EPIDEMIOLOGICAL HIGHLIGHTS ON SOME VIRUSES ASSOCIATED WITH INCREASED MORTALITY IN CHICKENS BRED IN SHARKIA GOVERNORATE.

EPIDEMIOLOGICAL HIGHLIGHTS ON SOME VIRUSES ASSOCIATED WITH INCREASED MORTALITY IN CHICKENS BRED IN SHARKIA GOVERNORATE.

BAYOUMIE, H.A.A.^* and HASANEEN, O.A.^**

^* Senior Researcher, Animal health Res. Inst. Zagazig (Poult. Dis. Dept.)

^** Lecture of Poult. Dis. (Dept. of Poult. and Rabbit Dis. Faculty. Vet. Med. Zagazig Univ.)

Email: heshambayoumie@yahoo.com                                                                   Assiut University Email: www.aun.edu.eg

INTRODUCTION

Eid (1994) stated that avian viruses causes severe economic losses in poultry beside other identified causative agents (Bacterial, mycotic, intoxication, etc.) and that the outcome of infection is influenced by many factors associated with the host, organisms and environment Bradbury )1984(, Dhillon and Kibenge )1987(, and Gelb )1989).

Morrow (2008) stated that demonstration of antibody simply shows the antigen that a bird has been in contact with at some time in the past., but this does not prove that a clinical disease syndrome is caused by the organism associated with the particular antigen., because vaccinated flocks will have antibody from vaccination., and because natural infection could have occurred earlier. For this Paired serum samples (taken at the time of clinical disease and then in convalescence) will provide a convincing evidence of seroconversion and association of an agent to the clinical signs seen. As for broilers testing the seroconversion is difficult because of their short life, and for possessing maternally derived antibodies which may be from vaccination of parent stock rather than wild strain infection. For this reason sentinel birds should be grown on to allow clearer seroconversion demonstration.

Comin et al. (2013) stated that the serological diagnosis of avian influenza (AI) can be performed using different methods, yet the haemagglutination inhibition (HI) test is considered the ‘gold standard’ for AI antibody subtyping.

This study aimed to highlight the potential existence avian of influenza infections circulating among chicken flocks in Sharkia beside NDV, IBV and EDS₇₆^.

MATERIALS

Chicken flocks and Serum samples

1317 Serum samples were collected from different localities in Sharkia Governorate from 36 poultry flocks during the period of January 2014 to November 2014., they represent (6) broiler, (2) breeder, and (28) layer chicken flocks. These flocks were suffering increased mortality, drop in egg production or assessing immune titers post vaccination. The obtained sera were stored at −20 °C in HI plates until used. Table (1) shows the vaccination history, source and number of the collected samples.

Washed CRBCS

Chicken RBCS were obtained from 28 day old specific antibody negative chickens (SAN).

Saline

Sodium chloride 0.9% (ADWIC) ®.

Viral antigens

IB viral antigen

IB viral antigens for HI test were obtained from GD Holland,

• § (mass- 41- VLDA 035) lot 12639-020412 exp. 4/2022,
• § (793 B Designated 4/91 – VLDIA 186) lot 13695-280613 exp.6/2023,
• § (D-274 VLDIA 032) lot 12645-020812 exp. 8-2022,

ND viral antigen

Allantoic fluid from chicken embryos inoculated with Lasota NDV{Intervet, batch-12636jj01, Exp.1-2015 }was used as HA antigen for HI-test.

EDS₇₆ viral antigen

Adeno 127 Designated EDS₇₆­ viral antigens for HI test were obtained from GD Holland. (VLDIA 038) lot 13773-020813 exp. 8-2022.

AIV viral antigens

• § H5N1 (Kindly obtained from Dr. Souzan Tolba NDV department El-Abassia)
• § H5N2 (lot 101111A pro. 10/11/2011 exp. 10/11/2015). Kindly obtained from Profarm for vaccine distribution.
• § H5N3 (VLDIA 240 GD Holand) lot 7605-010607 exp. 11-2023.
• § H9 (VLDIA 113 GD Holand) lot 14672-080414 exp. 4-2024.
• § H7 (VLDIA 98 GD Holand) lot 10604-260110 exp. 1-2020.

Negative serum         

Sera from one day old SPF chicks were used.

METHODS

HI for IBV, NDV, EDS ₇₆

HI tests, for the fore mentioned antigens were performed as described by Villegas (2006).

HI for AIV.

OIE Manual of Diagnostic Tests and Vaccines for Terrestrial Animals 2010.

Bacteriological Examination:

Bacteriological samples were examined according to Osbaldiston (1973).

Statistical analysis.

Data were statistically analyzed as described by Snedecor and Cochran (1967) using SPSS.16 computer program, value was used to determine significance.

Table 1: History of investigated samples.

RESULT

Results of the present work are illustrated in tables (2-4).

Table 2: Results of Serological investigations (After Cut-off values)

       * Average                          ** Standard Deviation                             *** Variance                           0 = Not Done

Table 3:Simultaneous viral affections in the examined samples

Table 4: Viral Concurrency in relation to El- Sharkia localities

DISCUSSION

Morrow (2008) stated that., demonstration of antibody through seroservillance simply shows what a bird has been in contact with at some time in the past., but this does not prove that a clinical syndrome is caused by a certain antigen., because vaccinated flocks will have antibody from vaccination., and natural infection could have occurred earlier and was not associated with this clinical syndrome. For these reasons a paired serum samples, taken at the time of clinical disease and in convalescence, provides the most convincing evidence of seroconversion and association of an agent to the clinical signs seen. He also mentioned that, testing the seroconversion in broilers is difficult because of their short life span, and for possessing maternally derived antibodies. For this reason sentinel birds should be grown on without neglicance., to allow clearer seroconversion demonstration.

During interpretation of serologic data It is usually impossible to differentiate between antibodies that are produced by vaccination or those resulting from field exposure to a given infectious agent. The only difference that may be observed is that the antibody titer following a field challenge may be higher than that observed following vaccination. A valid interpretation of serologic results requires a complete knowledge of the flock’s vaccination history and disease situation. In the present study a detailed vaccination history is illustrated in table (1).Morrow (2008) stated that., the number of serum samples needed depends on the reason for the testing and test characteristics i.e. for diagnosis., 10–60  serum samples per group should be taken., smaller numbers can be taken from sentinel birds during paired sampling for demonstration of the seroconversion., more samples are required to show an overall decrease in the number of seronegative birds., and for confirmation of freedom from infection a maximum of 60 samples per group is needed to give 95% confidence that infection of 5% of the animals would be detected. In this case the observation of one positive result defines the group as infected. This sort of testing can be a part of an eradication programme. He also mentioned that for post vaccination response evaluation a 20–30 sera per group are enough.

Newcastle disease virus (NDV) is one of the highly contagious diseases contained in the list A of the Office International des Epizooties (OIE). The disease is caused by the avian paramyxovirus serotype-1 (APMV-1). It affects a variety of avian species but causes most severe disease and economic losses in domestic poultry Kaleta (1992). Infection with different strains of NDV may result in a broad variation in severity and spread of the disease, even in a single host species.

Beard and Hanson (1981). Grouped NDV strains into five pathotypes based on the induced clinical signs in infected chickens (i) viscerotropic velogenic (associated with high mortality and intestinal lesions). (ii) Neurotropic velogenic (associated with high mortality following central-nervous signs). (iii) mesogenic (associated with low mortality, respiratory and nervous signs). (iv) lentogenic (associated with mild or clinical inapparent respiratory infections). (v) Asymptomatic enteritic (associated with inapparent intestinal infections).

ND is almost indistinguishable from HPAI Alexander (1997), Easterday et al., 1997). The possibility of misdiagnosing HPAI as ND couldn’t be ruled out in the field. When NDV infects chickens, antibody titers rise within 6 – 10 days and subsequently diminish slowly to zero Alexander (1997). Velogenic strains usually elicit a higher titer than mesogenic and lentogenic strains Alexander (1997), Alexander et al. (2004). In non-vaccinated chickens, HI titers specific to NDV can be taken as a sign of previous infection, and titers as high as 10 log₂ suggests field exposure to pathogenic NDVChrysostome et al. (1995), Alexander et al. (2004).

From another point of view Allan et al. (1978) were using ND- HI titer (using 4 HA units) as a predictive tools for the expected mortality and drop in egg production upon challenge as follow.

• When All individual HI values was 2² or less (100% mortality on challenge is expected).
• When all individual HI values was 2² to 2⁵ log mean 2^3.75 STDV 0.4 (10% mortality on challenge is expected).
• When all individual HI values was 2⁴ to 2⁶ log mean 2^5.2 STDV 0.35(0% mortality on challenge is expected).
• When all individual HI values was 2⁶ to 2⁸ log mean 2^6.5 STDV 1.2 (A serious drop in egg production, with no deaths could be expected and convalescent HI titer 2¹⁴ or greater could be reached).
• when All individual HI values was 2⁹ to 2¹¹ log mean 2^10.5 STDV 1.4 (No drop in egg production, no deaths,  and a convalescent titers 2¹¹ to 2¹²  could be obtained),
• And finally if the range was 2¹¹ to 2¹³ log mean 2^11.2 STDV 1.3 (A flock that will remain free from any risk of NDV).

In the present study., the ND-HI titers were ranging from (0 up to 10) log 2 (table-2). The titer of sample (6,7,14,19,23,24,27,29,30,32,and 35) were ranging from (9.1 up to 10) log₂ (table -2)., this could justify a NDV affection especially if we consider that the vaccines applied for these flocks is not sufficient to elect such titer.  

AIV-(H5)Comin et al. (2013) found that HI test has a near perfect accuracy that might be considered as a gold standard test. The test enables processing large amounts of samples in a short time, if the reference viral antigen is close enough to the virus isolate to be tested when panels of different antigens are used. In the present study, we had used H5 with three distinct neuraminidases i.e. (H5N1, H5N2, and H5N3) these represent the AIV commercially available viral antigens in the Egyptian market.

Timm Harder (2012): In a personal communication mentioned that, the neuraminidases has a significant role in HI as well. He explained., that neuraminidases are enzyme that binds to its substrate, the sialic glycans that are also used as the cellular receptor for influenza A virus HA proteins. He also gave a further interpretation as follow. (Given the situation of a duck holding that had experienced an H₁₁N₁ infection. Serum taken from these birds three weeks later will react in HI with the H₅N₁ and the H₇N₁ antigens but none of them will react with the H₅N₃ or the H₇N₇ ones. Thus such sera are considered H₅/H₇ negative despite their reactivity with one H₅/H₇ antigen!). In addition, he mentioned that HI assay are difficult to perform and more difficult to interpret! This is because HA and NA are always linked to diagnostic antigens (inactivated viruses). On the other hand, HI assays are highly specific. Thus, even antibodies raised against one of the Egyptian H₅N₁ cluster 2.2.1.1 viruses will probably not react with (European) H₅N₁ antigen due to the large antigenic distance between these HA antigens. In the present study, samples were collected from vaccinated bird. Vaccines in Egypt represent these viral categories (H₅N₁, H₅N₂, H₅N₃) in a reassortant form., so it became very difficult for us to evaluate to obtained results and because poultry holders had stopped the inclusion of sentinel birds in the breaded poultry batches., we thought that the high HI titer for the H₅N₃antigen observed in samples (3,6,8,13,17, 18,28,29,30,35 and 35) was  relevant to the vaccinal strain used as mentioned byTimm Harder (2012)., but on second thought  we had found that flock sample (3,6,8 and 30) were applying vaccination against AIV using the  H₅N₁ reasssortants and flock sample (13,28) were totally not vaccinated against AIV and that flock samples (17,18,29,35 and 36) had anonymous history., this may reflect H₅N₃ circulation in the fore mentioned flock samples.

AIV-(H7) Abdel whab et al. (2013) mentioned that Avian influenza viruses of H₅ and H₇ subtypes exhibit two different pathotypes in poultry: infection with low pathogenic (LP) strains results in minimal, if any, health disturbances, whereas highly pathogenic (HP) strains cause severe morbidity and mortality. LPAIV of H₅ and H₇ subtypes can spontaneously mutate into HPAIV. Ten outbreaks caused by HPAIV were preceded by circulation of a predecessor or LPAIV in poultry. Three of them were caused by H₅N₂ subtype and seven involved H₇ in combination with N₁, N₃, or N₇. Abdelwhab et al. (2014) mentioned that., H₇ subtype HA gene has been found in combination with all nine NA subtype genes. Most exhibit low pathogenicity and only rarely high pathogenicity in poultry (and humans). During the past few years infections of poultry and humans with H₇ subtypes have increased markedly. In the present study., fortunately H₇ vaccines is not yet allowed to Egypt., this had facilitated our mission in interpreting the obtained results, which aim to ascertain its existance in Sharkia., although the GMT of the HI against H₇N₁was ranging from (.1±.01 – 4.54±.32) (table -2). It worth to mention., that examined samples revealed positive seroreactive values that exceeded the cut off values which could be interpreted surely as infection in samples (14, 17, 18,19,20, 21,28, 29,30,34,35 and 36) had a positive seroreactive that exceeded the cutoff values OIE (2010). This gives a sure incidence of AIV-H7 in different localities of sharkia governorate but this incidence was milder than the previously recorded incidence by Afifi et al. (2013). The incidence of the disease in sharkia based on a seropositive reactors could be speculated to start in (June, July, Sept., Oct., and November 2014) in the locations from which samples were collected such as (El-Salhia, 10th of Ramadan, El-Ibrahemia and Abo Hammad) (table 2 figure 1), it also worth to mention that the positive seroreactives were totally from commercial layers except for flock (sample 29) which were collected from a broiler breeder flock. It should be also noted that the total seropositive samples exceeding the cutoff values were (63/1317 with a percent of 4.78%).

Abdel whab et al. (2014) mentioned that wild birds are the natural reservoir of the H₇ virus. Geographically, the most prevalent subtype is H₇N₇, which is endemic in wild birds in Europe and was frequently reported in domestic poultry, whereas subtype H₇N₃ is mostly isolated from the Americas. In humans, mild to fatal infections were caused by subtypes H₇N₂, H₇N₃, H₇N₇ and H₇N₉.

While, infections of humans have been associated mostly with exposure to domestic poultry, infections of poultry have been linked to wild birds or live bird markets. Fred Leung (2012) in a personal communication commented on the role of wild migratory bird in AIV disease transfer as follow (although the virus movements were recorded in the known migratory bird routes but in the opposite direction for the migratory bird flow route). Generally, depopulation of infected poultry was the main control tool. In contrast to recent cases caused by subtype H₇N₉, human infections were usually self-limiting and rarely required antiviral medication. Close genetic and antigenic relatedness of H₇ viruses of different origins may be helpful in development of universal vaccines and diagnostics for both animals and humans. Due to the wide spread of H₇ viruses and their zoonotic importance more research is required for better understanding of the epidemiology, pathobiology and virulence determinants of these viruses.

AIV-(H9) Positive seroreactive samples were recorded in the submitted samples from different localities of Sharkia governorate starting from (Jan 2014). The GMT of HI titers was ranging from (.32 up to 10). Even. In (sample15) 2 / 24 sample exceeded the cutoff values. Observing these results and considering that only flock sample (1 and 9) had received vaccines against H₉N₂ lead us to conclude., a wide distribution of H₉ in Sharkia governorate ,it also should be pointed for the fact that the distance between vaccination in flocks (1) and sample collection exceeded 100 day this could justify infection and with flock (9) we can observe that birds were simultaneously vaccinated with (AIV H₉+NDV) and that the serologic response for NDV is very low compared to the serologic response for AIV-H9 which suggests superinfection with H9. The total seropositive sample for H9 were (736 out of 1317 examined) i.e.(55.8%).

Afifi et al. (2013). Examined the potential existence of H₇ and H₉ AIV circulating among chicken flocks in Egypt. Serum samples were collected from chicken flocks that experienced respiratory distresses and/or variable mortality rates. H₇ and H₉ virus infections were screened by HI assay. Concerning Sharkia governorate. A 133 serum samples were collected from one broiler, one breeder and two layer flocks. 52 out of 133 examined sera, seropositve i.e. (39 %) were recorded for H₇ .As for H₉ A 113 out of 133 seropositive sample were recorded i.e. (84 %). Prevalence of both H₇ and H₉ antibodies were higher in layer followed by breeder then broiler flocks. Afifi et al. (2013) concluded that special consideration should be paid to control influenza viruses in Egypt, as pandemic influenza strains may develop unnoticed given the presence of subclinical infections, and the possibility of re-assortment with the prevailing endemic H₅N₁ virus strains exciting in Egypt.

Afifi et al. (2013)mentioned that., they were able to isolate H₉N₂ from broiler flock from Alexandria Governorate in the northern part of Egypt (unpublished data) however, only a broiler flock from Beni-Suef was found seropositive to H₉. Interestingly, another recent report for the isolation of H₉N₂ from quail in Egypt was also reported El-Zoghby et al. (2012). Meanwhile, the isolation of AIV subtype H₇ from Egypt was only recorded from migratory birds Aly et al. (2010) and Soliman et al. (2012) as follow., on 2004 (H₇N₁). On 2004, 2005, and 2006 (H₇N₇). On 2006 (H₇N₉), and on 2007 (H₇N₃). Interestingly, H₇ serological results on backyard from 11 villages in the nearby areas were negative Aly et al. (2008). To the best of our knowledge, no recent report revealed isolation of H₇ from commercial or backyard poultry populations. But historically, LPAIV A/turkey/ Egypt/88 (H₇N₁) was isolated Khafagy et al. (1992); however, they reported the absence of H₇ seropositive sera when testing 6124 chicken and 92 turkey sera Khafagy et al. (1995). However, the presences of antibodies against AIV H7 were reported Afifi et al. (1999). Egypt is located in the pathway of migratory birds and represents a hinge zone of wild bird migration, where the East Africa–West Asia and Black Sea–Mediterranean’s flyways overlap and large diversity of species migrating to and from South Africa, Europe, and Central Asia were detected in Egypt Soliman et al. (2012). It was recorded that the migratory birds plays a role in the introduction but not the spread of AIV to other wild and domestic species that are present in their migratory pathways Feare (2010), Soliman et al. (2012). Increased numbers of seropositive were observed in farms located within the migratory route of wild birds Al- Natour and Abo-Shehada (2005).

Avian adenovirus (AAV) associated with clinical disease was isolated from an outbreak of respiratory disease in quail Olson (1950). Since that time, AAVs were seen in all types and breeds of chickens and from a variety of other avian species. AAV were frequently isolated from the respiratory and/or intestinal tracts of the apparently healthy chickens, and their role in the etiology of clinical disease was regarded as insignificant Yates et al. (1976) and Winterfield (1984). Sometimes., AAVs were  associated with a variety of specific disease such as (quail bronchitis, inclusion body hepatitis (IBH), egg drop syndrome (EDS), turkey hemorrhagic enteritis (THE), marble spleen disease and respiratory manifestation) Du Bose et al. (1958), Du Boes and Grumbles (1959), Ismail (1966), Ahmed and El-Sisi (1969), Fadly and WinterfieId (1973), Rosenberger   et al. (1974), Hoffmann et al. (1975), McFerran (1981), McFerran (1989), McFerran (1991) and McFerran and Stuart (1990). Papanikoloau et al. (1985) and McFerran and Stuart 1990). Specific pathogenicity for poultry and vertical transmission had led AAVs to receive more attention McFerran and Stuart (1990). AAV should not be neglected as complicating factors in the course of some poultry diseases e.g Mycoplasmosis and IBV Monereal and Ahmed (1963),  Monereal (1966), Monereal (1968), Awad et al. (1973), Dhillon and Kibenge (1987)., and in birds vaccinated with spray IBV at day-old or immune-suppressed by IBDV Mousa et al. (1984), McFerran and Stuart (1990).

Egg drop syndrome 1976 (EDS₇₆) is an economically important viral disease characterized by a severe drop in egg production as well as the production of shell less, thin-shelled, discolored or misshapen eggs Van Ecz (1982). Although, the occurrence of EDS₇₆ in layers has been reported worldwide but there is limited information on its occurrence in broiler chickens Meulemans et al. (1979).

Hemagglutinating AAV is represented by EDS_76. There are three evident patterns of EDS₇₆. Classical EDS₇₆ following the introduction of virus into primary breeding stock, probably through a vaccine grown in duck cells. Spread was through the embryo and the resulting reactivation of virus at peak of egg production gave an apparent breed and age susceptibility. The second pattern is the endemic form where lateral spread between flocks via contaminated eggs occurs at any age of laying hens. The third (sporadic) form is again seen in any age or breed of birds resulting from infection from ducks and geese or any infected wild bird McFerran and Stuart (1990).

To our knowledge, reports on seroprevalence of EDS₇₆ in chickens in commercial farms in Sharkia are scanty. Although killed vaccines against EDS₇₆ such as (IBV+NDV+EDS₇₆) or (IBV+NDV+EDS₇₆ +AIV) are used in commercial layer flocks.In the present study three broiler flocks (sample 2), (sample 11) and (sample 13) were serologically positive and had HI titre of (6.75 ± 0.16, 5 and 7) respectively after neglecting the cutoff values., these flocks were located in (zagazig, Belbees and El-salhia). On the other hand., layer flocks in (sample 6), (sample 7) and sample 8) had a seropositive HI titer at age of 20wk, 26wk, and 26wk of age that was {7.98 ± 0.17, 7.09 ± 0.3 and 3.25 ±0.45} respectively., these flocks had  received the triple killed vaccine  at age around 11 WK of age during the rearing period in integrated plants but the layer flocks in small holders such as flock  (sample  25), (sample  26), (sample 27), (sample 28), (sample 29) and (sample 31) had  HI titer ranging from (6.62±0.9 up to 9.94±.06) which could be referred to as an infection. These flock were located in (Belbees, 10^th of Ramadan, El-Ibrahemia and Abo-hammad).

Singh et al. (1995) screened 22 broiler flocks using 347 serum samples examined with the (HI) test., they found that 114 sample i.e. (32.9%) were positive for antibodies to (EDS₇₆).,the HI titres of these serum samples ranged from 2 to 9 log2 and the overall geometric mean titre was 3.9 log2. All of the examined flocks in the present study were positive for antibodies suggesting widespread EDS'₇₆ infection in this region of Egypt. The presence of high HI antibody titres suggests that infection was acquired at an early age and this could not be due to maternal antibodies as the present study was limited to birds 5 to l0 weeks old and maternally derived antibodies are known to persist only up to 3 weeks of age McFerran (1981), McFerran and Stuart (1990).Vertical transmission is common in EDS'₇₆ virus infection but congenitally infected birds may not become serologically positive until 25-28 weeks of age VanEcz (1982). Because it has been observed that congenital infection remains dormant until sexual maturity or in response to stress of production Nawathe and Abegunde (1980). Contrary to this the present observation of HI antibody in broiler birds provides evidence for horizontal spread of the virus. This is supported by other workers who have confirmed the transmission of the virus at different stages throughout the rearing of chickens Cook and Darbyshire (1981). McFerran (1979) also reported the lateral spread of the virus because small farmers raise broilers and laying chickens on the same premises. An infected flock of broilers may be a potential risk to laying chickens and hence regular monitoring of broiler flocks for infection should be an essential component in the control of the disease.

Infectious Bronchitis Virus (IBV) is a highly contagious acute viral disease of the upper respiratory tract of chickens, it can also replicate in epithelial tissues of kidneys, gonads and oviduct of chickens causing their pathology and affecting the performance Lee et al. (2004).

Prevention of IB is achieved mainly through vaccination. Although in most cases IBV strains within a geographic region are distinct as mentioned byCallison et al. (2001), Gelb et al. (2005), Ignjatovic et al. (2006). Because IBV undergoes frequent changes in the viral genome, mainly in the S1 gene which result in point mutations promoting the emergence of new antigenic variants Bochkov    et al. (2007), Ammayappan et al. (2008) and Lee       et al. (2008). The multiple IBV serotypes and its antigenic variation adds complexity to the proper selection of vaccination protocol and proper selection of  serologic method to analyses the test results Jackwood and De Wit (2013). Vaccine strains should be selected to represent the antigenic spectrum of isolates in a particular region, because attenuated vaccines are known to have a limited range of protection, confined in many cases to homologous strains, rendering vaccination partially successful Lin et al. (2005). The use of heterologous vaccine strains, either simultaneous or sequentially, has broadened the protection spectrum in some cases Cook et al. (1999), but it is difficult to predict which combinations may confer the best protection. On the other hand, more virulent vaccine strains may have a broader range of protection, but their use is not recommended to avoid the risk of a disease outbreak Darbyshire (1985).

In the present study HI for IBV using the D-274 antigen of the German variant led us to conclude that the majority of the examined flocks were seropositive after neglecting the cutoff values and considering that., these flocks didn’t receive such protective vaccines as seen with sample (2, 3, 8, 10, 11, 14, 17, 18,19, 21,24, 26, 27and  30). On the other hand Infection with Mass type IBV was noticed in (sample 8)., the  history of this flock shows a drop in egg production and deformed egg quality. This flock was re-examined in (sample 20) at 51 week of age where a high immune titers was still detectable.

Abdelwhab and Abdel-Moneim (2015) had wondered! Will Egypt be the epicenter of the next influenza pandemic? We think yes especially if we consider viral Concurrency as seen in (table 3-4) beside neiglecance in poultry operation.

During investigation of the increased morality or dropped egg production in the examined flocks bacteriological work was performed parallel to the serosurvillance surprisingly a very resistant bacteria was detected, these resistant bacteria was initially identified as klebsilla spp. further examination is ongoing.

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