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<h1 class="Hdr1">

Research Article


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Research Journal for Veterinary Practitioners. 2 (2): 36 – 39


</p>


</div>


<div>
<h2 class="Hdr2">


Seroprevalence of Antibodies against Newcastle Disease in Layer Chicken at Cox’s Bazar, Bangladesh

</h2>

<p class="p3">


<strong>Mohammed Arif Uddin</strong><span><sup>1</sup></span>, 


<strong>Kamrul Islam</strong><span><sup>1</sup></span>, 


<strong>Shaharin Sultana</strong><span><sup>4</sup></span>, 


<strong>Mohammed Ashif Imtiaz</strong><span><sup>2*</sup></span>, 


<strong>Abul Kashem Muhammad Saifuddin</strong><span><sup>2</sup></span>, 


<strong>Mukti Barua3 </strong><span><sup></sup></span>
</p>


</div>

<div class="p4">

<ol>
   
<li>Department of Microbiology, Chittagong Veterinary and Animal Sciences University (CVASU)</li>

<li>Department of Physiology, Biochemistry and Pharmacology, CVASU</li>


<li>Department of Animal Science and Animal Nutrition, CVASU, Chittagong, Bangladesh</li>

<li>Department of Microbiology, Bangladesh Agricultural University (BAU), Mymensingh, Dhaka, Bangladesh</li>

</ol>




</div>


<div>        
<p><span class="Author">*Corresponding author:ashif.shawn@gmail.com </span></p>

</div>
<div id="ColorID"><p>
<span><b>ARTICLE CITATION:</b></span> 

Hassan MM, Amin KB, Ahaduzzaman M, Alam M, Faruk MSA, Uddin I (2014). Antimicrobial resistance pattern against E. coli and Salmonella in layer poultry. Res. j. vet. pract. 2 (2): 30 – 35. 


<br>
<span><b>Received: </b></span>2013–12–27, <span><b>Revised: </b></span>2014–01–30, <span><b>Accepted: </b></span>2014–02–02<br>

The electronic version of this article is the complete one and can be found online at 

(<a href="http://dx.doi.org/10.14737/journal.rjvp/2014/2.2.36.39">
http://dx.doi.org/10.14737/journal.rjvp/2014/2.2.36.39 

</a>)
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited </p>
</div>

<div>
     <h3 class="Hdr3">ABSTRACT</h3>

    <p>
    
    This research work was conducted to screen out the seroprevalence and immune status of layer chicken against Newcastle Disease Virus (NDV) in Cox’s Bazar, Bangladesh. From 17 different commercial layer farms, a total of 179 serum samples were collected and divided into three age groups such as 20–27 weeks, 36–42 weeks and 48 to above weeks. Haemagglutination Inhibition (HI) test was performed and calculated geometric mean titers for layer groups of 20–27 weeks, 36–42 weeks and 48 to above weeks of age were log28.39, log27.67 and log210.66, respectively. Though all birds showed positive specific immunity of 100%, means good mean titers that was protective to ND, still the birds remained susceptible to ND. So vaccination should be intensified by screening immune status of the flock against ND through analyzing the HI titers to NDV.   

    </p>
 
</div>




<div>
  
  <h3 class="Hdr3">INTRODUCTION</h3>
    <p>
    
Newcastle Disease (ND) is a highly contagious viral disease that attacks many species of domestic and wild birds which also locally known as Ranikhet Disease (<a href="http://www.nexusacademicpublishers.com/table_contents_detail/13/201/html/#Barman2002" target="_self">Barman, 2002</a>). The causal agent is Newcastle Disease Virus (NDV) which is a negative–sense single–stranded (ss) RNA virus belonging to the family Paramyxoviridae (<a href="http://www.nexusacademicpublishers.com/table_contents_detail/13/201/html/#Hossain2010" target="_self">Hossain et al., 2010</a>). Based on the pathogenicity, NDV is classified into highly virulent (velogenic), intermediate (mesogenic) or avirulent (lentogenic) strain (<a href="http://www.nexusacademicpublishers.com/table_contents_detail/13/201/html/#Lamb2000" target="_self">Lamb et al., 2000</a>). The infections of poultry range from latent to rapidly fatal depending upon the pathotype of virus involvement (<a href="http://www.nexusacademicpublishers.com/table_contents_detail/13/201/html/#Alexander2003" target="_self">Alexander, 2003</a>). Transmission of ND occurs by inhalation or ingestion through direct contact between healthy birds and discharges from infected birds through feces and secretions from nose, eyes and mouth (<a href="http://www.nexusacademicpublishers.com/table_contents_detail/13/201/html/#NABC2007" target="_self">NABC, 2007</a>).
</p><p>It is noticed as ND is the most significant viral disease of poultry in the world including developing countries. In Africa and Asia, it is a major constraint against the progress of both industrial and village poultry production (<a href="http://www.nexusacademicpublishers.com/table_contents_detail/13/201/html/#Pazhanivel2002" target="_self">Pazhanivel et al., 2002</a>; <a href="http://www.nexusacademicpublishers.com/table_contents_detail/13/201/html/#Alders2001" target="_self">Alders et al., 2001</a>). The disease causes high economic losses due to high mortality, morbidity, stress, decreased egg production and hatchability (<a href="http://www.nexusacademicpublishers.com/table_contents_detail/13/201/html/#Hossain2010" target="_self">Hossain et al., 2010</a>).
</p><p>Vaccination has been reported as the only safeguard against endemic ND (<a href="http://www.nexusacademicpublishers.com/table_contents_detail/13/201/html/#Orajaka1999" target="_self">Orajaka et al., 1999</a>; <a href="http://www.nexusacademicpublishers.com/table_contents_detail/13/201/html/#Saha1998" target="_self">Saha et al., 1998)</a>. The current vaccination schedule in Bangladesh directed by the Directorate of Livestock Services (DLS) includes administration of a live vaccine Baby Chick Ranikhet Disease Vaccine (BCRDV) of lentogenic F–strain by intra–ocular instillation to chicks followed by a live vaccine Ranikhet Disease Vaccine (RDV) of mesogenic Mukteswar (M)–strain by intramuscular injection at 21days old chicks and 60days old chicken which is repeated at every 2months interval. The infection still occurs in Bangladesh every year in the form of epidemic and appears to cause up to 40–60% of the total mortality in poultry population creating one of the major problems in the development of poultry industry in Bangladesh (<a href="http://www.nexusacademicpublishers.com/table_contents_detail/13/201/html/#Biswas2005" target="_self">Biswas et al., 2005</a>). 
</p><p>It is significant to elucidate the immune status of NDV among chickens in order to formulate appropriate vaccination schedule and control measures. So, the study was conducted to determine the seroprevalence and screen immune level of the birds to Newcastle Disease Virus (NDV) among layer chickens in Cox’s Bazar district, Bangladesh.


</p>
  
</div>


<div>
<h3 class="Hdr3">MATERIALS AND METHODS</h3>
<p>
The study was carried out at three upazillas of Cox’s Bazar from June to August, 2012. A total of 17 commercial layer farms which comprised at least thousand birds were randomly selected with the diagnosis of ND. Data were recorded according to clinical signs exhibited by individual bird during illness, strains of birds, number of birds in farm, age, vaccination schedule and date of last vaccination, number of dead birds, treatment measures if taken and information about management system of farm.
</p>
<p>
<strong>Diagnosis</strong><br>
In Upazilla Veterinary Hospital, poultry diseases were diagnosed on the basis of post mortem findings, clinical signs and as well as various laboratory tests such as Haemagglitination Inhibition (HI) test, Enzyme Linked Immune Sorbent Assay (ELISA) technique, Viral Neutralization (VN) test. Currently HI test is most widely used technique to detect antibodies to NDV in poultry sera.
</p>

<p>
<strong>Post Mortem Findings</strong><br>
These findings were varied but generally include haemorrhagic lesions on the proventriculus and intestinal tract which include pin point haemorrhage on the tip of proventriculas and small ecchymotic haemorrhages on the lymphatic nodule, Payer’s patches followed by intestinal mucosa. Edematous, haemorrhagic and degenerative ovaries, button ulcer on the caecum and spleenomegaly were observed (Figure 1). Other lesions accordingly hyperemia and congestion in respiratory tract, serous or catarrhal exudates in larynx and trachea, thickened air sacs containing yellow exudates were also frequent.
</p>
 </div>

<div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/imagesfiles/Fig1_280.PNG" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/imagesfiles/Fig1_280.PNG" width="80" height="80"></a>
<p class="Txt_Dis" ><span><b><a href="http://nexusacademicpublishers.com/uploads/imagesfiles/Fig1_280.PNG" target="new">Figure 1: </a></b><a href="http://nexusacademicpublishers.com/uploads/imagesfiles/Fig1_280.PNG"><span>A. Haemorrhage on the tip of the proventriculas; B. Severe haemorrhage on glandular tip and button ulcer in the intestinal mucosa; C. Haemorrhagic intestinal tract; D. Spleenomegali</a></p>
</div>
 
 
 
 <div>
<p>
<strong>Clinical Signs</strong><br>
Signs of ND followed as sudden death without sign, ruffled feathers, depression, prostration, edematous head and wattles, nervous signs as paralysis, torticollis and respiratory signs as gasping and coughing.
</p>

<p>
<strong>Collection of Samples</strong><br>
The sample, 1ml of blood from 179 birds (8 samples per thousand birds) was collected from each bird through wing vein puncture; Sera were separated and stored at –20°C until analyzed. Avinew® ND, VG/GA antigen lentogenic strain (Advance Animal Science Co. Ltd. Dhaka, Bangladesh) was used in this study to conduct HI test to detect the immune status against NDV.
</p>


<p>
<strong>HI Test to Screen Level of Antibodies in Blood Sera</strong><br>
HI test was performed in V–bottomed microtitration plates. Test procedure was conducted according to the methodology of <a href="http://www.nexusacademicpublishers.com/table_contents_detail/13/201/html/#OIE2002" target="_self">OIE Manual (2002)</a>. For preparation of 1% chicken Red Blood Cells (RBCs) used in the test, 5ml blood was taken from chicken that had not been primed with Newcastle Disease vaccine. RBCs were collected by centrifuging blood at 1500rpm for 15minutes and these collected RBCs were pooled in an equal volume of Alsever’s solution. After that, RBCs were washed thrice in phosphate buffered saline (PBS) before using as 1% (packed cell v/v) suspension. Serum was tested in two fold serial dilutions upto 10th well in microtiter plate. For virus suspension 4 Haemagglutinating Units (HAU) Avinew® ND, VG/GA antigen lentogenic strain was added upto 11th well. After keeping the plates at room temperature for 30minutes 1% chicken RBC suspension was added to each well. The 11th well contains antigen and RBCs as the positive control and the 12th well contains only RBCs as the negative control. After gentle mixing, the RBCs were allowed to settle at room temperature for 40minutes and agglutination was assessed by tilting the plates. Finally, at the end of 40minutes samples were showing central button shaped settling of RBCs which were recorded as positive and maximum dilution of each sample causing Haemagglutination Inhibition (HI). This was used to estimate the HI titer (Figure 2). The HI titer of each serum sample was expressed as reciprocal of the serum dilution.
</p>
</div>

<div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/imagesfiles/Fig2_280.PNG" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/imagesfiles/Fig2_280.PNG" width="80" height="80"></a>
<p class="Txt_Dis" ><span><b><a href="http://nexusacademicpublishers.com/uploads/imagesfiles/Fig2_280.PNG" target="new">Figure 2: </a></b><a href="http://nexusacademicpublishers.com/uploads/imagesfiles/Fig2_280.PNG"><span>Haemagglutination Inhibition test (HI test) (Buttoning)</a></p>
</div>
 
 
 <div>
<p>
<strong>Data Analysis</strong><br>
Data that were primarily stored at MS excel (Microsoft office excel–2007, USA). Descriptive analysis was through by STATA version 11 (STATA Corporation, Texus, USA) and results were expressed as proportion ± SEM, 95% Confidence interval (CI) and also followed as logarithm table.
</p>
</div>





<div>
<h3 class="Hdr3">RESULTS</h3>
<p>
From the total 179 serum samples of 17 different commercial layer farms, 99 serum samples were ranging from 20–27weeks of age, 53 samples 36–42weeks of age and other 27 samples were 48 to above weeks of age. Birds of all ages were found positive for specific immunity with positive percentage of 100 (Table 1).
</p>
</div>

<div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/imagesfiles/Table1_280.PNG" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/imagesfiles/Table1_280.PNG" width="80" height="80"></a>
<p class="Txt_Dis" ><span><b><a href="http://nexusacademicpublishers.com/uploads/imagesfiles/Table1_280.PNG" target="new">Table 1: </a></b><a href="http://nexusacademicpublishers.com/uploads/imagesfiles/Table1_280.PNG"><span>Serum samples of layer chicks showing immune response to NDV using Haemagglutination Inhibition (HI) test</a></p>
</div>



<div>
<p>But the HI antibody titer varied from log24 to log212 with a GMT of log28.39 at the age of 20–27weeks of age; log23 to log212 with a GMT of log27.67 at the age of 36–42weeks of age and log29 to log212 with a GMT of log210.66 at the age of 48 to above weeks of age (Table 2).

</p>
</div>


<div class="pt" > <a href="http://nexusacademicpublishers.com/uploads/imagesfiles/Table2_280.PNG" target="new"><img class="img_display" src="http://nexusacademicpublishers.com/uploads/imagesfiles/Table2_280.PNG" width="80" height="80"></a>
<p class="Txt_Dis" ><span><b><a href="http://nexusacademicpublishers.com/uploads/imagesfiles/Table2_280.PNG" target="new">Table 2: </a></b><a href="http://nexusacademicpublishers.com/uploads/imagesfiles/Table2_280.PNG"><span>Distribution of layer birds on the basis of Haemagglutination Inhibition (HI) titers obtained against NDV</a></p>
</div>











<div>
<h3 class="Hdr3">DISCUSSION</h3>
<p>
All samples irrespective of age represents positive for specific immunity with a positive percentage of 100 where <a href="http://www.nexusacademicpublishers.com/table_contents_detail/13/201/html/#Tariq2010" target="_self">Tariq and Taib (2010)</a> found 26%, 54% and 75% positive for specific immunity at 24–36, 36–48 and more than 48 weeks in chicken which is quite lower than this one. HI test has been widely used for the estimation of titers of specific antibody in the sera of individuals infected with certain viruses including NDV, Influenza virus etc as these viruses can agglutinate erythrocytes (<a href="http://www.nexusacademicpublishers.com/table_contents_detail/13/201/html/#Serrão2012" target="_self">Serrão et al., 2012</a>). Generally ND–HI titer of log23 and above is accepted as positive for specific immunity (<a href="http://www.nexusacademicpublishers.com/table_contents_detail/13/201/html/#Sa'idu2006" target="_self">Sa’idu et al., 2006</a>; <a href="http://www.nexusacademicpublishers.com/table_contents_detail/13/201/html/#OIE2002" target="_self">OIE 2002</a>; <a href="http://www.nexusacademicpublishers.com/table_contents_detail/13/201/html/#Alexander2000" target="_self">Alexander, 2000</a>). <a href="http://www.nexusacademicpublishers.com/table_contents_detail/13/201/html/#Numan2005" target="_self">Numan et al. (2005)</a> stated ND–HI titer as log27 to log28 in layer of 17–47 weeks of age that is closer to the present study. This shows that the serum antibody titer is low in some cases and enough to protect the birds from ND infection. There are several possible reasons for the lower level of protection in birds such as poor vaccine quality, not properly maintained vaccination schedule or techniques, impaired immune competence due to immunosuppressive substances in the feed or to immunosuppressive diseases and therefore, unable to protect the chicks from NDV infection.
</p><p><a href="http://www.nexusacademicpublishers.com/table_contents_detail/13/201/html/#Hossain2010" target="_self">Hossain et al. (2010)</a> found 98.35%, 92.86%, 97.46% and 97.98% layer chickens had protective HI titers in the summer, rainy, autumn and winter, respectively; <a href="http://www.nexusacademicpublishers.com/table_contents_detail/13/201/html/#Sa'idu2006" target="_self">Sa’idu et al. (2006)</a> analyzed log27.6 ± 1.62 in parent stock of layer; this result is similar to the present findings at the age of 36–42 weeks. Birds of this group showed lower antibody levels than the earlier group and showed relatively decreased susceptibility to clinical infection. One of the causes for outbreaks in vaccinated chickens might be the introduction of new ND virus strains against which the local birds have no or very low immunity, leading to vaccine failure.
</p><p>Group of birds age 48 to above showed higher antibody level than the previous two groups. Similar agreement has been described by <a href="http://www.nexusacademicpublishers.com/table_contents_detail/13/201/html/#Numan2005" target="_self">Numan et al. (2005)</a> where 100% of layer chickens were positive for specific immunity against NDV in Pakistan. In the present study, the birds in 48 to more weeks of age showed the highest level of antibody titers (GMT 10.66) and showed relatively low susceptibility to clinical infection. The HI titers obtained in the present study were higher than those reported by <a href="http://www.nexusacademicpublishers.com/table_contents_detail/13/201/html/#Biswas2006" target="_self">Biswas et al., 2006</a> who recorded that 64%, 47.4%, 62.6% and 56.3% of sonali chickens in southern part of Bangladesh had protective HI titers against NDV in autumn, winter, summer and rainy seasons respectively. Other factors like poor vaccine quality is a common problem in developing countries and results poor manufacturing standards, lack of adequate storage facilities, application of expired vaccine batches, faulty administration and handling during transportation (<a href="http://www.nexusacademicpublishers.com/table_contents_detail/13/201/html/#Vui2002" target="_self">Vui et al., 2002</a>). Heat stress and water deprivation also lead to production of steroids and thus result an immunesuppression. Quality of water which is offered to the birds was also found questionable which might hinder the development of specific immunity. Inappropriate vaccination schedule also leads to the neutralization of maternally derived antibodies and resultantly making the birds more susceptible to the infection.
 

</p>
</div>








<div>
<h3 class="Hdr3">CONCLUSION</h3>
<p>
The prevalence of Newcastle Disease is higher level (postmortem findings 45.6%) than the other viral diseases in Bangladesh. Although birds of commercial layer farms had mean titers that was protective to ND, still the birds remain susceptible to ND. To maintain good farm practices it is very important to vaccinate the birds of the flock at proper time with proper dose and schedule regularly. So vaccination should be intensified by monitoring immune status of the flock against ND through analyzing the HI titers to NDV. 
</p>
</div>





<div>
<h3 class="Hdr3"> ACKNOWLEDGEMENT	</h3>
<p>
Authors are grateful to DR. Shubhagata Das, Assistant Professor, Department of Pathology and Parasitology, CVASU; farmers and Veterinary Clinicians, Veterinary Surgeon and all the staff of Upazilla Veterinary Hospital, Cox’s Bazar for their cordial cooperation in the study.
</p>
</div>









<div>
<h3 class="Hdr3">REFERENCES</h3>
 
 <p class="p2"> <a name="Alders2001"></a>
Alders RG, Costa R, Dias P, Fringe R, Fumo A, Lobo Q, Mata BV, Silva A and Young MP (2001). Investigations into the Control of Newcastle Disease in Village Chickens in Mozambique: Review of work done in Mozambique. Report on the ACIAR/INIVE Newcastle Disease Control Project Coordination Meeting, Tofo, Inhambane Province, Appendix 3.
</p>

<p class="p2"> <a name="Alexander2000"></a> 
Alexander DJ (2000). Newcastle disease and other avian Paramyxoviruses. In: Revue Scientifique et Technique de l'OIE, 443–462pp.
</p>

<p class="p2"> <a name="Alexander2003"></a> 
Alexander DJ (2003). Newcastle disease, other avian paramyxoviruses and pneumovirus infections In: Diseases of Poultry 11th Edn. Edited by Saif YM, Barnes HJ, Glisson JR, Fadly AM, McDougald LR and Swayne DE. Iowa State Press, USA, 63–87pp.
</p>

 <p class="p2"> <a name="Barman2002"></a>
Barman LR (2002). An epidemiological and experimental study of Newcastle disease in village chickens of Bangladesh. M. Sc Thesis. The Royal Veterinary and Agricultural University, Dyrlaegevej 2, 1870, Frederiksberg C., Denmark. http://www.poultry.kvl.dk/upload/poultry/master_theses/lalita.pdf (Accessed on 29.01.2014)
</p>

 <p class="p2"> <a name="Biswas2005"></a>
Biswas PK, Biswas D, Ahmed S, Rahman A and Debnath NC (2005). A longitudinal study on the incidence of major endemic and epidemic diseases affecting semi–scavenging chickens reared under the Participatory Livestock Development Project areas in Bangladesh. Avian Pathology. 34(4): 303–312.<br>
<a href="http://dx.doi.org/10.1080/03079450500178972" target="_blank">http://dx.doi.org/10.1080/03079450500178972</a><br>
PMid:16147566
</p>

 <p class="p2"> <a name="Biswas2006"></a>
Biswas PK, Uddin GMN, Barua H, Roy K, Biswas D, Ahad A and Debnath NC (2006). Immune status of semi–scavenging Sonali chickens in Bangladesh against Newcastle disease. Livestock Rural Development. 18 (6). http://www.lrrd.org/lrrd18/6/bisw18074.htm (Accessed on 29.01.2014)
</p>

 <p class="p2"> <a name="Hossain2010"></a>
Hossain MMK, Ali YM and Yamato I (2010). Antibody Levels against Newcastle Disease Virus in Chickens in Rajshahi and Surrounding Districts of Bangladesh. Int. J. of Biol. 2 (2): 102–103. <br>
<a href="http://dx.doi.org/10.5539/ijb.v2n2p102" target="_blank">http://dx.doi.org/10.5539/ijb.v2n2p102</a>
</p>

 <p class="p2"> <a name="Lamb2000"></a>
Lamb RA, Collins PL, Kolakofsky D, Melero JA, Nagai Y, Oldstore MBA, Pringle CR and Rima BK (2000). Family Paramyxoviridae In: Virus Taxonomy, Edited by van Regenmortel MHV. 7th report of the International Committee on Taxonomy of Viruses. Academic Press, New York. 549–561pp.
</p>

 <p class="p2"> <a name="NABC2007"></a>
NABC (National Agricultural Biosecurity Center) (2007). Exotic Newcastle Disease Fact Sheet. Kansas State University. http:/nabc.ksu.edu/content/factsheets/category/Exotic%20Newcastle%Disease (Accessed on 29.01.2014)
</p>

 <p class="p2"> <a name="Numan2005"></a>
Numan M, Zahoor MA, Khan HA and Siddique M (2005). Serologic status of Newcastle diseae in broilers and layers in Faisalabad and surrounding districts. Pakistan Vet. J. 25(2): 55–58.
</p>

 <p class="p2"> <a name="OIE2002"></a>
OIE 2002. Office International Des Epizooties. Manual of Standards for Diagnostic Tests and Vaccines. http://www.oie.int/fr/normes/mmanual/ancien_manuel/a_00004.htm (Accessed on 28.01.2014)
</p>

 <p class="p2"> <a name="Orajaka1999"></a>
Orajaka LJE, Adene DF, Anene BM and Onuoha EA (1999). Seroprevalence of Newcastle disease in local chickens from Southeast derived savannah zone of Nigeria. Revue Elevage Medecine Veterinaire Pays Tropicaux. 52 (3–4):185–188.
</p>

 <p class="p2"> <a name="Pazhanivel2002"></a>
Pazhanivel N, Balsubramaniam GA, George VT and Mohan B (2002). Study on natural outbreak of Newcastle disease in and around Namakkal. Indian Vet. J. 79(3): 293–294.
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