018.1492089/20180921/images/medium/cavp_a_1492089_f0003_ob.jpg"})
Figure 3. Viral load (as log10 of copy number) in cloacal swabs. (A) CIAV vaccine virus loads at 7 DPI from non-vaccinated (NVx) and vaccinated (Vx) groups. The means between groups, analysed by unpaired t test, were significantly different (P = 0.0039). (B) CIAV vaccine virus loads at 14 DPI from non-vaccinated (NVx) and vaccinated (Vx) groups. NVx group was significantly different from the Vx group (P = 0.0002). Superscript (*) indicates whether the difference between groups was statistically significant (P < 0.05). Statistical analysis was performed with unpaired t test.
Table 3. Tissue distribution of CIAV in hens from both groups at different time points before and after vaccine inoculation.
The average CIAV viral load in various organs is summarized in Figure 2(A–C). Overall, there was a significantly higher viral load in the tissues at 7 DPI (P < 0.05) compared with 14 or 21 DPI. Individually, the viral loads in the thymus, spleen and bone marrow samples were the highest (P < 0.05) when compared with any other organs at 7 DPI (Figure 2(A)). The highest viral loads were observed in the thymus and spleen samples throughout the experimental period (P < 0.05). CIAV viral load in the bone marrow and liver samples was lower at 7 than at 14 DPI (P < 0.05); however, CIAV DNA was undetectable at 21 DPI in the same tissues (Figure 2(C)). The viral load in feather pulps was similar (P > 0.05) at 7 and 14 DPI with no detection of viral genome at 21 DPI.
Discussion
The present study was set up to resemble a critical period of a flock; that is, around the time when hens mature sexually after having been reared in high biosecurity conditions while remaining serologically negative to CIAV. The hens involved in this study were shown to be serologically negative to CIAV and also negative by qPCR. To be sure, all the birds in the trial were properly inoculated; Del-Ros virus vaccine strain of CIAV was introduced by live vaccination in experimental birds not only by the oral but also by the intramuscular route. In addition, the pullets were transferred to a laying facility around the time when they usually mature sexually, which introduced an added stress factor. The purpose of the oral administration of vaccine was to reach a high infection and colonization of the gastrointestinal tract and therefore high virus shedding into the faeces. The virus was recovered at 7 and 14 DPI only from cloacal swabs, but not from environmental samples, using a more sensitive commercial kit from Roche (showed in Table 3). During the 3-week period of experimentation, the virus was able to infect and colonize target organs or tissues such as the thymus, liver, spleen, bone marrow and feather pulp, where it was detected on days 7, 14 and 21 after the vaccination. This was not surprising since all strains of CIAV can be detected by qPCR (Toro et al., 2006). The qPCR technique was proved effective for the detection of CIAV DNA in infected chickens. Relative to the ELISA antibody test that detected seroconversion in 100% of the infected chickens as early as 14 days post-infection, qPCR was able to detect infection one week sooner as it was also described by other authors (Cardona et al., 2000a, 2000b; van Santen et al., 2004a). However, it must be kept in mind that the detection of antibodies in this study was performed with a serum dilution of 1:100 (as suggested for monitoring purposes), while in the cited references (Cardona et al., 2000a; van Santen et al., 2004a) the sera were diluted at 1:10 (which improves the sensitivity of the ELISA for detection). By 14 DPI, qPCR and antibody ELISA results were comparable. Thus, there may be a short period of time when CIAV infection would remain undetected while the birds seroconvert. This finding suggests that ELISA (used under the conditions of this study) as the sole testing criterion may leave a diagnostic window. During such period, when the infection is not yet detected, SPF eggs could be collected, distributed and used for vaccine production, diagnostic work or research purposes, increasing the chance of spreading the CIAV.
A high viral DNA concentration (viral load) was detected at all the sampling times in most of the organs tested. The highest viral load (P < 0.05) occurred at 14 DPI in all the tissues sampled. Even so, at 7 DPI the viral loads were considerably high in almost all the tissues (excepting the liver and plasma). Taking together the viral load and the ELISA results, we may consider that 7 DPI is the best time for virus detection using qPCR. Interestingly, CIAV remained detectable in tissues like thymus and spleen from 7 DPI to 21 DPI despite the presence of ELISA antibodies which were detectable from 14 DPI. For other tissues like the liver and bone marrow, even though they were positive at earlier times, CIAV DNA was not detected at 21 DPI, possibly influenced by the presence of neutralizing antibodies. Persistence of CIAV in tissues has been reported previously by Cardona et al. (2000b) who found longer persistence after seroconversion in the reproductive tissues of males and females, while other organs like the spleen showed less persistence. The significance of their findings is related with the pathogenesis of the vertical transmission of CIAV in infected birds, and thus the results of the present study are consistent with the findings of other authors, who observed persistence of the virus in tissues after seroconversion in infected flocks (Cardona et al., 2000b; van Santen et al., 2004a; Brentano et al., 2005). The non-infected negative controls maintained a negative status throughout the experimental period.
Previous studies have shown that experimentally infected males transmitted CIAV through their semen to their offspring until they developed CIAV antibodies between 8 and 14 DPI (Cardona et al., 2000b); however, we could not detect virus in the reproductive organs of males or hens from the vaccinated group when sampled at 21 DPI. The absence of CIAV genome in the ovaries and testicles by qPCR reaffirms the fact that the birds in this study were originally negative to the presence of CIAV since studies have shown that the virus can be present in the reproductive tissues in the absence of seroconversion of the birds (Cardona et al., 2000a). Unfortunately, the testicles and ovaries of inoculated birds were not tested at early times of infection in this study.
A non-invasive type of sample is more suitable for the industry of SPF eggs and embryos, products needed for poultry and human vaccine production and virus research. SPF flocks maintained under maximum-security management conditions, filtered-air and positive-pressure houses are intensively monitored in an effort to detect any change in disease status as soon as possible. Based on our results, feather pulp was the tissue of choice for non-invasive CIAV detection (considering plucking feathers as a non-invasive type of sampling), as previously indicated by Davidson et al. (2008). In any case, further work is needed to optimize DNA extraction and CIAV detection from other non-invasive samples such as cloacal swabs and environmental swabs. We also suggest the possibility of using qPCR as an alternative testing method for monitoring of CIAV in SPF flocks, fundamentally because qPCR allows the analysis of a high number of samples in a short time compared with regular PCR.
| Primer/Probe | Sequence | Locationa |
|---|---|---|
| Forward primer | 5′-CACTGGTTTCAAGAATGTGCC-3′ | 686–866 |
| Reverse primer | 5′- GCTCGTCTTGCCATCTTACAG-3′ | |
| 3′-end labelled probe | 5′-CTCCGAGTACAGGGTAAGCGAGCT(6-FAM)-3′ |
aNucleotide numbers in CIAV Cux-1 sequence, Genbank accession number M55918.
| Primer/Probe | Sequence | Locationa |
|---|---|---|
| Forward primer | 5′-GGGAACTGGAGAACCCAATTTT-3′ | |
| Reverse primer | 5′-CGTGCCGCTGTCTCTACCAT-3′ | 2360–2435 |
| 3′end labelled probe | 5′-TxR-CCCTTAACTGAGTTCCCCAGCTACTGCAG-BHQ2-3′ |
ªReported in Islam et al. (2004).
| N° of CIAV positive/total tested birds (%) | ||||||||
|---|---|---|---|---|---|---|---|---|
| 2 DPI | 7 DPI | 14 DPI | 21 DPI | |||||
| Vx | NVx | Vx | NVx | Vx | NVx | Vx | NVx | |
| ELISA + | 0/10 | 0/10 | 0/10 | 0/10 | 10/10 | 0/10 | 10/10 | 0/10 |
| (0) | (0) | (0) | (0) | (100) | (0) | (100) | (0) | |
| Thymus | 0/10 | 0/10 | 10/10 | 0/10 | 10/10 | 0/10 | 9/10 | 0/10 |
| (0) | (0) | (100) | (0) | (100) | (0) | (90) | (0) | |
| Liver | 0/10 | 0/10 | 6/9 | 0/10 | 6/9 | 0/10 | 0/10 | 0/10 |
| (0) | (0) | (67) | (0) | (67) | (0) | (0) | (0) | |
| Spleen | 0/10 | 0/10 | 9/9 | 0/10 | 4/8 | 0/10 | 5/9 | 0/10 |
| (0) | (0) | (100) | (0) | (50) | (0) | (56) | (0) | |
| B. Marrow | 0/10 | 0/10 | 9/9 | 0/10 | 9/10 | 0/10 | 0/10 | 0/10 |
| (0) | (0) | (100) | (0) | (90) | (0) | (0) | (0) | |
| Ovary | ND | ND | ND | ND | ND | ND | 0/10 | 0/10 |
| (0) | (0) | |||||||
| Testis | ND | ND | ND | ND | ND | ND | 0/10 | 0/10 |
| (0) | (0) | |||||||
| Feather P. | 0/10 | 0/10 | 8/10 | 0/10 | 7/10 | 0/10 | 0/10 | 0/10 |
| (0) | (0) | (80) | (0) | (70) | (0) | (0) | (0) | |
| Plasma | 0/10 | 0/10 | 0/10 | 0/10 | 0/10 | 0/10 | 0/10 | 0/10 |
| (0) | (0) | (0) | (0) | (0) | (0) | (0) | (0) | |
| Cloaca | 0/10 | 0/10 | 0/10 | 0/10 | 0/10 | 0/10 | 0/10 | 0/10 |
| (0) | (0) | (0) | (0) | (0) | (0) | (0) | (0) | |
| Cloacaa | ND | ND | 5/10 | 0/10 | 7/10 | 0/10 | ND | ND |
| (0) | (0) | (0) | (0) | |||||
| Boot swabs | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 |
| (0) | (0) | (0) | (0) | (0) | (0) | (0) | (0) | |
| Boot swabsa | ND | ND | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 | 0/2 |
| (0) | (0) | (0) | (0) | (0) | (0) | |||
Note: ND: Not done; DBI: Days before inoculation; DPI: Days post-inoculation.
aDNA used as template for the qPCR of these samples was obtained with the alternative method of DNA extraction (see Materials and Methods)
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Disclosure statement
No potential conflict of interest was reported by the authors.
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