The Paramyxovirus is classified under the Avulavirus genus and the Paramyxoviridae family. it associated development of several health problems in more than 235 species of wild and domestic birds, including industrial poultry farms. Viruses include three groups, known as Metaavulavirus, Orthoavulavirus, and Paraavulavirus [1,2]. The International Committee on Taxonomy of Viruses classified 21 APMV viruses in the 2020 edition of the Taxonomy of Viruses [3]. The pathogen viruses possess a single-stranded, negative-sense, non-segmented RNA genome, which has a length ranging from 13 to 17 kilobases. The viral genome has six distinct structural proteins that do not interact with each other. These proteins are the nucleocapsid (NP), phosphoprotein (P), matrix (M), fusion (F), hemagglutinin-neuraminidase (HN), and large polymerase (L) [3]. The presence of mono/polybasic amino acids at the protein cleavage site F is an essential marker of virulence. Mucosal viruses can be defined as velogenic, mesogenic and lentogenic strains  based on the characteristics of the cleavage site [1,2]. this virus causes sever damage to hens resulting in significant economic losses for  the poultry industry. The incubation  period  typically  ranges  from 3 to 6. However, not all of these viruses significantly impact the poultry industry.SomeAPMV viruses  can cause illness and reprodutive issues  in other avian species [4]. The APMV-13 virus has been isolate from wild avian  species in countries like China, however its non-pathogenicity to chickens has been confirmed [5]. Wild birds are a natural reservoir for the Avian Paramyxovirus (APMV) and play an important part in its transmission among poultry. Indigenous strains of Avian Paramyxoviruses originate from poultry and can be transmitted from  ducks and domestic chickens to wild birds [6]. Highly virulent strain of APMV can also be transmitted from poultry to waterfowl [7].The Tigris and Euphrates Basin in Iraq is a major wintering area  for migratory water bird traveling  between East Asia and Africa. Numerous migratory bird species  winter in Iraq. Wild birds play a crucial role  in the spread of infectious diseases  through  their  natural  movement [6]. Veterinerian and health institution  are interested  in monitoring  viral diseases, such as Avian  Influenza  Virus (AIV) in wild ducks. Epidemiological  studies  have shown the transmission  of viral  infection  from domestic and rural  environments  to chicken farms [8]. Egg  inoculation is leading methode  for isolating  the Newcastle virus [9]. The virus's widespread transmission and it is harmful  effects on embryos  necessitate this method for isolating the virus. Various traditional and modern diagnostic methods are used to identify isolates."" Among them, the serum neutralization test stands out as the most crucial. Serum neutralization is a crucial test for identifying isolates. This highly efficient test provides reliable serological identification. The virus is isolated on tissue culture medium, and its detrimental impact on these cells is observed, The disease affects susceptible hosts such as poultry, other avian species, and egg embryos’ PCR testing technique was used to ascertain the genetic sequence of isolates, and this method necessitates a source. Agglutination inhibition and ELISA can aid in diagnosing isolates [9,11]. Monitoring infections and examining local strains provides crucial data for controlling disease transmission in breeding areas. This study investigated the APMV virus and strains obtained from domestic birds in Mosul city between January and May 2024. The accuracy, specificity, and probability of local isolate transmission were examined using serum neutralization and hemagglutination inhibition tests in an epidemiological investigation.

Isolation Avian Paramyxo Virus
The Avian paramyxovirus was obtained from an infected bird with clinical signs, including congested trachea, congested lung, and ulcerated proventriculus, from a backyard in Mosul city(suggested Avian Paramyxovirus- 1). The infected lesion was ground in a mortar with sterile sand, and 1-2 ml of saline was added to create a thick paste. The paste was diluted 1:9 in sterile nutrient broth, centrifuged, and filtered through a Seitz filter. The filtrate was stored at -80°C for use as a stock virus solution. 0.1 ml aliquots of the stock virus were inoculated into the allantoic cavity of 12-day-old specific pathogen-free chicken eggs and incubated for 5-6 days.
 

All embryos died at the end of the first 24-hour were regarded as non-viral fatalities and subsequently discarded. All eggs were candled twice daily. Dead embryos were opened and inspected for lesions. Also, one living embryo was opened each day. From these observations it was determined that the lesions were completely formed on the fifth day than at any other time and this would be the most satisfactory incubation period. Allantoic fluid collected by sterile syringe with lesions were harvested, ground in a sterilized mortar, and the samples were centrifuged and filtered. These viruses were used for the process of titration. Serial ten-fold dilutions were made. Twelve day embryonated eggs per dilution were inoculated by the allantoic cavity with 0.1 ml of virus dilution. The eggs were incubated lying flat with the point of inoculation upward, and the eggs were not turned during incubation. The embryos were again checked twice daily, and the dead ones were examined for the presence of lesions. All remaining embryos were opened after five days. In replicated studies six similar passages and inspections were made. The stock virus titrated by Reed, and Muench [12].

Commercial Vaccines
A commercial vaccine strain of Avian paramyxo virus-3 obtained from Jordanian Center for Biological Industries. These commercial vaccines failed to induce pathological lesion when inoculated into allantoic cavity of chick embryo [13].

Preparation of Hyperimmune Sera
Hyperimmune serums were prepared according to the method mentioned somewhere [14,15]. 20-day-old broiler chickens were subcutaneously injected with a dose of 0.25 ml of a commercial vaccine studied in our paper. Revaccination was done in the same way, two, and three weeks after administration. First time vaccination: Ten days after the last administration, the chicks were slaughtered and blood was collected to obtain serum, and aliquots were stored at -20°C in sterilized plastic tubes until use. This serum considered specific antisera. And used to diagnose the isolated virus by serum neutralization test using conventional technique, and (HA/HI)

Neutralization Test
Titrated virus stock from the infected chicken was prepared in serial dilutions ranging from 10-1 to 10-10. The dilution was mixed with 0.25 ml. of undiluted specific antiseraandthe mixture was allowed to stand for one hour at room temperature. Five 12-day embryonated eggs per dilution were inoculated with 0.1 ml of the antiserum-virus mixture onto the allantoic cavity to determine neutralization index. Using this test alsoto detect paramyxovirus antibody and its titrations in the suspectable chickens. (unknown serum with our diagnosed isolate)

Haemagglutination HA and Haemagglutination inhibition HI
The HA and HI assays were used for determining the APMV-2   [16].  In  summary,  allantoic  fluids  obtained from chicken embryos that were infected with APMV-1 were used to do HA and HI assays. The HI test was run using the diluted serum constant-virus containing technique, with each dilution4HA units of virus [17]. Both positive and negative controls were included in each test. The HA and HI tests were conducted according to the previously provided protocol [18]. In the HA test, the fluid from the allantois, which was obtained 48-72 hours after infecting the eggs with paramyxovirus, was collected. Following the preparation of ten-fold dilutions in saline, a 0.1 suspension of chicken erythrocytes was introduced. The same technique was carried out using allantoic fluid obtained from an egg that was not injected with paramyxovirus. Following the detection of HA activity in the fluid obtained from injected eggs, an HI test was conducted in a microtiter plate. This test included the use of produced paramyxovirus hyperimmune serum and paramyxovirus negative chicken serum to ascertain if paramyxovirus was responsible for the agglutination. The Hemagglutination Inhibition (HI) test used allantoic fluid carrying paramyxovirus, which had been previously employed as the  Hemagglutination (HA) antigen .Each well test used 4HA units of antigen. A 0.5% chicken red blood cell (RBC) suspension was made by combining 0.5 ml of concentrated washed RBCs with 100 ml of phosphate-buffered saline (PBS) at a pH range of 7.0-7.2. The serum samples underwent heat treatment at 56°C for 30 minutes in a water bath to deactivate complements. The atypical β process, also known as the Diluted-Serum-Constant-virus procedure, was carried out on 96 well round bottomed microtiter plates. Following the creation of successful dilutions of the tested serum, the antigen was introduced and allowed to incubate. Subsequently, a 0.1% solution of chicken erythrocytes was added. The plates were kept at room temperature until the wells containing known HI-positive samples showed a distinct, well-defined button of unagglutinated, settled red blood cells. The HI titer is the value obtained by taking the reciprocal of the greatest dilution of serum that completely inhibits haemagglutination.

Using HaemagglutinationHA and Haemagglutination inhibition HI
To detect paramyxovirus antibody and its titrations in the suspectable chickens.in addition to detect commercial vaccine activity. we chose Hemagglutination and Hemagglutination Inhibition  test because they are not expensive and easy to use.

Titrations 
Calculated according to Reed, and Muench, method [19]. The  viral  titer  is  determined  by the reciprocal of the highest dilution that still causes a lethal Dose on egg embryo (LD50/ 1ML).

Local paramyxovirus was isolated by serial passage in embryo allantoic cavity inoculation method and identified by a serological neutralization test using hyper immune serum obtained from the injection of an active commercial vaccine in adult chickens. this examined by HA test with titer 104 EID50/1ML. viral isolate titration which obtained from the embryos had 105.2 titer calculated by Reed and Muench (1938). Subsequently, the sera of birds designated for analysis in the four locations were evaluated using our indigenous strain, and the presence of the illness was confirmed by two examinations: the serological neutralization test and the agglutinin inhibition test. The findings are as follows. The study revealed that in the first region, 4 animal sera samples were diagnosed as positive for the sero-neutralization test, accounting for 33.33% of the total samples. Additionally, the number of positive samples in the same region doubled during the sero-neutralization test, reaching a rate of 66.67% out of the 12 samples studied. This accounts for 21.82% of the total number of samples diagnosed with the disease, which amounted to 55 samples. In the second area, 8 samples were diagnosed positive in the serological neutralization test, accounting for 32% of the total samples. Additionally, 9 samples were diagnosed positive in the second test, accounting for 68% of the 17 samples and 30.09% of the total positive samples. The findings in the third area indicated an equal distribution of samples, with 8 samples each, accounting for 50% of the total 16 samples, which represents 29.09%. In the fourth area, the first inspection yielded 4 positive samples, representing 40% of the total. Additionally, out of 10 samples, 6 were positive, accounting for 60% and 18.18% of the total diagnosed samples, which amounts to 55 samples as previously said. The antibody standards achieved rates of 102.7 EID50/0.1 ml and 102.841 EID50/0.1 ml in the serological neutralization test and the agglutination inhibition test, respectively, across all areas.

Table 1: No positive sample and percentage

Chi-square= 2.56, P = 0.109

Statistical Analysis
The statistical test results indicate that there is no statistically significant variation between the groups in the studied variables. because the p-value is more than the significance level set at 0.05. so, we accepted the null hypothesis. And there is no evidence to support the existence of a statistically significant difference between the groups in the studied variables.

It is essential to obtain local isolate for diagnosis avian Paramyxovirus-1 which is characterized by moderate respiratory and neurological symptoms [1] and measurement immune response, particularly humoral response [20]. with identifying the causative agent and its serological or genetic relationship. In our study we applied two methods which are considered Various methodsto achieve this goal. Serological neutralization assay has been employed to diagnose the local isolate by virus inoculation in chick embryo with disease-causing agents, including congested trachea and congested lung. Infected proventriculus, as it described by Office International des Epizooties (OIE) [21,22]. Serological neutralization test is performed using the hyperimmune serum to confirm the diagnosis. This method is considered one of the most important and accurate serological tests in diagnosing viruses [1,23]. Among the disadvantages that accompany this test is the long time required to perform the procedure, in addition to the difficulty of injecting embryos and the need for large quantities of fertilized eggs. In addition to provides biosafety requirements. Therefore, it is currently being replaced by modern tests such as polymerase chain reaction technology due to its accuracy and ease when the necessary supplies are available [2]. Several laboratory techniques have been used to conduct serological tests with the aim of diagnosing and measuring modern immune responses, including the various types of ELISA test and the agglutination inhibition test.(Office International des Epizootic [21,24]. The agglutinin inhibition test is considered one of the most famous and most widespread serological tests in the diagnosis of paramyxovirus due to its specificity and high sensitivity. In our study, isolation was achieved by inoculation embryos into the allantoic cavity of infected samples from four different regions in Ninawa province, to obtain a local isolate specific to the study and avoid suspicion of non-existent isolates in the area. This method has been commonly used before. The diagnosis was confirmed by serological neutralization test using the immune serum prepared from injection of four adult chickens. the commercial vaccine's efficiency has been confirmed and the purity has been reassured. by the important indication of H.A test which reach 105 standard during this test. The virus titration reached a level of 104, as examined by same test, which is a recognized measure established in past studies [22]. 60 samples were collected from different areas in the city of Mosul for the purposes of the study. Samples were randomly selected from each individual household. Serums of birds suspected of being infected with the virus were taken and two serological tests were performed. These two tests were compared to our study isolate, namely the serological neutralization test and the hemagglutination inhibition test. The results showed that the infection rate in the second region was higher than the other regions by 30.91%, with a total of 17 positive samples out of 55 samples. The third region had a 29.09% infection rate, with 16 positive samples out of the total samples. Meanwhile, the first and fourth regions had lower rates of 21.82% and 18.18% respectively. The incidence rates are within the normal range of disease spread in any region worldwide and do not indicate the occurrence of abnormal disease outbreaks [25]. According to the study, it was found that the serological neutralization test diagnoses 33.33% of the samples, while the agglutination inhibition test diagnoses 66.67% of the samples from the first region. In the second region, the first examination identified 32% of the samples, while the second examination identified 68% of the samples. As for the samples in the third and fourth regions, the first examination identified 50% and 40% of them respectively, while the second examination identified 50% and 60% of the samples respectively. Through statistical analysis, it was found that there are no significant differences between the two tests when using the chi-square test. Therefore, either test can be used for the required examinations. These results align perfectly with researchers [25-27]. After measuring the antibody titer in the sera of the studied animals, it was found that the average titer ranges between 102.7and 102.841, which is a moderate average that does not indicate the occurrence of a disease (not high). It also excludes the possibility that this average is due to the use of vaccination programs, as home breeding systems lack regular preventive programs as in industrial breeding. The study has demonstrated the potential for disease transmission through the infection of migratory wild birds and the subsequent spread of infection to industrial projects. This necessitates intensive measures to protect birds from contracting the disease through this route. The study revealed the presence of a common local isolation between regions, possibly resulting from the transmission in this manner.
 

The recommendations suggest using modern methods for isolating viral pathogens for ease and speed, especially during outbreaks of infectious diseases. It also advises conducting regular routine tests to monitor communicable diseases, using the tests studied in this paper as well as modern routine tests. The study also recommends establishing a relationship between isolates from poultry farms and comparing them with isolates from outside. Finally This study underscores the importance of monitoring APMV infections to mitigate potential impacts on poultry health and the economy. Future research should focus on understanding the transmission dynamics and developing effective vaccination strategies.

The author is very thankful to the University of Alnoor/which helped improve the quality of this work.

Conflict of Interest
The authors revealed that there was no potential conflicts of interest.

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