INCIDENCE OF CHICKEN ANEMIA VIRUS IN SHARKIA GOVERNORATE CHICKEN FLOCKS

A.M. HEGAZY^*; F.M. ABDALLAH^**; L.K. ABD-EL SAMIE^*** and A.A. NAZIM^****

^* Prof. at Dept. of Avian & Rabbit Med., Faculty of Vet. Med., Zagazig University.

^** Assistant Prof. at Dept of Virology, Faculty of Vet. Med., Zagazig University.

^*** Colleague at Vet. Hospital, Faculty of Vet. Med., Zagazig University.

^**** PhD. at Dept. of Food Hygiene., Zagazig University, Hospitals.

E.mail: asmhegazey@gmail.com

INTRODUCTION

Chicken anemia virus (CAV) is an economically important pathogen with a world-wide distribution. The virus infections are manifested by either clinical or subclinical signs (Schat and Santen 2008). The clinical disease is mainly noticed in young chicks of 10–14 days of age which usually acquire the infection vertically. Chickens older than 2–3 weeks of age are also susceptible to infection but only develop a subclinical disease evidenced by poor vaccine response, increased severity of other infections and decreased cell mediated immune response (McConnell et al., 1993b, Adair 2000, Markowski and Schat, 2003, Schat and Santen 2008). Outbreak of the disease is characterized by anemia, thymus atrophy, bone marrow aplasia and immunosuppression (Adair, 2000, Yuasa et al., 1987). The immunosuppression effect whether directly by itself as it causes severe depletion of lymphocytes from primary and secondary lymphoid organs (Yuasa et al., 1979, Taniguchi et al., 1982 & 1983) consequently destruction of erythroblastoid cells in the bone marrow and thymocytes in the thymic cortex of the newly hatched chickens (Kato    et al., 1995) or indirectly as it participates other immunosuppressive viruses such as infectious bursal disease virus (IBDV), Marek’s disease virus (MDV) or reticuloendotheliosis virus (REV). In dual infections, anemia was produced even when the chickens were inoculated at two or more weeks of age or in chickens with maternal antibody (Yuasa et al., 1980, Bülow et al., 1986a).

The first report of CAV was from contaminated vaccines in Japan (Yuasa et al., 1979). In China, it was first isolated in 1996 (Zhou et al., 1996), Argentina (Craig, 2009), India (Natesan et al., 2006), Nigeria (Ducatez et al., 2005), South Korea (Kim     et al., 2010) and other countries have reported outbreaks in commercial flocks. Virus isolates were thought to belong to a single serotype and were antigenically indistinguishable by serum neutralization tests (Yuasa and Imai, 1986).

Detection of CAV using molecular techniques including polymerase chain reaction (PCR) provides an alternative to conventional testing as it is more sensitive and specific (USda. Gov.).

The study aimed to reflect the prevalence of CAV among suspected chicken farms at different localities of Sharkia governorate by using serological survey, histopathological changes and molecular identification.

MATERIALS and METHODS

Examined birds: Out of five different localities at Sharkia governorate, Chickens were sourced from 27 farms with complains of high mortalities, poor performance, recurrent infections and vaccination failure.

Samples: Performing postmortemexamination of selected birds, 360 blood sampleswere collected for determination of hematocrit values and serum for ELISA serological assay. Organs (Thymus loops, bone marrow, bursa of Fabricius, liver, and spleen) were collected and stored at (-70°C) to be used for PCR analyses and hitopathological examination.

Packed cell volume (PCV): was carried out according to (Duncan and Prasse, 1986) as preliminary diagnosis of CAV.

ELISA assay: Commercial chicken anemia virus antibody ELISA kit (ProFLOK^â PLUS, Symbiotic Corporation, San Diego, CA, USA; Cat. No. 92127-800-228-4305) was used under the manufacturer instructions for specific prevalence of the virus.

Histopathological examination: Visceral organs (thymus, bursa of Fabricius, liver, and spleen) were fixed with 10% buffered neutral formalin solution. Femurs were split longitudinally to expose bone marrow then fixed and decalcificated in 5% formic acid for 2 weeks. Samples dehydrated, embedded in paraffin and sectioned (4 micron thickness), and stained with hematoxylin and eosin (H&E) for microscopic examination (Haridy et al., 2012).

PCR identification: Tissues were prepared according to (Zhou et al., 1997) and DNA was extracted by using commercial extraction kits (QIAGEN^® EZ1 Virus Mini Kit Version 2.0(48), Cat. No. 955134) in automated extractor (QIAGEN^® EZ1 (BioRobot), Serial No. 0502f0707) according to manufacturer instructions.

Amplification of DNA was performed in the thermocycler (T3- Thermocycler, Biometra) using:

1- Reddy-Mix^™ PCR Master Mix (THERMO Scientific^®, Cat. No. AB-0575/LD/A, Lot No. 1404/15)

2- Two primers, CAV A1.1 (5` AAT GAA CGC TCT CCA AGA AG 3`) and CAV A1.2 (5` AGC GGA TAG TCA TAG TAG AT 3`), (MWG-Biotech AG^®, Batch No. 523470 & 523471 respectively) were used to amplify a 583 base pair (bp) DNA fragment(Tham and Stanislawek, 1992).

3- DNA Ladder of 100 Pb (QIAGEN ^®, Lot No. 3)

PCR assay was performed in a final volume of 50 µl containing 25 µl Reddy-Mix™ PCR Master Mix, 18 µl PCR Grade Water, 1 µl of each Primer, and 5 µl template.

The amplification was performed under the following conditions: one cycle of initial denaturation step at 95°c for 15m, followed by 30cycles of 95°c for 1m, 56°c for 1m, and 72°c for 1m representing denaturation, annealing, and extension steps respectively. Finally, one cycle of final extension step at 72°c for 5m.

The amplified products were analyzed using electrophoresis unit. It was loaded to 2% agarose, stained by ethidium bromide, visualized under ultraviolet light, and photographed by a gel documentation system using Canon Power-Shot^® G10 camera and the data was analyzed using computer software (Sambrook and Russel, 2001).

RESULTS

1. Clinical signs and lesions:

Examined birds showed signs of anemia, generalized weakness, depression, droopy appearance, pale comb and wattles, stunting, growth retardation and high mortality rate. The necropsy findings were watery blood, yellow fatty bone marrow, markedly atrophied thymus glands, atrophied bursa of Fabricius and enlarged liver and spleen. Subcutaneous and intramuscular hemorrhages were reported in some cases and the average PCV was 17% - 22% indicating reduced hematocrit value.

2. ELISA:

Out of 360 tested serum samples, 305 (84.72%) showed positive result to CAV specific antibodies, (Table 1).

3. Histopathology:

Histopathological examination, of the naturally infected tissues which were positive to CAV DNA by PCR assay, showed marked hypoplasia in bone marrow (Fig. 8) and generalized lymphocytic depletion in thymus (Fig. 7) and spleen (Fig. 5). Bursa of Fabricius showed moderate depletion in lymphoid follicles with presence of perifollicular edema (Fig. 6). Liver showed cellular swelling and apoptosis with marked apoptotic bodies (Fig. 2, 3, 4).

4. Identification of CAV by PCR:

Analysis of DNA extracted (thymus, bone marrow, bursa of Fabricius, liver, and spleen) from tissues of diseased chickens by agarose gel electrophoresis yielded positive reactions of correct size as primers (583 base pairs in size), (Fig. 1).

Fig. 1: PCR products (583 bp in size) of amplified CAV-DNA extracted from tissues of examined chickens.

Lanes 2, 3, 10, and 11 are positive

Lanes 4, 5, 6, 8, and 9 are negative

Lanes 1 and 7 are control negative and positive respectively

M: size marker (100 base pairs ladder)

Fig. 2: Liver showing congestion of portal blood vessels with perivascular fibrosis (1) and proliferation of the bile duct (2). H&E., X 1200.

Fig. 3: Liver showing portal proliferation of fibroblasts aggregation of round cells and (arrows). H&E., X 300.

Fig. 4: Liver showing hepatocyte apoptosis with marked apoptotic bodies (arrow). H&E., X 1200.

Fig. 5: Spleen showing depletion of the white pulps (arrow) and disarrangement of the normal architecture. H&E., X 300.

Fig. 6: Bursa of Fabricius showing moderate depletion of lymphoid follicles (arrows) with presence of perifollicular edema. H&E., X 1200.

Fig. 7: Thymus showing marked lymphoid depletion (arrows). H&E., X 1200.

Fig. 8: Bone marrow section showing markedly hypocellular marrow (arrow). H&E., X 1200.

Table 1: Results of serological assay (ELISA) for CAV in chicken flocks at Sharkia

*Age of different flocks ranged from between 3 weeks to 80 weeks

DISCUSSION

The investigated chicken flocks showed generalized weakness, depression, droopy appearance, pale comb and wattles, stunting, growth retardation, and high mortalities. The packed cell volumes measured were markedly reduced (average PCV was between 17% and 22%). Necropsy findings of the sampled chickens revealed watery blood, yellow fatty bone marrow, markedly atrophied thymus glands, atrophied bursa of Fabricius, and enlarged livers and spleens. The clinical signs, postmortem lesions, and PCV values agreed with the findings of (Yuasa et al., 1979, Taniguchi et al., 1982 & 1983, Aly 2001) and (Pope 1991, Ramadan et al., 1998) who stated that a case of hematocrit value below 27% with yellowish changes in bone marrow and thymic atrophy may be indicative to CAV infection beside other means of diagnosis.

The serologic survey performed in this study involved 5 different localities of Sharkia province, Egypt representing different breeds, ages and types of production. Bird's age was intended to exceed the age of 3 weeks to exclude maternally derived immunity that persists for about 3 weeks. The overall findings of the study proved that CAV is widely distributed among chicken flocks where 84.72% of the tested serum samples reacted positive at different localities of Sharkia governorate, Egypt. (Zaki and El-Sanousi, 1994) reported that in Egypt, the incidence of CAV antibodies in serum samples collected from broiler breeder, layer, and day old broiler flocks was 70%, (Amin et al., 1998) stated that it was 97.4% among 21 native and foreign grandparent, parent, and broiler flocks in 8 provinces, (Sabry et al., 1998) recorded that 1916 tested serum samples from 118 flocks of different breeds and ages in different governorates showed a high degree of positivity for CAV antibodies in both imported and locally produced broiler parent, broiler, and layer flocks, while (Islam 2003) reported that CAV seroprevalence was 74.6% and 67.3% in commercial broiler and broiler breeder flocks respectively in Sharkia province. In other countries, CAV serological findings was 85.7% in commercial layer flocks in Turkey (Kuyucuoglu       et al., 2003), 86% in commercial broiler flocks in Nigeria (Owoade et al., 2004), 67.3% in commercial layer flocks in Khartoum state, Sudan (Ballal et al., 2005), 87.7% in commercial broiler flocks in Iran (Mahzounieh et al., 2005), 82.61% in commercial broiler flocks in Northern Jordan (Dergham 2006) and (96.15%) in unvaccinated commercial broiler chicken farms in Malaysia (Hailemariam et al., 2008).

Detection of CAV antibodies in tested flock's sera indicates either vertical/horizontal infection or passive acquired immunity from breeders via yolk. Passively acquired immunity was excluded depending on age factor where sera were collected after 3 weeks of age, at which maternal antibodies decayed as mentioned by (McNulty et al., 1988). The presence of CAV antibodies in tested flock's sera with no history of clinical signs, lesions or vaccination certainly indicates horizontal infection through direct and indirect contact with virus-contaminated dust, water or feed with feces (Rosenberger 1991) specially that the virus shows extreme physical and chemical resistance to inactivation and so persists for long period in poultry houses (Yuasa 1992).

Histopathological examination go with the findings of (Sakr and Talaat, 1991) who observed marked depletion of the lymphocytes in the thymus and bursa of Fabricius beside severe hypoplasia in hematopoietic cells in bone marrow. In addition, (Hussein et al., 2002) reported some bursal changes with various degrees of atrophy in the lymphoid follicles with scattered necrotic foci, which were probably attributed to secondary infections. These findings similar to (Goryo et al., 1987, Smyth et al., 1993) that proved immunosuppressive effect of the virus may be attributed directly to its destructive effect of hematopoietic and lymphopoietic tissues leading to impaired immune response. Moreover (Adair et al., 1991) stated that CAV infection causes severe defects in splenic T-lymphocyte functions in form of decreased responsiveness to phytohemagglutinin, concanavalin A and fall in interleukin production. Macrophage concentration and functions are also severely reduced after in vivo or in vitro exposure to the virus such as interleukin-l (IL-1) production, Fc receptor expression, phagocytosis and bactericidal activity (Cloud et al., 1992; McConnell et al., 1993a & b). The adverse effects of the virus on lymphocyte and macrophage functions have substantial negative effects on immune response leading to enhancement of the concurrent infection with other pathogens and vaccination failure (Adair, 2000). On the other hand (Goryo et al., 1989, Smyth et al., 1993) reported that bursa of Fabricius was not susceptible to CAV infection and (Haridy et al., 2012) stated that there were no prominent or remarkable lesions in bone marrow and appeared fatty with no inclusions in hematopoitic cells of SPF chickens experimentally infected at 4 weeks old with CAV.

PCR assay is proved to be specific and definitely more sensitive than cell culture isolation of the virus, especially that DNA can be extracted from the same tissues as used for virus isolation (Miller et al., 2001; Soiné et al., 1993; Drén et al., 1994). In this study, PCR yielded positive reactions with correct size as primers (583 bp) for confirmation of CAV infection in chicken flocks that showed clinical signs, reduced hematocrit value and postmortem lesions. Similar result was reported by (Tham and Stanislawek, 1992). In all PCR positive flocks, the thymus and bone marrow samples were positive, this is probably because CAV targets erythroid and lymphoid progenitor cells in the bone marrow and thymus respectively (Adair, 2000). While (Hailemariam et al., 2008) recorded that out of 60 commercial broiler breeder hens tested for the presence of CAV DNA by nested PCR assay, the highest percentage of positive samples was detected in spleen where 45 samples out of 60 (75%) were positive. Duodenum was found to be an organ with the least distribution of CAV DNA in which 28 organs out of 60 (46.7%) were positive. There is no statistical significant (P < 0.05) difference in the percentages of CAV DNA between spleen, bone marrow, thymus and ovary. However, the distributions of viral DNA in liver, duodenum and oviduct were significantly less (P < 0.05) from the rest of the organs.

It is confirmed that CAV is widely distributed among chicken flocks in Sharkia governorate, Egypt by using PCR. Moreover, ELISA is a sensitive test for detection of clinical and subclinical forms of the virus infection. The virus has immunosuppressive effect due to destruction of lymphoid organs consequently causes huge economic losses. Adoption of breeder immunization programs against CAV is an urgent demand to avoid vertical transmission of the virus and protect the progeny via maternal antibodies.

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