Subclinical circulation of Chicken infectious anaemia virus- A Sero-molecular study

Abstract

Chicken infectious anaemia—an important immunosuppressive viral disease of chicken—gained much attention in the recent past. Based on huge mortality and production loss observed in the fast-growing poultry sector, the present study aimed to find out the current status of the chicken infectious anaemia virus (CIAV), among chicken flocks in the Punjab state of India by sero-molecular study. The sera from the blood samples were tested for anti-CIAV antibodies by indirect ELISA and also compared with haematological parameters. DNA from sero-positive samples underwent PCR amplification, sequencing and phylogenetic analysis of the most conserved genomic region (VP3 gene) to detect viraemia in asymptomatic birds. The serological study using indirect ELISA showed a high sero-positivity of 77.27% in chicken flocks. Additionally, the present study also revealed the high molecular evidence (72.54%) of CIAV in apparently healthy birds. Genetic analysis showed that all CIAVs have conserved VP3 genes without any nucleotide substitutions, indicating presence of CIAV and its subclinical circulation among apparently healthy flocks. The wide distribution of CIAV among birds may be the reason for huge mortality and production loss. Further, it is suggested that studies be conducted to find out the co-involvement of CIAV with other immunosuppressive microbial agents and the immunosuppressive effect of CIAV in apparently healthy birds. Also, its role in vaccine failure and outbreaks of various other avian diseases needs to be explored.

Introduction

Chicken infectious anaemia (CIA), resulting from a viral infection with chicken infectious anaemia virus (CIAV), is a well-known immunosuppressive disease of chicken and it possesses a potential threat to the poultry sector throughout the world. Commercial or intensive chicken production involves rearing birds at higher density (less floor/cage space per bird) which puts stress on birds. Stress and chicken infectious anaemia virus are close allies in birds, especially in younger ones leading to breaking the immune system of the body and making birds prone to acquire secondary (other) infections as the CIA virus shows synergy with some other viral diseases [1–4]. Chicken infectious anaemia virus is one of the smallest viruses (23–25 nm) among avian pathogens which are ubiquitously present in poultry farms worldwide. Complete elimination of the virus from the farm is a despairing task as it has remarkable chemical, thermal and physical stability [5, 6]. The outcome of the CIAV infection is dependent on various factors like vertical or horizontal transmission, the virulence of the virus strain, the dose and route of the infection and host factors. An age-related resistance occurs in older birds which are closely related to the ability of the bird to produce antibodies due to an active infection [7, 8].

Various studies on CIA have been done globally showing a high prevalence of the CIAV in almost all the continents [9–11]. Ledesma et al. [12] conducted a serological survey in Mexico which suggested that the virus is widespread in the Republic of Mexico. The results showed sero-conversion in both layers and broilers from all the states under the study (all birds were unvaccinated) which indicated the endemicity of CIA. Toro et al. [13] conducted a retrospective, serological survey in the USA which showed that the chickens could have been infected long before their first isolation during 1979. Karimi et al. [14] suggested that lower growth performance and inadequate response to vaccination observed frequently in broiler chicken farms of the Shahrekord region can be due to CIAV infection. Simeonov et al. [15] from Bulgaria reported 88 to 100% seroprevalence in backyard poultry with a significant association between the sero-positivity and age of birds under the study. Experimental infection of birds with CIAV and other important pathogens like infectious bursal disease virus or Marek’s disease virus resulted in high morbidity and mortality [4, 16, 17,]. Various studies on the genetic characterization of the CIAV show that the genetic makeup of CIAV is slowly changing from the original virus reported during the late twenty-first century [17–20]

No specific treatment exists and needs time-to-time surveillance of CIAV among the flocks for proper containment of CIA. Therefore, the present study was envisaged to screen for sero-conversion and the concurrent presence of CIAV DNA in the blood (viraemia) by natural infection in asymptomatic unvaccinated birds. A comparative study was done on haematology, serology and molecular biology for screening of CIAV among various flocks. Production loss, poor performance and a history of previous mortality were the selected criteria. Hence, a purposive random sampling technique was used to cover different regions (north, south, east, west and central) of the Punjab state of India. As far as our knowledge, this is the first detailed study on non-experimental birds having viraemia without showing any clinical symptoms. The findings will give awareness about the subclinical circulation of chicken infectious anaemia virus among healthy chicken flocks and can be given considerable attention to contain other poultry diseases. Since its subclinical nature and immunosuppression, the actual economic impact of CIAV needs to be studied.

Materials and methods

Sample collection

Blood samples (n = 120) were collected from unvaccinated apparently healthy birds or CIAV suspected/clinically ill birds from 27 organized farms located in the different regions of Punjab state of India (Moga, Ludhiana, Sangrur, Fazilka, Tarantaran, Hoshiarpur, Patiala, Pathankot, Mansa, Malerkotla, Jalandhar and Ahmedgarh). Flocks with the unknown serological or antigenic status of CIAV were collected randomly from various farms during the period 2015 to 2018. Production loss, poor performance and history of previous mortality were the selected criteria for preliminary screening of the farm for anti-CIAV antibodies. Two millilitres of blood was collected aseptically from the wing vein of selected birds. The whole blood and the serum collected were processed as per the standard protocol. These samples were stored at − 20 °C and used for further analysis as and when required.

Blood parameters measured

Packed cell volume (PCV) by micro-capillary method, haemoglobin (Hb-cyanmethemoglobin method) and differential leukocyte count (DLC) were estimated by the method described by Al-Hassani [21]. Total erythrocyte count (TEC) and total leukocyte count (TLC) were calculated by the method described by Natt and Herrick [22].

Indirect enzyme-linked immunosorbent assay (I-ELISA)

Serum samples were tested for anti-chicken anaemia virus antibody employing a commercial I-ELISA kit (BioCheck, UK) having > 98% sensitivity and specificity. Protocol followed as per the manufacturer’s instruction. The result was valid when the mean negative control absorbance was < 0.3 and the difference between mean positive control and mean negative control was > 0.15. Antibody titre was also calculated and an S/P ratio of 0.35 or greater/or a titre range of 725 or greater was considered positive.

DNA extraction and polymerase chain reaction (PCR)

DNA was extracted from all sero-positive blood samples using a DNA extraction kit (HiPura™, Himedia). The purity and concentration of the DNA were checked spectrophotometrically (Nanodrop, ThermoScientific) and the extracted DNA was stored at − 20 °C for further use. The DNA extracted was amplified using VP3 gene-specific primers which bind at the position of 486–507 bases and 827–848 bases to amplify a 364 bases product (complete VP3 gene). VP3 forward primer (5′ATG AAC GCT CTC CAA GAA GAT A 3′) and VP3 reverse primer (5′ CAG TCT TAT ACA CCT TCT TGC G 3′) set were used to amplify the VP3 gene of CIAV as described previously [23]. PCR protocol was optimized using GoTaq® Hot Start Green Master Mix (Promega) and amplified the whole sequence of the VP3 gene. A 25 μl reaction volume composed of 12.5 µl of PCR master mix, 1 µl of each forward primer and reverse primer (10 pmol) and 0.45 μg of template DNA and nuclease-free water (NFW) was used to optimize the PCR amplification. A positive control with a known template (from the vaccine strain) and a negative control were also kept along with the sample during amplification. The amplified products were then visualized by agar gel electrophoresis. The PCR products were gel-purified using a Wizard® SV Gel and PCR Clean-Up System (Promega) according to the manufacturer’s protocol. Purified products were sequenced by commercial outsourcing (SciGenom, Cochin, India).

Sequencing and phylogenetic analysis of VP3 gene

The DNA sequences of the VP3 gene were obtained by Sanger’s sequencing method. The sequences obtained were analyzed and aligned with other published sequences (GenBank, NCBI) using the ClustalW algorithm of Mega Align programme (DNASTAR, US) and phylogenetic analysis was done using the maximum likelihood method (MEGA7, USA) [24].

Statistical analysis

The haematological, serological and molecular data obtained were statistically compared using Tukey’s one-way ANOVA and the results were documented.

Results

Health status of the birds

Most of the birds included in the study were healthy/apparently healthy (n = 91). During the time of blood collection, a few samples (n = 29) showed a change in the consistency of blood (very thin blood with reduced viscosity, increased clotting time, etc.). Clinical signs noticed in unhealthy/ill birds were dullness, droopiness, respiratory distress, etc. The blood samples were collected randomly from both broilers and layers with different age groups ranging from 1 week to greater than 15 weeks with flock size ranges between 3000 and 45,000 birds. All of them were raised in conditions of intensive production with food and water provided ad libitum. Birds under treatment for other microbial infections and flocks with a history of sudden death/high mortality were also included in the present study.

Analysis of data

Analysis of haematological parameters

Haematocrit values of birds showed significant changes in PCV and Hb (P < 0.01). A significant decrease was observed in serologically positive birds when compared with sero-negative birds. No significant change was observed in other haematological parameters (Table 1). Few young birds under the study showed a severe decrease in PCV (< 6%) and Hb (3%) and they were also found serologically positive for CIAV infection.

Table 1.

Comparative blood profile of birds screened for anti-CIAV antibody. A significant reduction in haemoglobin (Hb) and packed cell volume (PCV) among sero-positive and negative birds

Parameters	Sero-positive (n = 66)	Sero-negative (n = 54)	Overall (n = 120)	P value
Haemoglobin (%)**	8.19 ± 0.29	9.67 ± 0.32	8.83 ± 0.23	0.001
PCV (%)**	27.32 ± 0.83	30.63 ± 0.76	28.81 ± 0.59	0.005
TEC (106/µl)	2.29 ± 0.11	2.31 ± 0.09	2.30 ± 0.07	0.874
TWC (103/µl)	18.15 ± 0.73	18.74 ± 0.67	18.42 ± 0.50	0.560
Heterophil (%)	43.36 ± 1.58	42.67 ± 1.72	43.05 ± 1.16	0.766
Lymphocyte (%)	49.77 ± 1.45	50.52 ± 1.71	50.11 ± 1.10	0.738
Monocyte (%)	4.89 ± 0.43	4.48 ± 0.38	4.71 ± 0.29	0.486
Eosinophil (%)	1.53 ± 0.24	1.43 ± 0.29	1.48 ± 0.19	0.782
Basophil (%)	0.53 ± 0.21	0.96 ± 0.20	0.73 ± 0.14	0.138

Values (Mean ± S.E.) and parameters with ** as superscript differs significantly (P < 0.01)

Indirect ELISA data analysis

Out of 120 serum samples analyzed, 66 {percentage positivity (PP) − 55%} were found positive for anti-CIAV antibody while the remaining 54 birds were found negative. All the age groups under the study showed the presence of antibodies to the chicken anaemia virus. 34.21% in 0–5 weeks and 57.14% in 5–10 weeks were found positive for anti-CIAV antibodies. In the present study, an average S/P ratio of 1.22 and 0.11 was observed among the sero-positive and sero-negative birds respectively. Similarly, an average antibody titre of 2873 and 183 was observed among the sero-positive and sero-negative birds respectively. The titre values of the birds which fell within 0–10 weeks were having the lowest (909) and highest (5000) range of antibody titre. The birds under the younger age (< 10 weeks) group had higher antibody titre with an average titre value of 3413.76 than older ones which showed an average titre value of 2198.31. The birds of age group greater than 10 were shown to have moderate antibody titre (Fig. 1). Among the 66 sero-positive birds, 51 (77.27%) were found apparently healthy with no signs/or symptoms of CIA and the remaining 15 birds were ill with characteristic clinical symptoms.

Fig. 1.

Antibody titre profile of birds screened for anti-CIAV antibody. All the age group were found positive for anti-CIAV antibody. Birds under 0 to 10 weeks had higher and lower antibody titre. Birds greater than 10 weeks found with moderate antibody titre values

PCR and sequence analysis

VP3 gene (complete gene) was amplified as it is the highly conserved region of the CIAV genome to confirm the presence of virus circulation in the blood. A positive control from the CIAV vaccine strain and a negative control (NTC) were also included in the PCR run. The primers amplified the VP3 gene sequence of CIAV to produce 364 bases as expected. The NTC did not show any amplification (Fig. 2).

Fig. 2.

Agarose gel electrophoresis of PCR amplifying VP3 gene. a Lane 1: positive control; lane 2: non-template control (NTC); lanes 3, 4: negative samples; lanes 5, 6 and 8: positive samples (364 bases); lane 7: marker (100 bp). b Lane 1: marker (100 bp); lanes 2–5: negative samples; lanes 6–15: positive samples

Out of the 66 DNA samples from sero-positive birds, 49 (PP-74.24%) were amplified VP3 gene. Among the 51 sero-positive apparently healthy birds, 37 (PP-72.54%) were amplified VP3 gene. Among the 15 sero-positive clinically ill birds, 12 (80%) were found positive for the CIAV gene. The rest of the samples were found negative. Six highly positive DNA samples (by DNA concentration) were sent for Sanger’s sequencing. Initially, the VP3 gene sequences obtained from the present study (GenBank Accession Numbers—MK385597, MK385598, MK385599, MK385600, MK385601 and MK385602) were analyzed by the Basic Local Alignment Search Tool (BLAST) method which showed high similarity with other CIAV VP3 gene sequences available in the GenBank. The sequences were aligned with published VP3 gene sequences retrieved from GenBank (NCBI). The nucleotide alignment report showed high sequence similarity with reference sequences. Multiple sequence alignment and genetic analysis of all six sequences from the present study with reference sequences revealed that the highest percentage identity with sequences from Punjab, Namakal, Haryana, Maharashtra and Ludhiana (99.7%) and lowest with Tamil Nadu (96.7%). The nucleotide identity among the six isolates from the present study showed 100% similarity except for one sequence (Patiala) which was 99.7%. The sequence from the present study also showed high similarity with other isolates reported globally. The deduced amino acid did not show any amino acid substitution in the VP3 protein of CIAV.

Phylogenetic analysis

Phylogenetic analysis to deduce the evolutionary relationship among the virus isolate was carried out by using MEGA7 software and the ClustalW algorithm. The maximum likelihood (ML) tree was constructed using the Jukes-Cantor model (Fig. 3). Based on the complete VP3 gene sequence analysis, the six isolates from the present study did not form any distinct Clad/cluster with the reference isolates. The isolates from the present study were branched close to the other reference isolates from India and various global isolates. Isolates from Tamil Nadu and Haryana branched distantly from Punjab isolates.

Fig. 3.

Molecular phylogenetic analysis of VP3 gene by maximum likelihood method. The evolutionary history was inferred based on the Tamura-Nei model. The bootstrap consensus tree inferred from 1000 replicates is taken to represent the evolutionary history of the taxa analyzed. Red colours indicate isolates from the present study except accession number MH269370

Discussion

The current study investigated the subclinical circulation of chicken infectious anaemia virus among various chicken flocks using sero-molecular methods and confirmed its presence. We have screened unvaccinated birds from the field, both serologically and molecularly, for the presence of CIAV especially in apparently healthy birds. All the farms screened were found positive for the presence of viral genome as well as for the anti-CIAV antibody. Since there was the presence of antibodies to CIAV (in India, regular vaccination of birds with CIAV is not practised by poultry farmers and all the samples were from unvaccinated birds), they might have sero-converted either from natural exposure or from maternal antibodies transferred from an infected parent. Our findings were also comparable with the previous reports which show a high prevalence of CIAV among unvaccinated birds [25, 26].

In young birds, susceptible to CIAV, the destruction of erythroid progenitor cells in the bone marrow results in severe anaemia, as well as depletion of granulocytes and thrombocytes [27, 28]. The present study also found that the birds with antibodies to CIAV had significant changes in their PCV, Hb and TEC levels. Since the virus can infect all the age groups, both the layers and broilers included in the present study tested positive for CIAV antibodies. A high seroprevalence in unvaccinated chickens suggests that the virus is circulating among the flocks, either clinically or subclinically. The apparently healthy birds included in the present study did not show any signs of CIA during the sample collection. Dai et al. [4] experimentally found that the body weight, thymus and bursa indices were significantly decreased at 21 dpi, which indirectly affects immunity and may induce immunosuppression in infected chickens.

An important finding in the present study was the presence of anti-CIAV antibodies along with DNA of the virus in sero-positive apparently healthy birds. Owoade et al. [29] suggested that the adult birds may seroconvert without any obvious symptoms which were in agreement with our study in apparently healthy birds. According to BioChek CAV antibody ELISA guidelines, an anti-CAV antibody titre ranges of no protection, moderate protection and protection are 724, 724–2295 and > 2296, respectively [30, 31]. Therefore, the birds which had no antibody titre or lower antibody titre (< 724) may be at risk of being infected from natural infection. In the present study, the birds under < 3 weeks of age were found negative for anti-CIAV antibodies. The birds which were found negative in the age group of 2–3 weeks are at risk of getting the infection and further development of clinical disease as it can cause severe anaemia in young birds.

Detection of viral DNA in the blood of birds reveals that the virus may be circulating through the blood, even after a long period of sero-conversion. In our study, the majority of the sero-positive birds were also found positive for viral DNA in their blood. Regardless of their antibody titre status, all the age groups were (< 5 to > 25 weeks) found positive for the virus genome. Previous reports suggest that maternal antibodies may protect at a younger age but as it diminishes, the bird may become susceptible to infection but not disease [32–34]. Therefore, we assume that the presence of the virus in the blood may be from a recent natural infection which induced antibody production or a latent form of the virus circulating among the chicken flocks. An in vivo laboratory study reported that even though the genome of CIAV is single-stranded DNA, the double-stranded replicative form may integrate into the host cell genome and lead to the presence of latent episomal DNA in tissues (gonads). Later, it can be vertically transmitted to the next generation [35]. This may lead to the carrier stage and in the course of life, episomal DNA may be spontaneously activated and virus particle is released into the circulation. Although birds carry viruses in their blood, none of the birds from the current study showed any clinical symptoms of CIA and this was consistent with haematological parameters. Another experimental study by Erfan, et al. [36] reports that the pre-infection/co-circulation of CIAV with other microbes like the Avian influenza virus and infectious bronchitis virus can contribute to the spread and evolution of these viruses. Furthermore, a study was conducted by Sreekala et.al. [37] to isolate and characterize CIAV’s genome from the sample(s) collected from the same farm was presented elsewhere (data not disclosed). This study once again confirmed the presence of the live virus in birds. Therefore, the birds with a history of poor performance, production loss and poor immune response to other avian vaccines are suggestive of CIAV presence in the flock [24, 38]. Thus, it is important to identify and exclude the poultry pathogens, especially immunosuppressive microbial agent like CIAV from the poultry premises for the better poultry production.

VP3 gene is highly conserved and can be used for confirmation of CIAV by PCR method when compared to VP1 and VP2 genes. The full-length VP3 gene amplified did not show any mutation or variation at the nucleotide level. The deduced amino acid also showed 100% identity with various other isolates reported globally. The PCR amplification of the VP3 gene with the same amplicon size was also reported by Wani et al. [23]. This study also confirms (along with various other reports) the robust conserved nature of the CIAV-VP3 gene. Therefore, viral persistence and vertical transmission may be the reason for the presence of the virus among chicken flocks [39]. Phylogenetic analysis of the isolate from the present study did not form a clear-cut clad/cluster and closely branched with other Indian isolates. This suggests that CIAV circulating among chicken flocks in the Punjab region may have originated from other Indian isolates. But, its proximity to other isolates from the world also needs to be considered.

Conclusion

Microbial agents especially bacterial and viral origins are causing a devastating economic loss in the avian industry throughout the world. The majority of these agents can impair the immune system which indirectly increases the risk of birds getting infected with other pathogens, vaccine failure, reduced performance, growth and production. The present study provides an updated insight into the subclinical circulation of CIAV among chicken flocks especially apparently healthy birds. It is one of the important immunosuppressive viruses affecting all age groups, especially at younger ages. The presence of CIAV may be the reason for the production loss and poor performance reported by various poultry farmers. Also, its role in vaccine failure and outbreaks of various other avian diseases needs to be explored for further formulation and implementation of preventive measures in the poultry sector. Further, it is recommended for studies in the area of improved avian vaccines to evoke the impaired/suppressed immune system. As it is one of the fast-growing sectors, the economic impact of CIAV along with other avian pathogens needs to be thoroughly investigated.

Abbreviations

CIAV

Chicken infectious anaemia virus

CIA

Chicken infectious anaemia

DNA

Deoxyribonucleic acid

IAEC

Institutional Animal Ethics Committee

PCV

Packed cell volume

DLC

Differential leukocyte count

Hb

Haemoglobin

TEC

Total erythrocyte count

TLC

Total leukocyte count

I-ELISA

Indirect enzyme-linked immuno sorbent assay

NFW

Nuclease-free water

NTC

Negative control

BLAST

Basic Local Alignment Search Tool

ML

Maximum likelihood

Author contribution

S. S.M.: funding acquisition. The idea for research or hypothesis/article generation. Administration of project. Data and sample collection, planning methods, validation, responsibility for conducting a literature search, responsibility for the logical interpretation and presentation of the results. Writing—the original draft. G. K.: supervision and responsibility for the organization and course of the project. Responsibility for writing review and editing of article. D. P.: conceptualization, supervision, validation of the project. Providing resources, tools, instruments and personnel vital to the project. Reviewing the final article before submission for intellectual content.

Funding

This study was supported by the Department of Science and Technology (DST) project under the Women Scientist scheme -A (No. SR/WOS -A/LS -253/2016) -Government of India.

Data availability

The datasets generated during and/or analyzed during the current study are available in the Nucleotide database repository (Accession Numbers MK385597 to MK385601) of GenBank, NCBI (https://www.ncbi.nlm.nih.gov/nuccore/MK385597).

Declarations

Ethical approval

Animal studies have been approved by the institutional animal ethics committee (vide Memo No: IAEC/2016/310–335) for the collection of blood samples from live birds and have therefore been performed following ethical standards.

Competing interests

The authors declare that they have no known conflict of interest. Sreekala S.M. reports financial support, equipment, chemicals or supplies, and travel were provided by the Department of Science and Technology (Ministry of Science & Technology), India.