Seroprevalence of mycoplasmosis in broiler, layer, and native chickens in Giza, Egypt

We aimed to investigate Mycoplasma infections among chicken flocks (Ross, Lohmann and native) in Giza, Egypt, using serological tests, including the slide plate agglutination (SPA) test, hemagglutination inhibition (HI) test, and enzyme-linked immunosorbent assay (ELISA). The slide plate agglutination examination, a serological test, indicated the prevalence of Mg and Ms infections of 10.9% and 13.2%, respectively. On 91 SPA test positive serum samples for either Mg or Ms, a passive hemagglutination/hemagglutination inhibition (HI) test was performed. The SPA and HI test findings were found to be comparable. On 90 SPA test positive samples, an ELISA was performed using commercial kits for Mg and Ms serodiagnosis. According to the ELISA data, only 83.33% and 18.88% of SPA test positive samples were confirmed as positive for Ms and Mg infections, respectively. The prevalence increased to 84.44% and 77.77%, respectively, when suspected samples were deemed positive.


Introduction
Avian mycoplasmosis is one of the diseases that have a negative impact on the health and productivity of domestic chickens. Mycoplasma gallisepticum (Mg) and Mycoplasma synoviae (Ms) are the major pathogens that cause the disease. Mycoplasma infection induces significant economic losses in poultry by reducing; body weight gain, meat quality, feed conversion rate; in broilers, causing a significant decline in egg output in layers, and increasing embryo mortality in breeders [1]. Mycoplasma propagation is transmitted both horizontally and vertically in poultry flocks, with the former facilitated by intense industry and stress causes [2]. Mycoplasma is a genus of prokaryotes belonging to the Mollicutes family, which includes the smallest recognized self-replicating prokaryotes. Ms induces infectious synovitis or mild upper respiratory disease in chickens, while Mg triggers chronic respiratory disease in chickens and infectious sinusitis in turkeys. Clinical specimens of Mycoplasma species are difficult to cultivate. This is attributable to their meticulous disposition, close links to their hosts, and slow growth on artificial media. Clinical signs of Mycoplasma infection can be detected, and Mycoplasma can be isolated and identified by culturing on Mycoplasma media [3]. Biochemical, serological, or molecular tests a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 are used to identify Mycoplasma isolates, as well as serological analysis of host sera using the slide agglutination test (SAT), hemagglutination inhibition (HI) test, or enzyme-linked immunosorbent assay (ELISA) [4]. Nonetheless, since the pathogen has multiple strains, culturebased diagnosis is inadequate for mycoplasmosis [5]. Where concurrent infections are reduced and optimal environmental conditions are provided, controlling the clinical manifestations of Mycoplasma infections is easier. Serological examinations are crucial for assessing and tracking the infection status of chicken flocks with Mycoplasma [6,7]. Serodiagnosis is one of the most efficient and accurate approaches for deciding whether or not birds have been subjected to numerous infectious agents. ELISA has been found to have better sensitivity and precision than SAR, although SAR is used as a screening test. Although HI has a high specificity, it is not commercially available and has a low sensitivity. Recently, ELISA with high sensitivity and specificity has become available [8]. The most commonly used tests are slide agglutination, ELISA, and HI, but others have been found, including radioimmunoassay, microimmunofluorescence, and IP assay. Poultry companies that use ELISA technology to screen large quantities of serum samples for virus antibodies can find this assay useful for Mycoplasma testing. Serological tests in general can lack accuracy and/or sensitivity; they are better used for flock tracking rather than individual bird research. Diagnosticians who choose to use those instruments should first determine the test's sensitivity and specificity in their own lab [9]. The aim of the present study was to use the SPA test, HI test, and ELISA to screen broiler, layer, and native chicken flocks of various breeds and ages for serodiagnosis of chicken mycoplasmosis.

Chickens and serum samples
The Department of Microbiology, Faculty of Science, Cairo University approved all animal experiments (CU-04-20), which adopted all national standards for the use of animals in scientific testing, and the basic procedure defined by the OIE. Blood samples from a conscious bird are suitable for diagnostic or testing purposes, according to the AVMA Guidelines for Animal Euthanasia (2020 Edition). In this study, three chicken breeds (Ross (broiler), Lohmann (Layer), and native (baladi)) were investigated. Samples were collected from clinical suspected birds. A total of 1000 blood samples were drawn from the wing veins or during the slaughter of chickens (666 Ross, 95 Lohmann, and 239 native). By centrifugation at 900 x g, serum was isolated from blood, aliquoted into clean tubes, and frozen at -20 ⁰C until required.

Mycoplasma antigens and antisera
The stained Mg and Ms antigens supplied by Intervet International B.V. (Netherlands) were used in the SPA examination. Antisera against Ms and Mg were purchased from Animal Health Service Ltd. (Netherlands). Antigens for Mycoplasma HI were prepared from Mg and Ms using the protocols suggested by Cho et al. 1976 [10]. After washing the bacterial harvest, the pellet was resuspended in the glycerine buffer (50.0% Cho buffer and 50.0% glycerine) to achieve a hemagglutination titer of 1:32 or 1:64. When the pellet was mixed with the glycerol buffer at a ratio of 2.0% of the total culture volume of Mg and 0.5-1.0% of the total culture volume of Ms, this titer was normally produced. The antigen was divided into 2.0 mL aliquots and kept at -70˚C.

Slide plate agglutination (SPA)
Before use, antigens and sera were warmed to room temperature. Thirty microliters of each serum sample was combined in a circular pattern with an equivalent amount of mycoplasma antigen inside 4 cm 2 squares on a ruled glass plate. The plate was shaken for 5 s. The plate was no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing interests:
The authors have declared that no competing interests exist.
shaken again for 5 s at the end of the first minute, then read 55 s later. A positive reaction was suggested by the forming of distinct clumps, which normally began at the mixture's periphery. Positive and negative control sera were analyzed concurrently with the testing of tested samples [5].

Hemagglutination inhibition (HI)
The HI titers were calculated as the reciprocals of the highest dilutions which resulted in a hemagglutination sheath on the bottom wall of the well. The HI titer of each sample was the highest serum dilution exhibiting complete inhibition of hemagglutination as indicated by formation of a loose button and flowing of cells when the plate was tilted. Antigen titration and HI were carried out following the protocols of USDA (1984) and OIE (2018).

Enzyme linked immunosorbent assay (ELISA)
The presence of distinct antibodies in the sera of tested chickens was used to diagnose Mg and Ms infection using commercial indirect ELISA kits (Synbiotics Corp., CA, USA). The manufacturer's guidelines were followed precisely. The plates were read using an ELISA reader at 405 nm. For MG, samples with OD values less than 0.2 (titer = 0) were considered negative and those with OD values greater or equal to 0.6 (titer = 744 or greater) were considered positive while samples with OD values between 0.2 and 0.599 (titer = 149 to 743) were considered probable (suspect). Concerning Ms, samples with OD values less than 0.3 (titer = 0) were considered negative and those with OD values greater or equal to 0.6 (titer = 744 or greater) were considered positive while samples with OD values between 0.3 and 0.599 (titer = 270 to743) were considered probable (suspects).

Prevalence of Mg and Ms by serum slide plate agglutination
Out of 1000 tests, 109 were positive for Mg infection (10.9%), and 132 were positive for Ms infection (13.2%), as seen in Table 1. Native chickens had a higher frequency of Mg and Ms illnesses, with rates of 34.72% and 28.03%, respectively. Both Mg and Ms infections were observed in 2.25% and 5.40% of Ross chickens, respectively, and 11.57% and 27.36% of Lohmann chickens.  represented in the tables in comparison with the frequency of Mg and Ms infection. Fig 1 shows that the youngest ages yielded the lowest percentage of positivity within Lohmann layers. In the case of Mg infection, no consistent relationship between age and infection occurrence was found, and the same was found in the case of mixed infection. Ms infection was more prevalent in older birds, with a 60% prevalence rate among 67-week-old birds, and comparable findings were found in Ross and native breeds (Figs 2 and 3). In terms of total prevalence, Ms infection was more common in the Lohmann and Ross breeds, whereas Mg infection was more common in the native breed.

Hemagglutination inhibition (antibody titers)
Mg and Ms plate agglutination positive samples tested positive for HI utilizing the HI-Mg and HI-Ms antigens. Both antigens could hemagglutinate chicken RBCs in the antigen control wells (without serum). Table 2 shows that no sample had a negative response to the HI test using either the Mg or Ms antigens. The HI titer; however, differed between tests, varying from 2 3 to 2 6 HI units. A clear correlation was observed between the HI titer and the agglutination degree for the same sample as compared with the findings of the SPA examination. It was also discovered that Ms antigen produced higher HI titers than Mg antigen. Indirect ELISA. Indirect ELISA was used to evaluate 90 serum samples from Ross, Lohmann, and local chicken breeds for the existence of Mg and Ms antibodies. Table 3  Meanwhile, the percentage of samples with OD values indicating probable (doubtful) results was low in the Ms test (7.77%). In contrast, Mg infection prevalence (18.88%) was slightly lower than that of Ms. Also, the Mg test produced further tests with possible findings (20%).
Of 90 chicken serum samples, examined for Mg and Ms infection by ELISA and SPA tests, similar results were obtained in 71.11% and 70% of the samples with Mg and Ms, respectively. When the probable results were considered positive, the respective percentages increased to 84.44% and 77.77%. When the prevalence of infection was considered with both tests, ELISA prevalence rates were 90% and 38.88% while SPA prevalence rates were 74.44% and 43.33% for Ms and Mg infections, respectively. As a result, the ELISA findings may be more dependent on than the results of the other two serological studies included in the study. There is no doubt; however, that serum plate agglutination is a fast, low-cost, and reliable test that can be used to initiate a mycoplasmosis serosurvey.  The Lohmann breed showed prevalence rates of 11.57% and 27.36%, and the Ross breed showed the least prevalence rates of 2.25% and 5.4% for Mg and Ms infection, respectively. The high rates of Mg and Ms infection in native chickens could be attributed to the owners' rearing and management practices, which include fewer hygienic precautions and therapeutic approaches. Furthermore, the majority of the native flocks used to gather the samples were held in open systems (backyards) with more stressful environmental conditions. When compared with either native or Lohmann chickens, Ross samples had the lowest rate of infection. However, the majority of the samples came from Ross broilers, where SPA tests are unlikely to reveal positive cases of Mg and Ms. Serum samples were collected from Lohmann chickens aged 16 weeks and up, which had a higher risk of being positive. Jordan, 1979 [11] stated that the significance of age on tolerance to Mycoplasma infection in chickens is unclear when it comes to the age-related occurrence of Mg and Ms infection. Chickens aged 2 to 20 weeks; however, were found to be vulnerable to experimental infection. The detection of Ms infection in Ross chickens aged 5-8 weeks indicates that the antibodies were found in the sera of infected chickens after a period of time (2-3 weeks). Positive responses to both Mg and Ms antigens were observed in certain Lohmann and native chicken tests, suggesting either mixed infection or cross reactivity. It was reported that serum from Mg-infected birds did not react with Ms antigen. In contrast, sera from Ms-infected birds reacted with the Mg antigen. Cross reactions; however, did not pose a concern in other experiments, such as the HI test [12,13]. Positive SPA test reactions for both antigens may also be called Ms infection while mixed infection is unlikely. Other tests, such as HI, can confirm this. In the HI test, only 12 of 18 samples reacted positively with Ms antigens, while 6 reacted positively with both antigens. This supports the finding that cross reactions in the serum SPA test do not represent a problem with the HI test [12,13]. The Ms detection HI test tends to be reasonably specific, and it can be used in combination with the Mg detection HI test to differentiate between Ms and Mg infection. For detecting antibodies against Mg, the HI test was suggested as an official test and as the most reliable serological test [14]. The positive response of a sample to a serum plate test when the same sample was negative to an HI test utilizing the same antigen was traditionally due to the bird's treatment of immunosuppressive drugs including chlorotetracycline in different doses. Early antibodies (IgM agglutinins) may be inhibited to a lesser degree by certain medications than HI late IgG antibodies [15]. The diagnosis of avian mycoplasmosis was achieved using ELISA. The assay was described as a successful test for that purpose [16,17]. In the present study, concordance rates between ELISA and serum SPA were 71.11% and 70% with Mg and Ms infection, respectively. When questionable ELISA results are considered positive, the rates rise to 84.44% and 77.77%, respectively. Apart from concordance, Mg and Ms infection rates with ELISA were 38.88% and 90%, respectively, and with the serum SPA test were 43.33% and 74.44%. This confirms once more the possible cross reaction of serum samples from Ms-infected birds with Mg antigen in the serum SPA test [12,13].
As a more precise measure, ELISA may differentiate between false and true positive reactions. ELISA was identified as an easy, effective, and specific assay in related studies, and it was used to track the appearance of specific anti-Mg IgG in chicken sera at different intervals after the onset of mycoplasma-induced respiratory disease [18]. Antibodies to MS were also detected using ELISA in the egg yolks of commercial layers as proof of MS infection. The assay showed a prevalence of egg antibody to MS of 78.6%, and it was identified as a sensitive and specific test [19]. When compared to other serologic studies, ELISA was found to be more accurate than HI in confirming Mg infection in fair and exhibition birds. In birds experimentally infected with Mg by Ewing et al. [20], there were no significant differences between HI and ELISA.
The Mg serum SPA assay cross-reacted with 15% of 195 sera obtained from flocks that were reported positive for Ms by culture. Neither the Mg ELISA nor the HI revealed any cross-reactions. In populations with a low prevalence of Mg infection, the findings revealed no differences between ELISA and HI as confirmatory tests. In a population with moderate levels of Mg infection; however, ELISA was superior to HI [20]. Feberwee et al. (2005) [21] reported that ELISA and the rapid SPA test using undiluted serum showed a relatively high number of falsepositive results for mycoplasmosis serosurvey. Every serological test, according to the findings, should predict a certain level of false-positive results. Because the prevalence of false-positive results varied between serologic studies, relying solely on one test method is not recommended. Serum samples were diluted in ELISA but not in SPA or HI experiments in this analysis. As a result, the findings of the ELISA may be relied on more than the results of the other two serological experiments included in the analysis. There is no doubt; however, that serum SPA is a quick, low-cost, and reliable test that can be used to initiate a mycoplasmosis serosurvey.