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Molecular analysis was conducted on 36 Mycoplasma gallisepticum DNA extracts from tracheal swab samples of commercial poultry in seven South African provinces between 2009 and 2012. Twelve unique M. gallisepticum genotypes were identified by polymerase chain reaction and sequence analysis of the 16S-23S rRNA intergenic spacer region (IGSR), M. gallisepticum cytadhesin 2 (mgc2), MGA_0319 and gapA genetic regions. The DNA sequences of these genotypes were distinct from those of M. gallisepticum isolates in a database composed of sequences from other countries, vaccine and reference strains. The most prevalent genotype (SA-WT#7) was detected in samples from commercial broilers, broiler breeders and layers in five provinces. South African M. gallisepticum sequences were more similar to those of the live vaccines commercially available in South Africa, but were distinct from that of F strain vaccine, which is not registered for use in South Africa. The IGSR, mgc2 or MGA_0319 sequences of three South African genotypes were identical to those of the ts-11 vaccine strain, necessitating a combination of mgc2 and IGSR targeted sequencing to differentiate South African wild-type genotypes from ts-11 vaccine. To identify and differentiate all 12 wild-types, mgc2, IGSR and MGA_0319 sequencing was required. Sequencing of gapA was least effective at strain differentiation. This research serves as a model for the development of an M. gallisepticum sequence database, and illustrates its application to characterize M. gallisepticum genotypes, select diagnostic tests and better understand the epidemiology of M. gallisepticum.

21Low Atmospheric Pressure Stunning (LAPS) is a novel approach to pre-slaughter 22 stunning of chickens using progressive hypobaric hypoxia by the application of gradual 23 decompression (280s cycle) according to a set of prescribed pressure curves. LAPS 24 produces a non-recovery state. Concerns have been raised relating to the possible 25 pathological and welfare consequences of expansion of air in the body during LAPS. 26In a randomized trial we compared the gross pathology of broilers exposed to LAPS 27 with a control group euthanized by intravenous injection of pentobarbital sodium (60 28 mixed sex broilers per treatment). The birds were exposed to each treatment in triplets 29 and all birds were subject to necropsy examination to detect and score (1 to 5, minimal 30 to severe) haemorrhagic lesions or congestion for all major organs and cavities (e.g. 31 air sacs, joints, ears and heart) as well as external assessment for product quality (e.g. 32 wing tips). Behavioural data (latency to loss of posture and motionless) and chamber 33 cycle data (temperature, humidity, pressure and oxygen availability) confirmed that 34 LAPS had been applied in a manner representative of the commercial process. All of 35 the organs observed were structurally intact for both treatment groups. No lesions 36were observed in the external ears, oral cavity, tracheal lumen, crop, and air sacs of 37 birds from either treatment group. There was no difference between treatments in the 38 wingtips, nasal turbinates, thymus, biceps femoralis, and colon. Haemorrhagic lesions 39 were observed in the calvaria, brains, hearts and lungs of both treatment groups, but 40 lesions in these areas were more severe in the LAPS treatment group. It was not 41 possible to distinguish between pathological changes induced by decompression or 42 recompression. In the barbiturate group, more severe haemorrhagic lesions were 43 observed in the superficial pectoral muscles as well as greater congestion of the 44 infraorbital sinuses, liver, spleens, duodenum, kidneys and gonads. These findings 45 provide evidence that LAPS did not result in distension of the intestines and air sacs 46 sufficient to cause changes, which were grossly visible on post mortem examination. 47There was also no evidence of barotrauma in the ears and sinuses. The pathological 48 changes observed in the barbiturate treatment were as expected based on barbiturate 49toxicity. LAPS appears to produce pathological changes by a variety of well-50 established mechanisms, and while these pathological data have limited value as 51 welfare indicators, the results confirm that organ integrity was not compromised by the 52 process. 53 54

Live Mycoplasma gallisepticum vaccines are used for the control of respiratory disease, egg production losses and egg transmission associated with M. gallisepticum infection in long-lived poultry. The first field case of apparent increased virulence and vertical transmission of ts-11, a live M. gallisepticum vaccine, has been reported. In that study a M. gallisepticum isolate from the broiler progeny of ts-11-vaccinated breeders was genotyped as ts-11 by sequence analysis of four different genetic targets and Random Amplified Polymorphic DNA and found to be significantly more virulent than ts-11 vaccine. The objective of the current study was to evaluate the rate of egg transmission and pathogenicity of ts-11 vaccine and isolates recovered from ts-11-vaccinated breeders (K6222B) and their broiler progeny (K6216D) which had been genotyped as ts-11. Groups of 28-week-old specific pathogenfree chickens at 87% average weekly egg production were inoculated with sterile broth media (negative controls), ts-11 vaccine, K6222B, K6216D or R strain (positive controls) by eye-drop and aerosol. K6216D transmitted via the egg at an average rate of 4.0% in the third and fourth weeks post-infection, while egg transmission of K6222B and ts-11 vaccine was not detected. M. gallisepticum was isolated from the air sacs, ovaries and oviducts of hens infected with K6216D and K6222B, but not from those infected with ts-11 vaccine. K6216D and K6222B both induced respiratory signs and significantly more tracheal colonization and more severe tracheal and air sac lesions than ts-11 vaccine (P ≤ 0.05). There were no substantial differences in the egg production of ts-11, K6216D and K6222B infected groups. These results provide the first conclusive evidence of transovarian transmission of an isolate genotyped as ts-11 and indicate that isolates genotyed as ts-11 vary in their virulence and ability to transmit via the egg.