Assessment of predisposing effect of Chicken Anaemia Virus (CAV) in Gangrenous Dermatitis (GD) outbreaks in commercial layer chicken Arulmozhi A.1*, Balasubramaniam G.A.1, Goplakrishnamurthy T.R.2, Sivaseelan S.1 1Department of Veterinary Pathology, Veterinary College and Research Institute, Namakkal - 637002, Tamilnadu, India. 2Poultry Disease Diagnosis and Surveillance Laboratory, Tamil Nadu Veterinary and Animal Sciences University, Namakkal - 637002, Tamilnadu, India. Address for Correspondence Dr A. Arulmozhi, Department of Veterinary Pathology, Veterinary College and Research Institute, Namakkal - 637002, Tamil Nadu, India, E-mail: arulvinod2000@yahoo.co.in
Abstract Gangrenous dermatitis (GD) is an economically important disease in commercial layer chicken and its severity have increased for the past few years despite effective farm management system and suitable antibiotic treatments. This study was designed to explore the immunosuppressive role of Chicken Anaemia Virus (CAV) in gangrenous dermatitis outbreaks in Namakkal region of Tamil Nadu, India. Thirty commercial white leghorn layer flocks were investigated in and around Namakkal District of Tamil Nadu State, India. Detailed farm history was collected. Serum and blood samples were collected to screen the antibody titer and haemogram, respectively. Heart blood, liver samples from dead birds and cutaneous swabs from ailing and dead carcasses were taken to identify the bacterial etiology. Detailed necropsy was conducted and the gross lesions were recorded. Thymus, bone marrow, liver and muscle were collected for screening of CAV by Polymerase chain reaction and histopathological studies. The sera samples showed 100 per cent positivity for CAV and the blood profile showed anaemic picture. The GD affected birds showed anaemia, severe serosanguinous, haemorrhagic and gangrenous lesions in wings, suncutaneous tissue and interdigital space of toes. Necropsy revealed severe gangrene, extravasation of serosanguinous fluid from the skin, hypoplasia of thymus and paleness of bone marrow. Microscopic examination showed hypoplasia / atrophy of thymus and replacement of erythropoietic tissue of bone marrow with fat cells. Cultural examination exhibited the presence of Closrtidium perfringens, Staphylococcus aureus and E. coli. The significance of the study was that CAV is the major culprit in GD outbreaks in poultry which invites the diverse etiological agents by its immunosuppressive action. Top Keywords Chicken anaemia virus, Commercial layers, Gangrenous dermatitis, Immunosuppression, Polymerase chain reaction. Top |
Introduction Gangrenous dermatitis (GD) is an economically important disease in commercial layer chicken caused by Clostridium perfringens type A1, Clostridium septicum2, E.coli and Staphylococcus aureus3 either singly or in combination. This disease is responsible for severe economic losses in poultry industry worldwide4 and placed constantly as a top priority disease list for the poultry industry5. GD is also called blue wing disease in chicken and cellulitis in turkeys6. It is characterized by rapid onset of acute and huge mortality, necrosis of the skin and subcutaneous tissue usually involving the breast, abdomen, wing or thigh due to rapid and fatal toxaemia by clostridial organisms7. It occurs due to invasion of ‘normal’ wounds by bacteria in immunosuppressed birds due to infectious bursal disease, chicken infectious anemia, reticuloendotheliosis, inclusion body hepatitis and poor litter conditions. |
Chicken Anaemia Virus (CAV) has the potential to induce immunosuppression8 and leads to enhanced susceptibility to other avian pathogens. It causes co-infections with other viral, bacterial and fungal agents as the pathogens mutually act and enhance their effects. Among such CAV co-infections, gangrenous dermatitis is the first and foremost infectious disease which causes increased mortality and huge economic loss in the poultry industry9. |
In Namakkal Poultry Zone (second largest egg production centre in India), both the incidence and severity of gangrenous dermatitis have increased for the past few years despite improvement in poultry production method, effective farm management system and suitable antibiotic treatments. This trend indicates that it is likely to pose a dreadful challenge to poultry farmers to control mortality as well as to meet out the treatment expenditure during GD outbreaks. |
Hence, the present study was designed to explore immunosuppressive and predisposing effect of CAV which could be a hidden/ underlying cause in Gangrenous dermatitis apart from bacterial etiologies. |
Top Materials and Methods Flock history Thirty commercial white leghorn layer flocks with a flock size of 25,000 to 1,00,000 chickens exhibiting the clinical signs of gangrenous dermatitis were investigated in and around Namakkal District of Tamil Nadu State, India. There were sudden and high mortality of layer chicken between 6 and 20 wks of age. Necropsy was carried out on both fresh carcasses and ailing chicken (270 Nos.). Detailed data regarding breed and strain of chicken, flock strength, age, method of rearing, vaccination schedule, source of feed and water, production performance including time of peak production, percentage of production, production drop and mortality were collected. All the flocks were vaccinated according to the standard protocols including vaccinations against Marek’s disease, Newcastle disease, infectious bursal disease, infectious bronchitis, infectious coryza and fowlpox. Serology and haemogram Serum samples were collected randomly from ten birds (6 to 20 weeks) from each flock affected with gangrenous dermatitis and examined for the presence of antibodies to chicken anaemia virus by conducting competitive ELISA using commercial kit (M/S.IDEXX Laboratories, USA). Blood samples were also collected from these birds to assess the anaemic status of GD affected birds by haematological parameters like Haemoglobin (Hb), Packed cell volume (PCV), Red blood cell count (RBC), White blood cell count (WBC), heterophil, lymphocyte, monocyte, eosinophil and basophil10. Blood samples from normal healthy birds (ELISA negative) were also collected to compare the haemogram values of CIA affected birds. Necropsy and histopathological examination Necropsy was conducted on eight to ten dead birds per flock totaling 270 birds and was thoroughly examined for gross pathological changes as per approved procedure11. Skin, muscle and all internal organs were examined carefully for its colour, consistency, nature of its exudates and size of the lymphoid organs. Necrosed and hemorrhagic feathers areas were removed and impression smears made from the gangrenous area of the skin and subcutaneous tissue to screen bacteria. Skin, muscle, thymus, bursa, liver and kidneys were collected and fixed in 10% neutral buffered formalin. Likewise, bone marrow was collected by cut open the long bones and exposed the marrow tissues to fixatives for hardening. The paraffin embedded sections were cut at 5 μm thickness and stained with hematoxylin and eosin for histopathology 12. Isolation of bacterial agent Heart blood, liver samples from dead birds and cutaneous swabs from ailing and dead carcasses were collected for screening of bacterial agents. The samples were inoculated into brain heart infusion broth and incubated overnight at 37°C. Then the broth culture were streaked on to selected agar plates viz. Mannitol salt agar (MSA), Eosin-methylene blue agar (EMBA) and Clostridium perfringens agar plates to identify Staphylococcus aureus, E. coli and Clostridium perfringens organisms respectively. The plates were incubated under aerobic and / or anaerobic condition at 37°C for 48 hours. The organisms were identified based on colony morphology, growth characteristics, sugar fermentation and biochemical characteristics 13. Confirmation of viral etiology by molecular method Sample from thymus, bone marrow, liver and muscle were taken from 10 birds affected with gangrenous dermatitis. One gram of pooled tissue samples were grinded with 9 ml of phosphate buffer solution (PBS) by mortar and pestle and transferred to test tube for DNA extraction. Genomic DNA purification kit (M/s Promega, USA) was used for the extraction and purification of genomic DNA from tissues. The amplification of VP2 gene of CAV genome14 produced at 419 bp fragment by polymerase chain reaction (PCR) using Forward primer: 5'-CTA AGA TCT GCA ACT GCG GA-3’ and Reverse primer: 5'- CCT TGG AAG CGG ATA GTC AT - 3’. The PCR products were subjected to electrophoresis at 80 volts for 45 min in 1% agarose gel and detected by staining with ethidium bromide (0.5μg/ml). PCR products with a molecular size of 419 bp were considered as positive. Statistical analysis The data obtained were statistically analysed by one way ANOVA15 and the data collected were analysed using SPSS 10® software package. Top Results Age and season-wise incidence of Gangrenous Dermatitis The clinical and pathological manifestation of the gangrenous dermatitis in thirty commercial layer flocks showed a sudden increase in mortality, vaccination failure and repeated outbreaks of various bacterial and viral diseases. Though the incidence was recorded from 6 to 20 weeks of age, the mortality rate was high during 12–14th weeks of age and it gradually decreased as the age advanced (Table 1). The mortality rate varied between flocks from 3% to 27%. Season wise analysis of GD revealed that the incidence was highest during December (13 flocks; 43.4 per cent) followed by January (8 flocks; 26.6 per cent), February (6 flocks; 20 per cent) and November (3 flocks; 10 per cent). Clinical signs The clinical signs were anaemia with pale comb, wattle and shanks; reddish black discolouration with moist edematous and exudative lesions in the medial aspect of wing, breast, back, interdigital space and various parts of the body. Serology The serosurveillance study of CAV in GD affected flocks by competitive ELISA detected 100 per cent positivity with the high antibody titre ranged from 4566 to 27429 and the grand mean total value was 8855. Haemogram The mean (± SE) haematological values of normal unaffected and gangrenous dermatitis affected chicken are presented in Table 2. The GD affected flocks showed the anaemic picture viz., reduction in Haemoglobin (Hb-6.8 ± 0.11 g/dl), Packed cell volume (PCV 22.71 ± 0.53 per cent) and total erythrocytic count (TEC 1.86 ± 0.06 x 106/cmm) when compared to normal healthy flocks Hb (10.77 ± 0.73 g/dl), PCV (32.00 ± 1.98 per cent) and total erythrocytic count (3.62±0.53 x 106/cmm). There were highly significant increase in heterophils (51.06 ± 0.74) and decrease in lymphocytes (42.82 ± 0.75) of GD affected flocks when compared to heterophil (36.33 ± 1.15) and lymphocytes (56.17 ± 1.68) of normal healthy flocks. Gross pathology On postmortem examination, the carcasses were anaemic, emaciated and had pale breast muscles. Skin lesions were more severe and characterized by bluish to greenish discolouration and extravasation of serosanguinous fluid from the skin lesions especially from wings, subcutaneous tissue (Fig. 1) and inter digital space of toes. There were petechiae to ecchymotic haemorrhage and numerous necrotic foci with thin fibrin covering on the hepatic parenchyma noticed in mixed infection of CIA with Clostridium perfringens and E.coli. Thymus revealed variable degrees of atrophy, hypoplasia (Fig. 2) and even complete visual absence of thymic lobes in some cases. Bone marrow appeared pale, yellowish-pink in colour and fatty appearance when compared to pink to a dark red coloured bone marrow (Fig. 3) of normal healthy birds. Histopathology Microscopical examination of subcutaneous tissue revealed inflammatory exudates, haemorrhage, edema and dermal necrosis with clusters of organism (Fig. 4). The thigh and breast muscles exhibited necrosis and haemorrhage between the myofibres. Thymus showed severe hypoplasia of thymic lobules, fibrous tissue hyperplasia (Fig. 5) and thinning of cortex. Bone marrow showed severe hypoplasia with decreased cellularity and atrophy of the haematopoietic tissues which were scattered within the aplastic areas. Erythrocytic and granulocytic series of the bone marrow was replaced by adipose tissue (Fig. 6) in the intra and extravascular spaces. Liver and kidney revealed dilated blood vessels with fibrin substances and thrombi. Concurrent infection of CAV with bacterial etiologies Chicken anemia virus concurrently infected with bacterial agents and causing GD (Table 3). The genomes of CAV were detected in pooled samples of thymus, liver and spleen obtained from infected chicken by amplification of a specific product of 419 bp of VP2 gene (Fig. 7). The notable feature in this study is the chicken anaemia virus specific nucleic acid was detected in all the 30 flocks affected with GD. The CAV co-occurred with single bacterial agents or mixed bacterial infection in GD outbreaks. Among the bacterial etiology, Closrtidium perfringens plays a vital role in GD outbreak along with CAV (45.18%). The samples from skin, muscle, liver and kidneys showed these bacterial organisms which was confirmed by stormy clot formation in skim milk media; black shiny colonies in Clostridium perfringens agar plates and zone of opalescene on egg yolk agar medium (Nagler’s reaction). The second major bacteria co-infected with CAV in GD cases was Staphylococcus aureus (27.03%) and was identified by circular smooth and shiny surface colonies (2-3 mm in diameter) on Mannitol salt agar; Positive for catalase, urease, citrate, coagulase, VP and methyl red; negative to oxidase, hydrogen sulphide, indole and gas. The influence of E. coli (4.07) with CAV in GD outbreaks was less and was identified by black metallic sheen colonies on EMBA agar; lactose fermenting pink glistening colonies on MacConkey’s agar; indole production at 44 °C and gas production in Eijkmarvn’s test. Mixed bacterial infection with CAV also noticed in considerable number of farms (4.81% to 18.88%). Top Discussion The susceptibility to GD was noticed from 6 to 20th week of age and mortality rate was high in 12–14* weeks of age. The skin injuries occurred during the transfer of chicks from deep litter to cage system might have provided anaerobic environment which favours the proliferation of Clostridium spp.16. It also enhanced by poor hygienic conditions, poor ventilation and lower quality feed 17. Similar age susceptibility was also recorded by earlier authors 18,19. The varied morbidity and mortality patern (3 to 27 %) between farms are depending upon the severity of disease 20 and health status of the birds 21. |
The increased susceptibility of birds to GD during winter season might be due to cold temperature and reduced air circulation in poultry houses in winter which favours the virus survival and bacterial multiplication22. In addition, the relative humidity was high during winter months and increased seasonal contamination of clostridium in feed 23. |
The affected birds were severely anaemic with pale comb and wattle, gangrenous lesion in the skin, wing and interdigital space of legs24. Competitive ELISA was used to detect antibodies against CAV in GD affected flocks and the results showed 100 per cent positivity with high antibody titre (grand mean total 8855). Similar higher antibody titre and seroprevalence (83-100%) against CIA was also recorded in layer flocks of Nigeria 25. |
The haemogram of the affected flocks showed the anaemic picture viz decreased Hb, PCV and TEC. It was also evidenced by the depletion of haemocytoblast of the bone marrow in microscopy. The CAV targets haemocytoblasts of the bone marrow which are the specialized progenitors for erythroid and myeloid series 26. Grossly, diffuse subcutaneous and intramuscular haemorrhage over the thigh, breast and pectoral muscles were noticed in most of the chicken 27. The endothelial injury by the bacterial toxins might at ribute the haemorrhagic, necrotic and gangrenous lesions in skin, subcutaneous tissue and muscle28. The bacterial toxins from these lesions entry into visceral organs via hematogenous route and causing necrotic to haemorrhagic lesions29. The hypoplastic thymic lobules and pale bone marrow were suggestive of immunosuppression by CAV 30. |
Histopathologically, thymus revealed severe lymphoid depletion and thinning of the cortex 31 as the CAV selectively infect the cortical immature T cells. The cortical lymphocytes were first cells to be destroyed and viral antigen has been demonstrated in large number of thymocytes in the cortical region 32. Likewise, fibrinous type of exudates with severe vascular pathology in visceral organs indicated the endothelial damage by the bacterial toxins of Closrtidium perfringens, Staphylococcus aureus and E. coli. |
The study revealed chicken anaemia virus was the main viral pathogen detected in all the 30 GD affected flocks and was co-occurred with various bacterial pathogens either individually or combined bacterial infection33. Among the bacterial etiology, Closrtidium perfringens was the major bacterial pathogen associated with GD outbreaks (45.18%) followed by Staphylococcus aureus and E. coli. |
The immunosuppression by CAV leads to susceptibility of birds to secondary bacterial, viral and fungal infection. It causes reduction in vaccine immunity and lower the production performance in the field situation34. |
Most of the time, treatment strategies for GD mainly focused on the bacterial etiology and neglect the underlying causes like CAV. It results in misdiagnosis, persist outbreak and improper control strategies. Hence, devising control strategies for GD should include strict bio-security measures at the farm level; immunostimulants in the therapeutic measures and proper vaccination against CAV at the breeder stock level is warranted. |
Top Acknowledgment The authors are highly thankful to Tamil Nadu Veterinary and Animal Sciences University for providing necessary facilities to conduct the research. |
Top Figures | Fig. 1.: GD: CAV + Cl. perfringens + S. aureus: Skin - Subcutaneous tissue of breast region showing severe gangrene with sero-sanguinous fluid;
|  | |
| | Fig. 2.: GD: CAV + Cl. perfringens-Thymus showing hypoplasia of thymic lobes;
|  | |
| | Fig. 3.: GD: CAV + Cl. perfringens: Bonemarrow of femur bone showing paleness when compared to the normal bird;
|  | |
| | Fig. 4.: GD: CAV + Cl. perfringen + E. coli + S. aureus - Subcutaneous tissue of breast region showing necrosis with clusters of rod and cocci organisms. H&E x400.
|  | |
| | Fig. 5.: GD: CAV + Cl. perfringens - Thymus showing atrophic lobules and fibrous tissue hyperplasia. H&E ×400;
|  | |
| | Fig. 6.: GD: CAV + Cl. perfringens - Bone marrow of femur showing replacement of erythropoietic tissue with fat cells. H&E ×400;
|  | |
| | Fig. 7.: CAV - Amplification of VP2 gene (419 bp) of Chicken anaemia virus. Lane 1: DNA ladder (100 bp); Lane 2 to 8: Positive sample for CAV; Lane 9: Negative control and Lane 10: Positive control.
|  | |
|
Tables Table 1.:
| | S.No. | Age | No. of flocks | Percentage | | 1. | 6 - 8 | 05 | 16.67 | | 2. | 9 -11 | 08 | 26.67 | | 3. | 12 - 14 | 14 | 46.67 | | 4. | 15-17 | 02 | 6.66 | | 5. | 18-20 | 01 | 3.33 | | Total | | 30 | 100 |
| | Table 2.:
| | S.No. | Parameters | Affected | Normal | | 1. | Hb (g/dl) | 06.80±0.11P | 10.77±0.73Q | | 2. | PCV (%) | 22.71±0.53P | 32.00±1.98Q | | 3. | RBC (x106/cmm) | 01.86±0.06P | 03.62±0.53Q | | 4. | WBC (x103/cmm) | 10.58±0.39P | 13.99±0.45Q | | 5. | Heterophil (%) | 51.06±0.74P | 36.33±1.15Q | | 6. | Lymphocyte (%) | 42.82±0.75P | 56.17±1.68Q | | 7. | Monocyte (%) | 03.41±0.21p | 04.50±0.43q | | 8. | Eosinophil (%) | 02.71±0.17 | 02.83±0.31 | | 9. | Basophil (%) | 00.00±0.08 | 00.17±0.17 |
|
| Column-wise group means with different superscript (pq) differ significantly ; (p<0.05) and those bearing upper case (PQ) differ highly significant (p<0.01) | | Table 3.:
| | S.No. | Concurrent infection | No. of samples | Percentage | | Tested | Positive | | 1. | CIA + Cl. perfringens | 270 | 122 | 45.18 | | 2. | CIA + Staphylococcus aureus. | 270 | 73 | 27.03 | | 3. | CIA + E.coli | 270 | 11 | 4.07 | | 4. | CIA + Cl. perfringens+ Staphylococcus aureus | 270 | 51 | 18.88 | | 5. | CIA + Cl. perfringens+ Staphylococcus aureus + E.coli | 270 | 13 | 4.81 |
| |
|