The study was conducted in 24 layer flocks from 14 different commercial farms, located in and around Namakkal District of Tamil Nadu from April 2016 to March 2017. Birds in all investigated flocks had been vaccinated according to the standard vaccination schedule including Marek’s disease, Newcastle disease, infectious bursal disease, fowl pox, infectious bronchitis and infectious coryza.

During visit to the farm EAA symptoms were explained to the farmer for recognition in the present flock or in some of their past flocks. A flock was considered EAA positive if their egg production had symptoms such as increased fragility of shells, increasing the number of downgraded eggs (rough appearance of the apex of the eggs shelland very fragile eggs).

Data on the flock size, strain, age at which EAA symptoms begun, type of management, vaccination and antimicrobial treatment, mycoplasma monitoring programme during the production cycle of commercial layer flock, morbidity, mortality and previously observed pathologies were collected from the investigated flocks at the time of visit.

Six choanal cleft swabs were collected randomly from live birds of each flock. Necropsy was conducted on dead birds from the EAA investigated flocks. Pooled samples of trachea, air sac, and lung swabs were collected aseptically. The outer surface of the oviduct was seared with a hot scalpel blade; an incision was made with a sterile scalpel and sterile cotton swabs used to swab both the isthmus and the uterus.Swabs taken from the live and dead birds were used for general bacteriology and mycoplasma culture.

Mycoplasma culture was performed as previously described (Marois et al., 2000). The swabs were placed into 2ml modified Frey’s medium and transported to the laboratory then agitated on a vortex mixer for 30 sec and then swabs discorded. Broth medium was incubated under microaerophilic condition with 90 % relative humidity at 37 °C until the phenol red indicator changed from red to yellow (4 - 5 days).

Good eggshell quality is the primary concern of poultry industry and economic viability of the egg industry. If the eggshell has defects on its surface, it is prone to damage, dehydration, and microbial infection. Many factors have been identified as having direct or indirect effects on egg shell quality including strain of chicken, nutrition, management and disease (Roberts, 2004). The eggs examined from 24 commercial poultry flocks from 14 different farms showed shell defect at the apex (pointed end), which was thin, soft and easily broken with a sharp zone of demarcation from the rest of the shell, which appeared normal. These findings were consistent with the previous reports describing EAA (Faberwee et al., 2009a).

In the present study, samples collected from 7 out of 24 flocks showed colour change (red to yellow) in Frey’s mycoplasma broth, which were subjected to PCR for confirmation by specific amplification of 16S rRNA gene (185 bp for Mg and 215bp for Ms). The results obtained by PCR were in agreement with early workers (OIE, 2008) who used a primer pair complementary to 16S rRNA gene designated for the detection of Mg and Ms at 185 bp and 215 bp respectively. Among the 7 flocks 4 (16.6 %) were positive for MS alone and 3 (12.5%) were positive for both Ms and Mg. The results demonstrated overall avian mycoplasma prevalence in EAA affected flocks was 29.1%. These findings concurred with earlier studies that found 27 % (Tomar et al., 2017) and 33 % (Rajkumar et al., 2018) positivity for avian mycoplasma in Indian poultry. Since vaccination against mycoplasma is not currently carried out in commercial layer flocks of Namakkal poultry zone, cultural and molecular confirmation indicate natural infection.

In the investigated flocks, eggs with EAA ranged from 0.66 to 1.35% (Table 1) which was in agreement with Catania et al. (2010) since they also reported EAA incidence of 1.3% in an intensive layer farm in Italy, however Faberwee et al. (2009b) reported 25% in layers housed on the floor in Netherlands and 22.9% in challenged SPF layers (Feberwee et al., 2009a). The low prevalence in the present study might be due to a pure Mycoplasmal infection and absence other factors such as IBV which contributed strongly to a higher number of abnormal eggs (up to 25%) in the earlier studies.

It is not clear how M. synoviae affects the normal eggshell calcification process or why the defect is confined to a distinct zone at the apex of the egg. M. synoviae may affect the composition and concentration of eggshell matrix proteins in the uterine fluid, which are needed for the regulation of the growth of calcite during eggshell calcification (Gautron et al, 1997; Hinke et al, 1999 and 2003). M. synoviae may also affect ciliary motility in the oviduct, which could lead to changes in the uterine fluid content affecting the deposition of calcium carbonate crystals (Dominquez-Vera et al., 2000). Preferential colonization of a specific area of the uterus might also offer an explanation for the localization of the eggshell defects.

The flock size may also affect the prevalence of Mycoplasma by promoting its circulation between birds. Mycoplasma associated egg shell apex abnormalities was noticed in flock size above 10,000 birds (100 %) and absent in flock size below 10,000 birds (Table 1). Highest infection rate in larger flocks might be correlated with poor management and bio-security measures in addition to horizontal transmission of the organisms (Dufour-Gesbert et al., 2006).

Comparing the frequency of mycoplasma associated EAA in different strains (Table 1), results showed that highest incidence in strain 1 (71.4%) comparing to strain 2 (14.3%) and strain 3 (14.3%). This might be due to highest placement of strain 1 (60 to 70%) in Namakkal poultry zone. Among the various age groups of flocks examined 25 to 40 wk showed highest (75%) incidence of EAA (Table 1). Well maintained and disease free commercial layer flocks reach its peak production at 28^th wk (97 per cent) and it will be maintained at above 90 per cent up to 65 wk. It was opined that stress of peak egg production might cause an immunosuppressive effect (Srinivasan et al., 2012) which can facilitate the mycoplasmal infection and might induced EAA in commercial layer chicken.

Birds in the egg shell apex abnormality affected flocks were apparently normal and no evidence of clinical disease was observed. One of the main characteristics of mycoplasmosis, especially the one caused by Ms, is an asymptomatic manifestation, although it induces damages to the infected host’s health, and may even cause suppression of their immune system (Stipkovits and Kempf, 1996).

Mortality, egg production drop and seasonal incidence of EAA in commercial layer chicken were presented in Table 2. Mortality in the affected flocks was ranged from 0.05% to 0.35%. Similar findings have been reported by earlier workers (Srinivasan et al., 2014). Egg shell apex abnormalities affected flocks showed a production drop varied from 2.0 to 7.7 per cent from the expected production as prescribed by the breeder. Santos et al, (2014) observed 10 to 23 % drop in egg production in free range chicken and Sumitha et al. (2017) reported 10 to 20% drop in 60 to 70 wk old flocks affected with EAA.

In the present investigation, highest incidences of EAA was recorded in the months of October and November (2 each) followed by August, September and December (each One). Similar results were published earlier (Srinivasan et al., 2014). Prevalence of rain, cold weather and sudden changes in temperature in the above said months favour the growth and transmission of mycoplasma.

Persistence of egg shell apex abnormalities in the affected flock varied from 9 to 41 days. Present investigation revealed 04 out of the 14 farm revealed EAA like defect in their previous flocks. It has also been reported that infection was more frequent in multiage farm (Kleven, 2008). In our study all the mycoplasma positive flocks were maintained in multiage production complexes within a restricted geographical area which facilitated persistence of infection in above said flocks and transmission to subsequent flocks by horizontal transmission (Stipkovits and Kempf, 1996; Kleven, 2008).

Investigated flocks were periodically treated with antimycoplasmal agents such as tiamulin and oxytetracyclin through feed as recommended by the poultry health consultant. Although medication can reduce the clinical signs and economic impact of mycoplasma infection, it cannot completely eliminate mycoplasmal infection (Kelvin, 2008) and birds become persistently infected with mycoplasma and remains carrier for life. This can be one of the reasons for inability to eradicate mycoplasma in commercial layer flocks that stay a long period in rearing and production sites. In Namakkal poultry zone, commercial layer flocks were not routinely monitored for avian mycoplasmosis during their production cycle. A consistently applied monitoring system is essential for prevention of mycoplasma infection.

The present study revealed 0.66 to 1.35 per cent of eggs produced showed egg shell apex abnormalities (EAA) in commercial layer chicken. Among the mycoplasma positive flocks M. synoviae in four and Ms and Mg as a concurrent infection in 3 flocks were detected by PCR. EAA was most commonly noticed in the age group of 25 to 40 wk (75%) of age. EAA affected flocks showed 2.0 to 7.7 per cent drop in production and 0.05 to 0.35 per cent mortality without any clinical signs. Hence poultry health keepers should be aware of the EAA-like presentations and consider the syndrome in the differential diagnosis of eggshell quality problems in layer flocks. The early diagnosis could help to decrease economic losses caused by these infection.These findings underline the increasing importance of M. synoviae in commercial layer chicken despite the absence of clinical signs in infected birds.

The research work is funded by the Science and Engineering Research Board, Department of Science and Technology, Government of India. Authors are also thankful to Tamil Nadu Veterinary and Animal Sciences University for providing necessary facilities to conduct the research.