Shell abnormalities were observed from eggs produced by 55-week-old Hyline brown layers of the SH farm located in Gyeonggi-do that were submitted to the Avian Disease Laboratory of Chungbuk National University (Korea) for diagnosis. Rough and dark patches of various sizes with different densities were found on the blunt end of the eggs. A clear demarcation zone separating the patches from the normal part of the shell was clearly identified when a light was held up to the eggs. Discoloration, specks, and cracks were also observed in the eggs with EAA (panels A-D in Fig. 1).

Egg production rates of 54-week-old layers from the SH farm where EAA was observed decreased from 86% to 79% during a 3-week period. Cracked egg rates also increased from 2.6% to 8.3%, reaching a peak along with increased mortality at 57-week-old age (Fig. 2). While changes of eggshell quality may be attributed to non-infectious factors [13], an experimental analysis was conducted to confirm the existence of infection. Factors such as sudden changes in egg production or concentration of specific lesions on the apex of the eggs strongly suggest the possibility that egg quality has been altered by infectious agents.

Eggshell samples were obtained from 55-week-old chickens that produced eggs with EAA and 77-week-old chickens in which EAA was not observed after molting that resided at the SH farm. The serum samples were screened for antibodies specific for reproductive diseases to evaluate the possibility of infection. A hemagglutination inhibition test was conducted to monitor for avian influenza (AI), Newcastle disease (ND), and egg drop syndrome (EDS) according to Office International des Epizooties (OIE; France) standards. The titers of antibodies against AI, ND, and EDS fell well under vaccine-induced titers. Commercial ELISA kits (IDEXX Laboratories, USA) were used to screen for infectious bronchitis (IB), Mycoplasma gallisepticum (MG) infection, and MS infection. An additional rapid serum agglutination test was also conducted to survey for MG and MS infection using antigens provided by the Animal and Plant Quarantine Agency (Korea).

MS-specific antibody titers measured by the ELISA were 13,563 ± 5,945 for the 55-week-old layers and 6,053 ± 2,709 for the 77-week-old layers. These data indicated that the layers were infected by wild-type MS and reflected the fact that MS vaccines are not routinely administered in Korea. Anti-MG antibody was not detected by ELISA. An IB-specific ELISA was also conducted for 60 serum samples. The mean antibody titers for 55-week-old layers and 77-week-old layers were 5,871 ± 1,981 and 5,777 ± 2,744, respectively. Considering the fact that the flocks were vaccinated with IB oil emulsion vaccines before reaching the egg-laying age, the level of titer showed infection by wild-type IB did not occur [9].

To confirm infection by isolating wild-type MS, sterilized cotton swabs were collected from the choanal cleft, trachea, and oviduct. MS was cultured in Mycoplasma broth (BD, USA) as previously described by Catania et al. [2]. Samples for which the Mycoplasma broth color changed (yellow) were subjected to PCR for amplification of the vlhA gene [7]. DNA was extracted using a Viral Gene Spin kit (iNtRON Biotechnology, Korea). PCR-positive samples were submitted for gene sequence analysis (Cosmogenetech, Korea). MS was isolated from the respiratory tract of 41 birds among a total of 60 layers sampled at the age of 55 weeks. Additionally, 20 birds were selected from the 55-week-old layers to isolate MS from both the isthmus and uterus. Only one bird was MS-positive for both the isthmus and uterus. At the age of 77 weeks, 60 birds that did not produce eggs with EAA were surveyed for MS and 35 were found to be MS-positive in the respiratory tract. Among the 20 birds selected for MS isolation from the oviduct, none were positive. Samples from both groups (55 weeks or 77 weeks old) were cultured in blood agar medium (Hanil Komed, Korea). All cultures produced negative results.

Oviduct tissue samples were obtained from the infundibulum, magnum, isthmus, and uterus. The samples were processed by general methods for histologic examination [11] and subjected to hematoxylin and eosin (H&E) staining (Sigma-Aldrich, USA). No significant findings including evidence of inflammation were observed.

Previous studies revealed that pathologic lesions may be absent in the oviduct despite the presence of eggshell abnormalities, especially in cases of primary MS infections [4,5]. vlhA gene sequence analysis results showed that the strains (KM985992) isolated from the SH farm were identical, lacking 39 nucleotides (nt 133~171) compared with the EAA strain (FJ495803) previously documented by Feberwee et al. [5]. The isolated strains also had 100% resemblance with the enrofloxacine intermediate-resistant KC832815 strain previously isolated from broilers by Lysnyansky et al. [10].

To verify EAA, eggshell quality was evaluated for eggs collected from 55- and 77-week-old layers of the SH farm. The eggshells were tested for strength, thickness, and ultrastructure integrity. Scanning electon microscope (SEM) samples were prepared as previously described by Feberwee et al. [4] using Karnovsky fixative. The shells were fixed for 1~2 weeks. A statistical analysis was conducted using Fisher's least significant difference test. A p value less than 0.05% was considered statistically significant. The average shell thickness of EAA eggs from 55-week-old birds was 0.34 ± 0.05 mm. These data were compared that for eggs from both 77-week-old birds without signs of EAA and the negative control group of 54- and 64-week-old birds sampled from Hyline layer farm located in Gyeonggi-do without MS infection or EAA that produced eggs with a shell thickness ranging from 0.40 to 0.42 mm. Results of the analysis demonstrated that decreased shell thickness of the EAA eggs was statistically significant (p < 0.01). Similar results were recovered from the eggshell strength tests. The strength of the shells of eggs from 55-week-old birds with EAA (26.95 ± 6.15 N/cm²) was low compared to that of eggs produced by 77-week-old birds without EAA as well as negative control chickens without MS infection or EAA (34.55~36.95 N/cm²). Shell strength of the EAA eggs was significantly decreased (p < 0.01, Table 1).

To evaluate shell ultrastructure, SEM was performed. A loss of mammillary knob layer detail was observed for EAA eggs. These findings coincide with data from Feberwee et al. [4]. In addition to assessment of the mammillary knob layers, evaluation of mammillary alterations was conducted. The majority of lesions fell into the three categories proposed by Bunk and Balloun [1]. Specific changes such as the presence of mammillary bodies, multinucleation, waves in the nucleation zone, and deformation mammillary knobs (cavernous tips) were observed in the EAA eggs of 55-week-old birds (panels E-G in Fig. 1). Specific EAA-associated lesions were also present in the ultrastructure of non-EAA eggs produced by 77-week-old birds, which did not display any gross lesions. In contrast, shell thickness and strength of the non-EAA eggs from 77-week-old birds were normal.

In conclusion, MS infection of 55-week-old birds from the SH farm that produced eggs with EAA was confirmed by serologic tests and MS isolation. Egg quality evaluation and SEM also demonstrated the existence of pathologic changes. The current case study describes the first domestic EAA cases in Korea and revealed the fact that eggs which appeared to recover from EAA are still defective at the ultrastructure level. Further studies are required to fully understand the correlation between MS oviduct infection and EAA.