Windows server 2008 slow network issue

Perplexing question as to what has happened.

Situation is 3 windows 2003 servers (32bit) one was DC, one was a TS and the other was a ISA (& default gateway). Introduced 4 new boxes. One new DC W2k8 , 2 W2k8 servers acting as virtual hosts for 4 virtual servers each and one w2k3 box running isa 2006. All network connections a gigabit.

All new servers are 64 bit apart from the one for the ISA server.

Transferred all DC roles to new DC and network speed was fine before the default gateway was switched from old isa server to new isa server then the following has occurred.

File transferred from new DC to any other server (except new isa) fine (500mb in approx ten seconds). Files transferred between any virtual server and another virtual server fine with the same speeds as file from the DC. Files transferred from new ISA server to any other server except new DC also fine.

However files transferred to new DC or new isa from any other server crawl. Transferring the same 500mb takes about 10 minutes.

I have already tried:
disable receive-side scaling
disable chimney offload state
disable receive window auto tuning
disable offloading in the network adapter properties
disable ipv6 in the network adapter

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